Background: Breast cancer remains a prominent cause of mortality and morbidity in the female population. Despite advances in terms of novel compounds, exhibiting chemotherapeutic activity successful delivery remains challenging. Particulate-based system such as Nanostructured Lipid Carriers (NLCs) a new generation carrier allow encapsulation of drug and targeting of desired tissue. Resveratrol (RES) is a chemotherapeutic drug limited by its physiochemical properties (i.e. poor solubility and bioavailability), encapsulation into NLCs is potentially viable solution to the aforementioned issues. Aim: The main aim of the present work is to develop a nanostructured lipid carriers based drug delivery system of RES in order to overcome its physicochemical and pharmacokinetic limitations and impart suitable functionalities for targeting breast cancer cells. Methods: Box-Behnken experimental design (BBD) was used to understand the effect of three independent factors (amount of liquid lipid, amount of drug and surfactant concentration) and interactions between these factors for each of the six liquid lipids on the selected response variables particle size (PS), polydispersity index (PDI), zeta potential (ZP), drug encapsulation efficiency (%EE), and drug loading (%DL). The formulated resveratrol nanostructured lipid carriers (RES-NLCs) were subjected to a series of physicochemical characterization including particles size, zeta potential measurements, in-vitro drug release while the morphology of RES-NLCs was confirmed through various microscopic methods, differential scanning calorimetry, x ray diffraction and Fourier transfer infrared studies was carried out in order to understand the interactions between RES and the components of the formulation. Undertake stability studies for the formulated RES-NLCs, at various storage conditions in order to select the most stable formulation and take it for further functionalization with targeting ligands hyaluronic acid; HA-NLCs, folic acid; FA-NLCs and dual targeting system using both hyaluronic acid and folic acid; HAFANLCs, and optimization of ligand density and quantify the amount of amine groups present on the surface on RES-NLCs. The efficacy of RES-NLCs was demonstrated through various in-vitro cell lines studies carried on three breast cancer cell lines entailed: MCF-7, MCF-10A and MDAMB-231 cells. Finally, the permeability of the formulations was evaluated through Caco-2 monolayer and Caco2/HT29 co-culture cell lines in order to understand the intestinal barrier transport mechanism. Results: The employed Box Behnken design resulted in the formulation of particles which were <100nm in size, PDI <0.3, a negative surface charge (-24), high entrapment efficiency (91-99%) and drug loading of 3-4%, with tuneable characteristics within the design space. Differential scanning calorimetry and x-ray diffraction indicated amorphous nature of the drug in nanoparticles indicating its entrapment. Upon exposure to acidic medium (pH 1.2), <20% drug release was observed in 4 hours, with 100% drug release observed upon the increase of pH to 5 over a period of 24 hours. Upon stability testing of NLCs, nanoparticles formulated with GTO as a liquid lipid showed good stability and therefore was taken forward for further PEGylating and surface modification with three ligands; hyaluronic acid, folic acid and combination of both ligand in order to impart targetability for breast cancer cells. Surface modification drastically reduced drug release to < 2 % at pH 1.2, however at pH 5, drug release time plots indicated slower overall release from the surface modified NLCs. In-vitro cytotoxicity studies showed that RES-NLCs were effective against both non-TNBC; MCF-7 and TNBC; MDAMB-231 breast cancer cell lines. Dual ligand appended nanoparticles showed 2.7 folds higher toxicity in MCF-7 and 3.6 fold higher toxicity in MDAMB-231 cells as compared to RES-NLC-GTO-PEGS40 demonstrating its potential for treatment in the aggressive triple negative cancer. None of the RES-NLCs formulations containing different liquid lipids or their blanks were cytotoxic to healthy MCF-10A cells demonstrating safety of the formulations. All bare, PEGylated and surface modified RES-NLCs showed time dependent cellular uptake on both MCF-7 and MDAMB-231 cell lines. Surface modification lead to 3 fold increase in the cellular uptake confirming the targetability potential of the ligand appended formulations toward overexpressed receptors on the surface of both cancer MCF-7 and MDAMB-231 cells. Upon examination of endocytosis mechanisms when cells were treated with the formulations, it was noted that two mechanisms were prominent. Clathrin-mediated endocytosis was identified as the primary method of endocytosis for all formulations. However, specifically for surface modified NLCs receptor mediated endocytosis was also found to be responsible for uptake of nanoparticles. Bidirectional transport study demonstrated that the permeability was sensitive to the type of liquid lipid incorporated with the highest permeability exhibited for PGML based formulation, when studied using a Caco-2 monolayer. On comparison to Caco-2/HT29 co-culture free resveratrol showed higher permeability when compared to the formulated NLCs. Conclusion: The aforementioned research has demonstrated that resveratrol NLCs are a viable potential product for use in the treatment of breast cancer, exhibiting high versatility and specificity.

No description available.

This study aims to compare the efficiency of conventional liposomes and surfactant-enriched vesicles (surfactosomes) using the hydrophilic drug salbutamol sulphate (SBS) and the hydrophobic drug beclometasone dipropionate (BDP) for pulmonary delivery via nebulisation. Initially liposomes and surfactosomes with or without cholesterol were prepared using thin film method and were compared for their VMD, span and drug entrapment. Their drug retention on extrusion through 5µm, 2µm, 1µm and 0.4µm polycarbonate membrane using mini-extruder was also studied. It was observed that liposomes were more stable than surfactosome. Particulate based proliposome technology was also used to study their potential for generating stable and inhalable dispersions. Mannitol was used as the carbohydrate carrier and on hydration; proliposomes and prosurfactosomes have generated liposomes and surfactosomes respectively. The VMD, span and zeta potential of the vesicles, and drug entrapment and drug retention on extrusion were studied. It was seen that lower proportions of SBS were entrapped using proliposome technology; hence, further extrusions through 5µm and 2µm were avoided. In vesicle with BDP, inclusion of cholesterol has decreased the drug entrapment and crystallisation of mannitol was observed. Nebulisation of liposomes and surfactosomes with and without cholesterol was studied using PARI LC sprint air jet nebuliser, Aeroneb pro and Beurer iH50 vibrating mesh nebulisers. Two stage (Twin) impinger was used to study the potential suitability of the generated vesicles for inhalation. VMD, span and zeta potential of vesicles before and after nebulisation was studied. BDP delivery and retention in both stages of the twin impinger was also studied. It was found that surfactosomes without cholesterol delivered maximum BDP to the twin impinger. Nebulisers suitable for all four formulations were also studied. Beurer iH50 delivered maximum BDP via liposomes with and without cholesterol, Aeroneb Pro delivered maximum BDP via surfactosomes with cholesterol to upper impinger while PARI LC sprint delivered maximum BDP via surfactosomes with cholesterol. VMD and span of aerosols generated from all three nebulisers were also studied. Stability of liposomes and surfactosomes prepared using proliposome technology was studied. VMD, span, zeta potential and BDP retention before and after spray drying and freeze drying were investigated. It was concluded that liposomes and surfactosomes were equally stable when spray drying was used whereas liposomes were more stable that surfactosomes when freeze drying was conducted. X-ray diffraction, scanning electron microscopy and transmission electron microscopy were used to analyse the characteristics of proliposomes and prosurfactosomes. A reduction in size and crystallinity was observed after spray drying and freeze drying of the formulations. Stability was also studied on storing proliposome and prosurfactosome in different environmental conditions like 5-6°C, room temperature and 40°C for a period of 3 months. It was concluded that both proliposomes and surfactosomes were most stable in 2-8°Cwhereas least stable in 40°C. Proliposomes were more stable than prosurfactosomes regardless of the storage temperature. Formulation and characterisation of novel prosurfactosomes and comparing it with conventional liposomes for pulmonary drug delivery is the novelty of this thesis.

Locum community pharmacists (‘locums’) constitute a significant proportion of the community pharmacy workforce in the UK, and have been identified as isolated practitioners who work outside existing quality assurance processes. This study examines professional engagement of locums in terms of their networking with pharmacist colleagues and their professional identity as pharmacists. With a constructivist, inductive approach, the study consisted of a series of five focus groups with a total of 25 participants in 2013, which were thematically analysed to yield a series of themes around professional engagement. The focus groups confirmed the isolation felt by locums and the effort undertaken by them to develop and maintain networks with colleagues. Locums used their networks for obtaining information, benchmarking their practice, decreasing personal stress, problem solving, sharing opinion on moral and ethical issues and promoting professional growth Next, the LocumVoice online forum for locum pharmacists was observed for a two month period in 2014, with the data being examined using an adaptation of Bales’ interaction process analysis, integrated with thematic analysis of the content. The interactions and content of the forum support it being considered a pharmacy community of practice, with locums’ interactions developing professional identity concepts via storytelling, sharing opinions and information. In particular, views on the nature of the role of the pharmacist were prominent in the discussions. The study contributes to knowledge of UK locum community pharmacists in that it describes the purpose and value of networking as perceived by locums and examines in detail the interactions occurring on an online community of practice that contribute to locum professional engagement and identity development.

Cytokines are proteins produced by cells of the immune system. The working hypothesis of the thesis is to show that the lymphocytes and glial cells in Glioblastoma Multiformae patients have an altered pattern of secretion of cytokines compared with low-grade and non-cancerous patients. Lymphocyte subset analysis was performed using flow cytometry. Serum specimens taken from both high and low grade glioma patients were analysed with samples taken from control patients with no history of cancer. Cerebro-spinal fluid was analysed from high-grade glioma patients. Comparisons of high grade pre-surgery serum samples, high grade post-surgery samples, control pre-surgery samples and control post-surgery samples were included. The analysis was completed utilising a luminex immunoassay. This technology is able to measure 34 cancer-associated analytes simultaneously. Immunohistochemistry of candidate biomarkers was done using primary tumour tissue. The results show that there were significant differences in several analytes in the sera and CSF of the different groups. These were follistatin, fibroblast growth factor (FGF), granulocyte – colony stimulating factor (G-CSF), soluble human epidermal growth factor receptor 2 neural (sHER2neu), soluble interleukin-6 receptor alpha (sIL-6R alpha), platelet-derived growth factor - AABB (PDGF-AABB), platelet and endothelial cell adhesion molecule (PECAM-1), stem cell factor (SCF), prolactin, soluble vascular endothelial growth factor receptor 1 (sVEGFR-1) and urokinase plasminogen activator (uPA). The data also show that tumour tissue revealed increased expression of follistatin and G-CSF with little expression of prolactin. In conclusion, these results suggest that potential candidate biomarkers can be used to enable diagnosis of glioma and moreso to distinguish between different grades of glioma using a panel of biomarkers.

Volume 1 (A/B) of the thesis investigated the innervation pattern, the cellular mechanism(s) of stimulus-secretion coupling (SCC), the effects of age and the in eraction between calcium and magnesium signalling in secretory cells taken from the thyroid gland, the parotid land, the ancreas, the stomach and the lacrimal gland. 1n addition, the study investigated the roles of anti-ulcer and anti-secretory drugs in the stomach and calpains in relation to vision. The study was designed to understand diseased states ( eg diabetes mellitus, pancreatitis, gastric ulcers, cataractogenisis and the dry eye syndrome) compared to healthy conditions. The results have demonstrated that, in addition to parasympathetic nerves, the pancreas, parotid and the lacrimal glands are innervated with adrenergic and non-cholinergic, non-adrenergic (peptidergic) nerves. Stimulation of these intrinsic secretomotor nerves can result in the secretion of proteins and enzymes from the pancreas, the parotid and lacrimal glands. Exogenous application of either acetylcholine (ACh), noradrenaline (NA), or a number of neuropeptides (vasoactive intestinal peptide (VIP), substance P, neuropeptide Y (NPY) and several others), can elicit protein and enzyme secretion. Apart from nervous control, exocrine pancreatic secretion is regulated by both endocrine and paracrine hormones. Cholecystokinin-octapeptide (CCK), secretin and histamine can stimulate protein and enzyme secretion from the pancreas. In addition to innervation pattern, this study also investigated the cellular mechanisms of stimulus-secretion coupling. The results have shown that there are four functionally distinct intracellular signalling pathways via which secretagogues (neurotransmitters, hormones and drugs) can elicit their responses in secretory epithelial cells. These include the utilization of such second messengers as calcium (Ca2+), adenosine 3, 5 cyclic monophosphate ( cyclic AMP), protein kinase C (PKC) and tyrosine kinase (TK).

Gliomas are the most common primary tumours of the central nervous system with a lethality rate approaching 80% in the first year of glioblastoma diagnosis. Their highly invasive nature renders local therapies such as surgery and radiation ineffective; whereas novel approaches such as immunotherapy are being actively studied as possible adjuncts in the treatment of patients with malignant gliomas. A number of factors have been proposed to play a role in glioma immune escape, including their poor immunogenicity since they do not express specific glioma antigens; their location within the CNS, which is considered to be an immune-privileged organ and some indications that glioma cells are capable of releasing various immunosuppressive cytokines, which inhibit the cytotoxic function of T cells. Cytokines are multifunctional pleiotropic proteins, which are involved in intercellular communication and cellular activation, and they may exert their effects in the CNS both directly and indirectly. Since they may originate either from peripheral immune organs, and cross the blood-brain barrier or they may be produced by the neuronal cells within the CNS, their properties are both immunoregulatory and neuromodulatory. This thesis investigated gliomas for the expression of cytokines and implicated their expression in the malignancy and the angiogenesis of the tumours. Cytokine secretion was evaluated from isolated peripheral blood mononuclear cells (PBMCs) of 33 patients with malignant gliomas (grade IV) immediately before surgery, in parallel with a control group of 23 age-matched individuals, and in 5 primary glioma cell cultures using the sensitive ELISPOT methodology. Thi cytokines tumour necrosis factor (TNF-a) and interferon (IFN7) were markedly reduced compared to control levels (P=0.01 and P < 0.001, respectively). In contrast, Th2 interleukins IL-(4) and IL-(10) were strongly expressed in both peripheral lymphocytes and glioma cell cultures (P < 0.05 and P < 0.001 respectively). Immunohistochemical localisation of IL-6, IL-8, COX, IL-lO, VEGF (vascular endothelial growth factor) and CD34 expression levels was performed in formalin-fixed paraffin-embedded tissue sections taken from 23 patients. Microvessels highlighted by means of anti-CD34 irnmunohistochemistry were enumerated with computer assisted image analysis. IL-6 was identified immunohistochemically in all specimens and showed an elevation in expression as necrosis grade increased (p=O.00S). IL-6 was also increased as the histological grade of the tumour increased (p0.020). IL-S expression was detected in all cases of gliomas with a mean of 19.5% of the cells. Expression was not related to either necrosis grade or increase in tumour grade. COX immunoreactivity was detected in all cases with an average of 29.1% positive cells. However, COX immunoreactivity was not significantly correlated with either necrosis or tumour grade. VEGF immunoreactivity occurred in 58.3% of cells and demonstrated a positive correlation with tumour grade (pzr0.035). The expression level of IL-10 was low both in terms of staining intensity and percentage of positive cells, and did not correlate with either necrosis or histological grade. CD34 immunoreactivity indicated that vascular volumetric parameters were not affected by either tumour or by necrosis grade. The expression of soluble CD95 as measured using a specific ELISA was not significantly (p > 0.05) reduced in the serum of 43 glioma patients compared to normal levels. In conclusion, these results indicate that patients harbouring malignant gliomas exhibit a broad suppression of cell-mediated immunity possibly due to the marked dysregulation in their cytokinetic profile. Glial tumours seem to induce a Thi (stimulatory) to Th2-type (inhibitory) cytokine shift which further supports humoral immunity at the expense of cell-mediated immune responses, contributing to the inefficient anti-tumour responses generated in these patients.