Cytoactivity And Cytocompatibility Of The Water-soluble Chitosan And Chitosan Thermosensitive Hydrogel

Chitosan is a unique polysaccharide derived from chitin by partial deacetylation with alkali. Chitin and chitosan are recommended as suitable functional materials, for their excellent biological properties such as non-toxicity, biodegradation, immunological, antibacterial and wound-healing activities. Despite of advantages above, poor solubility of chitin and chitosan in neutral water and in common organic solvents have prevented them from being applied widely. To improve the solubility of chitosan, chemical modification, such as a partial N-acetylation, PEG-grafting, sulfonation, quaternarization, N, and O-hydroxylation, and carboxymethylation, have been studied. Carboxymethyl chitosan (CMCH) is one of the water soluble derivatives of chitosan which has been studied widely. CMCH has unique chemical, physical and biological properties such as high viscosity, large hydrodynamic volume, low toxicity, biocompatibility and film, gel-forming capabilities, all of which make it an attractive option in biomedical and pharmaceutical formulations.Injection of in situ gel-forming biopolymer is becoming increasingly attractive for the development of therapeutic implants and vehicles. Chitosan thermosensitive hydrogels prepared with different methods are of great interest in drug delivery, cell encapsulation, tissue engineering and so on. Chitosan solutions containing glycerol-β-phosphate which has temperature-controlled solution–gel transition at a temperature close to 37°C has recently been proposed as a suitable vehicle for the extravascular parenteral administration of drugs. However, there are no reports on the preparation and influence of preparing conditions of chitosan-αβ-mixtured- glycerol-phosphate (CS-αβ-GP) thermosensitive hydrogel. Furthermore the cost ofαβ-mixtured-glycerophosphate (αβ-GP) was far lower than that of glycerol-β- phosphate.In this paper, CMCHs with different molecular structural parameters are prepared. The cytoactivity of CMCHs is investigated by using MEF, MPM and Hela cell lines. The new thermosensitive hydrogel of CS-αβ-GP is prepared with chitosan andαβ-GP. In addition, the effect of the CS-αβ-GP hydrogels on sustained release of Adriamycin is studied, and the biocompatibility of CS-αβ-GP hydrogels is investigated by using the method of the MTT assay.The main contents and conclusions are as follows:1. A series of carboxymethyl chitosan samples (CMCHs) with different molecular structural parameters are prepared with chloroacetic acid and chitosan in alkaline condition and all samples have water solubility, similar molecular structure, high DS (1.12-2.39).2. All the CMCHs stimulate MEF proliferation and CMCHs with different molecular weight (MW) have the similar effect on fibroblasts proliferation. The least concentration for CMCHs (degree of deacetylation, DD 70.3-79.9%, the degree of substitution, DS 1.12-1.26) exhibiting acceleratory effect on fibroblasts proliferation is 50μg ml-1. As the DD increased from 70.3% to 93.6%, CMCH's ability of stimulating fibroblasts proliferation increased significantly. CMCH possessed much higher proliferation rate with the DS increasing to 2.39. CM40 with 92.4% DD and 2.39 DS had the strongest acceleratory fibroblasts proliferation at the range tested.3. CMCHs can activate MPM and induce nitric oxide production. After 24h incubation with CMCHs, viable MPM cells are assayed by the method of MTT, and there is no cytotoxic effect on MPM cells. Phagocytosis of MPM treated with CMCHs become more efficient than controls via the neutral red assay. MPM produce a large amount of nitric oxide when they are incubated with CMCHs.4. CMCHs can slightly inhibit the proliferation of Hela in vitro. The inhibition effect on the proliferation of Hela increased when the concentration of CMCHs was increasing from 10μg ml-1 to 100μg ml-1.5. A novel thermosensitive hydrogel (CS-αβ-GP) is prepared with chitosan andαβ-mixtured-glycerophosphate (αβ-GP) which could be transformed into gelation at 37℃. The CS-αβ-GP hydrogel is an ideal sustained release system according to the results of adriamycin release from the hydrogel which was only 60% to 70% during 24 hours. MEF cultured with 24h and 72h leachates of CS-αβ-GP are investigated by the MTT assay and RGR is calculated and the cytotoxicity is graded by generally accepted standard. Vitro cytotoxicity test has shown CS-αβ-GP hydrogel has good biocompatibility which provides possibilities and foundations for the further research.