Anticoagulant Sulfated Fibroin Research

Anticoagulant materials are widely used in pharmaceutical medicine, such as leading blood coagulation in the use of artificial dialysis and surgery or prevent blood coagulation in blood storage. At present, many medical devices and artificial organs also required the materials with anticoagulant. In recent years, heparin, low molecular weight heparin, proteolytic enzyme inhibitors, antiplatelet agents are commonly used as anticoagulant in medicine caring. Many medical devices and materials of artificial organs mainly produce through the blending of such pharmaceutical and raw materials. However, its high cost and side effects dive us to develop a novel anticoagulant material. After previous study, man finds that SO₄^2- is an essential element in the anticoagulant effect of heparin. Accordingly, if can add SO₄^2-, it also can create a similar structure of heparin, generating anticoagulant effect. Recently, people have developed a sulfated synthetic polymers or polysaccharides, which have good anti-clotting effect. Its biocompatibility is better, but a rather poor stability and are not easily used as bio-medical materials. In comparison, the SF has many advantages, such as good oxygen, ventilation and other unique physical and chemical properties, good biocompatibility. As protein material, it is easy to use protein engineering to produce additional function. The discovery of functions in sulfated fibroin makes a better study prospects.The purposes of this study include the method of sulfated fibroin simplification, preparation of sulfated fibroin with good anticoagulant effects and biocompatibility. We using cocoon shell as raw material, apply pyridine sulfur trioxide to make sulfuric acid of fibroin, and then detect its sulfur content, anti-clotting effects biocompatibility and other indicators. The main results are as follows:1. Preparation of soluble fibroin Chemical method is using lOg cocoon shell to gain 6.5g soluble fibroin after some treatment. Physical method is extracting 0.1g soluble fibroin perl silkworm on average. The soluble fibroins are similar in structure producing from these two methods. When choosing raw materials to start preparation, we need according to the characteristics of soluble fibroin and actual requirement to choose suitable method.2. Preparation of sulfated fibroinAs time passed by and increasing of the quality of sulfur trioxide-pyridine, the color is also darkening. At this time, the molecular weight of fibroin is gradually reducing and the amount of sulfuric acid content of silk fibroin is also in reduction correspondingly. In order to get more samples and a good quality, we must choose a optimal condition.3. Detection of sulfated fibroinThe sample which add more than 5g pyridine sulfur trioxide and more than 2h treatment can combine to fibroin in branch as long as being detected having SO42 -When the addition amount of sulfur trioxide pyridine reaches 10g, processing time is 6h, the sulfur content of sulfated fibroin samples reaches the maximum with 1.26 mmol/g.4. Sulfated fibroin anticoagulant effect detectionIn optimal conditions for different concentrations of sulfated fibroin solution test:the blood in test tube have added fibroin and 0.2 mg/mL sulfated fibroin coagulate in 3 min; 30 min later, the blood with 0.02 mg/mL heparin and 0.4,0.6 mg/mL sulfated fibroin also coagulate; 1 h later, the 0.04 mg/mL heparin sodium solution one also in coagulation, and the 0.8 mg/mL sulfated fibroin reveal a significant anti-clotting effect.5. Biocompatibility testAccording to providing method in GB/T 16886, we conduct a testing and find that the pyrogen source contained in sulfated fibroin is in line with regulation and never cause skin sensitization, no local stimulatory on integrated or damaged skin, no toxicity and no hemolysis phenomenon.