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