Nisin adsorption to polyethylene oxide layers and its resistance to elution in the presence of fibrinogen

Abstract

The adsorption and elution of the antimicrobial peptide nisin at silanized silica surfaces coated to present pendant polyethylene oxide chains was detected in situ by zeta potential measurements. Silica microspheres were treated with trichlorovinylsilane to introduce hydrophobic vinyl groups, followed by self assembly of the polyethylene oxide–polypropylene oxide–polyethylene oxide (PEO–PPO–PEO) triblock surfactant Pluronic® F108, or an F108 derivative with nitrilotriacetic acid end groups. Triblock-coated microspheres were γ-irradiated to covalently stabilize the PPO-surface association. PEO layer stability was evaluated by triblock resistance to elution by SDS, and layer uniformity was evaluated by fibrinogen repulsion. Introduction of nisin to uncoated or triblock-coated microspheres produced a significant positive change in surface charge (zeta potential) as a result of adsorption of the cationic peptide. In sequential adsorption experiments, the introduction of fibrinogen to nisin-loaded triblock layers caused a decrease in zeta potential that was consistent with partial elution of nisin and/or preferential location of fibrinogen at the interface. This change was substantially more pronounced for uncoated than triblock-coated silica, indicating that the PEO layer offers enhanced resistance to nisin elution.

Introduction

Nisin is a small (3.4 kDa) amphiphilic peptide with five lanthionine rings. It is cationic at neutral pH, due to an isoelectric point above 8.5. Nisin is an effective inhibitor of Gram-positive bacteria, including the two most frequently encountered biomaterial-associated pathogens Staphylococcus aureus and Staphylococcus epidermidis [1], [2], [3], and holds potential for use as an anti-infective agent in medical device coatings [4], [5].

Tai et al. [6] reported results of an ellipsometric analysis of nisin adsorption and elution at surfaces coated with the PEO–PPO–PEO surfactant Pluronic® F108. Those results suggested that nisin adsorption occurred via penetration of and entrapment within the PEO layer, as opposed to adsorption onto the mobile PEO chains. It is generally understood that PEO resists protein interactions, and the protein repellent properties of the F108 layer, if retained after nisin adsorption or integration, would inhibit displacement of the antimicrobial peptide by blood proteins. In this way, nisin loading could impart an active protective function, and increase the effectiveness of such a coating. In this regard Tai et al. [7] evaluated the antimicrobial activity of nisin-loaded, F108-coated polystyrene microspheres and polyurethane catheter segments after incubation with blood proteins for up to 1 week. F108-coated surfaces were observed to retain more antimicrobial activity than uncoated surfaces, suggesting that the pendant PEO chains inhibited displacement or elution of nisin by contact with blood proteins.

The F108 triblocks used by Tai et al. were bound to the base substrates only by hydrophobic association of the polymer and PPO centerblock. It is thus possible that adsorbing nisin dislocated the adsorbed Pluronic at the surface, rather than being integrated into the brush layer itself. Important conclusions relating to nisin entrapment among PEO chains, as well as the enhanced resistance to elution of nisin bound in this way, have thus remained somewhat tentative. In this paper we describe the individual and sequential adsorption of nisin and fibrinogen at silanized silica surfaces coated with covalently-bound PEO–PPO–PEO triblocks. Zeta potential was recorded after protein adsorption to microsphere suspensions coated with F108, or with F108 that had been end-activated with nitrilotriacetic acid groups (EGAP-NTA).

While an abundant literature describes the protein repelling mechanisms of material surfaces presenting pendant PEO, there are very few reports describing the adsorption of small proteins to PEO layers. It has been argued that once a sufficiently high chain density is achieved, the rejection capacity of the pendant polymer phase is determined by protein size, relative to the average distance between polymer chains [8], [9]. Archambault and Brash [10] suggested that grafting densities consistent with the brush configuration would be required before protein discrimination based on size would become evident. Halperin [11] formulated a model for protein adsorption in a PEO brush based on kinetic and thermodynamic considerations, and predicted two possible modes of protein adsorption: primary adsorption (at the surface itself) and secondary adsorption (at the periphery of the grafted PEO chains). Multilayer formation or integration of protein within the PEO chains is not predicted by this simplified model. However, based on surface force experiments involving compression of PEO brushes by protein-coated surfaces, Sheth and Leckband [12] suggested that polymer chains in a PEO brush may exhibit coexistence between an inner, dense, hydrophobic phase and a dilute hydrophilic phase at the outer edge of the brush. Such coexistence would give rise to an inner region “attractive” for protein adsorption. Nisin adsorption within PEO layers may thus be attributable to its high amphiphilicity, in addition to its small size.

Fang et al. [13] formulated a model for protein interaction with PEO brushes based on a generalized diffusion approach. Their model showed that adsorption and desorption kinetics depend on protein size and brush layer thickness. In particular, when the pendant chain layer thickness is greater than the size of the protein, adsorption and desorption kinetics both decrease with increasing chain length. In fact, their model indicated that the adsorption time is so large that, for any practical purpose, protein adsorption is negligible. A particularly interesting outcome of their approach was that proteins may become “trapped” between the surface and the barrier presented by the pendant chains. Increasing the chain length increases the steric barrier to elution, and the rate of protein desorption is thus decreased. Based on that result, they suggested that such a trapping mechanism could be used in the design of strategies for the controlled release of proteins from surfaces.

Some studies have shown that protein adsorption is insensitive to PEO end group chemistry while others have reported significant effects. Mathematical models of PEO in the brush configuration indicate that it is highly unlikely that end group chemistry would affect interaction with proteins. For example, Halperin [11] showed that chain ends are statistically distributed throughout the brush, with a maximum occurring at a distance about 70% of the chain length from the surface. Unsworth et al. [14] showed experimentally that protein repulsion at PEO brushes was uniquely determined by chain density, independent of chain length and end group chemistry. However, beyond a critical chain density, it was observed that brushes with OH end groups were observed to remain nonfouling, while brushes with OCH₃ end groups promoted protein adsorption. The authors suggested that the high densities of terminal methoxy groups may have resulted in increased inter-chain association and/or adsorption-induced protein denaturation. The formation of terminal OCH₃ “islands” and defects in the brush layer are also predicted theoretically in a random-sequential-adsorption model advanced by Katira et al. [15].