Modification Of Chitosan And Its Effect On Coagulation Properties

Chitosan,a kind of positively charged natural polymer,is obtained from deacetylation of chitin.It has a wide range of source and has active chemical properties,also can be broken down by microorganism.The good biocompatibility,antibacterial activity,adsorption property and hemostatic ability made chitosan favorable in biology,medicine,chemical,food,cosmetics and other fields.In order to study the hemostatic properties of chitosan derivatives,two kinds of modified product were prepared.Firstly,a series of allkylated chitosan with different degree of substitution and allkyl chains were synthesized by reductive amine reaction.Secondly,the guanidine chitosan was made by catalytic reaction of chitosan and arginine,with MES buffer,EDC and NHS as crosslinking agent.For the alkyl modified chitosan,the infrared spectroscopy and elemental analysis was used for characterization,which proved that the allkyl chain had been successfully grafted onto the chitosan backbone,and the substitution degree(SD)was related to the mole proportion of alkyl aldehyde and chitosan.By MTT assay,cell staining and traumatized mouse model,the cytotoxicity of alkylated chitosan was studied and results showed that the material could promote cell proliferation and tissue repairing.which declared a low cytotoxicity and good biocompatibility for wound use.To explore the hemostatic property and mechanism of alkylated chitosan,the in vitro coagulation tests including BCT,APTT,PT and TT was carried out.The results showed that the alkylated chitosan could significantly shorten the clotting time of blood and improve the hemostatic ability of chitosan.The improvement was related to the SD and grafted group of alkylated chitosan,declaring that the derivative with longer alkyl chain and lower SD got better performance on blood clotting.Moreover,the coagulation caused by alkylated chitosan did not work via the traditional pathways.In the platelet related experiment of intracellular calcium ion(Ca2+)and P-selectin,we found that the alkylated chitosan did not cause an obvious increase of the concentration change of Ca2+ and expression change of P-selectin,which meant that the promoting behavior of alkylated chitosan may not rely on platelets.The following platelet adhesion test also confirmed this view.Based on the above results,a hypothesis was proposed to describe the possible role that the alkylated chitosan had played in the coagulation process.Moreover.guanidine chitosan was synthesized.The cytotoxicity was also assessed via MTT and cell staining,which showed good results in biocompatibility.To study its hemostatic efficiency,the in vitro whole blood and blood plasma clotting time was tested.Results showed that the guanidine chitosan could shorten the whole blood clotting time,but couldn't promote the activation of plasma proteins.The promotion of coagulation was mainly depend on the chitosan backbone because of the adjacent time of chitosan and guanidine derivative.The grafting modification of guanidine group cannot improve the hemostatic capability of chitosan.