Preparation and Evaluation of Diphtheria Toxoid-Containing Microspheres

Preparation of chitosan (CS) microspheres as a novel drug delivery vehicle for intranasal immunization using high, medium and low CS molecular weight (MW) was investigated in this study. Diphtheria toxoid (DT) was used as a model antigen. The emulsion-solidification method was adopted for microencapsulation of DT.  In the first step, following the purification of semi-crude DT by the ion-exchange column chromatography technique, the antigenicity and biological characteristics of DT were investigated by the bicinchoninic acid protein assay, ELISA and western blot techniques. Results showed that the purification process was successful and the purified toxoid gave an activity of 1500 Lf/ml; which was three times more than that of the semi-crude toxoid. Next, DT-loaded microspheres were prepared and characterized for their surface morphology, particle size distribution, loading efficiency and in-vitro antigenically active DT release.  This study showed that the loading efficiency of CS microspheres depends on the MW, as well as the type of cross-linker used, such that, microspheres prepared by high MW CS and glutaraldehyde (cross-linking agent) had the highest DT loading level (95.61±3.57 percent). Size distribution studies showed that the particle size of microspheres prepared by low and medium MW CS solutions with a concentration of 1 %w/v was below 10 µm.  These microspheres also had a smoother surface morphology than those prepared using high MW CS solutions with concentrations above 1 %w/v. In addition, by investigating the antigenicity of the prepared CS microsphere, no significant reduction in the activity of DT before and after microencapsulation was noted.  Finally, in-vitro release studies showed an initial burst effect followed by an extended release of antigenically active DT over a period of 15 days.