Preparation Crosslinked Carboxymethyl Chitosan And Study On Its Biocompatibility And Immunological Adjuvant Activity

Purpose:Carboxymethyl chitosan(CM-CTS) is one of the water soluble derivatives of chitosan which has been widely studied. CM-CTS has unique chemical, physical and biological properties such as low toxicity, biocompatibility film and gel-forming capabilities, all of which make it a successful material that attracted more and more attentions. The preparation of CM-CTS need several time consuming protocols in a constant alkaline and heating environment, so the molecular mass and viscosity have remarkably reduced. The property of fast degradation and absorption restrict the application of CM-CTS. Present studies indicated that CM-CTS has immunity adjust activity. The purpose of this study is to explore a applicable preparation method which can maintain the high viscosity of CM-CTS, and to study the immunological adjuvant activity of materials prepared by this method.Methods:1. CM-CTS is prepared from chitosan and monochloroacetic acid in a alkaline and heating environment. The cell proliferation activity, cytotoxicity and biocompatibility of CM-CTS is measured.2. The optimum condition of applied GA to crosslink CM-CTS is established : the cincentration of CM-CTS is 2.0%, the reaction time is 3h ,the reaction pH is 8.0 and reaction temperature is at 25oC. C-CM-CTS is synthesized, and then deoxidized to eliminate the schiff base structure in branches.3. The effects of the C-CM-CTS and D-CM-CTS on the proliferation of L929 cells in vitro are studied. Their biocompatibilities are tested.4. D-CM-CTS is evaluated for its effect on mice splenocyte proliferation in vitro , haemolytic activity and adjuvant potentials on the cellular and humoral immune responses of mice to ovalbumin (OVA).Results: 1. CM-CTS is purified and prepared for its germ-free and pyrogen-free qualities. Cell culture assay shows that CM-CTS does not have cytotoxicity effect on L929 fibroblasts. On the other hand, CM-CTS can promote L929 fibroblast proliferation. The optimal concentration is 100μg/mL.2. C-CM-CTS and D-CM-CTS are synthesized with Glutaraldehyde crosslinking method. Both of them are dissolvable. Compared with CM-CTS, the viscosities of both are increased by 170 times.3. D-CM-CTS performs better in biocompatibility test4. D-CM-CTS does not show haemolytic activity. D-CM-CTS enhaneed the Con A- and OVA-stimulated splenocyte proliferation in OVA-immunized mice. The OVA-specific IgG antibody levels in serumare enhanced especially at the concentration of 2.5% compared with OVA control group.Conclusions:Compared with CM-CTS, the viscosity of D-CM-CTS which is synthesized with Glutaraldehyde crosslinking method is increased by 170 times, but still dissolvable. D-CM-CTS has good biocompatibility but no cytotoxicity at all. Besides, D-CM-CTS also has good Immunological adjuvant activity. These information will be useful for developing D-CM-CTS biomedical materials and new drug delivery carriers.