Fluorescent imaging technologies that offer new ways to visualise and quantify fluorescently labelled molecules are increasing, necessitating the development of fluorescent molecules that can efficiently and specifically label targets in vitro and in vivo. The first aim of this thesis was the study of human neutrophil elastase. Human neutrophil elastase is an important enzyme in the regulation of inflammation but if over expressed can become part of the cause of inflammation itself. To elucidate this dual function and have a greater understanding of this enzyme, an imaging probe for neutrophil elastase was designed. Firstly, the syntheses of fluorescently labelled three branched dendron core structures were optimised, and studied in neutrophils. The selected core structure was functionalised with an elastase specific peptide sequence and fluorescently labelled. The probe was specifically cleaved by neutrophil elastase in an enzymatic assay and in the presence of activated neutrophils (Chapter 1). Fluorescein and rhodamine are dyes that are readily available, are affordable and have convenient wavelengths for microscopy and flow cytometry. Carboxyfluorescein diacetate N-succinimidyl ester (CFDA-SE) is a commonly used fluorescein derivative, widely used in cell proliferation assay. It is mainly used as a mixture of isomers and its synthesis is not reported. Herein a short and simple synthesis of the two individual isomers of carboxyfluorescein diacetate N-succinimidyl ester as well as the equivalent rhodamine variation (carboxytetraethylrhodamine N-succinimidyl ester) is reported (Chapter 2). The labelling properties of these probes were studied in proliferation assays on mouse and human T lymphocytes. Finally, the nuclear penetration of the dendron structure combined with nuclear localisation sequences (NLS) was investigated. Attachment of nuclear localisation sequences to the probe in the presence of fluorescein demonstrated successful entry into the nucleus in human alveolar adenocarcinoma cell line (A549) (Chapter 3).

Niosomes are synthetic microscopic vesicles consisting of an aqueous core enclosed in a bilayer consisting of cholesterol and one or more nonionic surfactants. They are made of biocompatible, biodegradable, non-toxic, non-immunogenic and non-carcinogenic agents which form closed spherical structures (self assembly vesicles) upon hydration. With high resistance to hydrolytic degradation, niosomes are capable of entrapping many kinds of soluble drugs while exhibiting greater vesicle stability and longer shelf life. In this work, a potential drug delivery system has been designed, synthesized and characterized. For the synthesis of niosomes, a hydration process was developed with varying design parameters such as mass per batch, angle of evaporation, rotation speed of vacuum rotary evaporator and nitrogen flowrate to produce uniform thin film in 50 ml round bottom flask. The rehydration process was developed by varying the choice of solvents (H2O, phosphate buffer solution (PBS) and PBS/5(6)-carboxyfluorescein (CF) as a drug model) and hydrating temperature of below and above gel transition temperature. Lastly, a sonication process to produce unilamellar vesicles was partially optimized based on the particle distribution and the number of vesicles formed with sonication time. As a result of this process, unilamellar and multilamellar vesicles were formed with the combination of different nonionic surfactants (sorbitan monostearate-Span 60, sorbitan monopalmitate-Span40 and sorbitan monolaurate-Span20), cholesterol and an electrostatic stabilizer (dicetyl phosphate). The vesicles were examined using light scattering optical microscopy and UV microscopy. Optical sensing technology (Particle Sizing System) is used to determine the vesicles' size distribution. Gel exclusion chromatography (GEC) is discussed as a method to separate unencapsulated CF while retaining vesicle integrity. Particle Sizing System and luminescence spectrophotometer were used to determine CF encapsulation percentage and leakage. Result: Span 20, Span 40 and Span 60/Niosomes were made with mean particle size of 0.95-0.99 micro (mu)m. Typical concentrations of vesicle per ml/per mass of surfactant used were in the range of 1.46-1.79x108 . Typical encapsulation efficiencies were in the range of 48.8-62.9% for all three Span/Niosome systems. Niosomes were found to be stable for 9 days. The largest vesicles were observed with Span 60 with highest entrapment efficiency as compared to Span 20 and Span 40.