Evaluation of Eudragit-coated chitosan microparticles as an oral immune delivery system

https://doi.org/10.1016/j.ijpharm.2005.04.008Get rights and content

Abstract

Chitosan microparticles containing ovalbumin (OVA), OVA-containing chitosan microparticles (Chi-OVA), were prepared, coated with Eudragit L100 (ER), and evaluated as oral vaccine. Chi-OVA with an OVA content of 34.4% (w/w) and a mean particle size of 2.3 μm were used for experiments in vitro and in vivo. ER-coated Chi-OVA (ER-Chi-OVA) contained 3.6–20.5% (w/w) OVA and had a particle size of 47.9–161.1 μm. Chi-OVA dissolved readily in JP 14 first fluid, but not in JP 14 second fluid. The release of OVA from Chi-OVA was suppressed extensively in JP 14 second fluid. ER-Chi-OVA did not dissolve in JP 14 first fluid, and the release of OVA was suppressed greatly in JP 14 first and second fluids. OVA solution, Chi-OVA and ER-Chi-OVA (200 and 800 μg OVA/mouse) were administered to Balb/C mice twice at a 1-week interval. At 7 d after the second administration, plasma OVA-specific IgG and fecal OVA-specific IgA levels were measured. OVA-specific IgG tended to be enhanced in Chi-OVA and ER-Chi-OVA, but was the highest in OVA solution. OVA-specific IgA was induced significantly more efficiently by ER-Chi-OVA than the others. These suggested that ER-Chi-OVA should be possibly useful to induce an intestinal mucosal immune response.

Introduction

Many infections occur via the mucosal surface. However, most of vaccinations are performed by parenteral administration, in which systemic immune responses are induced but mucosal immune responses are not. Parenteral vaccinations require trained personnel and sterilized materials, and infusion often causes non-compliance to patients. Oral vaccinations have no such limitations and can induce both systemic and mucosal immune responses (O’Hagan et al., 1989, Tabata et al., 1996, Trolle et al., 1998, Wikingsson and Sjoholm, 2002, Minato et al., 2003). The induction of mucosal immune responses is also important for the prevention of infection; that is, mucosal immunization can prevent the entry of infectious matter. Thus, much attention has been paid to mucosal immunization, especially oral vaccination, as an easy and acceptable approach.

However, the simple oral administration of antigens elicits little immune responses because gastric acid, various hydrolytic enzymes, thick mucus, etc. degrade or remove the ingested antigens (Walker and Taylor, 1978, Fushiki et al., 1985, Kunisawa et al., 2001, Takagi et al., 2003). Therefore, various delivery systems have been investigated in order to make oral immunizations more effective. In particular, microparticules with a diameter of less than 10 μm are reported to show good adjuvant effects (Eldridge et al., 1990, Uchida and Goto, 1994, Tabata et al., 1996, Nakamura et al., 1998). Such particles can protect the antigens from degradation in the stomach and intestine, and deliver them efficiently to the gut associated lymphoid tissue (GALT) located in the lower portion of the small intestine. When such microparticles reach the Peyer's patches in the GALT, they can be taken up by M-cells located between the epithelial cells. The internalization by M-cells appears to be an important initial step of an oral vaccination (Tabata et al., 1996). Also, the mucoadhesive properties and release control of antigens are associated with the effectiveness as well as the protection of the antigens, and the mucoadhesion to the middle and lower part of the small intestine appears to facilitate the delivery of the antigens to Peyer's patches due to closer contact with luminal surface (Kunisawa et al., 2001). Further, the antigens seem to need to be released from the delivered sites, the Peyer's patches; at that time, a gradual release is effective in enhancing the immune response (Uchida and Goto, 1994, Nakamura et al., 1998, Kunisawa et al., 2001). The effect of particle size on the degree of induction and pattern of the immune responses has been reported. PLA or PLGA microspheres with a diameter of 4 μm best induced systemic immune responses (Tabata et al., 1996, Uchida and Goto, 1994), and PLA microspheres of 7 μm best induced the mucosal immune responses (Tabata et al., 1996). Gelatin microspheres with a diameter of a few micrometers also greatly enhanced the mucosal immune response (Nakamura et al., 1998).

Recently, chitosan microparticles have been used for the delivery of antigens to Peyer's patches (Van der Lubben et al., 2001a, Van der Lubben et al., 2002). Chitosan itself exhibits mucobioadhesive properties to the mucosal membrane probably because of its cationic and viscous properties, and is considered suitable for the delivery to specific sites of the intestine (Takeuchi et al., 1996, Takishima et al., 2001, Takishima et al., 2002). Chitosan microparticles with a diameter of several micrometers were reported to enhance the uptake of a model antigen ovalbumin (OVA) in Peyer's patches (Van der Lubben et al., 2001b, Van der Lubben et al., 2001c). However, as chitosan is easily dissolved in the acidic stomach, simple oral administration results in the dissolution or collapse. In this study, OVA-containing chitosan microparticles (Chi-OVA) are prepared, coated with Eudragit L100 (ER) to protect chitosan microparticles from dissolution or collapse under gastric conditions, and characterized in vitro in terms of physical stability and dissolution. Further, systemic and mucosal immune responses are examined in vivo after oral administration to mice.

Section snippets

Materials

Chitosan 10 (Chi), used as chitosan throughout the study, and polysorbate 80 (Tween 80) were purchased from Wako Pure Chemical Industries, Ltd. (Japan). Ovalbumin was obtained from Calbiochem (USA). Eudragit L100 (ER) was obtained from Röhm GmbH (Germany). Sorbitan sesquioleate (SO-15) was purchased from Nikkko Chemicals Co. Ltd. (Japan). The BCA Protein Assay Kit was purchased from Pierce (USA). Mouse IgG and IgA ELISA Quantitation Kits were obtained from Bethyl Laboratories Inc. (USA).

Particle characteristics of Chi-OVA and ER-Chi-OVA

Chi-OVA were prepared by ionic gelation and emulsification–solvent evaporation. Although an aqueous suspension of Chi-OVA was obtained by ionic gelation using sodium sulfate, the resulting microparticles (Chi-OVA(1)) tended to aggregate after lyophilization of the aqueous suspension, which was observed with a scanning electron microscope. Therefore, their particle size is not described in Table 1. Chi-OVA(1) exhibited a very low OVA content and encapsulation efficiency. On the other hand,

Discussion

The purpose of the present study is to develop an oral mucosal immune drug delivery system using chitosan which is less toxic, mucoadhesive and biodegraded by the intestinal bacteria. A well-characterized protein antigen OVA was used as a model antigen. The potential for immunization is dependent on the physicochemical and biological characteristics of the system. In microparticulte systems, the particle size and release profiles of antigens play an essential role in the immunization

Conclusion

Emulsification–solvent evaporation gave Chi-OVA which had a size adequate for uptake by Peyer's patches and a high OVA content. The coating with ER protected Chi-OVA from the dissolution in JP 14 first fluid (pH 1.2). Chi-OVA released OVA very slowly in JP 14 second fluid, and ER-Chi-OVA suppressed the release of OVA from JP 14 first fluid and JP 14 second fluid (pH 6.8). Chi-OVA and ER-Chi-OVA tended to show a higher OVA-specific IgG level, though OVA solution overall exhibited the highest

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