Alginate fibres modified with unhydrolysed and hydrolysed chitosans for wound dressings

Abstract

A range of commercial chitosans were sourced, subjected to controlled acid hydrolysis, and their molecular size profiles and degrees of acetylation (DA) determined (pre- and post-hydrolysis) by High Performance Size Exclusion Chromatography and ¹H-NMR spectroscopy, respectively. Unhydrolysed and hydrolysed chitosans were subsequently utilised for modification of sodium alginate/alginic acid fibres (prepared using a range of different fibre spinning conditions), and levels of chitosan incorporated onto/into base alginate fibres were estimated by elemental analysis. Tensile properties (% elongation and tenacity) of resultant chitosan/alginate fibres were determined in order to assess their suitability for potential application in wound dressings. A broad range of chitosan contents (∼0–6% w/w) and hydrolysed chitosan contents (∼7–25% w/w) were obtained using a variety of alginate and chitosan starting materials. Modification of fibres with unhydrolysed chitosans generally resulted in a significant reduction in tenacity (and a reduction in % elongation if a water washing stage was not used), i.e. no increase in fibre strength was observed, implying that the unhydrolysed chitosan is more like a coating rather than penetrating/reinforcing the alginate fibre. Reduction of chitosan molecular weight had a positive effect on its ability to penetrate the alginate fibres, not only increasing fibre chitosan content, but also reinforcing fibre structure and thus enhancing tensile properties (compared with unhydrolysed chitosan/alginate fibres). Hydrolysed chitosan/alginate fibres demonstrated an antibacterial effect (in terms of bacterial reduction) with initial use, and had the ability to provide a slow release/leaching of antibacterially active components (presumably hydrolysed chitosan fragments). The overall aims of maximising chitosan content (in order to provide satisfactory antibacterial activity) whilst retaining desirable physical properties (i.e. satisfactory textile processing ability) were therefore achieved, with respect to the fibres having potential application as wound dressing materials.

Introduction

Fibres have been extensively used in wound dressing applications because of their unique/advantageous properties, such as high surface area, softness, absorbency and ease of fabrication into many product forms. Fibres made from natural sources, especially polysaccharides, have been considered the most promising due to their excellent biocompatibility, non-toxicity, and potential bioactivity at the wound surface and beyond. Many commercial wound dressing products (woven and non-woven dressings, and hydrogels) are made from such natural polymers and their derivatives, the simplest being retention bandages, support and compression bandages, absorbents, gauzes, tulle dressings, and wound dressing pads produced from woven cellulose fibres (cotton and viscose) (Kennedy et al., 1996, Lloyd et al., 1998, Kennedy et al., 2001, British National Formulary, 2001).

Among the various fibrous and hydrogel products, alginate-based products are currently the most popular ones used in wound management, since they offer many advantages over traditional cotton and viscose gauzes (Horncastle, 1995, Qin and Gilding, 1996). They are biocompatible and form a gel on absorption of wound exudate. This eliminates fibre entrapment in the wound, which is a major cause of patient trauma/discomfort during dressing removal. Such gelation prevents the wound surface from drying out, which is beneficial since a moist wound environment promotes healing and leads to a better cosmetic repair of the wound (Winter, 1962). Performance requirements for such gelled dressings (which often aim to replicate the inherent permeability/water content of natural skin) are obviously higher than mere absorbent coverings in order for the wound to remain moist during the contact period (which could be more than several days) (Thomas, 1990). Hence, it is also reported that alginate-based dressings have haemostatic properties and can enhance the rate of healing of skin wounds (Jarvis et al., 1987, Attwood, 1989).

Commercial alginate-based dressings include Algisite^® M (non-woven calcium alginate fibre, Smith and Nephew), Algosteril^® (calcium alginate, Beiersdorf), Kaltocarb^® (calcium alginate fibre, ConvaTec), Kaltogel^® (calcium/sodium alginate gelling fibre, ConvaTec), Kaltostat^® (calcium alginate fibres in non-woven pads, ConvaTec), Melgisorb^® (calcium/sodium alginate gelling fibre, Molnlycke), Seasorb^® (calcium/sodium alginate gelling fibre, Coloplast), Sorbalgon^® (calcium alginate, Hartman), and Sorbsan^® (calcium alginate fibres in non-woven pads, Maersk) (Kennedy et al., 2001, British National Formulary, 2001 ). Consequently there are numerous patents detailing the production of alginate fibres and dressings (Tong, 1985, Thompson, 1996, Griffiths and Mahoney, 1997, Fenton et al., 1998, Mahoney and Howells, 1998, Barikosky, 1999, Kershaw and Mahoney, 1999, Mahoney et al., 1999, Mahoney and Walker, 1999, Horsler, 2000, Qin and Gilding, 2000).

Another type of natural polysaccharide of interest with respect to wound management products is chitin, and its partially deacetylated derivative, chitosan. The presence of chitin/chitosan in a dressing is reported to promote fibroblast growth and affect macrophage activity, which accelerates the wound healing process (Balassa and Prudden, 1978, Muzzarelli et al., 1989, Technical textiles, 1995, Hon, 1996, Mattioli-Belmonte et al., 1997, Muzzarelli et al., 1999). Chitosans are biocompatible (since their biodegradation products are natural metabolites), and are used in a wide variety of commercial application areas, such as cosmetics, haemostatic agents, drug delivery vehicles, wound dressings, etc (Reports Group, Technical Insights, 1989, Berscht et al., 1995, Pittermann et al., 1997, Skjåk-Bræk et al., 1989). Although chitosan can be produced in powder, film, bead, fibre and fabric forms (Qin et al., 1997, Qin and Agboh, 1998), products made from pure chitosan fibres have not been commercially viable due to the high processing costs involved (deproteination, demineralisation and deacetylation processes are required to produce chitosan materials of adequate purity) and the availability of such purified material is still insufficient for large industrial scale fibre production. Poor textile processing properties of resulting fibres has also been a major problem.

The chemical structures of sodium alginate/alginic acid and chitin/chitosan are displayed in Fig. 1 (Collins, 1998). Alginic acid is obtained from the cell walls of brown algae (Phaeophyta) such as the seaweeds Laminaria sp. and Ascophyllum sp (Clare, 1993). It is a linear block copolymer composed of uronic acid residues, namely β-d-mannuronic and α-l-guluronic acid, linked by (1→4)-linkages. The distribution of the uronic acids along the chain is non-random and involves relatively long sequences of each uronic acid. In the presence of divalent cations, such as calcium, alginate gels can be formed due to ionic cross-linking via calcium bridges between l-guluronic acid residues on adjacent chains (McDowell, 1974). Chitin is a naturally occurring polysaccharide found in the outer shell of crustaceans, and is composed of 2-acetamido-2-deoxy-β-d-glucopyranose residues (N-acetyl-d-glucosamine residues), linked by (1→4)-linkages. Chitosan is partially deacetylated chitin and is therefore composed of 2-amino-2-deoxy-β-d-glucopyranose (d-glucosamine) and N-acetyl-d-glucosamine residues.

Alginate fibres are generally prepared by injecting a solution of water-soluble alginate (usually sodium alginate) into a bath containing an acidic solution and/or calcium salt solution to produce the corresponding alginic acid and/or calcium alginate fibres, respectively, which can be used to produce yarns and fabrics for medical applications (Qin et al., 1997, Chen et al., 2001, Miraftab et al., 2001, Miraftab et al., 2002). Many of chitosans properties rely on its cationic nature, which allows it to interact with negatively charged biomolecules such as proteins, anionic polysaccharides and nucleic acids, many of which are located in skin. Therefore, under certain conditions, alginate and chitosan have opposite and therefore mutually attractive charges, making the use of mixed dope solutions of suitable concentrations unfeasible, with respect to chitosan/alginate fibre production, since rapid coagulation/gelation of dope solution can occur.

Chitosan has been used to coat calcium alginate filaments (utilising the cationic interaction of the chitosan with the anionic nature of the alginate to produce a tight interaction) (Tamura, Tsuruta, & Tokura, 2002). However, due to its high molecular weight the chitosan must be used in very low concentrations, as precipitation occurs in the presence of calcium ions, resulting in very low levels of chitosan incorporation into the fibres (<0.2% w/w). Problems in the direct production of chitosan/alginate fibres have been overcome using a variety of different approaches. Alginate and chitosan fibres have been separately produced and subsequently blended, and chitosan has been utilised as the insolubilising cation for production of an alginate fibre (Cole and Nelson, 1993, Pandit, 1998).

The approach adopted for the production of fibres presented in this paper was the use of an initial core fibre produced using one of the polysaccharides, and subsequently applying the other polysaccharide by absorption into/coating onto this core fibre. The obvious route was to use an alginate core fibre (since the required methodologies for the production of fibres with suitable physicochemical characteristics are well known). The aim of the investigations was to produce fibres that combine the biomedical properties of both alginate and chitosan, and that have good textile processing ability and relatively low production costs. Alginate would essentially manage excess liquid/exudate and chitosan would provide antibacterial, haemostatic and wound healing properties. It was predicted that use of hydrolysed chitosans should result in higher levels of chitosan incorporation, since fragments (molecules of lower molecular size than the parent unhydrolysed molecules) should be able to more easily penetrate the base alginate fibre structure (as illustrated in Fig. 2). It was also hoped that penetration of hydrolysed chitosan fragments into base alginate fibres would result in some reinforcement and thus increase/enhance tensile properties (perhaps via ionic interactions as illustrated in Fig. 3).

The comparative analysis of a range of commercial chitosans and their hydrolysates are therefore reported, along with the production and analysis of a range of chitosan/alginate fibres, produced by treating freshly extruded alginate fibres with unhydrolysed or hydrolysed chitosan solutions. The tensile properties (% elongation and tenacity) of the produced fibres were also evaluated in order to evaluate their suitability for potential use in wound dressings applications.