Hydrogels and Nanoparticles for the Detection and Treatment of Pathogenic Bacteria

Abstract

The aim of this research project was to develop various systems such as hydrogels and nanoparticles to detect and possibly treat pathogenic bacteria. Particularly, the bacteria virulence factors were targeted.
In Chapter 1, an introduction provides general information about bacteria, their resistance mechanisms, and their presence in wound infections. General aspects of hydrogels and nanoparticles are also inspected. Finally, a review on peptide hydrogels, hyaluronic acid hydrogels and nanoparticles, as well as their potential targets such as protease and hyaluronidase enzymes, is presented.
In Chapter 2, information on materials and methods used for the different synthesis, characterisation, enzyme, and bacteria testing is provided. The basic theory behind the main instrumentation used in this project can also be found.
Chapter 3 presents the work done on genipin-crosslinked gelatin hydrogels. The synthesis of the hydrogels, and their optical and mechanical properties, characterized with Ultra-Violet-Visible (UV-Vis) and fluorescence spectroscopy techniques and rheology, are included in this chapter. Protease sensitivity of the system is also explored with weight monitoring experiments and Scanning Electron Microscopy (SEM) pictures investigating the presence of pores. Rheology is used again to monitor in real time the degradation of the peptide hydrogel in presence of trypsin protease enzyme. Bacterial supernatant tests are also performed with 36 bacterial strains to investigate whether the hydrogels are degraded by one or several bacteria species. Further protease inhibitor test and bacterial mutants are used to identify the protease family responsible for the hydrogel degradation. Genomic investigation also gives an insight on what type of bacteria do and do not induce degradation.
Chapter 4 explores in a similar way the potential of genipin-crosslinked soy protein isolate (SPI) hydrogels as a protease susceptible system. Rheology is used to characterize the hydrogels and swelling ratios are assessed. Protease sensitivity is also investigated with rheology, weight monitoring experiments and SEM pictures. Bacterial supernatant tests are also followed by protease inhibitor tests to research the potential bacteria responsible for the degradation of the hydrogels.
In Chapter 5, multilayer systems are investigated. Kirby-Bauer test and bacteria counting multilayer experiments are both performed with genipin-crosslinked gelatin and soy protein isolate (SPI) hydrogels. The introduction of antibiotics or phage in another agarose layer allows the system to be consider for both detection and treatment of bacteria. Minimum Inhibitory Concentration (MIC) of polymyxin B and phage susceptibility to different bacteria are studied and diffusion of antibiotics through the agarose layer is also evaluated. Both methods, namely Kirby-Bauer and bacteria counting multilayer experiments are compared
as their outcomes are respectively based on visual observation of a zone of inhibition or bacteria counting experiments.
Chapter 6 introduces a 1,4-butanediol diglycidyl ether (BDDE)-crosslinked hyaluronic acid (HA) hydrogel which is a simpler hyaluronidase sensitive system than commonly used Reissig assay. The chapter presents an easy and straightforward synthesis of the hydrogel. The hydrogel mechanical properties are characterized with rheology to understand the impact of different crosslinker concentrations. Rheology is also used to monitor the hydrogel degradation when subjected to a hyaluronidase enzyme solution compared to a tris buffered saline solution. Weight monitoring experiments of the hydrogel soaked in hyaluronidase
solution are also shown, and cryo-SEM pictures were taken to observe the potential formation of pores after enzyme treatment. The assay consists of a large disc of hydrogel where several smaller discs are removed and where different solutions can be pipetted in. If the solution contains hyaluronidase enzyme, the discs get larger as degradation is happening. This assay is tested with different concentrations of hyaluronidase as well as with ten bacterial supernatants from different species. The results are then compared with a Reissig assay performed in literature with the same bacterial strains to evaluate the
performance of our assay. It was found our assay was easier to reproduce but with potentially lower sensitivity to hyaluronidase than the Reissig assay.
Chapter 7 also aims to develop a hyaluronidase sensitive system. This time oleylamine-grafted hyaluronic acid conjugates are self-assembled into nanoparticles and a rhodamine 6G dye is encapsulated. The chapter presents the synthesis of these nanoparticles (NPs) and proves the successful grafting of the oleylamine onto the polysaccharide using Nuclear Magnetic Resonance (NMR) techniques. The Critical Micelle Concentration (CMC) of the conjugate is calculated using a fluorescent probe method with pyrene. The presence of
spherical NPs is also shown with Dynamic Light Scattering (DLS) and Transmission
Electron Microscopy (TEM) pictures. The NPs are then shown to be hyaluronidase sensitive by mixing them with the enzyme and measuring the fluorescence intensity. The encapsulated dye is supposed to be self-quenched, while after degradation of the structure by the enzyme, the dye is released and a higher fluorescence signal can be observed. The NPs are also tested with the same bacterial supernatants as in Chapter 6 and the results are compared once again with the Reissig assay. It was shown our assay successfully detect hyaluronidase and potentially another compound released by S. aureus bacteria.
Chapter 8 presents the conclusion of this project, what is achieved, and what can be done in the future to improve the systems.

Date of Award	19 Feb 2025
Original language	English
Awarding Institution	University of Bath
Supervisor	Petra Cameron (Supervisor) & Maciek Kopec (Supervisor)