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

Mesoporous matrices for the delivery of the broad spectrum bacteriocin, Nisin A

Mesoporous matrices of different pore size and chemical composition were explored as potential delivery matrices for the broad spectrum bacteriocin, nisin A. The adsorption of nisin A onto two mesoporous silicates (MPS - SBA-15, MCM-41) and two periodic mesoporous organosilanes (PMO - MSE, PMO-PA) was examined. It was found that hydrophobic interactions dominated in the adsorption of this peptide to the matrices, lending the highest adsorption to MCM-41 with a small pore size of 2.8 nm. The hydrophobic ethylene-bridged MSE (6 nm pore) improved the loading and protection of nisin A from degradation by a non-specific protease pepsin, over un-functionalised SBA-15 which had a slightly larger pore size and less hydrophobic moieties. Nisin A did not adsorb onto an amine-functionalised PMO. Upon suspension in modified fasted state simulated gastric fluid (pH 1.6), the highest release of nisin A was observed from MCM-41, with a lower release from SBA-15 and MSE, with release following Higuchi release kinetics. No release was detected into modified fasted state simulated intestinal fluid (pH 6.5) but despite this, the suspended matrices loaded with nisin A remained active against Staphylococcus aureus

Optimization of Organotin Polymers for Dielectric Applications

Recently, there has been a growing interest in developing wide band gap dielectric materials as the next generation insulators for capacitors, photovoltaic devices, and transistors. Organotin polyesters have shown promise as high dielectric constant, low loss, and high band gap materials. Guided by first-principles calculations from density functional theory (DFT), in line with the emerging codesign concept, the polymer poly­(dimethyltin 3,3-dimethylglutarate), p­(DMTDMG), was identified as a promising candidate for dielectric applications. Blends and copolymers of poly­(dimethyltin suberate), p­(DMTSub), and p­(DMTDMG) were compared using increasing amounts of p­(DMTSub) from 10% to 50% to find a balance between electronic properties and film morphology. DFT calculations were used to gain further insight into the structural and electronic differences between p­(DMTSub) and p­(DMTDMG). Both blend and copolymer systems showed improved results over the homopolymers with the films having dielectric constants of 6.8 and 6.7 at 10 kHz with losses of 1% and 2% for the blend and copolymer systems, respectively. The energy density of the film measured as a <i>D</i>–<i>E</i> hysteresis loop was 6 J/cc for the copolymer, showing an improvement compared to 4 J/cc for the blend. This improvement is hypothesized to come from a more uniform distribution of diacid repeat units in the copolymer compared to the blend, leading toward improved film quality and subsequently higher energy density

Additional file 1: Figure S1. of Exercise-induced mitochondrial p53 repairs mtDNA mutations in mutator mice

Endurance exercise confers complete phenotype protection, suppresses early mortality, mitigates mitochondrial ROS-mediated oxidative damage, increases cellular antioxidant capacity, and 4 prevents cellular senescencmutator mice. Figure S2. Endurance exercise prevents dysregulated mitochondrial-induced apoptosis and reduces nuclear p53-mediated repression of PGC-1ι and promotes mitochondrial biogenesis in mutator mice. Figure S3. Endurance exercise promotes systemic mitochondrial biogenesis in mtDNA mutator mice. Figure S4. Magnitude of mitochondrial ROS (physiological vs. pathological) regulates p53 subcellular localization. Figure S5. Pre-treatment with exogenous antioxidant preferentially shuttles p53 to mitochondria in response to stress. Figure S6. Endurance exercise-mediated attenuation of sarcopenia, increase in endurance capacity, skeletal muscle mitochondrial biogenesis, and repair of muscle mtDNA mutations is p53 dependent. Table S1. WT, PolG-SED, and PolG-END Skeletal Muscle Microarray IPA-GO Analysis. Table S2. Real-time PCR primer sequences. (PDF 1601 kb

Vertically Resolved Measurements of Nighttime Radical Reservoirs in Los Angeles and Their Contribution to the Urban Radical Budget

Photolabile nighttime radical reservoirs, such as nitrous acid (HONO) and nitryl chloride (ClNO₂), contribute to the oxidizing potential of the atmosphere, particularly in early morning. We present the first vertically resolved measurements of ClNO₂, together with vertically resolved measurements of HONO. These measurements were acquired during the California Nexus (CalNex) campaign in the Los Angeles basin in spring 2010. Average profiles of ClNO₂ exhibited no significant dependence on height within the boundary layer and residual layer, although individual vertical profiles did show variability. By contrast, nitrous acid was strongly enhanced near the ground surface with much smaller concentrations aloft. These observations are consistent with a ClNO₂ source from aerosol uptake of N₂O₅ throughout the boundary layer and a HONO source from dry deposition of NO₂ to the ground surface and subsequent chemical conversion. At ground level, daytime radical formation calculated from nighttime-accumulated HONO and ClNO₂ was approximately equal. Incorporating the different vertical distributions by integrating through the boundary and residual layers demonstrated that nighttime-accumulated ClNO₂ produced nine times as many radicals as nighttime-accumulated HONO. A comprehensive radical budget at ground level demonstrated that nighttime radical reservoirs accounted for 8% of total radicals formed and that they were the dominant radical source between sunrise and 09:00 Pacific daylight time (PDT). These data show that vertical gradients of radical precursors should be taken into account in radical budgets, particularly with respect to HONO