The formulation of artificial reference standards for use within the ELISPOT assay

Whether to assess the functionality of equipment or as a determinate for the accuracy of assays, reference standards are essential for the purposes of standardisation and validation. The ELISPOT assay, developed over thirty years ago, has emerged as a leading immunological assay in the development of novel vaccines for the assessment of efficacy. However, with its widespread use, there is a growing demand for a greater level of standardisation across different laboratories. One of the major difficulties in achieving this goal has been the lack of definitive reference standards. This is partly due to the ex vivo nature of the assay, which relies on cells being placed directly into the wells. Thus, the aim of this thesis was to produce an artificial reference standard using liposomes, for use within the assay. Liposomes are spherical bilayer vesicles with an enclosed aqueous compartment and therefore are models for biological membranes. Initial work examined pre-design considerations in order to produce an optimal formulation that would closely mimic the action of the cells ordinarily placed on the assay. Recognition of the structural differences between liposomes and cells led to the formulation of liposomes with increased density. This was achieved by using a synthesised cholesterol analogue. By incorporating this cholesterol analogue in liposomes, increased sedimentation rates were observed within the first few hours. The optimal liposome formulation from these studies was composed of 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), cholesterol (Chol) and brominated cholesterol (Brchol) at a 16:4:12 µMol ratio, based on a significantly higher (p<0.01) sedimentation (as determined by a percentage transmission of 59 ± 5.9 % compared to the control formulation at 29 ± 12 % after four hours). By considering a range of liposome formulations ‘proof of principle’ for using liposomes as ELISPOT reference standards was shown; recombinant IFN? cytokine was successfully entrapped within vesicles of different lipid compositions, which were able to promote spot formation within the ELISPOT assay. Using optimised liposome formulations composed of phosphatidylcholine with or without cholesterol (16 µMol total lipid) further development was undertaken to produce an optimised, scalable protocol for the production of liposomes as reference standards. A linear increase in spot number by the manipulation of cytokine concentration and/or lipid concentrations was not possible, potentially due to the saturation that occurred within the base of wells. Investigations into storage of the formulations demonstrated the feasibility of freezing and lyophilisation with disaccharide cryoprotectants, but also highlighted the need for further protocol optimisation to achieve a robust reference standard upon storage. Finally, the transfer of small-scale production to a medium lab-scale batch (40 mL) demonstrated this was feasible within the laboratory using the optimised protocol.

Development and evaluation of a novel intranasal spray for the delivery of amantadine

The aim of this study was to develop and characterize an intranasal delivery system for amantadine hydrochloride (AMT). Optimal formulations consisted of a thermosensitive polymer Pluronic® 127 and either carboxymethyl cellulose or chitosan which demonstrated gel transition at nasal cavity temperatures (34 ± 1°C). Rheologically, the loss tangent (Tan δ) confirmed a 3-stage gelation phenomena at 34 ± 1°C and non-Newtonian behavior. Storage of optimized formulation carboxymethyl cellulose and optimal formulation chitosan at 4°C for 8 weeks resulted in repeatable release profiles at 34°C when sampled, with a Fickian mechanism earlier on but moving toward anomalous transport by week 8. Polymers (Pluronic® 127, carboxymethyl cellulose, and chitosan) demonstrated no significant cellular toxicity to human nasal epithelial cells up to 4 mg/mL and up to 1 mM for AMT (IC50: 4.5 ± 0.05 mM). Optimized formulation carboxymethyl cellulose and optimal formulation chitosan demonstrated slower release across an in vitro human nasal airway model (43%-44% vs 79 ± 4.58% for AMT). Using a human nasal cast model, deposition into the olfactory regions (potential nose-to-brain) was demonstrated on nozzle insertion (5 mm), whereas tilting of the head forward (15°) resulted in greater deposition in the bulk of the nasal cavity.

Tail gas catalyzed N2O decomposition over Fe-beta zeolite. On the promoting role of framework connected AlO6 sites in the vicinity of Fe by controlled dealumination during exchange

A novel route to prepare highly active and stable N2O decomposition catalysts is presented, based on Fe-exchanged beta zeolite. The procedure consists of liquid phase Fe(III) exchange at low pH. By varying the pH systematically from 3.5 to 0, using nitric acid during each Fe(III)-exchange procedure, the degree of dealumination was controlled, verified by ICP and NMR. Dealumination changes the presence of neighbouring octahedral Al sites of the Fe sites, improving the performance for this reaction. The so-obtained catalysts exhibit a remarkable enhancement in activity, for an optimal pH of 1. Further optimization by increasing the Fe content is possible. The optimal formulation showed good conversion levels, comparable to a benchmark Fe-ferrierite catalyst. The catalyst stability under tail gas conditions containing NO, O2 and H2O was excellent, without any appreciable activity decay during 70 h time on stream. Based on characterisation and data analysis from ICP, single pulse excitation NMR, MQ MAS NMR, N2 physisorption, TPR(H2) analysis and apparent activation energies, the improved catalytic performance is attributed to an increased concentration of active sites. Temperature programmed reduction experiments reveal significant changes in the Fe(III) reducibility pattern with the presence of two reduction peaks; tentatively attributed to the interaction of the Fe-oxo species with electron withdrawing extraframework AlO6 species, causing a delayed reduction. A low-temperature peak is attributed to Fe-species exchanged on zeolitic AlO4 sites, which are partially charged by the presence of the neighbouring extraframework AlO6 sites. Improved mass transport phenomena due to acid leaching is ruled out. The increased activity is rationalized by an active site model, whose concentration increases by selectively washing out the distorted extraframework AlO6 species under acidic (optimal) conditions, liberating active Fe species.