Designing biomaterials for the development of bacteriocin based antimicrobial therapies

The antimicrobial resistance crisis presents a huge challenge to public health, with the continued rise in resistance to commonly prescribed antibiotics by a range of pathogens such as methicillin resistant S. aureus (MRSA), vancomycin resistant Enterobacteriaceae (VRE) and Carbapenemase producing enterococci (CPE). Bacteriocins are a class of antimicrobial peptide that show a high degree of inhibitory activity against a range of clinically relevant pathogenic bacteria, both Gram-positive and Gram-negative. Bacteriocins, like nisin, present a myriad of physicochemical and biophysical challenges in terms of their development into medicines. In fact, while bacteriocins have been reported in the literature for about a century, there remains a large gap in the literature with regards to their solution concentration data, and effective pre-formulation strategies including potential biomaterials to effectively deliver them to a site of infection. In this thesis, the susceptibility of nisin and the antimicrobial biopolymer epsilon polylysine (EPL) to degradative enzymes was determined. Antimicrobial synergism was investigated between nisin and EPL, and nisin and a deacetylated chitosan, glycol chitosan (GC) against Staphylococcus aureus. The physicochemical properties of nisin were determined showing poor solution concentrations at physiological pH’s (PBS, pH 7.4, and Fasted State Simulated Intestinal Fluid, pH 6.5). In an attempt to improve the solution concentration of nisin and EPL, solubilisers were screened. Tween® 80 was found to significantly increase the solution concentration of nisin. This was used to attempt to improve the in vitro activity of nisin in a dynamic dissolution assay that mimics the oral administration process. While an increase in activity was observed, the antimicrobial activity was still poor in FaSSIF (pH 6.5). However, nisin combined with either GC or EPL and Tween® 80 showed a three fold increase in antimicrobial activity against S. aureus in FaSSIF (pH 6.5) in the presence of pancreatin (enzyme extract) and porcine bile extract. In order to protect nisin from digestive enzymes, mesoporous silicates (MPS) SBA-15 and MCM-41, and the periodic mesoporous organosilane (PMO) MSE were investigated. Adsorption of the bacteriocin to the varied pore sizes of the SBA-15, MSE and MCM-41 was dominated by hydrophobic interactions, whereby the highest absorption was observed onto the MCM-41. All three matrices provided a high degree of protection of nisin from the digestive enzyme pepsin, whereby the matrices prevented the globular enzyme from penetrating the matrices pores where nisin was retained, and thus prevented degradation of the nisin. Varied release profiles were obtained from the three matrices, whereby MCM-41 showed the highest release over 72 h into FaSSGF (pH 1.2). Lower release was observed from the SBA-15 and MSE. However, difficulty in easily tuning the properties and potential biocompatibility issues outside of oral administration applications, as well as their lack of biodegradability, all present limitations in their use as delivery platforms for bacteriocins. Thus, polysaccharide based hydrogels, that cross link in situ upon injection and which can offer easily tuneable, highly biocompatible, biodegradable and highly versatile biomaterials for the delivery of biologics like bacteriocins to sites of infection were investigated. Here, oxidised dextran (dextran di aldehyde) and hydrazine functionalised alginic acid (alginate-hydrazine) were produced. Upon combination of the two polymer solutions in a 21 gauge needle, in situ cross linking leads to the generation of hydrazone bonds (Schiff base, covalent). Nisin is encapsulated in these gels by solubilisation of the dextran-dialdehyde in a nisin solution. Glycol chitosan (GC) was substituted into gels in place of the alginate hydrazine. The addition of this higher Mwpolymer allowed for modulation of the gels elastic modulus, whereby increasing concentrations of GC increased the elastic modulus. This increase in GC, and simultaneous reduction in alginate hydrazine content also generated gels with a higher degree of swelling (in PBS, pH 7.4). The release of the bacteriocin nisin A was also controlled, where it was hypothesised that higher GC content, and lower cross link density based on the reduced hydrazone bonding, allowed nisin to penetrate gels deeper, and interact more with the gel matrix thanks to the lower degree of chain entanglement, slowing release. Following this study, the effect of the degree of oxidation of dextran on the properties of the hydrogels was investigated. As the degree of dextran oxidation increases, the weight average molecular mass decreases. This impacts the gels elastic modulus, whereby lower Mw dextran gels exhibit lower Young’s moduli. The antimicrobial activity of the encapsulated nisin was altered, whereby lower dextran Mw gels (higher oxidation degree) showed more sustained inhibition over an 8 day period, in vitro, against S. aureus. Addition of 3% w/v GC to lower dextran oxidation gels (Dex14%) allowed for an increase in antimicrobial activity, presumably due to synergistic inhibitory effects. The study shows another means of tuning the properties of these dextran-aldehyde and alginate-hydrazine injectable hydrogels,