In vitro evaluation of the mucoadhesive properties of chitosan microspheres

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Abstract

The mucoadhesive properties of chitosan and chitosan microspheres were evaluated by studying the interaction between mucin and chitosan in aqueous solution by turbidimetric measurements and the measurement of mucin adsorbed on the microspheres. A strong interaction between chitosan microspheres and mucin was detected. Adsorption studies were carried out for the adsorption of mucin to chitosan microspheres with different crosslinking levels. The adsorption of type III mucin (1% sialic acid content), to chitosan microspheres followed Freundlich or Langmuir adsorption isotherms. When the contents of sialic acid was increased (i.e. type I-S mucin, 12% sialic acid content), the adsorption type followed more closely an electrostatic attraction type of isotherm. The heat of the adsorption was found to be 13–23 kJ/mol. A salt-bridge effect has been proposed for the interaction of the positively charged mucoadhesive chitosan microspheres with the negatively charged mucus glycoprotein. The extent of mucus adsorption was proportional to the absolute values of the positive zeta potential of chitosan microspheres and negative `zeta potential' of mucus glycoprotein. Factors leading to a reduction or a reversal of these absolute values (e.g. different crosslinking levels of chitosan microspheres, different types of mucin, different pH, or ionic strength of the medium used) led to a reduction in the amount adsorbed. The extent of this reduction depended upon the decreasing extent of the repective zeta potentials. Biological studies showed that chitosan microspheres were retained by a biological tissue; rat small intestine.

Introduction

Chitosan (poly[β-(1-4)-2-amino-2-deoxy-D-glucopyranose]) is a cationic polysaccharide, derived by the deacetylation of chitin, which is the most abundant polysaccharide in the world, next to cellulose. As early as the 1980's, chitosan was evaluated as a matrix for sustained release granules (Miyazaki et al., 1981; Hou et al., 1985), tablets (Kawashima et al., 1985; Akbuga, 1993), and more recently as a component of gels (Knapczyk, 1993; Kristl et al., 1993), and membranes (Thacharodi and Rao, 1993) as well as a film matrix (Chandy and Sharma, 1991; Angelova et al., 1995).

Pharmaceutical applications of chitosan in the form of beads, microspheres and microcapsules were developed in the early of 1990's. Large chitosan microspheres and beads (up to thousands of microns) have typically been used for the prolonged release of drugs (Hou et al., 1994; Wan et al., 1994; Sezer and Akbuga, 1995; Acikgoz et al., 1996) and proteins such as bovine serum albumin (Jameela et al., 1994), DNA (Alaxakis et al., 1995), and brain derived neurotrophic factor (Mittal et al., 1994). Small particle size (<5 μm) chitosan microspheres, containing anticancer agents such as 5-fluorouracil (5-FU) (Ohya et al., 1993), and magnetic microspheres (Hassan et al., 1992) have been described for site specific delivery.

Chitosan is a hydrophilic, biocompatible and biodegradable polymer of low toxicity. It is commercially available in a range of molecular weights, degrees of deacetylation and types of salts such as glutamate, hydrochloride and lactate. Chitosan possesses OH and NH2 groups that can give rise to hydrogen bonding and the linear molecule expresses a sufficient chain flexibility, the conformation of which is highly dependent on ionic strength. These properties are considered essential for mucoadhesion (Smart et al., 1984; Peppas and Buri, 1985; Robinson et al., 1987; Robinson and Mlynek, 1995). Furthermore, the cationic polyelectrolyte nature of chitosan could provide a strong electrostatic interaction with mucus or a negatively charged mucosal surface. The importance of the mucoadhesive properties of chitosan has been demonstrated in earlier work by Lehr et al. (1992), Illum et al. (1994), Lueßen et al. (1994), Fiebrig et al. (1995)and Aspden et al. (1996). More specifically, chitosan has been used as a delivery vehicle for the nasal and peroral delivery of peptide drugs, in order to improve drug absorption (Illum et al., 1994; Lueßen et al., 1996Lueßen et al., 1997). Mucoadhesive tablets, containing chitosan, have been developed by Takayama et al. (1990), Miyazaki et al. (1994)and Nakayama et al. (1994). These tablets can be used for oral, intraoral or sublingual drug delivery. They have both adhesive and sustained release characters. It was also shown that the coating of liposomes with chitosan improved their adsorption to mucosal surfaces (Takeuchi et al., 1996). The application of chitosan in ocular mucoadhesive drug delivery systems has been reviewed by Greaves and Wilson (1993).

The in vitro evaluation of the mucoadhesive properties of a polymer or polymeric microsphere is a basic step in the development of a mucoadhesive drug delivery system. The aim of the present study has been to evaluate the mucoadhesive properties of chitosan, particularly, of the chitosan microspheres. Previously the aspect has been little discussed. Based on the fundamental principles of physical chemistry, these properties have been assessed by evaluation of the interaction between chitosan and mucin in aqueous solution, adsorption isotherms of mucus glycoprotein to chitosan microspheres, and the corresponding heat of the adsorption. Furthermore, using a biological approach, the adhesion of chitosan microspheres to mucosal tissue (rat small intestine) has also been evaluated.

Section snippets

Materials

Chitosan (Seacure CL 210) was obtained from Pronova A/S. Norway. Ethyl cellulose (EC) was purchased from Sigma (Dorset, UK). Mucin, Type III, and Mucin Type I-S, which contain approximately 1% and 12% sialic acid, respectively, were purchased from Sigma. Glutaraldehyde (50% aqueous solution) and poly(vinyl alcohol) (PVA, Mwt 50 000) were obtained from Aldrich (Dorset, UK). All chemicals, reagents and solvents used were of the highest grade available and used as provided.

Preparation of chitosan microspheres

Chitosan microspheres

Physicochemical characteristics of the microspheres

The physicochemical characteristics of the five different chitosan microspheres with different levels of cross-linking (batches no. 1–5) and the EC microspheres are shown in Table 1. The characteristics of chitosan microspheres are closely associated with the level of the cross-linking. The particle size of the chitosan microspheres ranged from 3–12 μm. The more of the cross-linking agent added, the less irregular were the microspheres formed, and the smaller the particle size of the

Conclusions

A strong interaction between chitosan and mucus glycoprotein in aqueous solution was measured. Positively charged chitosan microspheres, prepared by a spray drying method, had the ability to adsorb mucus glycoprotein.

Adsorption studies (adsorption kinetics, adsorption isotherms and heat of adsorption) were carried out for the adsorption of different types of mucin on chitosan microspheres with different crosslinking levels. The adsorption of type III mucin followed Freundlich or Langmuir

Acknowledgements

The authors would like to thank Dr Tony Fisher for his cooperation in the biological studies.

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