In VivoUptake of Chitosan Microparticles by Murine Peyer's Patches: Visualization Studies using Confocal Laser Scanning Microscopy and Immunohistochemistry

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Abstract
Although oral vaccination has numerous advantages over parenteral injection, degradation of the vaccine and low uptake by the gut associated lymphoid tissue (GALT) still complicate the development of efficient oral vaccines. However, previous studies in our laboratory demonstrated that chitosan microparticles can have suitable size, charge, loading and release characteristics for oral vaccination using ovalbumin as model vaccine. In this study, two different approaches were used to investigate the in vivo uptake of chitosan microparticles by murine Peyer's patches. Firstly, a confocal laser scanning microscopy (CLSM) study was performed to visualize the uptake of fluorescent-labeled chitosan microparticles in the Peyer's patches after intragastrical feeding. Subsequently, the intestinal epithelial uptake of ovalbumin loaded chitosan microparticles was visualized using immunohistochemical staining of ovalbumin. Because the microparticles are biodegradable, this entrapped ovalbumin will be released after intracellular digestion in the Peyer's patches. CLSM visualization demonstrated that chitosan microparticles enhance the uptake of fluorescent-labeled ovalbumin by the epithelium of the Peyer's patches. No ovalbumin uptake by the intestinal epithelium was observed when the protein was administered without microparticles. Moreover, immunohistochemical visualization studies revealed that ovalbumin could only be transported into the Peyer's patches after association to chitosan microparticles. Since uptake by Peyer's patches is an essential step in oral vaccination, these in vivo experiments demonstrate that chitosan microparticles are very promising vaccine delivery systems.