Construction And Immune Property Of Chitosan/Manganese Gel Microspheres Based On Sodium Alginate

Mn²⁺is a potential immune adjuvant with application value which can induce the production of type I interferon by enhancing the sensitivity of the cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon gene pathway,thereby promoting and regulating the innate and specific immunity of organisms.However,excessive accumulation of Mn²⁺is harmful to the central nervous system of human.It will lead to pathological immune activation and irreversible cell and tissue damage.Due to its excellent cell compatibility and biodegradability,chitosan is often used as a biopharmaceutical carrier.In recent years,studies have shown that chitosan also promotes innate immunity through the cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon genes pathway.In order to reduce the toxic and side effects of Mn²⁺and synergize its innate immunity with chitosan,chitosan/manganese gel microspheres based on sodium alginate was constructed in the study.The construction conditions and rules,the construction mechanism and structural characteristics of the gel microsphere were explored.The sustained release property of Mn²⁺and the synergistic effect of chitosan and Mn²⁺in innate immunity were studied.The research content and results are as follows:1.Study on the construction conditions of chitosan/manganese gel microspheres based on sodium alginateChitosan/manganese gel microspheres based on sodium alginate were constructed by external gelatinization,internal gelatinization and pregelatinization.The effects of sodium alginate,Mn Cl₂,chitosan concentration and p H of solution on the structure and particle size of microspheres were investigated,and the preparation conditions of microspheres were optimized.The results show that the external gelatinization is suitable for preparing millimeter microspheres,and the pregelatinization is suitable for preparing nanoscale microspheres.In external gelatinization,the specific surface area of millimeter microspheres expands with the increase of sodium alginate concentration.The concentration of Mn Cl₂ and chitosan,as well as the p H of the solution,have a significant influence on the size of nanospheres,while the dispersity of particle size is only affected by the p H of the solution.In the pregelatinization,the ratio of sodium alginate to chitosan is the key factor for the formation of nanospheres.The microspheres will agglomerate with the mass ratio of sodium alginate to chitosan is greater than 1.When the concentration of sodium alginate and chitosan is 0.50 mg/m L,and the concentration of Mn Cl₂ is 0.010 mol/L,the size and dispersibility of the nanospheres are most appropriate,which are 291.1 nm and 0.22.2.Study on the combination of chitosan/manganese gel microspheres based on sodium alginateThe combination of chitosan/manganese based on sodium alginate was explored by analyzing the formation process and disintegration process of microspheres.The results of infrared spectrum show that sodium alginate interacted with Mn²⁺and amine groups on chitosan by carboxyl group.Thermogravimetry curves suggest that chitosan can improve the thermal stability of manganese alginate.The release of Mn²⁺indicates that sodium alginate bind to Mn²⁺through a relatively stable coordination effect.The disintegration of chitosan/manganese microspheres based on sodium alginate in different salt solutions shows that the microspheres are acid resistant but not salt resistant,and sodium alginate is the framework in the gel structure.Chitosan is in the inner layer of microspheres by internal gelatinization;in the exogelatinized microspheres,chitosan was wrapped on the surface of manganese alginate.3.Study on the function of chitosan/manganese gel microspheres based on sodium alginateEffects of component concentration,construction method,and particle size on the sustained release function of microspheres were investigated.The results show that the microspheres can release Mn²⁺continuously,and the release time could be appropriately prolonged by increasing the concentration of sodium alginate and Mn Cl₂.The release of Mn²⁺in the externally gelatinized microspheres and nano particles is more,which has a better stimulation effect on cells.The relative expression of interferon beta 1 and interferon-beta and mean intensity of ⁵⁵Mn in Hela cells stimulated by different samples were measured.The results indicate that the combination of chitosan and Mn²⁺can promote the production of more interferon-beta in cells.It means that they have a synergistic promoting effect.And externally gelatinized microspheres and microspheres with small size are more conducive to this promoting effect.