title: A bioactive hyperbranched polymer disclosure system for bacteria
Creators: name: Balmford, Oliver
Publisher: name: University of Reading
Year: 2024

description: The disclosure of Biological Warfare Agents (BWAs) poses a considerable scientific and practical challenge as a result of the inherent lower reactivity afforded by natural products, viral and bacterial pathogens. In the 20th century throughout both the first and second world wars Bacillus anthracis, the causative agent of Anthrax, was studied for its potential use in biological warfare. B. anthracis is one of a limited number of bacteria capable of forming endospores. The endospore is a dormant and stable form of the organism in which all water is removed and cell metabolism ceases. Bacteria that can form these structures are capable of surviving thousands of years. Hence, land contaminated with pathogenic endospores can be rendered uninhabitable and, in some cases, impassable for decades. However, if the B. anthracis cell is in its vegetative state, the process of decontamination is made substantially easier by the increased susceptibility of the cells. It is, therefore, favourable to induce germination in spores before attempting decontamination. This thesis is a multidisciplinary approach to develop a formulation capable of inducing germination of B. anthracis endospores and disclosing their presence to an end-user such that appropriate action can be taken to decontaminate the surface. In this thesis Chapter 1 provides an overview to the obstacles that have so far restricted development of the desired formulation. It also encompasses the historical background, posed risks and detailed aims of this project. Chapter 2 reports the synthesis and optimisation of processes towards materials that can provide favourable environments to promote endospore germination in a controlled fashion. Investigations focussed on the promising characteristics of a class of low molecular weight gelators (LMWGs) known as supergelators because of their exceptionally ability to form rigid structures at concentrations as low as 0.9 mM as well as hydrophilic branched polymers based upon the monomer glycerol. Investigations into hydrophilic materials capable of providing sufficient humectant properties to sustain germination suggested the hyperbranched polyether polyglycerol could fulfil many of the desired requirements of a formulation suitable for storage and spray dispersal. The polymers were further optimised by introduction of orthogonal reactive groups of furfuryl alcohol capable of reacting via the facile thermoreversible Diels-Alder process to enable future bioactive sensors or dyes. The benefit of this method is that these reactions are typically conducted in very mild conditions and at low temperatures which allows for sensitive or less stable functional compounds to be attached to the polymer backbone without fear of degradation or damage to these materials. These materials were noted to possess LCST properties which were further investigated by means of DLS. Chapter 3 focussed on the development of a suitable disclosure agent for the B. anthracis biomarker calcium dipicolinate (CaDPA). Based upon the fluorescent carbazole moiety, a sensor originally developed by Curiel et al. was resynthesized using optimised procedures to evaluate its suitability for use in an aqueous formulation. Following this, steps were taken to attempt to increase the effectiveness of the sensor by altering its colourimetric response through extension of the π-system of the carbazole fluorescent backbone. Further optimisations to increase its water solubility were explored via introduction of charged imidazolium species. Chapter 4 details the steps taken to assess the suitability of the polyglycerol formulations prepared in Chapter 2 to assist in the germination of B. thuringiensis. B. thuringiensis endospores are used as a simulant for the more dangerous B. anthracis and were cultivated and purified before use. The degree of germination of the endospores was assessed through a plate counting method and compared to a rate of germination assessment carried out via optical density measurements. The studies described herein provide a strong foundation for further development of a novel formulation capable of rapidly disclosing the presence of B. anthracis contamination.