A more quantitative understanding of peptide loading and release from polyethylene oxide (PEO) brush layers will provide direction for development of new strategies for drug storage and delivery. The antimicrobial peptide nisin shows potent activity against Gram-positive bacteria including the most prevalent implant-associated pathogens, its mechanism of action minimizes the opportunity for the rise of resistant bacteria and it does not appear to be toxic to humans, suggesting good potential for its use in antibacterial coatings for selected medical devices. In this work, optical waveguide lightmode spectroscopy was used to record changes in adsorbed mass during cyclic adsorption-elution experiments with nisin, at uncoated and PEO-coated surfaces. PEO layers were prepared by radiolytic grafting of Pluronic® surfactant F108 or F68 to silanized silica surfaces, producing long- or short-chain PEO layers, respectively. Kinetic patterns were interpreted with reference to a model accounting for history-dependent adsorption, in order to evaluate rate constants for nisin adsorption and desorption, as well as the effect of pendant PEO on the lateral clustering behavior of nisin. Lateral rearrangement and clustering of adsorbed nisin was apparent on uncoated and F68-coated surfaces, but not on F108-coated surfaces. In addition, nisin showed greater resistance to elution by peptide-free buffer from uncoated and F68-coated surfaces. These results are consistent with shorter PEO chains allowing for peptide adsorption to the base substrate in the case of F68-coated surfaces, while adsorption to the F108-coated surfaces is apparently governed by the presence of a hydrophobic core within the brush layer itself. Further, these results suggest that while peptide location within the hydrophobic core provides stability against lateral rearrangement, the pendant PEO chains themselves provide no steric barrier to nisin rearrangement within the brush layer. / Graduation date: 2012

Nisin, an amphiphilic, antimicrobial peptide, has been shown to integrate into the hydrophobic inner region of poly(ethylene oxide) (PEO) brush layers; however, the presence of integrated nisin may compromise the protein repulsive character of the PEO layer. In particular, the introduction of fibrinogen to nisin-loaded brush layers has been observed to cause changes consistent with partial elution of nisin and/or location of fibrinogen at the interface. Questions surrounding the possibility of fibrinogen adsorption warrant further investigation, as the location of procoagulant proteins at a peptide-loaded PEO layer would significantly reduce the viability of a medical device coating based on such an approach. In this work, the preferential location of fibrinogen at PEO brush layers was investigated by: detection of FITC-labeled fibrinogen after sequential introduction of nisin and labeled fibrinogen; measurement of changes in the zeta potential of PEO coated and uncoated surfaces following nisin, fibrinogen, and/or buffer challenges; and evaluation of adsorption and elution kinetics in label-free, sequential adsorption experiments using optical waveguide lightmode spectroscopy (OWLS). PEO layers were constructed through radiolytic grafting of Pluronic�� F108 or F68 onto silanized silica surfaces producing long-chain or short-chain PEO layers, respectively. Adsorption results indicated that sequential introduction of nisin and fibrinogen to PEO brush layers, based on F108, does not result in fibrinogen adsorption beyond that expected for a nisin-free PEO layer. No evidence of nisin entrapment in fibrinogen-repellent F68 layers was recorded. Low-level fibrinogen adsorption observed at F68 layers following the introduction of nisin was determined to be a result of nisin adsorption at (uncoated) defect regions on the surface. In conclusion, retention of PEO layer capacity for protein repulsion after nisin entrapment is owing to a steric repulsive barrier provided by PEO segments extending beyond the level of entrapped nisin. It was then hypothesized that the immobilized, pendant PEO chains will inhibit exchange of entrapped nisin by competing proteins, and therefore prolong nisin activity retention. In order to evaluate nisin function following its entrapment, the antimicrobial activity of nisin-loaded, F108-coated silica surfaces was evaluated against the Gram-positive indicator strain, Pediococcus pentosaceous. The retained biological activity of these nisin-loaded layers was evaluated after incubation in the presence of bovine serum albumin (BSA), for contact periods up to one week. Surfaces were withdrawn at selected times and placed on plates inoculated with P. pentosaceous to measure kill zone radius in order to quantify nisin activity. In the presence of BSA, F108-coated surfaces retained more antimicrobial activity than the uncoated, hydrophobic surfaces. These results strongly suggest that PEO brush layers may serve as a viable drug storage platform due to the retained non-fouling character after bioactive peptide entrapment and the prolonged peptide activity in the presence of other proteins. / Graduation date: 2013

Biodesulfurization (BDS) is an alternative to hydrodesulfurization (HDS) as a method to remove sulfur from crude oil. Dibenzothiophene (DBT) was chosen as a model compound for the forms of thiophenic sulfur found in fossil fuels; up to 70% of the sulfur in petroleum is found as DBT and substituted DBTs; these compounds are however particularly recalcitrant to hydrodesulfurization, the current standard industrial method. My thesis deals with enhancing BDS through novel strains and through nanotechnology. Chapter highlights are: Chapter 2. My first aim was to isolate novel aerobic, mesophilic bacteria that can grow in mineral media at neutral pH value with DBT as the sole sulfur source. Different natural sites in Iran were sampled and I enriched, isolated and purified such bacteria. Twenty four isolates were obtained that could utilize sulfur compounds. Five of them were shown to convert DBT into HBP. After preliminary characterization, the five isolates were sent to the Durmishidze Institute of Biotechnology in Tbilisi for help with strain identification. Two isolates (F2 and F4) were identified as Pseudomonas strains, F1 was a Flavobacterium and F3 belonged to the strain of Rhodococcus. The definite identification of isolate F5 was not successful but with high probability it was a known strain. Since no new strains were apparently discovered, I did not work further in this direction. Chapter 3. In a second approach I studied the desulfurization ability of Shewanella putrefaciens strain NCIMB 8768, because in a previous investigation carried out at Cranfield University, it had been found that it reduced sulfur odour in clay. I compared its biodesulfurization activity profile with that of the widely studied Rhodococcus erythropolis strain IGTS8. However, S. putrefaciens was not as good as R. erythropolis. Chapter 4 and 5. I then turned to nanotechnology, which as a revolutionary new technological platform offers hope to solve many problems. There is currently a trend toward the increasing use of nanotechnology in industry because of its potentially revolutionary paths to innovation. I then asked how nanotechnology can contribute to enhancing the presently poor efficiency of biodesulfurization. Perhaps the most problematic difficulty is how to separate the microorganisms at the end of the desulfurization process. To make BDS more amenable, I explored the use of nanotechnology to magnetize biodesulfurizing bacteria. In other words, to render desulfurizing bacteria magnetic, I made them magnetic by decorating their outer surfaces with magnetic nanoparticles, allowing them to be separated using an external magnet. I used the best known desulfurizing bacterial strain, Rhodococcus erythropolis IGTS8. The decoration and magnetic separation worked very well. Unexpectedly, I found that the decorated cells had a 56% higher desulfurization activity compared to the nondecorated cells. I proposed that this is due to permeabilization of the bacterial membrane, facilitating the entry and exit of reactant and product respectively. Supporting evidence for enhanced permeabilization was obtained by Dr Pavel Grigoriev, Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino. In Chapter 6, to optimize attachment of the nanoparticles to the surface of the bacteria I created thin magnetic nanofilms from the nanoparticles and measured the attachment of the bacteria using a uniquely powerful noninvasive optical technique (Optical Waveguide Lightmode Spectroscopy, OWLS) to quantify the attachment and determine how the liquid medium and other factors influence the process.