Elsevier

Carbohydrate Polymers

Volume 55, Issue 4, 15 March 2004, Pages 437-453
Carbohydrate Polymers

Physicochemical and structural characterization of a polyionic matrix of interest in biotechnology, in the pharmaceutical and biomedical fields

https://doi.org/10.1016/j.carbpol.2003.11.013Get rights and content

Abstract

This paper reports on the swelling degree and the rheological and structural characteristics of a hydrogel composed by chitosan and xanthan. The latter is a polyionic hydrogel obtained by complexation between the both polysaccharides. The swelling degree has been found to be influenced by the time of coacervation, the pH of the solution of chitosan used to form the hydrogel and the pH of the swelling solution. The molecular weight and the degree of acetylation of the chitosan also influence the swelling degree of this matrix. The connectivity between chitosan and xanthan affects the swelling degree of this matrix. A rheological study has been carried out in order to understand the formation of the coacervate and of the subsequent hydrogel. The evolution of the storage modulus with time during the coacervation has allowed to optimize the time of coacervation required for a subsequently hydrogel, with desirable swelling degree. The kinetics has shown that (a) the coacervate is formed in two distinct steps and (b) the storage modulus of the hydrogel reaches a stable plateau. The values of the storage modulus have been correlated with the swelling degree. The microscopic characterization has shown the presence of a porous network with a fibrillar structure. To complete the characterization studies fine powder of this hydrogel has been used to determine the surface, perimeter, Feret diameter and sphericity factor distribution of dry and hydrated (swollen) particles.

Introduction

This hydrogel is formed by ionic bonding and van der Waals interactions between two biopolymers: chitosan and xanthan (Dumitriu et al., 1995a, Dumitriu et al., 1994).

Chitosan is a linear binary heteropolysaccharide composed of β-(1→4)-2-amino-2-deoxy-d-glucopyranose and of β-(1→4)-2-acetamino-2-deoxy-d-glucopyranose, obtained by alkaline deacetylation of chitin, poly(β-(1→4)-2-acetamino-2-deoxy-d-glucopyranose). Chitin, the second most abundant biopolymer after cellulose, is a natural polysaccharide found in the shells of crustaceans such as crabs and shrimps, the cuticle of insects and the cell walls of fungi. Due to their biocompatibility, biodegradability and other favourable properties, chitin and chitosan have found applications in several sectors: biomedical, pharmaceutics, food additives, antimicrobial agents, paper and textiles, environmental remediation and other industrial areas (Muzzarelli, 1998, Struszczyk, 2002a, Struszczyk, 2002b, Struszczyk, 2002c, Struszczyk et al., 1989).

Xanthan is an extracellular heteropolysaccharide produced by Xanthomonas campestris. It consists of d-glucose, d-mannose, d-glucuronic acid and pyruvate. Xanthan has a cellulosic backbone and a trisaccharide side chain on every second glucose residue: α-mannose, α-glucuronic acid and β-mannose compose the trisaccharide side chain. Pyruvic acid diketal groups are located at the 4,6-position of the terminal mannose (Stokke, Christensen, & Smidsrod, 1998). Xanthan is one of the most applied microbial polysaccharides in food (Dhami et al., 1995, Quemener et al., 2000, Wang et al., 2002), pharmaceutics (Gohel et al., 2002, Kondo et al., 1996, Talukdar et al., 1998) and in the biomedical field (Stokke et al., 1998).

Many polyionic complexes use chitosan as polycation. Kikuchi and Fukuda (1974) have shown that the chemical structure, the molecular weight, the flexibility, functional groups and hydrophobicity have an effect on the formation of complexes of chitosan–polyanion. Moreover, the conditions of the reaction such as pH, polymer concentration, ionic strength, temperature and the ratio between chitosan and polyanion influence the complexation (Kubota and Kikuchi, 1998, Yamauchi, 2001). Anionic polysaccharides, that have been reported as forming complexes with chitosan are sodium dextran sulfate (Kikuchi & Fukuda, 1974), heparin (Kikuchi & Noda, 1976), carboxymethylcellulose (Fukuda, 1980), carrageenan (Sakiyama, Chu, & Yano, 1993), sodium alginate (Daly & knorr, 1988), sodium carboxymethyldextran (Fukuda & Kikuchi, 1978), chondroitin sulfate (Denuziere, Ferrier, & Domard, 1996) and xanthan (Dumitriu et al., 1994).

Hydrogels are hydrophilic three-dimensional (3D) polymeric networks that can absorb much more water than their own weight so as to provide ideal aqueous conditions for biocompatible applications (Dumitriu & Chornet, 1998a) and for environmentally sensitive bioactive materials such as proteins and specific compounds (Park, Lu, & Crotts, 1995). The formation of a 3D network structure has an important advantage, in that, the gelation increases the mechanical and chemical stability (Dumitriu, Vidal, & Chornet, 1998).

The formation of the polyionic matrix is shown in Fig. 1. The formation of this hydrogel is carried out in many steps. The first one is the mixing of the two solutions of macromolecules. The second step is a modification of concentration of each polymer in the final solution due to the mixing and modification of the structure of the macromolecule. This is due to the change of the local pH (pH 6.8) since the solutions of chitosan and xanthan not have the same pH. The third step is the formation of polyionic interactions between the NH3+ in chitosan and COO in xanthan. The steps 2 and 3 correspond to coacervation, which is a well-known described phenomenon. (Tsung & Burgess, 1997). Coacervation is the separation of aqueous polymeric solutions into two immiscible phases: a coacervate phase (concentrated in polymers) and a dilute phase (Tsung & Burgess, 1997). The ionic interaction between chitosan and xanthan has been already proved by infrared spectrum (Dumitriu et al., 1994) and by the evolution of the rheological parameter of the system (Section 3.4). The coacervate obtain is composed of chitosan, xanthan and oriented and non-oriented water molecules. In fact, during the coacervation, the water molecules are entrapped between the macromolecules. The water molecules arrange themselves in layers. The first layer is at the surface of the macromolecules and in this layer the water molecules are oriented via hydrogen bonding. The second layer corresponds to the free (non-oriented) water molecules. This organization of water molecules is the same as that of the organization of water in silica gel (Brinker & Scherer, 1990). Finally, the last step corresponds to the structural modification of the macromolecular chains by mixing and results in the formation of the hydrogel. In this step, the non-oriented water is eliminated by the mixing strength.

Due to its structure and properties, this hydrogel is particularly attractive as a matrix for enzyme immobilization (Dumitriu and Chornet, 1998, Dumitriu et al., 1995b, Dumitriu et al., 1994, Magnin et al., 2002, Magnin et al., 2001). This matrix is a hydrophilic microenvironment favourable for the inclusion and the stability of the enzymes. In 1994, Dumitriu et al. (1994) demonstrated that the co-immobilization of protease and xylanase led to a synergistic effect (Dumitriu et al., 1994). In a more recent study, Magnin et al. (2002) have shown the influence of the concentration of enzyme, the storage temperature and the molarity of the buffer storage medium, on the catalytic activity. Lipase immobilization into this matrix doubles its enzymatic activity in aqueous media (Magnin et al., 2001). They are also active in organic media (Magnin et al., 2001). Moreover, the immobilization of lipase protects the enzyme against thermal degradation and excellent activity is obtained at 55 °C (Magnin et al., 2001). A distinct application of this matrix is in the pharmaceutical field (Jshizawa, 2002), where it has been used to enhance the dissolution rate of water insoluble drugs, and, consequently, increase their bioavailability. Such approach has been proven with fenofibrate, ursodeoxycholic acid, nifedipine and indomethacin (Jshizawa, 2002, Ishizawa et al., 2001). This polyionic hydrogel can also be used in dermocosmetics. Vitamins in this matrix are protected against rapid oxidation (Dumitriu & Chornet, 2000). Recently, the inclusion of an antibiotic polypeptide has been investigated in order to decrease the solubility of this drug (Magnin et al., 2002).

In this paper, we discuss the physicochemical and structural characteristics of this hydrogel. Its swelling properties are studied in relation with the fibrillar network connectivity within the hydrogel. The kinetic study of the coacervation is carried out via rheological measurements and in addition, microscopic characterization is used to characterize the porous structure and the internal microstructure of the matrix.

Section snippets

Preparation and characterization of chitosan and xanthan solution

Chitosan samples (CH 79 and CH 71P), from shrimp shells, were provided by Kemestrie, Inc. (Sherbrooke, Que., Canada) and (CH V) by Vanson, Inc. (Redmond, WA, USA). Chitosan was characterized by its molecular weight and degree of acetylation (DA). Chitosan preparation and solutions of chitosan and xanthan were prepared as described in the previous study (Dumitriu et al., 1994). An aqueous solution of chitosan of 0.65 wt% is used. The pH of the solution of chitosan was varied between 2 and 5.8.

The

Characterization of the chitosan

Table 1 shows the molecular weight of three chitosans, as determined by the viscometric and GPC methods. Pullulan was used to make the calibration curve in the case of GPC (Jshizawa, 2002). The chromatographic results were in agreement with the viscosity-average molecular weights.

Table 2 presents the DA of three chitosans, as determined by the infrared method and elemental analysis. The two methods provide results that show good agreement.

Influence of pyruvic acid of xanthan on the complexation

As shown in Table 3, the percent of pyruvic acid of

Conclusion

Physicochemical and structural methods have been used to characterize a potential matrix for pharmaceutical formulations and biotechnology applications. This hydrogel shows a swelling degree that can be easily modulated by the choice of the molecular weight and DA of chitosan, the pH of the solution of chitosan and the time of coacervation. The latter two parameters are very important and are operationally controllable. Moreover, it is possible to prepare this hydrogel in bulk or in the form of

Acknowledgements

Financial support from NSERC (Natural Sciences and Engineering Research Council of Canada) is gratefully acknowledged.

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