Chitosan microparticles for oral vaccination: preparation, characterization and preliminary in vivo uptake studies in murine Peyer's patches

Although oral vaccination has numerous advantages over parenteral injection , degradation of the vaccine in the gut and low uptake in the lymphoid tiss ue of the gastrointestinal tract still complicate the development of oral v accines. In this study chitosan microparticles were prepared and characteri zed with respect to size, zeta potential, morphology and ovalbumin-loading and -release. Furthermore, the in vivo uptake of chitosan microparticles by murine Peyer's patches was studied using confocal laser scanning microscop y (CLSM). Chitosan microparticles were made according to a precipitation/co acervation method, which was found to be reproducible for different batches of chitosan. The chitosan microparticles were 4.3 +/- 0.7 mum in size and positively charged (20 +/- 1 mV). Since only microparticles smaller than 10 mum can be taken up by M-cells of Peyer's patches, these microparticles ar e suitable to serve as vaccination systems. CLSM visualization studies show ed that the model antigen ovalbumin was entrapped within the chitosan micro particles and not only associated to their outer surface. These results wer e verified using field emission scanning electron microscopy, which demonst rated the porous structure of the chitosan microparticles, thus facilitatin g the entrapment of ovalbumin in the microparticles. Loading studies of the chitosan microparticles with the model compound ovalbumin resulted in load ing capacities of about 40%. Subsequent release studies showed only a very low release of ovalbumin within 4 h and most of the ovalbumin (about 90%) r emained entrapped in the microparticles. Because the prepared chitosan micr oparticles are biodegradable, this entrapped ovalbumin will be released aft er intracellular digestion in the Peyer's patches. Initial in vivo studies demonstrated that fluorescently labeled chitosan microparticles can be take n up by the epithelium of the murine Peyer's patches. Since uptake by Peyer 's patches is an essential step in oral vaccination, these results show tha t the presently developed porous chitosan microparticles are a very promisi ng vaccine delivery system. (C) 2001 Elsevier Science Ltd. All rights reser ved.