The physical properties and reactivity of 2,3-dihydro-1H-imidazo[1,2-f]phenanthridinium cations (DIPs) and analogous heterocycles have been investigated. The adapted syntheses developed were applied to a range of aryl amines and more elaborate substrates, such as bifunctional aminoquinolinium cations and amino functionalised polyoxometalates (POMs). 1,2,3,12b-tetrahydroimidazo[1,2-f]phenanthridines (TIPs), an intermediate in the 3-step cascade synthesis of DIPs, have also been isolated. Derivatisation of the TIP structure at the imidazo-N position enables control of the reactivity of the intermediate with respect to electronic potential and pKa, allowing isolation of a selection of TIP structures. Correlations between these parameters and reaction outcome have been made and other influences such as steric and solvent effects have also been investigated. Investigation of the structure and properties of the TIP framework led to the discovery of a pH dependent cyclisation between the ring-closed TIP form and the ring-open aminoethylphenanthridinium (AEP) form. The complementary TIP and AEP forms can be further manipulated by oxidation or reduction to convert them to their “pH-inert” forms, DIP and aminoethyldihydrophenanthridine (AEDP). These four interchangeable states formed the basis of a redox “lockable” molecular switch that could be useful for molecular level information processing and data storage. The pH dependent cyclisation between the TIP and AEP forms was also investigated as a targeting mechanism for potential intercalating antitumour agents. The system looks to exploit the pH difference between cancerous tissues and normal tissue to combine high cytotoxicity with high selectivity, a desirable trait that is unfortunately absent in most chemotherapeutic agents. Preliminary in vitro assays have shown the TIP/AEP frameworks to have IC50 values in the pM range in human ovarian cancer cell lines, comparable to cisplatin and the closely related DIP intercalaters.

The human metabolism and biotransformation of arsenic were studied by the analytical speciation of urinary arsenic excreted by volunteers in a series of laboratory-controlled experiments, the general population and several groups subject to enhanced environmental or occupational exposure to inorganic arsenic. In additon, the post-depositional remobilisation of arsenic in the aquatic environment was investigated by the establishment of the chemical forms of arsenic in the sediment porewaters of Loch Lomond and the Dubh Lochan and in the sediments of nearby Scottish sealochs. Total arsenic concentrations were measured by hydride generation-atomic absorption spectrometry (HGAAS) following acid digestion of human urine and biological material and dry ashing of sediment samples. Urine samples were rapidly directly screened for the approximate sum of the hydride-forming species arsenate (As(V)), arsenite (As(III)), monomethylarsonic acid (MMAA) and dimethylarsinic acid (DMAA), which were subsequently separated and accurately determined by ion-exchange chromatography/HGAAS, a procedure uninfluenced by the presence of the common dietary organoarsenicals from seafood. In two experiments, the ingestion of several hundred micrograms of arsenic as the stable organoarsenical in seafood and as inorganic As(V) in mineral water resulted in the rapid gastrointestinal absorption and major urinary excretion of arsenic in under a week. A two-component model showed that urinary arsenic excretion was faster in the former, as reflected in first and second component half lives of 6.9 - 11hr and 3 days compared with 17 - 24hr and 7.1 - 8.6 days for the latter. However, while the seafood organoarsenical was excreted unchanged in urine, inorganic As(V) was rapidly reduced to As(III) and further biotransformed into the less toxic methylated metabolites MMAA and DMAA. After only 12hrs, DMAA became the predominant species to such an extent that over 7 days As(V) constituted only 9 - 10% of the total eliminated arsenic, As(III) 12 - 15%, MMAA 9-18% and DMAA 57 - 69%. In a subsequent experiment involving regular oral intake of As(V), and equilibrium was established in which 40 - 60% of the daily dose of 60 - 70ug As(V) was excreted in urine. As(V), As(III), MMAA and DMAA were eliminated in a steady speciation pattern with mean proportions of 5.8/15.2/14.3.64.7% respectively. Some possible longer term tissue retention of As(III) was indicated. For the general population, the concentration of the sum of the hydride-forming species As(V), As(III), MMAA and DMAA rarely exceeds 10ug/g creatinine, of which DMAA is the dominant species (> 80%). With calculated intakes derived from urinary arsenic data comparable to M.A.F.F. dietary estimates and well below recommended limits, there is a negligible risk to the health of the general population from typical exposure to inorganic arsenic. In the mineralised south west of England, where it has been suggested that the highly enriched soil arsenic concentrations may at least be a cofactor in the increased incidence of skin cancer, urinary arsenic levels were only slightly elevated. The potential for increased uptake by the local population was, however, reflected in the more frequent occurrence of As(III) and MMAA in urine and, of especial significance, in two comparatively highly elevated sum of species concentrations of 48.7 and 20.8ugAs/g creatinine recorded for 2 pre-school children. Elsewhere, for workers occupationally exposed to inorganic arsenic, mean urinary arsenic excretion varied from < 10ug/g creatinine for those in the electronics industry to 67.4ug/g creatinine for timber treatment workers applying arsenical wood preservatives, to 79.4ug/g creatinine for a group of glassworkers using arsenic trioxide and 244.8ug/g creatinine for chemical workers involved in the manufacture of arsenical compounds. The maximum urinary arsenic (As(V)+As(III)+MMAA+DMAA) concentration recorded was 956.4ug/g creatinine. For the most exposed groups, the average species proportions ranged from 1 - 6% As(V), 11 -

Dendrimers are a novel class of highly branched polymers with a high degree of uniformity and monodispersity. As a result of the unique properties and characteristics of dendrimers, they have found a wide range of pharmaceutical applications. This study investigated the ability of polyamidoamine (PAMAM) dendrimer-based drug delivery systems to enhance the permeability of a drug that is a substrate of P-glycoprotein (P-gp) efflux transporter with low water solubility. This thesis described the syntheses, characterisations and biological evaluations of the dendrimer-based drug delivery system to overcome the cellular barriers. G3 PAMAM dendrimers were conjugated with lauryl chains via a carbamate bond. Paclitaxel, a low water solubility P-gp substrate and anti-tumour drug, was conjugated to G3 and/or lauryl-G3 PAMAM dendrimer conjugates via a glutaric anhydride linker. Unmodified G3 PAMAM dendrimer and all the dendrimer conjugates (lauryl-G3 and G3-drug conjugates) were labelled with fluorescein isothiocyanate (FITC) for quantitative detection by spectrofluorimetry in permeability studies. These conjugates were characterised using various techniques including thin layer chromatography, 1H and 13C NMR, ESI-MS and dynamic light scattering. Chemical stability studies showed that the tested dendrimer conjugate (FITC-G3L6-glu-pac) was stable at all test pHs (1.2, 7.4, and 8.5) after 48 h of incubation at 37oC. The ester bond of the conjugate was stable under several chemical conditions after 10 days of incubation. Biological evaluation of the dendrimer conjugates was initially conducted using Caco-2 cells. Lactate dehydrogenase (LDH) release assay showed that conjugation of lauryl chains and paclitaxel molecules on the surface of G3 PAMAM dendrimer significantly (p < 0.05) increased the cytotoxicity in Caco-2 cells. The permeation of G3 PAMAM dendrimer and drug conjugates was investigated by measuring the apparent permeability coefficient (Papp) in both apical to basolateral AàB and BàA directions across Caco-2 cell monolayers at 37oC. The BàA Papp of paclitaxel was found to be significantly (p < 0.05) higher than the AàB Papp, indicating active function of P-gp efflux transporter system in the cell model. Covalent conjugation of paclitaxel to G3 PAMAM dendrimer via a glutaric anhydride linker significantly (p < 0.05) increased its AàB Papp through Caco-2 cell monolayers. A more pronounced increase of paclitaxel permeation was observed when surface modified G3 PAMAM dendrimers with six lauryl chains were used as the carrier. L6-G3-glu-pac was found with highest permeability across the Caco-2 cell monolayers. The results suggested that G3 PAMAM dendrimer-based drug delivery systems enhance the permeability of paclitaxel and bypass P-gp efflux transporter system in Caco-2 cell monolayers, thereby overcoming the intestinal barrier. Further biological evaluation was carried out using porcine brain endothelial cells (PBECs) as a blood-brain barrier (BBB) cell model to examine the potential of G3 PAMAM dendrimer as a carrier for paclitaxel (P-gp substrate) to bypass the BBB. Cell culture conditions of PBECs were monitored and examined to establish optimal conditions for cell growth. PBECs were successfully cultured with characteristic elongated spindle-like morphology. Integrity of the cell monolayers was evaluated by measuring the transendothelial electrical resistance (TEER) across the cell monolayers. Successfully cultured cell monolayers that achieved TEER values of higher than 200 Ω.cm2 were used for permeability studies. Elevation of TEER was observed when PBEC monolayers were cultured in the serum-free medium with hydrocortisone and treated with the cAMP/RO-20-1724 solution mixture. G3 PAMAM dendrimer was found to be relatively non-toxic to PBECs compared to all other conjugates. Conjugation of lauryl chains and paclitaxel molecules on the surface of G3 PAMAM dendrimer significantly (p < 0.05) increased the cytotoxicity in PBECs, as assessed by LDH assay. Permeability studies of paclitaxel across the PBEC monolayers showed a similar transport profile to that of Caco-2 cell. The significant higher BàA Papp of paclitaxel compared to the AàB Papp indicated active function of P-gp efflux transporter system in the cell model. The AàB Papp of L6-G3-glu-pac was found to be approximately 12 fold greater than that of free paclitaxel across the PBEC monolayers, where lauryl chains were acting as permeability enhancer. The cytotoxicity and permeability results using PBECs were found to be in good agreement with the findings when Caco-2 cells were used as the cellular barrier cell model. From these studies, it can be concluded that G3 PAMAM dendrimers-based drug delivery systems are potential nanocarriers for low solubility and P-gp substrate drug to bypass the P-gp efflux transporter system and overcome cellular barriers.

This thesis investigates the effects of various inflammatory stimuli, including cytokines and air pollution particles, on the expression and secretion of various proinflammatory proteins in the lung epithelial cell line A549. The pro-inflammatory proteins investigated were C-reactive protein (CRP), fibrinogen, and heat shock protein 70 (Hsp70), all of which are known to be produced during inflammation and are also known risk factors for cardiovascular disease. These proteins were investigated because inhalation of particulate air pollution has been associated with increased cardiovascular disease and mortality. The production of these proteins in the lung may be involved in a systemic inflammatory response which increases the plasma levels of these proteins and other cardiovascular risk factors therefore increasing the susceptibility for cardiovascular events. Alternatively, localised CRP functions as an opsonin, while fibrinogen and its degradation products are involved in the recruitment of neutrophils and leukocytes to the lung. Both CRP and fibrinogen are known to be produced in hepatocytes in response to cytokines. Chapter 3 investigates the effect of cytokine treatment of the A549 cells which shows that CRP and fibrinogen can be induced in this cell line by various cytokines. CRP was found to be induced by the cytokines ILI P, EL6, IL8, TNF(x and IFN7 with increased effects shown by simultaneous treatment with two cytokines. Fibrinogen was found to be induced mainly by IL6, but fibrinogen levels were also slightly increased by ILlp; simultaneous treatments of IL6 with either ILlP or TNF(x reduced the effect of IL6 treatment. Treatment of A549 cells with IL6 and IL8 simultaneously induced a synergistic effect. After establishing that cytokines induce the expression of CRP in A549 cells, the effects of particulate air pollution on pro-inflammatory protein expression were then investigated (chapter 4). The cells were treated with carbon black (CB), ultrafine CB (ufCB), and iron chloride (FeC13) to find out what effect these air pollution components and PM10 would have on the expression of CRP, fibrinogen, Hsp70 and the transcription factor NFKB and its inhibitor IKB. NFKB is known be activated by PM10 and is involved in the signalling of pro-inflammatory responses. All the particulate treatments induced a pro-inflammatory response with expression and secretion of CRP, fibrinogen, and Hsp70, whereas the soluble metal treatment had little effect. The metal salt FeC13w as used to treat the cells since it has been suggested that PM10 may mediate its effect through the presence of transition metals which are implicated in oxidant generation. The particulate exposures were also associated with the activation and nuclear translocation of NFKB indicating the involvement of NFKB and IxB in the induction of the pro-inflammatory response. Investigations into the mechanisms by which the particles induced the proinflammatory response are discussed in chapter 5. Firstly, investigations into the effect of particles on the secretion of the pro-inflammatory proteins were carried out; these indicated that particles were capable of inducing the secretion of CRP, flbrinogen and Hsp70. Investigations into the transcriptional mechanisms of proinflammatory protein expression were carried out using specific inhibitors. CRP and fibrinogen were induced in an NFKB dependent manner, while Hsp70 was produced as a result of activation of the JAK/STAT pathway. The effect of oxidative stress being induced as a result of particle exposure was investigated using antioxidants. This showed a reduction in the amount of intracellular and secreted CRP, fibrinogen and Hsp70 in the presence of antioxidants, indicating that oxidative stress was involved. This correlated with a reduction in the levels of intracellular ATP. Finally in chapter 6 the effects of proteins secreted from A549 cells on the monocytelike cell line MM6 were examined. This was carried out as a model of the effects of secreted pro-inflammatory proteins from the lung epithelium on other cells. It was found that CRP and fibrinogen were able to induce the activation of NFkB in MM6 cells, indicating that secretion of these proteins during lung inflammation could have an effect on other resident lung cells. Conditioned medium from A549 cells was also found to induce the expression of pro-inflammatory proteins including CRP, fibrinogen, Hsp70, IL6, IL8 and TNFcc. The A549 conditioned medium also appeared to induce a stress response and cellular damage in MM6 cells, thereby potentially exacerbating the inflammatory response. These results indicate that pro-inflammatory proteins can be produced in lung epithelial cells in response to particle exposure as a result of oxidative stress. They may be involved in the progression of localised lung inflammation and in the systemic response to particulate air pollution.