Effect of chitosan on epithelial permeability and structure

Abstract

Numerous studies have shown that chitosan, a mucoadhesive polymer, is a potential enhancer for transmucosal drug delivery. To further understand the mechanisms involved in chitosan action on the mucosal barrier, the activity of chitosan on the function and structure of monolayers of intestinal epithelial cells was investigated. In Caco-2 cells, chitosan caused a reversible, time and dose-dependent decrease in transepithelial electrical resistance. The effect of chitosan on tight junctions was confirmed by an increased permeability coefficient for mannitol transport when cells were treated with 0.1–0.5% w/v chitosan solution for 60 min compared to control cells. Involvement of tight junctions was visualized by confocal scanning microscopy using occludin and ZO-1, tight junctional proteins. Following an incubation with 0.01 or 0.1% w/v chitosan, labeling of both proteins varied in localization and decreased in fluorescent intensity at the cell periphery. In addition, a focal condensation of actin was observed preferentially at areas of cell-to-cell contacts. However, after 24-h recovery, the cell structure resembled untreated control cells. Simultaneous addition of cycloheximide, a protein synthesis inhibitor, prevented full recovery. This implied that protein synthesis was required for the cells to return to baseline levels. Chitosan treatment appeared to slightly perturb the plasma membrane as assessed by an increased release of lactate dehydrogenase. However, addition of 0.5% chitosan for 60 min did not affect cell viability as shown by Trypan blue dye exclusion. These data suggest that chitosan increases cell permeability by affecting paracellular and intracellular pathways of epithelial cells, in a reversible manner.

Introduction

Chitosan, a high molecular weight cationic polysaccharide, has been reported to enhance the absorption of various compounds across the mucosal barrier. Illum et al. (1994) demonstrated the ability of glutamate chitosan to enhance the transport of insulin across the nasal mucosa of sheep and rat. The efficacy of chitosan as a nasal absorption enhancer was confirmed using salmon calcitonin in a rat model (Tengamnuay and Mitra, 1997). Chitosan hydrochloride has also been used to improve the bioavailibility of buserelin in rats, as described by Lueβen et al. (1996). The mechanism of action of chitosan was suggested to be a combination of mucoadhesion and an effect on the gating properties of tight junctions (TJ) (Artursson et al., 1994). It has also been shown that chitosan does not enhance drug absorption by reducing the metabolic activity of both intestinal proteases trypsin and carboxypeptidase B (Lueβen et al., 1997).

The objective of this study was to further elucidate mechanisms in a model of intestinal epithelium. Caco-2 cells represent a well-characterized in vitro transport model system for the small intestinal cellular barrier (Hidalgo et al., 1989). Therefore, we examined the effects of chitosan treatment at various times and concentrations on Caco-2 permeability and morphology by studying more complex aspects of the system not yet addressed in the limited data published on this subject (Borchard et al., 1996; Schipper et al., 1996; Lueβen et al., 1997). Namely, the requirement for protein synthesis was analyzed during the recovery process using cycloheximide as an inhibitor and plasma membrane perturbation was assessed by a lactate dehydrogenase (LDH) activity assay. In addition, an extensive study was performed by confocal scanning and electron microscopy to elucidate the effects of chitosan on cell morphology. In confocal scanning microscopy the label of three cell markers was followed: ZO-1, a TJ-associated protein (Stevenson et al., 1986), occludin, a transmembrane protein of the TJ (Furuse et al., 1993) and bodipy phalloidin to stain actin.