Studies On Cyclodextrin Complexed Ovalbumin Encapsulated Chitosan Microparticles Delivery System For Oral Immunization

Based on cyclodextrins and their derivatives can improve the stability of protein,also can be used as protein drug’s oral absorption enhancers and penetration enhancers,so take advantage of cyclodextrins and chitosan together to build a new oral vaccine delivery system.Ovalbumin(OVA)as a model protein drug,(3-cyclodextrin(β-CD)and its derivative carboxymethyl-hydroxypropyl-β-cyclodextrin(CM-HP-β-CD)as the host molecules,prepared OVA-β-CD and OVA-CM-HP-β-CD inclusion complexes by the stirring-freeze-dried method.And then load them into chitosan(CS)microspheres to research the immune response in Balb/c.CM-HP-P-CD,a novel β-cyclodextrin derivative was successfully synthesized and the degree of substitution was determined for carboxymethyl and hydroxypropyl,which was 5.7 and 2.9,respectively.OVA-(3-CD and OVA-CM-HP-β-CD inclusion complexes were prepared and the stability constants(Kc)were determined which were about 3.5×103 M-1 and 2.1×104 M-1 respectively.The optimal inclusion ratio was 10:1 obtained by the method of gradual change in concentration.The inclusion complexes were characterized by SEM and TGA.TGA revealed that OVA-CM-HP-β-CD and OVA-β-CD decomposed at 288.2℃ and 286.8℃,which was higher 30.9℃and 29.5℃than OVA,respectively.The inclusion position between OVA and(3-CD were simulated by AutoDock4.2.CS microparticles were prepared by a precipitation/coacervation method,then loaded OVA and its inclusion complexes.The different series of CS microparticles optimized by single-factor method,which had the particle size of 0.52～4.58μm and zeta potential of 0～30 mV.The loading efficiency of OVA/CS,OVA-β-CD/CS and OVA-CM-HP-(3-CD/CS microparticles were 30.9%.27.7%.and 20.4%,respectively.The in vitro release indicated that CS microparticles had a burst release of 20%and showed the sustained-release effect after 1 h.The drug cumulative release of CS microparticles was lower 33%at 72 h.The drug-loaded microspheres were administered orally Balb/c mice.Specific-OVA IgG levels in serum and sIgA levels in intestinal were determined by ELISA.The results shouwed that compared with OVA solution.specific-OVA IgG levels in serum of OVA/CS.OVA-β-CD/CS and OVA-CM-HP-P-CD/CS microparticles increased about 1.3-fold,1.7-fold and 1.9-fold and sIgA levels in intestinal increased about 1.9-fold,3.6-fold and 3.0-fold,respectively.It was speculated that the CD/CS carrier can protect the activity of OVA more efficiently.