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Title: Multi-metallic microparticles for the treatment of pulmonary tuberculosis
Author: Ellis, Timothy David
ISNI:       0000 0004 7655 6224
Awarding Body: University of London
Current Institution: Imperial College London
Date of Award: 2018
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Mycobacterium tuberculosis (M.tb) has the extraordinary ability to adapt to the administration of antibiotics through the development of resistance mechanisms. By rapidly exporting drugs from within the cytosol, these pathogenic bacteria diminish antibiotic potency and drive the presentation of drug tolerant tuberculosis (TB). The membrane integrity of M.tb is pivotal in retaining these drug-resistant traits. Silver (Ag) and zinc oxide (ZnO) nanoparticles (NPs) are established antimicrobial agents that effectively compromise membrane stability, giving rise to increased bacterial permeability to antibiotics. In this work, biodegradable multi-metallic microparticles (MMPs), containing Ag and ZnO NPs, were developed for use in the pulmonary delivery of antituberculous drugs to the endosomal system of M.tb-infected macrophages. Ag NPs were synthesised with a uniform size distribution, having a mean diameter of 15 nm; well suited for potent antitubercular activity against M.tb. ZnO NPs were successfully synthesised with a smaller mean diameter of 4 nm, providing NPs with a large surface area to volume ratio. ZnO NPs, with a large surface area to volume ratio, act as potent antimicrobials through direct NP interaction or through rapid dissolution of Zn2+ ions. Material characterisation data of ZnO NPs, in a powdered form, confirmed the association of stearate as a capping ligand, together with information of ZnO NP composition and purity. Building upon these data, DPPC-capped Ag NPs and stearate-capped ZnO NPs were incorporated into solubilised PLGA, in the formation of MMPs, with SEM and EDX providing quantitative information of MMP diameter and elemental composition. MMP fabrication effectively encapsulated Ag and ZnO NPs and formed microparticles with a mean diameter of 1.5 mm. Efficient uptake of MMPs by M.tb-infected THP1 cells was demonstrated using an in cellulo macrophage infection model, with direct interaction between MMPs and M.tb visualised with the use of electron FIB-SEM tomography, following 24 hours post-exposure. The release of Ag NPs and ZnO NPs within the macrophage endosomal system increased the potency of the model antibiotic rifampicin by as much as 76%, realised through an increase in membrane disorder of intracellular M.tb; quantified through the gold standard CFU assay and visualised by STEM. MMPs, associated near the extracellular face of M.tb-infected THP1 macrophages, were also shown to drive independent lysis of extracellular M.tb bacilli in the absence of rifampicin, demonstrating a MMP-specific antitubercular mechanism that was visualised using FIB-SEM. MMP(Ag) and MMP(Zn) were found to suppress M.tb-induced expression of IL-1b and TNFa, with MMP(Ag) also suppressing the M.tb-induced expression of IL-6. MMPs containing both Ag and Zn (MMP(Ag+Zn)) were shown to significantly inhibit the M.tb-induced formation of NO2-, a stable breakdown product of the inflammatory mediator, NO. The literature suggests that Ag and Zn inhibit the MAPK pathway, activated upon TLR2 and TLR4 association to M.tb. Inhibition of this pathway is thought to inhibit NF-kB-mediated expression of IL-1b, TNFa, IL-6 and iNOS. The MMP system developed in this thesis presents as an effective drug delivery vehicle that could be used for the transport of antituberculous drugs, such as rifampicin, to infected alveolar macrophages, whilst increasing drug potency. By increasing M.tb membrane permeability, such a system may prove effectual in improving treatment of drug-susceptible TB in addition to M.tb strains considered drug-resistant.
Supervisor: Porter, Alexandra ; Ryan, Mary ; Dexter, David Sponsor: EPSRC
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral