Abstract
Tuberculosis (TB) remains one of the deadliest infectious diseases globally, claiming millions of lives every year and posing a significant health burden. A major contributor is its treatment challenges, characterised by complex, prolonged treatment regimens and high systemic toxicity, particularly with first-line drugs such as isoniazid (ISO). Drug-resistant TB strains prevalence also exacerbate these issues, making treatment increasingly difficult with conventional antibiotics. Therefore, there is a critical need for a sustainable solution involving treatment agents that can overcome the limitations of current TB therapies. Nanotechnology, particularly the use of silver nanoparticles (AgNPs), offers an innovative approach. It leverages AgNP’s enhanced drug delivery, drug toxicity reduction, intrinsic antibacterial effects, and diverse bacterial killing mechanisms that can hinder the emergence of drug resistance. Traditional methods of AgNP synthesis often rely on harmful synthetic chemicals, making them environmentally harmful and biologically toxic. Consequently, there is growing interest in sustainable and eco-friendly alternatives for synthesising nanomaterials from natural sources, including plants. Using plants as biofactories for AgNP synthesis offers a sustainable approach, leveraging natural reducing and stabilising agents in plant phytochemicals and avoiding toxic chemicals. The overall aim of this thesis is to develop innovative TB treatment agents by extensively optimising a unique Spinacia oleracea (S. oleracea) leaf extract-mediated method for producing AgNPs, focusing on the strategic loading of anti-TB drug ISO, both alone and in combination with metformin (MET) to explore MET’s repurposing potential in TB therapy. The details of this investigation are reported through a systematic approach.In Chapter 3, an optimised, inexpensive one-pot method for synthesising AgNPs using S. oleracea leaf extract as a reducing and stabilising agent was developed, which revealed the significance of leaf source and extract concentration in the synthesis process. The synthesised AgNPs were characterised spectrophotometrically using ultraviolent-visible spectroscopy (UV-VIS). Morphology, size, and elemental characterisations were also conducted using dynamic light scattering (DLS), scanning electron microscopy (SEM) and Inductively coupled quadrupole plasma mass spectrometry (ICPQMS). Results confirmed the effective synthesis of monodispersed spherical AgNPs well in the nano range, obtaining AgNP-2% (173nm), AgNP-3% (211nm), AgNP-4% (148nm), AgNP-7% (120nm), and AgNP-10% (109nm) v/v S. oleracea leaf extract. Regarding antimicrobial activity, the lower concentration S. oleracea leaf extract AgNPs showed no antimicrobial activity; however, all the higher concentration AgNPs fully inhibited Escherichia coli, Staphylococcus aureus, Streptococcus pyogenes and Candida albicans. Thus, the superiority of the higher concentration S. oleracea leaf extract AgNPs was established, with the AgNP-10% offering the most favourable physiochemical properties.
In Chapter 4, the AgNPs synthesised using 10% v/v S. oleracea leaf extract (AgNP-10%) were functionalised with ISO and further stabilised polyvinylpyrrolidone (PVP). To achieve maximum ISO loading efficiency, two loading methods were explored: ISO was added either before or after PVP i.e. PVP1 (AgNP + ISO + PVP1) and PVP2 (AgNP + PVP2 + ISO). PVP concentration was also varied: PVPa 0.0125 and PVPb 0.025mg/ml. Similarly, the synthesised AgNPs were extensively characterised, and additional morphology, size and elemental characterisations were also conducted using transmission electron microscopy (TEM), DLS, TEM – Energy Dispersive Xray (TEM-EDX) and nuclear magnetic resonance (NMR), enabling confirmation of drug loading. The results confirmed the successful synthesis of spherical ISO-loaded AgNPs with sizes ranging from 73 – 131nm and polydispersity (PDI) values below 0.3. Notably, the PVP loaded AgNPs exhibited superior performance, facilitating optimum drug loading efficiency, especially in the PVPb samples, ~56% (PVP1b) and ~62% (PVP2b). Additional antimicrobial evaluation against TB strains, Mycobacterium aurum (M. aurum) and Mycobacterium bovis (M. bovis), showcased their mycobacteria-killing capabilities.
Lastly, in Chapter 5, the ISO-loaded AgNPs were further functionalised by co-loading with MET, denoted as AgNP ISO:MET (IM), and the two PVP loading methods were investigated: PVP1 IM (AgNP + ISO:MET + PVP1) and PVP2 IM (AgNP + PVP2 + ISO:MET), using the lower PVPb 0.025mg/ml concentration. Likewise, the ISO:MET AgNPs were thoroughly characterised using similar techniques, revealing their spherical shapes, monodispersity (PDI<0.3) and nano sizes of 120nm (AgNP IM), 113nm (PVP1 IM) and 123nm (PVP2 IM). Similarly, the PVP stabilised particles facilitated increased loading efficiency with ~63% ISO/ 81% MET in PVP1 IM and ~63% ISO/68% MET in PVP2 IM. Moreover, antimicrobial analysis of these ISO:MET AgNPs also proved their antimycobacterial effects against M. aurum and M. bovis. Overall, this PhD thesis demonstrates plant-mediated AgNPs' potential as a promising alternative for TB treatment and proposes novel treatment agents. The findings highlight the prospects of sustainable and eco-friendly approaches to combat chronic infectious diseases, paving the way for further research and development in nanomedicine.
| Date of Award | 1 Oct 2024 |
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| Original language | English |
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| Supervisor | Bahijja Raimi-Abraham (Supervisor) & Rocio Martinez Nunez (Supervisor) |