Studies On REPO Loaded Colon-bioadhesive Thiol-modified Chitosan Microshperes

EPO is becoming more and more important due to its significant effection on treating anaemia diseases. But unfortunately, just like other proteins and peptides, EPO only has injections on the market, which brings much inconvenience to the patient. So it is necessary to develop a new and acceptable administration route for EPO.In this paper, thiolated chitosan with perfect bioadhesive property was synthesized by a novel method using cheap chitosan as material. The preparation formulation conditions were primarily optimized by considering the effects of pH value, amount of chitosan, TGA and EDAC on the concentration of TGA in thiolated chitosan. The IR analysis results indicated that the thiolated chitosan was obtained. The molecular mass of chitosan and thiolated chitosan was 85.9±11.2 and 93.6±13.1 kDa, respectively. The viscosity of thiolated chitosan was much higher than that of chitosan under different concentration. The ionic concentration affected the viscosity of thiolated chitosan, but under the same ionic concentration, the thiolated chitosan had a much higher viscosity than unmodified chitosan. The pKa of chitosan and thiolated chitosan was 6.5 and 8.8 , respectively. The higher concentration of thiol group, the higher oxidation degree had the thiolated chitosan, which means the lower concentration of thiol. The degradation speed of thiolated chitosan was much lower than that of chitosan and it decreased with the increasing of concentration of thiol group. The water absorption property of chitosan and thiolated chitosan was related to the thiolated degree. The amount of water absorbed by thiolated chitosan with different thiolated degree was different, and it increased with the increasing of thiolated degree.The bioadhesive properties of thiolated chitosan were systemically studied in this paper. The results of rotary testing indicated that under the same pH value, the viscosity of thiolated chitosan was much higher than that of unmodified chitosan. The results of tensile indicated that there is significant difference between the viscosity of thiolated chitosan and unmodified chitosan, and the difference will be more significant with the decreasing of pH value. The amount of GSH added into solution could affect the bioadhesive property of thiolated chitosan, ie, the bioadhesive propersity of thiolated chitosan decreased with the increasing of amount of GSH. The bioadhesive propersity of thiolated chitosan could increase with the increasing of molecular mass of chitosan but decrease significantly with increasing of pH value. With the increasing of concentration of thiol group in thiolated chitosan, the bioadhesive, elasticity, and absorption enhancement property and the gelling process of this polyer could increase significantly.The optimized formulation of the preparation of thiolated chitosan microspheres was as following: the concentration of thiolated chitosan-2.0%, CaCl₂-2.0%, alginate-1.5%. After being coated by Eudragit S, both the particle size and zeta potential of the microspheres increased significantly. The microspheres with and without enteric coating were observed by SEM to be good sphere shape, but there was much pore on the surface before coating and more compact after that. The swelling property of thiolated chitosan in water was very good, and much beater than that of unmodified chitosan, but there was no significant difference in artificial intestinal liquid. The concentration of thiol group was 85.6±19μmol/g, 153.9±27μmol/g and 301.4±32μmol/g, respectively. The pH value could affect the bioadhesive property of thiolated and unmodified chitosan microspheres, the TWA of both thiolated chitosan and unmodified chitosan microspheres significantly decreased with the increasing of pH value. But under the same pH value, TWA of thiolated chitosan microspheres was much higher than that of unmodified chitosan microspheres. The results of intestinal perfusion indicated that there was significant difference between the bioadhesive percentage of thiolated chitosan microspheres and unmodified chitosan microspheres. The remaining percentages of both microspheres in stomach were lowest, and significantly increased in intestinal, especially in colon. Within half an hour after oral administration, the labeled microspheres loaded capsule still stayed in stomach, and then were transported to intestinal and stayed there up to 2-6 hours, finally they could stayed in colon for more than 12 hours.After oral administration of enteric coating EPO loaded thiolated chitosan microspheres to rats, the anti-anaemia effects in injecting group was significantly different to that in control group, and the effective time was also prolonged. Also the anti-anaemia effects was positively related to the dosage, ie it would increased with the increasing of dosage. After multiple oral administration of enteric coating EPO loaded thiolated chitosan microspheres to rats, the anti-anarmia effects were similar to single dosage administration, there was no significant difference. But the anti-anaemia effect could significantly increase with increasing of frequencies of administrations. Also, the anti-anaemia effect of enteric coating EPO loaded thiolated chitosan microspheres prepared with different concentration of thiol group were positively related to the thiol concentration, ie, it could significantly increased with increasing of concentration of thiol group.