Preparation and evaluation of sucrose stabilized tetanus toxoid encapsulated into chitosan microspheres

Immunization is the most cost effective weapon for disease prevention in developing countries, and advanced molecular and genetic technologies are making new types of vaccines feasible. Here, the utility of both in vitro and in vivo methods to assess the release pattern of chitosan microspheres containing tetanus toxoid (TT) vaccine were evaluated. TT was stabilized and encapsulated in chitosan (TTCH) with a water-in-oil-in-water (W/O/W) multiple emulsion method using sucrose as a protein stabilizer. The TTCH prepared was smooth and spherical in shape with a diameter of around 10 μm. The in vitro release efficiency of TTCH was evaluated by differing stabilizer (sucrose) concentration (5%, 7%, 10% and 12% w/v) for a period of 70 days. The antigen release rates from the microspheres were determined by enzyme-linked immunosorbent assay. In these TTCH microspheres, a 10% w/v sucrose concentration gave good sustained antigen delivery for the period of 70 days. Based on the results of in vitro release, the in vivo studies were carried out using alum-adsorbed TT (from the Central Research Institute) as the standard. The antibody level was measured after 6 months, 9 months and finally, with one booster dose, after 12 months. In these in vivo studies, the TTCH antibody level rose up to 3.5 IU/mL of guinea pig serum; this compared with 2 IU/mL of guinea pig serum using the alum-adsorbed TT after 12 months with a second booster dose. The TTCH approach would be helpful to replace the existing adjuvant alum in the future.