Tuberculosis is one of the most prevalent infectious diseases globally due to the successful survival mechanisms displayed by Mycobacterium tuberculosis (Mtb). Mtb primarily infects alveolar macrophages (AMs) and is able to live intracellularly for extended periods of time due to a number of virulence factors which inhibit the antibacterial mechanisms of the AMs. This aspect of the Mtb life cycle means TB treatments suffer from poor bioavailability and efficacy. Additionally, the rise in resistant strains of Mtb means the use of higher doses and the use of alternative second and third line drugs which increase the risk of systemic toxicity. Drug encapsulation is a novel approach that can provide more favourable drug pharmacokinetics and pharmacodynamics. The aim of this project was to develop a liposomal drug delivery system to target Mtb infected alveolar macrophages. The system involved the encapsulation of two drugs; the antibiotic gatifloxacin (GFLX) and Mtb virulence factor inhibitor CV7. The hypothesis was that the two different antibacterial mechanisms would work in synergy and increase the efficacy of the treatment. AM targeting and receptor-mediated endocytic uptake was encouraged by the presence of a ligand attached to the surface of the liposome. Furthermore a pH-sensitive release mechanism was to be incorporated into the liposome to encourage the release of the encapsulated drugs in the vicinity of the intracellular bacteria. The intention was to produce a drug delivery system to enable a TB therapy regime of fewer, lower doses to increase compliance and reduce systemic toxicity by increasing efficacy through improved bioavailability. GFLX was successfully encapsulated using a weak base active loading method. To establish encapsulation efficiency, a homogeneous fluorescence assay able to quantify intra- and extra-liposomal gatifloxacin simultaneously was developed. pH-sensitive release of the payload could be achieved using a pH-sensitive peptide with a novel design based on chimeric structure, namely P3. CV7 was successfully encapsulated using a weak acid active loading method. CV7 liposomes were able to be functionalised by the incorporation of a mannose ligand on the surface of the liposome. An inhibition assay using the target enzyme of CV7, MptpB, was optimised to assess efficacy of liposomally encapsulated and released CV7. Flow cytometry and confocal microscopy studies confirmed that the liposomal formulations were internalised by the target macrophage cell line, J774a.1. Mannose liposomes conveyed superior uptake kinetics. Further confocal microscopy showed that after internalisation the liposomes entered the endolysosomal pathway and colocalised with BCG. A BCG-macrophage infection model was used to determine the intracellular efficacy of the liposomal formulations. Encapsulated CV7 displayed increased efficacy over free CV7, while encapsulation in functionalised liposomes showed better efficacy still. The encapsulation of GFLX did not increase the efficacy of GFLX and synergy between the two drugs was not achieved. In conclusion, the liposomal encapsulation of CV7 increased uptake of the drug by the target cell line and facilitated colocalisation of the drug with the target pathogen thereby increasing efficacy. Such a formulation could potentially increase bioavailability and efficacy in vivo for a more tolerable TB therapy.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:713597 |
| Date | January 2017 |
| Creators | Hopkinson, Devan |
| Contributors | Aojula, Harmesh |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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