Sulfation Of Agarose And Its Biocompatibility

As one of the seaweed polysaccharide, agarose has drawn considerable interest for its biocompatibility. However, its biological inertia limited its applications in the biomedical field, and it needs to be modified with some functional groups with biological activity and expand its application fields.With formamide as dispersing agent, agarose was sulfated with the method of chlorosulfonic acid-pyridine. The orthogonal test result showed that the optimal reaction condition was reaction time of 4h, the reaction temperature of 65℃, and the ratio of chlorosulfonic acid to agarose was 4(mL/g).The substitution degree of the product in this optimal reaction condition was 1.10.Two different products with low degree of substitution and high degree of substitution were obtained in different reaction conditions. The high degree of substitution product was purified and graded with dialysis bag. Two kinds of sulfated agarose (AGS) with different molecular weights were obtained. The molecular weight was measured by viscosity, degree of substitution (DS) was determined by elemental analysis, infrared spectroscopy (IR) and 13C nuclear magnetic resonance (13C-NMR) spectroscopy were used to characterize the chemical structure, and their anticoagulant properties were checked by assays of the activated partial thromboplastin time ((APTT, tAPTT) and thrombin time (TT, tTT). Results showed that they had same degree of substitution and sulfation position, whereas their molecular weight was different. The bioassay results of coagulation index demonstrated that the anticoagulant activity of agarose improved with increasing of molecular mass within a certain range.For the low degree of substitution products, their structures were characterized by IR. The adsorption properties of Hb and BSA in different media were determined by coomassie brilliant blue method. Acute toxicity test and thermal test were used to examine its biocompatibility, and their blood compatibility was characterized with haemolysis ratio, the erythrocyte morphology and dynamic blood clotting test. Results revealed that with increasing of DS of the sulfated agarose, its BSA adsorption capacity was enhanced, while the adsorption of Hb was reduced. The products were not toxic and pyrogenic, and the hemolysis rates were all less than 5%. It proved that the anti-clotting properties of the products were improved with addition of sulfuric acid ester. The sulfated agarose could protect the form of red blood cells well after adsorption of BSA.In conclusion, there is a wide application promise for sulfated agarose to be used as heparin-like material in anticoagulation or endothelial regeneration scaffold.