Lysozyme uptake into pharmaceutical grade fucoidan/chitosan polyelectrolyte multilayers under physiological conditions

Abstract

Polyelectrolyte multilayers composed of pharmaceutical grade fucoidan and chitosan have been assembled and studied in terms of their response to physiological solution conditions and the presence of lysozyme. The influence of phosphate buffered saline (PBS) solution on the multilayer was interrogated using attenuated total reflectance (ATR) Fourier transform infrared (FTIR) spectroscopy and atomic force microscopy (AFM). The combination of the techniques reveal that the polyelectrolyte multilayers swell when exposed to PBS after build-up and may include a small degree of mass loss as the film swells. The degree of swelling was influenced by the terminating layer of the multilayer. Upon exposure to lysozyme, it was observed that some deswelling occurred, as the enzyme adsorbed onto and permeated into the multilayer. The behaviour of the multilayer as a potential reservoir for lysozyme contrasts with the interaction with bovine serum albumin, which did not penetrate into the multilayer, indicating either exclusion by size or due to the overall net negative charge of the film.

Introduction

Polyelectrolyte multilayers (PEMs) are a subtype of soft matter surface coatings that can be influenced by various factors both during and after the formation of the coating, e.g. ionic strength and composition, pH, and temperature [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15]. These parameters effect PEM films owing to the nature of the layer-by-layer (LbL) method by which the films are produced. The LbL technique uses the interaction between the charged functional groups on polyelectrolytes to build the PEM by sequentially adsorbing layers of oppositely charged species that interact predominantly via direct ion pairing. The effect of pH and ionic strength of the background electrolyte is known to impact PEM properties [16], [17], [18], [19], [20], [21], [22], and recently several authors have investigated ion-specific effects on PEMs [23], [24], including investigating the Hofmeister series [25], [26], monovalent [13], [27] and divalent ion effects [28], [29], [30], [31] and ion transport [20], [21], [32].

In biomaterials research, PEMs intended for in vivo application are often formed in more ideal solution conditions (i.e. simpler and more dilute electrolyte composition, pH below 6) than those representative of the physiological environment (higher salt concentration, osmolarity ~0.15 M, and a pH that can vary depending on the fluid). In order to mimic physiological conditions, phosphate buffered saline (PBS) is often used as the fluid from which subsequent interactions can be studied, such as the loading of biological molecules into the PEMs [33], [34], [35], [36], [37], [38], [39], [40], [41], [42], [43]. The effect of phosphate ions on synthetic and biopolymer PEMs has been investigated, with PEM films observed to become more swollen and softer in the presence of phosphate [25], [28].

For PEMs aimed towards biological applications, the impact of changing the electrolyte solution is of interest as it changes their physical properties and their ability to interact with biomolecules. In this work, we have chosen to investigate the influence of electrolyte change (between formation and physiological conditions) on the multilayer itself, and the subsequent interactions with a relevant enzyme from a biomaterials perspective. The PEM used in this work was composed of Fucus vesiculosus fucoidan, a sulfated polyanion sourced from brown seaweed, and Protosan a type of chitosan (deacetylated chitin polycation) derived from fungi. Both polymers have potential application as anti-pathogenic materials [44], [45]. Both polymers used are pharmaceutical grade materials, and therefore are ideal for the study of surface coatings that have potential for clinical applications.

The enzyme used in this work is lysozyme (LYZ), a positively charged protein of approximately 14 kDa which is predominantly found in secretory fluids, including tears [46], [47]. Lysozyme is responsible for the catalytic hydrolysis of β(1 → 4) glycosidic linkages at the C₄ atom within an N-acetyl-d-glucosamine unit. These glycosidic linkages can be found within chitosan, a constituent of our PEM films. Chitosan has been shown to be degraded by lysozyme in chitosan/hyaluronic acid PEMs [48]. Fucoidan contains α(1 → 3) glycosidic linkages [49], and therefore cannot be hydrolysed by lysozyme. Catalytic hydrolysis is the reason behind the anti-bacterial nature of lysozyme; it degrades the cell wall of gram-positive bacteria through its action on peptidoglycan [47]. Creating a PEM film composed of anti-pathogenic polyelectrolytes with the ability to harvest lysozyme from tears may have an application as an antibacterial coating for contact lenses that act by exploiting and enhancing the body’s natural defenses.

To get a clear sense of the chemical and physical changes within the PEM during formation, electrolyte change, and exposure to an enzyme, we have chosen to use attenuated total reflection (ATR) FTIR. Critically, we have made use of ATR internal reflection element (IRE) materials that have allowed us to selectively study penetration and diffusion within the PEM, and separate this process from surface adsorption onto the PEM. In addition to obtaining information on the chemical changes that occur during PBS and enzyme exposure, we have used atomic force microscopy (AFM) imaging to determine the physical changes in the film, including morphology, thickness, and rigidity. These two techniques have provided a detailed picture of the evolution of the PEM during exposure to a simulated biological environment.