This thesis covers a range of important issues in hair analysis and includes 27 scientific works in which the name of the candidate was either listed as the first author or as the major contributor. The work presented in this thesis involved the development of a series of analytical methods to detect trace amounts of drugs in hair and also investigated the mechanisms by which drugs may be incorporated into hair. The major areas covered in this study can be summarized as follows: 1. The methods for the identification and quantification of opiates, amphetamines, ketamine, cannabis, cocaine, benzodiazepines, antidepressants, antipsychotics, and anabolic steroids in hair were developed using gas chromatography–mass spectrometry (GC-MS), liquid chromatography–tandem mass spectrometry (LC-MS/MS) and gas chromatography–tandem mass spectrometry (GC-MS/MS). With GC-MS methods, the limits of detection were 0.1-0.5 ng mg-1 of hair for antidepressants and antipsychotics. For illegal drugs, hair specimens were analyzed by GC-MS with limits of detection of 0.02-2ng mg-1. GC/MS/MS is more sensitive than GC-MS to detect these drugs in hair. The lower limits of detection ranged from 0.001 to 0.020 ng mg-1 for 21 anabolic androgenic steroids and their esters in hair using liquid chromatographic-tandem mass spectrometric method. And the limits of detection ranged from 0.2 to 5 pg mg-1 for benzodiazepines in hair. Tandem mass spectrometry is characterized by its sensitivity, selectivity and specificity, which makes it particularly suitable for the analysis of trace amount of target analytes in hair. 2. Usually, screening for drugs of abuse is the first step in clinical and forensic toxicology. There are a large number of controlled substances and doping agents and novel compounds, which have yet to be characterised. A series of screening methods for drugs of abuse in hair were developed using LC-MS/MS and GC-MS/MS. Using our own library of MRM transitions, the optimum collision energies selected for each transition and retention times were set up. These methods have been applied successfully in forensic casework. 3. Of growing importance to the field of hair analysis is the detection of metabolites related to the parent drugs. Demonstrating the presence of a metabolite of a drug (such as, heroin, amphetamines, cocaine, meperidine, ketamine, triazolam or psychotropic drugs) provides compelling evidence for exposure to the parent drug, and permits distinction between external contamination from ingestion and facilitation of the interpretation of results. The presence of antidepressant and antipsychotic drugs and their metabolites in the hair of psychiatric patients was investigated using GC-MS-EI and GC-MS-PCI. The parent drug and its major metabolite, such as opiates (morphine, 6-acetylmorphine), methamphetamine (methamphetamine, amphetamine), ketamine (ketamine, norketamine), cocaine (cocaine, benzoecognine), meperidine (meperidine, normeperidine), triazolam (triazolam, α-hydroxytriazolam), and clonazepam (clonazepam, 7-aminoclonazepam) were quantified in authentic hair samples simultaneously. The differences were finding in the ratio of parent drug to metabolite. For illegal drugs, the concentrations of parent drugs were higher than that of their metabolites. The results of triazolam and clonazepam were contrary. These data are suitable reference values and are the basis for the interpretation of results. 4. The mechanisms by which drugs are incorporated into hair are not fully understood. Based on experiments with guinea pigs with black, white, or brown hair, the mechanisms of incorporation of cocaine, methamphetamine, ketamine, triazolam and anabolic steroids into hair were investigated. The concentrations of drugs in hair were found to be related to physicochemical properties of drugs. The parent drugs were the predominant analytes in hair. There was an obvious relationship between the concentration of drugs in hair and hair pigmentation. The concentrations of drugs deposited in black hair was found to be higher than that in brown and white hair samples, even when comparing results using hairs on the same multicoloured animal body. This work confirmed that melanin affinity is a governing factor in drug incorporation into hair shafts. These studies on the distribution of drugs in the hair shaft and how their concentration changes along the shaft provide information relevant to the time of ingestion and substance use/abuse. 5. In recent years an increase in drug-facilitated sexual assault (DFSA) has been reported. Segmental hair analysis has proved useful in widening the window of detection, as blood and urine analyses are of limited use, due to the long delays between the actual assaults and obtaining samples from suspects that are frequently encountered in investigations of such crimes. In China, benzodiazepines are the most frequently observed compounds in cases of drug-facilitated crime. In a paper reported here, 14 volunteers ingested a single 1-6 mg estazolam tablet to permit the evaluation of segmental hair analysis after a single drug dosage. Hair was collected one month after administration of the drug. All the proximal segments tested positive for estazolam. With increased dosage, estazolam could be detected in the 2-4 cm segments nearest the hair root in some subject’s hair shafts. In some cases, the 4-6 cm segments also tested positive. Hair analysis was applied to samples from two authentic criminal cases. A significant variation was observed between those obtained from previous studies and the results presented here. The intersubject variability in segmental analysis can be explained mainly due to melanin content and diffusion from sweat or other secretions during formation of the hair shaft. However, more substantial procedural and interpretation guidelines are required to use segmental hair analysis in drug-facilitated crimes. On the other hand, the minimal dosage for detection, which is a critical but previously unknown threshold value of fundamental importance in hair analysis, was determined for triazolam and ketamine in guinea pig hair. 6. Doping with endogenous anabolic steroids is one of the most serious drug issues in sports today. The measurement of anabolic steroid levels in human hair permits the distinction between pharmaceutically produced steroids and naturally occurring steroids. Full-length hair samples were taken at the skin surface from the vertex of 39 males, 30 females and 11 children from China. None of the subjects were professional athletes. Testosterone and dehydroepiandrosterone were detected in all the hair segments. The physiological concentrations of testosterone were in the range 0.8-24.2 pg mg-1, 0.1-16.8 pg mg-1 and 0.2-11.5 pg mg-1 in males, females and children, respectively. However, the mean values of dehydroepiandrosterone were much higher than those for testosterone. This is the first investigation into the physiological concentrations of anabolic steroids in human hair in Chinese subjects. These data provide suitable reference values and form the basis for the interpretation of results from investigations into the abuse of endogenous anabolic steroids. In conclusion, the work presented in this study demonstrates that there was a good correlation between the concentration of drugs in hair and drug dosage. There was an obvious relationship between hair drug concentration and hair colour. Melanin affinity is shown to be a governing factor in determining drug incorporation into hair, and the concentration of drugs deposited in black hair was found to be higher than that in brown and white hair samples. This thesis provides data that will be useful in the application of hair analysis regarding drugs of abuse and in the interpretation of toxicological results.

Heat shock protein 90 (Hsp90), a highly conserved molecular chaperone, has been proposed to play a vital role in tumorigenesis. Hsp90 has two isoforms, of which Hsp90α is the major isoform of the Hsp90 complex and has an inducible expression profile. The molecular chaperone Hsp90α has been recognized in different cancers and it is implicated to play a role in cell cycle progression, apoptosis, regulates invasion, angiogenesis and metastasis. It is being recognized as a promising target in cancer treatment. Previous studies in our laboratory have demonstrated hsp90α expression in both primary glioma tissue and cell lines, but not in normal healthy brain tissues and cell lines. Enhanced chemosensitivity was observed upon specific inhibition of hsp90α expression by siRNA, suggesting that inhibiting hsp90α expression could possibly be a favourable therapeutic approach compared to conventional chemotherapies. In this novel study, Hsp90 was inhibited by either treatment with 17AAG or shRNA oligonucleotide targeting hsp90α (shhsp90α) in the U87-MG glioma cell line. The inhibition profile of Hsp90α was observed at the protein levels in control and treated cells by FACS analysis (quantitative) using a flow cytometer and Hsp90α ELISA kit. The results demonstrated a significant reduction of Hsp90α protein levels post treatment with 17AAG and shhsp90α. The activity of Hsp90α was assayed by quantifying the levels of Akt/PKB in the samples. Significant reductions (>50 %) of Akt/PKB levels were observed post hsp90α inhibition. Cell cycle analysis carried out reported S and G2 phase arrest, post Hsp90 inhibition by either 17AAG or shhsp90α. Interestingly, it was reported that 17AAG shows a better silencing profile compared to shhsp90α. To analyse the downstream effects of Hsp90 inhibition and to determine the client proteins affected, proteomic analysis was performed. Proteomic analysis identified several proteins which were either upregulated/downregulated post Hsp90 inhibition. IPA analysis further identified “cancer” as the top network significantly transformed post Hsp90 inhibition. Upregulated proteins include Hsp70 family members, Hsp27 and gp96, thereby suggesting the role of Hsp90 co-chaperones in compensating for Hsp90 function post Hsp90 inhibition. Moreover, members of the glycolysis/glucogenesis pathway were also upregulated, demonstrating increased dependency on glycolysis for energy supply by the treated glioma cells. Considering Hsp70 and its role in anti-apoptosis, it was postulated that a combination therapy involving a multi-target approach could be carried out. Subsequently, inhibition of both Hsp90 and Hsp70 in U87-MG glioma cell line was carried out resulting in 60 % cell death along with S and G2 phase arrest. Thus, in the effective treatment of glioma, the inhibition of multiple targets needs to be taken into consideration. Conclusion: It can be thus concluded that, combination therapy involving silencing of Hsp90 and Hsp70 could be of possible significance in glioma therapy.

Chromium(VI) is a reactive and highly toxic pollutant species which is present in certain industrial effluent streams as well as sea water and polluted ground water. Chromium(III), however, is relatively inert and less toxic. Conventional methods used to eliminate Cr(VI) from aqueous phase include its reduction to Cr(III) at acidic pH by reaction with strong reducing agents such as thiosulphate, FeSO4 and SO2, followed by precipitation as hydroxide in alkaline media. However, this procedure is not suitable for Cr(VI) elimination in dilute aqueous solutions. Thus, semiconductor photocatalysis has been studied as a possible alternative. Photocatalysis is the process of using light to promote catalysis of reactions and normally involves the photoexcitation of a semiconductor catalyst. During photocatalysis, light of a wavelength corresponding to an energy greater than that of the band gap, Eg, of the material is incident on the catalyst, resulting in electrons being excited from the semiconductor valence band to its conduction band. These electrons can then reduce the Cr(VI) to Cr(III). The semi-conductor used consists of nanoparticulate iron oxide embedded in a clay matrix. Clays are micro-crystalline layered minerals. This gives rise to interlayer spaces in which the iron oxide is ‘grown’. The clay structure restricts the growth of the iron oxide, resulting in nano-particulate sized semiconductor particles. To monitor the changes in Cr(VI) concentration, a new, stable potentiometric method has been developed that involves the successful use of a gold electrode to measure Cr(VI) concentration, that has to date, not been reported elsewhere in literature. Results show that the nanocomposite does photo-reduce Cr(VI) to Cr(III) and does photo-oxidise ethanol. Modelling of the time dependence of the measured (photo-induced) potential has allowed for the extraction of key rate parameters for the Cr(VI) reduction process with a view to system optimisation.

Microporous materials play an important role in a variety of industrial and domestic applications. While a diverse range of microporous materials have been identified, this thesis focuses on porous organic cages (POCs) because they have received much attention as synthetically tunable, solution processable, microporous materials. After introducing the latest developments in POC synthesis and the general application of microporous materials as selective sorbents, this thesis presents three developments in organic cage chemistry: a high-throughput workflow for the discovery of POCs, which yielded a novel organic cage compound; the measurement of selective adsorption by POCs, wherein the first instance of chiral selectivity by a POC was recorded; and the first instance of applying POCs as stationary phases for gas chromatography, which produced columns that separate racemic mixtures, alkylaromatic isomers, and alkane isomers. Chapter 2, discovering novel organic cages, presents attempts to use high-throughput and in-silico techniques to accelerate the discovery of novel organic cages. These methods were utilised to isolate a novel organic cage, CCX-S, which is characterised and discussed. Chapter 3, organic cages as selective sorbents, presents the development of approaches for measuring selective adsorption. These methods were used to identify the first reported instance of enantioselective adsorption by an organic cage. Further measurements to explain this separation behavior are also presented. Chapter 4, chromatographic separations with organic cages, presents one method of practically leveraging the presented separation behavior. In Chapter 4, the coating of capillary columns with CC3 is presented. These columns were used to successfully perform gas chromatographic separations, the first recorded instance of using a POC to do so. The columns were further improved by modifying the coating method and using prefabricated CC3 nanoparticles. This modification enabled difficult separations to be performed using the column; for example, the separation of hexane’s five isomers.

Nano-biotechnology combines recent advances in nanotechnology with biology. It is a relatively new discipline and full of promise. One such promise is the elucidation of complex bio-molecular reactions and interactions, the elucidation of which requires the development of reliable in-vitro models. Such models could be developed through the use of self-assembled monolayer’s (SAMs). Research into this competitive field has already started and there is currently a call to develop SAMs which present specific bio-molecules in a switchable fashion; switchable SAMs can have their surface properties switched between two states, i.e. they can be switched ‘on’ or ‘off’. Such switch-ability would help such models mimic the real time changes of the bodies’ bio-chemistry and is a vital development. This thesis addresses this current research need, through the employment of azobenzene based SAMs. Currently the switch-ability (isomerisation) of numerous azobenzene SAMs has been shown to be hindered by a lack of inter-surfactant space. This hindrance to isomerisation is addressed in Chapter 4. While Chapter 5 explores the design of an azobenzene based photo-switchable SAM, for use as in-vitro model for the study of bio-molecular interactions. The two chapters are not directly related and future work would aim to bring the findings together.

Multi-component crystallisation is investigated as a route to controlling crystalline forms of selected materials that possess pharmaceutical properties. This includes investigating the use of co-crystallisation methodology to selectively crystallise metastable polymorphs and solvated forms of these materials. This differs from the conventional use of co-crystallisation, as the aim of this aspect of the investigation is not to obtain a molecular complex of the two components, but instead for them to crystallise independently, while one component perturbs the solution environment to direct the crystallisation of the second component towards a different, often metastable, polymorph (or solvate). This co-crystallisation methodology is used as a route to crystallising new or elusive polymorphs (or solvates) of the active pharmaceutical ingredients paracetamol, piroxicam, gallic acid monohydrate and piracetam. It is also demonstrated that the use of this method can lead to crystal forms with otherwise unobtainable structural features. Co-crystallisation is also investigated as a route to controlling the ionisation state of piroxicam in the formation of molecular complexes. Molecular complexes were formed with a number of mono-substituted benzoic acids as well as with nitrogen-heterocycles and strong acids. In the molecular complexes formed, piroxicam was found to adopt the non-ionised, zwitterionic, anionic or cationic form, depending on the co-former used. Attempts are made to rationalise the occurrence of each ionisation state by consideration of the relative pKa values of piroxicam and the co-formers. The hydrogen bonded supramolecular synthons in these molecular complexes are also investigated. Co-crystallisation is also used as a route to obtaining molecular complexes of paracetamol and its derivative, 4-acetamidobenzoic acid, with nitrogen-heterocycles as co-formers. Molecular complexes of the two, with similar co-formers, are compared in terms of their hydrogen bonded supramolecular synthons. Despite having otherwise similar structural features, the phenolic hydroxyl group in paracetamol and carboxylic acid group in 4-acetamidobenzoic acid result in the formation of very different synthons and in some cases different component ratios. The susceptibility of 4-acetamidobenzoic acid to deprotonation is found to play a major role in the differences observed. Molecular complexes of paracetamol with co-formers containing multiple carboxylic acid groups are also investigated, with a view towards further crystal engineering approaches for molecular complexes of paracetamol. Piracetam complexes with carboxylic acids are investigated in a similar manner. The potential for transfer of a range of these multi-component crystallisations into a non-evaporative environment, with a view to implementing continuous crystallisation approaches, is also investigated. This transfer is found to be challenging for the systems investigated.

The success of forensic investigation very often depends on the establishment of the correct timeline of events. In the investigation of fatalities, this depends greatly on the estimation of the time since death of the victim. Current methods lead to inaccurate results and depend greatly on the experience of the investigator. Pathologists estimate the time since death based on visual inspection of the bodies as well as body temperature measurement. Only very short post-mortem intervals (PMIs) can be evaluated with some degree of certainty. This investigation used untargeted proteomic and metabolomic approaches to identify potential molecular markers (proteins, metabolites) which could help to quantify post-mortem changes and aid PMI estimation. Animal models were used in the initial stages of the project. Aged beef meat (stored at 4°C for 13 days) and rat muscle samples (intact cadavers stored at ambient temperature for 3 days) were sampled at 24 h time intervals. In the final stages of the project, human tissue samples were collected at the Forensic Anthropology Centre at Texas State University (San Marcos, Texas). Muscle samples were collected at various times post-mortem from 6 different subjects over the period of two weeks. For the proteomics experiment, 0.5g of tissue was homogenized in extraction buffer consisting of urea, thiourea and 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate (CHAPS). Protein separation was carried out using two-dimensional gel electrophoresis. Protein identification was performed using liquid chromatography-tandem mass spectrometry. For the metabolomics experiment, 0.5g of tissue was homogenized in chloroform/methanol/water solution. The extracted samples were analysed using liquid chromatography-mass spectrometry as well as gas chromatography – mass spectrometry. The investigation allowed the identification of potential biomarker candidates. The proteins of interest varied between the sampled mammals. However, myosin and actin appear as promising candidates for all three species. The metabolomics experiments yielded a large number of possible biomarker candidates. Both liquid and gas chromatography approaches were successfully applied, pointing towards various compounds. Proteogenic amino acids were identified as main compounds of interest in all species using both methods. The study has shown that both proteomic and metabolomic approaches can be successfully applied in forensic medical science and can help to find PMI markers. Using the untargeted approach gives the advantage of looking at a whole range of detected molecules and choosing the most appropriate ones for the task. Furthermore, the combination of these two approaches gives a deeper insight into the post-mortem biological processes. The biomarker candidates proposed in this study require further validation in a larger cohort of subjects.

Since its first use to solve the structure of sodium chloride in 1915 X-ray crystallography has developed significantly to become the premier technique for obtaining 3D structural information of small molecules and macromolecules alike. As the technique continues to develop and focus its attention on weak diffraction from the likes of micro-crystals and poorly packed crystals of membrane proteins and large protein complexes; as well as ultra-high resolution data and weak anomalous signal from native atoms, data quality is becoming more and more important. Data quality is particularly important in the wake of long wavelength macromolecular crystallography (MX) for phasing using anomalous signal from native sulphur and phosphorous atoms in proteins and DNA. This thesis first investigated the use of a new sample handling technique using a humidity controlled stream to preserve macromolecular crystals while excess surrounding solvent is removed (Chapter 2). Following the successful development of this technique the effects of excess surrounding solvent on data quality was assessed when collecting at standard MX X-ray wavelengths (~ 1 Å) and longer X-ray wavelengths (~ 2 Å). Datasets were collected from large populations of control and test crystals at standard and longer wavelengths to allow robust statistical methods to be applied; a practice not widely adopted in method development studies in X-ray crystallography. This made it possible to assess the small differences in data quality in the presence and absence of excess surrounding solvent. The effects of surrounding solvent at longer wavelengths appear to be protein dependent with some proteins tested showing no significant difference and others a significant decrease in data quality at longer wavelengths (Chapter 3). Originally this project aimed to use the new long wavelength in-vacuum MX beamline, I23, at Diamond Light Source UK to carry out phasing experiments using native sulphurs for structure solution. However, the considerable complexity involved in developing in-vacuum MX meant these experiments could not be carried out during the time frame of this thesis. Chapters 4 and 5 outline the production of a novel cancer protein (cancerous inhibitor of protein phosphatase 2A) and two protein targets from the Achromobacter xylosoxidans (Ax) genome intended for sulphur single wavelength anomalous dispersion phasing experiments on I23. Of these proteins the structure of Ax-α/β hydrolase was solved by conventional methods, the structure of which is discussed in Chapter 5. Of the protein crystals used in long wavelength data quality experiments in Chapter 3 the molecular biology of PA3825-EAL, a biofilm regulating protein essential to the swarming ability of Pseudomonas aeruginosa, was investigated further. The crystal structure of PA3825-EAL was solved in the resting, substrate bound and product bound states to high resolution. Comparison of the crystal structures of monomeric and dimeric PA3825-EAL with the inactive dimeric structure of MucR-EAL suggests dimerisation via helix 8 plays a role in inhibition of EAL domains. Prior to this, dimerisation was thought to be an activating factor in EAL domains. The product bound state of PA3825-EAL showed the presence of a previously unreported third metal binding site which may form an essential component of the reaction mechanism of EAL domains. Inability of MucR-EAL to incorporate this third metal due to dimerisation may explain the lack of activity despite possessing the conserved catalytic residues necessary. The fast detector technology and improvements in automated data processing software that allowed diffraction data for large populations of crystals to be collected in Chapters 2 and 3 have also been applied to development of a serial data collection technique. Of 159 datasets collected from 8 crystals of a copper nitrite reductase from Achromobacter cycloclastes, 45 datasets from a single crystal were analysed to observe the reaction mechanism using high resolution crystal structures. X-ray radiolysis initiated the reaction and high resolution data allowed the conversion of nitrite (NO2) to nitric oxide (NO) to be observed in the crystal. Other aspects of the reaction were investigated from the data series including a conserved water chain connecting the copper sites which may act as a proton wire to donate a proton and produce NO. This technique may have wide applications to the study of the reaction mechanisms of other metallo-proteins.

The transition from a fossil fuel-dependent society to a cleaner, more sustainable society will not be possible without renewable energy sources. Hydrogen holds great potential as an energy carrier as an alternative to fossil fuels in such society. However, the compact and safe storage of hydrogen are still major challenges. Solid state hydrogen storage offers the possibility to store hydrogen in solids offering high volumetric and high gravimetric energy densities, while reducing the risks associated when handling hydrogen gas. However, no single system has fully achieved the required properties for on-board mobile applications. Various approaches can be adopted with the aims of improving the kinetics and thermodynamics of hydrogen sorption. The nanostructuring of materials is one of the more promising strategies to achieve these aims. Reduction of the particle size of hydrides by nanoconfinement in forms of porous matrix leads to an increased surface area of the active material, and shorter diffusion distances for hydrogen atoms or ions to travel in the solid state. Kinetic barriers can be overcome and thermodynamics manipulated. An enhanced dehydrogenation rate and a reduced dehydrogenation temperature can be achieved by impregnating metal hydrides into porous scaffolds. Two complex hydrides are selected for study in this work; LiAlH4 and LiNH2. LiAlH4, is the lightest of the alanates, with a theoretical hydrogen storage capacity of 10.5 wt.%, and 7.9 wt.% H2 evolved below 220 °C. LiNH2 mixed with LiH, as part of the Li-N-H system, can reversibly desorb/uptake 6.5 wt.% H2 at 300 °C. When LiNH2 is heated alone, it releases ammonia (which is decomposed to N2 and H2 at higher temperatures > 400 °C). In this work, LiAlH4 has been impregnated in different types of commercial and synthesised porous carbon scaffolds for the first time. Nanoconfinement of the active material was achieved using solution impregnation with diethyl ether as a solvent. Analogously, the confinement of LiNH2 in porous carbon was achieved “in-situ” using lithium-ammonia solutions. Both confined composites showed lower dehydrogenation temperatures in comparison with the respective bulk materials. The influence of the design of the carbon scaffold (as manifested for example, by the surface area and the pore volume and pore size distribution) on the dehydrogenation behaviour of the impregnated complex hydrides is demonstrated. By judicious selection of an appropriate porous host, we show how it is possible to induce faster H2 desorption and substantially reduce the desorption temperature. The onset of hydrogen release for confined LiAlH4 decreased significantly in temperature, being reduced by 51 °C (in both porous hosts used, AX-21 and FDU-15) in comparison with pristine LiAlH4. The temperature at which the hydrogen release was maximised was also lowered (by 16 °C in FDU-15 and by 26 °C in AX-21) in comparison with as-received LiAlH4. The confined LiNH2 showed a much earlier release of hydrogen compared with as-received LiNH2. Normally LiNH2 would thermally decompose to Li2NH with ammonia evolution, but ammonia release was eliminated for the confined sample. Reaction with carbon led to irreversible Li2CN2 formation and hydrogen evolution. A set of experiments to establish the formation of Li2CN2 with physically mixed samples were performed. The physically mixed samples showed hydrogen release between 400 - 450 °C, producing a mixture of Li2NH and Li2CN2, suggesting two decomposition pathways were followed. In contrast, confined LiNH2 released hydrogen ca. 220 °C lower than the physically mixed sample, with no detectable trace of ammonia release.

Cardiovascular disease (CVD) is the most common cause of death worldwide, accounting for 31% of the annual deaths per year. The term cardiovascular disease refers to many different disorders of the heart and blood vessels, the most prolific of which is coronary heart disease (CHD). The root cause of CHD is atherosclerosis, which is the build-up of plaque in the intima of the arteries, restricting the blood supply which reaches the heart. If left untreated, the plaques lining the vessel can rupture and cause thrombosis and myocardial infarction (MI). Taking into account an individual’s risk-benefit ratio, a suitable treatment is considered with the aim to prevent MI from occurring. The two main revascularisation strategies are coronary artery bypass graft (CABG) and percutaneous coronary intervention (PCI), the latter being the most frequently used; within the UK 80% of revascularisation is performed by PCI. A PCI procedure is non-invasive and involves the deployment of an intravascular stent into the diseased vessel, which acts as a permanent scaffold that mechanically re-widens the vessel. However, despite the prolific use of intravascular stents within the clinic, there are significant problems associated with this revascularisation technique, such as in-stent restenosis (ISR) and late stent thrombosis (LST). ISR is the re-narrowing of arteries after stent deployment, characterised by a neointima growth within the lumen of the vessel. ISR is a significant clinical problem, because it can lead to repeat revascularisation procedures and often patients present frequently with unstable symptoms, fulfilling the criteria for an MI diagnosis. ISR occurs as a complex wound healing response caused by the stent deployment. Denudation and tearing of the endothelium and the mechanical stress endured by the vascular smooth muscle cells (VSMC) is a stimulus for hyperplasia. VSMC de-differentiate from the contractile to synthetic states and proliferate and migrate into the lumen of the vessel, secreting ECM which forms the bulk of the neointima. The neointima growth will continue to form until the endothelium has re-established itself. As a treatment to prevent ISR, drug-eluting stents (DES) were introduced which were coated with anti-proliferative drugs, to prevent ISR from occurring. DES proved to be very successful at preventing ISR; however elevated rates of LST were associated with these stents. This elevated event rate for DES by LST is thought to be caused as a result of delayed re-endothelialisation and by inflammatory reactions to the polymer used in the coating of DES. The aim of this study was consequently to investigate methods for delivery and coating of novel therapeutics, adenoviruses and miRNA onto stent surfaces. This would allow investigation into the prevention of ISR using these therapeutics, taking advantage of the localised stent setting. It was hoped that local delivery would provide significant advantages over systemic delivery by decreasing the dosage required, avoiding systemic side-effects and increasing delivery to the targeted area, thereby enhancing the therapeutic effect. For the development of coating methods for adenovirus eluting stents, the majority of the work conducted was done using Ad5 as a model virus vector. Three different methods were investigated and evaluated in vitro; deposition onto polyelectrolyte multilayer (PEM) surfaces, by direct conjugation with a modified poly(lactic acid) (PLA) surface through covalent bond formation and collagen gel entrapment. The development of coating methods for miRNA eluting stents focussed initially on collagen gel entrapment; however it was discovered that direct application onto a PLA surface provided a system whereby excellent delivery of the miRNA could be achieved. This methodology was extensively investigated and evaluated in vitro from stent material surfaces, and in vivo in both the porcine and murine stenting models. The results presented here have extended current methodology for both miRNA and adenovirus eluting stents. To the best of our knowledge, this is the first time that miRNA eluting stents have been used in these animal models and therefore contributes significantly to the field of miRNA-based therapeutics.