CN106730052A - A kind of anticoagulant fimbrin material and preparation method thereof - Google Patents

Abstract

The invention provides a kind of preparation method of anticoagulant fimbrin material, comprise the following steps：S1) silk fibroin protein solution, polyethylene glycol diamines are mixed with crosslinking agent, is dialysed after reaction, dried, obtain the fibroin material of polyethylene glycol diamines cationization；S2 during the fibroin material that the polyethylene glycol diamines are cationized) impregnated in into hirudin solution, anticoagulant fimbrin material is obtained.Compared with prior art; the preparation method that the present invention is provided protects the functional group with fibrin ferment calmodulin binding domain CaM not because again the domain for being influenceed to be combined with fibrin ferment by reaction can be such that hirudin is incorporated into the ionic bond of stronger adhesion on the fibroin albumen of polyethylene glycol diamine cationization; reach the effect that stabilization plays anti-freezing; so as to make the anticoagulant fimbrin material for obtaining that there is the function of significantly inhibiting thrombin activity, it is particularly applicable to prevent the formation of neointimal hyperplasia and postoperative thrombus such as artificial blood vessel.

Description

A kind of silk fibroin anticoagulant material and preparation method thereof

technical field

The invention belongs to the technical field of anticoagulant materials, and in particular relates to a silk fibroin anticoagulant material and a preparation method thereof.

Background technique

There are millions of deaths from cardiovascular and cerebrovascular diseases in my country every year, and it is increasing year by year. Therefore, blood-contact materials are currently the most scarce biomaterials (medical devices) in clinical practice, especially artificial blood vessel grafts. , Large-diameter artificial blood vessels are also very rare in my country, and the annual use ratio of domestic products is only about 20%, while the transplantation of small-caliber artificial blood vessels is still a clinical blank. The biggest problem is that it is easy to form thrombus, and the long-term patency rate is poor .

At present, artificial blood vessels used in medicine are mainly made of synthetic materials such as polyester and expanded polytetrafluoroethylene, which have clinical applications in large and medium-caliber artificial blood vessels, but these synthetic materials have poor cytocompatibility and are not conducive to endothelialization , easy to form thrombus, affecting tissue healing. Bombyx mori silk is a natural animal protein synthesized and secreted by silkworms. It has a wide range of sources. Its silk fibroin has good biocompatibility and is composed of 20 kinds of amino acids that can be absorbed by the human body. The final degradation products are amino acids or small peptides, which are easily absorbed by cells. Absorption or phagocytosis will not cause obvious immune response. A large number of literature studies have shown that silk fibroin materials can support the growth of various cells, and the research on tissue engineering materials has become more and more in-depth, and breakthroughs have been made. Recently, silk fibroin materials are more and more used in vascular tissue engineering. Having attention.

Although silk fibroin material itself has the advantages of cytocompatibility and tissue compatibility, as an exogenous material, it will stimulate the coagulation system and induce hemolysis or coagulation when it comes into contact with blood. In order to improve the anticoagulant performance of silk fibroin materials, some researchers at home and abroad have also paid attention to improving the anticoagulant performance of silk fibroin materials.

At present, the anticoagulant modification of silk fibroin mainly reports grafting polymers with anticoagulant effect and sulfated or heparinized methods. For example, She et al. added heparin to the silk fibroin/chitosan scaffold under mild conditions to enhance the anticoagulation (Polymer International, 2010,59(1):55-61); Liu et al. Sulfonic acid-treated silk fibroin was used to make silk nano-scaffolds, and the anticoagulant properties of sulfated nano-silk fibroin scaffolds were significantly enhanced (Biomaterials, 2011, 32(15): 3784-3793); Wang et al. prepared by electrospinning technology Heparin-modified silk fibroin nanomaterials, in vitro coagulation test results show that the anticoagulant property of silk fibroin nanomaterials after modification is much higher than that of pure silk fibroin (InternationalJournal of Biological Macromolecules, 2011, 48 (2): 345-353); The anticoagulant activity of the sulfated silk fibroin prepared by chlorosulfonic acid is much higher than that of the sulfated silk fibroin prepared by sulfuric acid (Biomaterials, 2004, 25(3): 377-383), but far inferior to heparin. The research on the anticoagulant properties of heparin-modified silk fibroin materials already has intellectual property rights (such as the preparation of anticoagulant dermal stents, the Chinese patent application number is CN200910223207.4; the nanofiber artificial blood vessel and its preparation method, the application number is CN200910228843.6 Chinese patent), so the introduction of heparin is currently the main method for anticoagulant modification of silk fibroin materials, but heparin is an indirect inhibitor of thrombin, and the anticoagulant effect depends on antithrombin and specific cofactors, so even if the material Blended or bonded heparins may or may not be anticoagulant.

Hirudin is a specific inhibitor of thrombin, which can directly inhibit the formation of thrombus, and can also have a certain thrombolytic effect on the formed thrombus. We have researched and developed a hirudin/silk fibroin anticoagulant material (an anticoagulant silk fibroin material and its preparation method, the Chinese patent application number is ZL201310250951.X; an anticoagulant silk fibroin film and its preparation method , Chinese patent with application number ZL201310251819.0; Journal of Biomedical Materials Research Part B, 2015:103B:556–562), but in-depth research found that the research results were combined with the mechanism of hirudin’s inhibition of thrombin activity. The modification method of valence bond will reduce the activity of hirudin.

Contents of the invention

In view of this, the technical problem to be solved by the present invention is to provide a silk fibroin anticoagulant material and a preparation method thereof. The silk fibroin anticoagulant material prepared by the method can efficiently inhibit thrombin activity and continuously have anticoagulant properties. Function.

The invention provides a preparation method of silk fibroin anticoagulant material, comprising the following steps:

S1) Mix the silk fibroin solution, polyethylene glycol diamine and a cross-linking agent, dialyze after the reaction, and dry to obtain a silk fibroin material cationized with polyethylene glycol diamine;

S2) immersing the polyethylene glycol diamine cationized silk fibroin material in a hirudin solution to obtain a silk fibroin anticoagulant material.

Preferably, the mass concentration of the silk fibroin solution is 3%-20%.

Preferably, the mass ratio of the polyethylene glycol diamine to the silk fibroin in the silk fibroin solution is A, 0<A≤0.5.

Preferably, the crosslinking agent is selected from 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, 2-morpholineethanesulfonic acid, carbodiimide One or more of amines, genipin and polyglycol glyceryl ether.

Preferably, the mass of the cross-linking agent is B% of the mass of silk fibroin in the silk fibroin solution, and B≥20.

Preferably, the reaction time in the step S1) is 10-30 minutes.

Preferably, the dialysis time is 12-48 hours.

Preferably, the concentration of hirudin in the hirudin solution is C U/ml, 0<C≤500.

Preferably, the soaking time in the step S2) is 2-10 hours.

Preferably, silk fibroin and hirudin ionized with polyethylene glycol diamine are included.

The invention provides a preparation method of a silk fibroin anticoagulant material, comprising the following steps: S1) mixing a silk fibroin solution, polyethylene glycol diamine and a cross-linking agent, dialyzing after the reaction, and drying to obtain a polyethylene glycol diamine Silk fibroin material cationized with diol diamine; S2) The silk fibroin material cationized with polyethylene glycol diamine is immersed in a hirudin solution to obtain a silk fibroin anticoagulant material. Compared with the prior art, the preparation method provided by the present invention protects the functional groups (-COOH, -NH ₂ , -OH) in the binding region to thrombin from being reacted to affect the binding domain to thrombin, and enables Hirudin binds to polyethylene glycol diamine cationized silk fibroin with a strong ionic bond to achieve a stable anticoagulant effect, so that the obtained silk fibroin anticoagulant material can significantly inhibit thrombin The active function can be especially applied to prevent the neointimal hyperplasia of artificial blood vessels and the formation of postoperative thrombus.

detailed description

The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Apparently, the described embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

The invention provides a silk fibroin anticoagulant material, which comprises silk fibroin and hirudin cationized by polyethylene glycol diamine and a cross-linking agent.

Wherein, the mass ratio of polyethylene glycol diamine to silk fibroin is preferably A, 0<A≤0.5, more preferably 0.01-0.5, more preferably 0.01-0.2, most preferably 0.05-0.1; In some embodiments provided by the invention, the mass ratio of polyethylene glycol diamine to silk fibroin is preferably 0.01; in some embodiments provided by the invention, the mass ratio of polyethylene glycol diamine to silk fibroin The mass ratio is preferably 0.05; in other embodiments provided by the present invention, the mass ratio of polyethylene glycol diamine to silk fibroin is preferably 0.1.

The cross-linking agent is a cross-linking agent well-known to those skilled in the art, and there is no special limitation. In the present invention, it is preferably -(3-dimethylaminopropyl)-3-ethylcarbodiimide, N - one or more of hydroxysuccinimide, 2-morpholineethanesulfonic acid, carbodiimide, genipin and macrogol glyceryl ether; the quality of the cross-linking agent is preferably silk fibroin The amount of B%, B ≥ 20, more preferably 20% to 50%.

The present invention also provides a preparation method of the above-mentioned silk fibroin anticoagulant material, comprising the following steps: S1) mixing the silk fibroin solution, polyethylene glycol diamine and a cross-linking agent, dialyzing after the reaction, and drying to obtain Polyethylene glycol diamine cationized silk fibroin material; S2) The polyethylene glycol diamine cationized silk fibroin material was soaked in a hirudin solution to obtain a silk fibroin anticoagulant material.

The present invention has no special limitation on the sources of all raw materials, which can be commercially available or self-made.

Wherein, the mass concentration of silk fibroin in the silk fibroin solution is preferably 3% to 20%; the silk fibroin can be silk fibroin well-known to those skilled in the art, and there is no special limitation. It is preferably silkworm silk fibroin; the silk fibroin solution is preferably prepared according to the following method: degumming and dissolving silk or silkworm shells, pouring them into a dialysis bag, and dialysis with deionized water to obtain a silk fibroin solution.

Wherein, the degumming method is a method well known to those skilled in the art, and there is no special limitation. In the present invention, silk or silkworm shells are preferably heated in an aqueous solution of sodium carbonate, washed with water, and loosened to obtain degummed silk or silkworm shells. Silk fiber; the sodium carbonate aqueous solution is an aqueous solution well known to those skilled in the art, and there is no special limitation. In the present invention, it is preferably an aqueous sodium carbonate solution with a concentration of 0.1% to 1%, more preferably 0.1% to 0.5%. , more preferably 0.2%～0.3%; the ratio of described silk or silkworm shell and sodium carbonate aqueous solution is preferably (0.1～10) g: 50ml, more preferably (0.5～5) g: 50ml, more preferably (0.5 ～2) g: 50ml, most preferably 1g: 50ml; the temperature of the heat treatment is preferably 98°C～100°C; the time of the heat treatment is preferably 20～40min; the number of times of the heat treatment is preferably 2～ 4 times.

Dissolving the degummed silk fibroin fiber to obtain a silk fibroin solution; the dissolving method is a method well known to those skilled in the art, and there is no special limitation. In the present invention, the degummed silk fibroin fiber is preferably mixed with chlorine The aqueous solution of calcium chloride-ethanol is mixed, and after heating and dissolving, silk fibroin solution is obtained; the ratio of the degummed silk fiber to the aqueous solution of calcium chloride-ethanol is preferably (0.1～5) g: 10ml, more preferably ( 0.5-3) g: 10 ml, more preferably (1-2) g: 10 ml; the molar ratio of calcium chloride to ethanol is preferably 1:2; the temperature for heating and dissolving is preferably 60°C-80°C, It is more preferably 65° C. to 75° C., most preferably 70° C.; the time for heating and dissolving is preferably 1 to 3 hours, more preferably 2 to 3 hours.

Pour the silk fibroin solution into the dialysis bag, and dialyze with deionized water to obtain a silk fibroin solution; the dialysis bag is a semipermeable membrane, and its molecular weight cut-off is preferably 12.0-16.0kDa; during dialysis, preferably every 1-3h , more preferably replace the deionized water used for dialysis with new deionized water or purified water every 2h; the time of the dialysis is preferably 3 days.

Mix the silk fibroin solution, polyethylene glycol diamine and a cross-linking agent, preferably first mix the silk fibroin solution and polyethylene glycol diamine, and then add the cross-linking agent; the polyethylene glycol diamine The mass ratio of mass to silk fibroin in the silk fibroin solution is preferably A, 0<A≤0.5, more preferably 0.01-0.5, more preferably 0.01-0.2, most preferably 0.05-0.1; the crosslinking agent is A cross-linking agent well known to those skilled in the art can be used without any special limitation. In the present invention, it is preferably 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide One or more of amine, 2-morpholineethanesulfonic acid, carbodiimide, genipin and polyethylene glycol glyceryl ether, more preferably 1-(3-dimethylaminopropyl)-3- Ethylcarbodiimide, N-hydroxysuccinimide and 2-morpholineethanesulfonic acid; the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide The mass ratio of imide to 2-morpholineethanesulfonic acid is preferably 2:1:2; the quality of the cross-linking agent is preferably B% of the silk fibroin in the silk fibroin solution, B≥20, more preferably 20% to 50%.

Then carry out the reaction; the reaction time is preferably 10 minutes to 1 hour, more preferably 15 to 45 minutes, and more preferably 20 minutes.

Dialysis after the reaction; the molecular weight cut-off of the semipermeable membrane used in the dialysis is preferably 12.0-14.0 kDa; the time of the dialysis is preferably 12-48 hours to obtain a polyethylene glycol diamine cationized silk fibroin solution.

After the polyethylene glycol diamine cationized silk fibroin solution is dried, the polyethylene glycol diamine cationized silk fibroin material is obtained; the drying can be heat drying or freeze drying; during drying, preferably Pour the silk fibroin solution cationized with polyethylene glycol diamine into a flat plate, and then heat-dry or freeze-dry; the flat plate is preferably a polystyrene plate; the temperature of the heat-drying is preferably 50°C to 70°C .

The silk fibroin material cationized with polyethylene glycol diamine is immersed in a hirudin solution; the concentration of hirudin in the hirudin solution is preferably CU/ml, 0<C≤500, more preferably 10～ 500U/ml, more preferably 10-300U/ml, more preferably 10-200U/ml, more preferably 10-100U/ml, most preferably 10-50U/ml; the polyethylene glycol diamine cationization The ratio of the silk fibroin material and the hirudin solution is preferably 1 to 10 ml of the hirudin solution per milligram of the material, more preferably 1 to 8 ml of the hirudin solution per milligram of the material, more preferably 1 to 5 ml of the hirudin solution per milligram of the material, and then It is preferably 2～5ml hirudin solution per milligram of material, most preferably 3ml hirudin solution per milligram of material; the time of said soaking is preferably 2～10h, more preferably 2～8h, more preferably 4～6h, most preferably 5h.

After dipping, it is preferable to take it out and air-dry it, and then rinse it with a buffer solution. After air-drying, the silk fibroin anticoagulant material is obtained; the buffer solution is preferably phosphate buffer saline (PBS); the number of times of the washing is preferably 3 to 4 times .

The preparation method provided by the present invention protects the functional groups (-COOH, -NH ₂ , -OH) of the binding region to thrombin from being reacted to affect the structural domain binding to thrombin, and enables hirudin to bind with stronger binding force. The ionic bond is combined with polyethylene glycol diamine cationized silk fibroin to achieve a stable anticoagulant effect, so that the obtained silk fibroin anticoagulant material has the function of significantly inhibiting the activity of thrombin, especially for the application of It is used to prevent the neointimal hyperplasia of artificial blood vessels and the formation of postoperative thrombus.

In order to further illustrate the present invention, a silk fibroin anticoagulant material provided by the present invention and its preparation method are described in detail below in conjunction with examples.

The reagents used in the following examples are all commercially available.

Example 1

1.1 Put silkworm silk or cocoon shells dried and stratified into 0.2% aqueous sodium carbonate solution at a bath ratio of 1:50 (g/mL), and treat them three times at 98°C to 100°C, each time for 30 minutes , and then fully wash the silk with deionized water, loosen it, and place it in an oven at 60° C. to dry to obtain degummed silkworm silk fiber.

1.2 Weigh the silkworm silk fibroin after degumming and dissolve it in an aqueous solution of calcium chloride-ethanol with a molar ratio of 1:2 (the molar ratio of ethanol to water is 1:4) at a bath ratio of 1:10 (g/mL), 70 ℃ for 2 hours to obtain silkworm fibroin solution.

1.3 Pour the silkworm fibroin solution into the dialysis bag. The wall of the dialysis bag is a semi-permeable membrane with a molecular weight cut-off of 12.0-16.0kDa. Place the dialysis bag filled with the silkworm silk solution into a container filled with deionized water Inside, the water in the container was replaced with new deionized water or pure water every 2 hours, and the dialysis was continued for 3 days to obtain the purified aqueous silk fibroin protein solution. Adjust the concentration of silk fibroin solution after dialysis to 4%.

1.4 Configure the mixed solution of silk fibroin and polyethylene glycol diamine according to the mass ratio of 100:1, stir evenly, add 1-(3-dimethylaminopropyl group whose mass ratio is 20% of silk fibroin to the above mixed solution )-3-ethylcarbodiimide was stirred evenly, 10% of N-hydroxysuccinimide and 20% of 2-morpholineethanesulfonic acid were reacted for 20 minutes and dialyzed with deionized water for 12 to 48 hours to obtain The polyethylene glycol diamine cationized silk fibroin solution is then poured into a flat polystyrene plate and dried at 60° C. to obtain the polyethylene glycol diamine cationized silk fibroin material.

1.5 Cut the silk fibroin material cationized with polyethylene glycol diamine into small pieces of a certain area, soak it in the hirudin solution of 10U/mL according to the ratio of 3ml hirudin solution per milligram of material, take it out after 5 hours and let it stand and air dry Rinse with PBS for 3 to 4 times, and finally air-dry to obtain silk fibroin anticoagulant material.

The silk fibroin anticoagulant material prepared in Example 1 was tested for antithrombin activity, and the absorbance value of thrombin activity at 450 nm was about 80% of that of pure thrombin activity, that is, silk fibroin anticoagulant The material has a certain inhibitory effect on the activity of thrombin, indicating that hirudin can be better combined with the silk fibroin material.

Example 2

2.1 Put silkworm silk or cocoon shells dried and stratified into 0.2% aqueous sodium carbonate solution at a bath ratio of 1:50 (g/mL), and treat them three times at 98°C to 100°C, each time for 30 minutes , and then fully wash the silk with deionized water, loosen it, and place it in an oven at 60° C. to dry to obtain degummed silkworm silk fiber.

2.2 Weigh the degummed silkworm silk fibroin and dissolve it in an aqueous solution of calcium chloride-ethanol with a molar ratio of 1:2 (the molar ratio of ethanol to water is 1:4) at a bath ratio of 1:10 (g/mL), 70 ℃ for 2 hours to obtain silkworm fibroin solution.

2.3 Pour the silkworm fibroin solution into the dialysis bag. The wall of the dialysis bag is a semi-permeable membrane with a molecular weight cut-off of 12.0-16.0kDa. Place the dialysis bag filled with the silkworm silk solution into a container filled with deionized water Inside, the water in the container was replaced with new deionized water or pure water every 2 hours, and the dialysis was continued for 3 days to obtain the purified aqueous silk fibroin protein solution. Adjust the concentration of silk fibroin solution after dialysis to 4%.

2.4 Configure the mixed solution of silk fibroin and polyethylene glycol diamine according to the mass ratio of 100:5, stir evenly, and add 1-(3-dimethylaminopropyl group whose mass ratio is 20% of silk fibroin to the above mixed solution. )-3-ethylcarbodiimide was stirred evenly, 10% of N-hydroxysuccinimide and 20% of 2-morpholineethanesulfonic acid were reacted for 20 minutes and dialyzed with deionized water for 12-48 hours to obtain The polyethylene glycol diamine cationized silk fibroin solution is then poured into a flat polystyrene plate and dried at 60° C. to obtain the polyethylene glycol diamine cationized silk fibroin material.

2.5 Cut the silk fibroin material cationized with polyethylene glycol diamine into small pieces of a certain area, dip it in the hirudin solution of 10U/mL according to the ratio of 3ml hirudin solution per mg of material, take it out after 5 hours and let it dry in the air Rinse with PBS for 3 to 4 times, and finally air-dry to obtain silk fibroin anticoagulant material.

The silk fibroin anticoagulant material prepared in Example 2 was tested for antithrombin activity, and the absorbance value of thrombin activity at 450 nm was about 45% of that of pure thrombin activity, that is, the silk fibroin prepared in Example 2 The fibroin anticoagulant material can obviously inhibit the activity of thrombin, indicating that hirudin is firmly bound to the silk fibroin material.

Example 3

3.1 Put silkworm raw silk or cocoon shells dried and layered into 0.2% sodium carbonate aqueous solution at a bath ratio of 1:50 (g/mL), and treat them three times at 98-100°C for 30 minutes each time. Then, the silk is fully cleaned with deionized water, loosened, and dried in an oven at 60° C. to obtain degummed silkworm silk fiber.

3.2 Weigh the degummed silkworm silk fibroin and dissolve it in an aqueous solution of calcium chloride-ethanol with a molar ratio of 1:2 (the molar ratio of ethanol to water is 1:4) at a bath ratio of 1:10 (g/mL), 70 ℃ for 2 hours to obtain silkworm fibroin solution.

3.3 Pour silkworm fibroin solution into the dialysis bag. The wall of the dialysis bag is a semipermeable membrane with a molecular weight cut-off of 12.0-16.0kDa. Place the dialysis bag filled with silkworm silk solution into a container filled with deionized water. Inside, the water in the container was replaced with new deionized water or pure water every 2 hours, and the dialysis was continued for 3 days to obtain the purified aqueous silk fibroin protein solution. Adjust the concentration of silk fibroin solution after dialysis to 5%.

3.4 Configure the mixed solution of silk fibroin and polyethylene glycol diamine according to the mass ratio of 100:5, stir evenly, and add 1-(3-dimethylaminopropyl group whose mass ratio is 20% of silk fibroin to the above mixed solution. )-3-ethylcarbodiimide was stirred evenly, 10% of N-hydroxysuccinimide and 20% of 2-morpholineethanesulfonic acid were reacted for 20 minutes and dialyzed with deionized water for 12 to 48 hours to obtain The polyethylene glycol diamine cationized silk fibroin solution is then poured into a flat polystyrene plate and freeze-dried to obtain the polyethylene glycol diamine cationized silk fibroin material.

3.5 Cut the cationized regenerated silk fibroin material into small pieces of a certain area, dip in 20U/mL hirudin solution at a ratio of 3ml hirudin solution per milligram of material, take it out after 5 hours, let it stand and air-dry, and rinse with PBS for 3-4 hours. times, and finally air-dried to obtain silk fibroin anticoagulant material.

The silk fibroin anticoagulant material prepared in Example 3 is tested for antithrombin activity, and the absorbance value of thrombin activity at 450 nm is about 33% of that of pure thrombin activity, that is, adding silk fibroin for anticoagulation The blood material can significantly inhibit the activity of thrombin, indicating that hirudin is firmly bound to the silk fibroin material.

Example 4

4.1 Put silkworm raw silk or cocoon shells dried and layered into 0.2% sodium carbonate aqueous solution at a bath ratio of 1:50 (g/mL), and treat them three times at 98-100°C for 30 minutes each time. Then, the silk is fully cleaned with deionized water, loosened, and dried in an oven at 60° C. to obtain degummed silkworm silk fiber.

4.2 Weigh the degummed silkworm silk fibroin and dissolve it in an aqueous solution of calcium chloride-ethanol with a molar ratio of 1:2 (the molar ratio of ethanol to water is 1:4) at a bath ratio of 1:10 (g/mL), 70 ℃ for 2 hours to obtain silkworm fibroin solution.

4.3 Pour the silkworm fibroin solution into the dialysis bag. The wall of the dialysis bag is a semi-permeable membrane with a molecular weight cut-off of 12.0-16.0kDa. Place the dialysis bag filled with the silkworm solution into a container filled with deionized water. Inside, the water in the container was replaced with new deionized water or pure water every 2 hours, and the dialysis was continued for 3 days to obtain the purified aqueous silk fibroin protein solution. Adjust the concentration of silk fibroin solution after dialysis to 5%.

4.4 Configure the mixed solution of silk fibroin and polyethylene glycol diamine according to the mass ratio of 100:10, stir evenly, and add 1-(3-dimethylaminopropyl group whose mass ratio is 20% of silk fibroin to the above mixed solution. )-3-ethylcarbodiimide was stirred evenly, 10% of N-hydroxysuccinimide and 20% of 2-morpholineethanesulfonic acid were reacted for 20 minutes and dialyzed with deionized water for 12 to 48 hours to obtain The polyethylene glycol diamine cationized silk fibroin solution is then poured into a flat polystyrene plate and freeze-dried to obtain the polyethylene glycol diamine cationized silk fibroin material.

4.5 Cut the polyethylene glycol diamine cationized silk fibroin material into small pieces of a certain area, dip it in a 40U/mL hirudin solution at a ratio of 3ml hirudin solution per milligram of material, take it out after 5 hours and let it stand and air dry Rinse with PBS for 3 to 4 times, and finally air-dry to obtain silk fibroin anticoagulant material.

The silk fibroin anticoagulant material prepared in Example 4 was tested for antithrombin activity, and the absorbance value of thrombin activity at 450 nm was less than 10% of that of pure thrombin activity, that is, the silk fibroin obtained in Example 4 The fibroin anticoagulant material can significantly inhibit the activity of thrombin, indicating that more hirudin is firmly bound to the silk fibroin material.

Comparative example 1

1.1 Put silkworm raw silk or cocoon shells dried and layered into 0.2% aqueous sodium carbonate solution at a bath ratio of 1:50 (g/mL), and treat them three times at 98-100°C for 30 minutes each time. Then, the silk is fully cleaned with deionized water, loosened, and dried in an oven at 60° C. to obtain degummed silkworm silk fiber.

1.2 Weigh the silkworm silk fibroin after degumming and dissolve it in an aqueous solution of calcium chloride-ethanol with a molar ratio of 1:2 (the molar ratio of ethanol to water is 1:4) at a bath ratio of 1:10 (g/mL), 70 ℃ for 2 hours to obtain silkworm fibroin solution.

1.3 Pour the silkworm fibroin solution into the dialysis bag. The wall of the dialysis bag is a semi-permeable membrane with a molecular weight cut-off of 12.0-16.0kDa. Place the dialysis bag filled with the silkworm silk solution into a container filled with deionized water Inside, the water in the container was replaced with new deionized water or pure water every 2 hours, and the dialysis was continued for 3 days to obtain the purified aqueous silk fibroin protein solution. Adjust the concentration of silk fibroin solution after dialysis to 5%.

1.4 Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide with a mass ratio of 20% silk fibroin to the silk fibroin aqueous solution and stir evenly, 10% N-hydroxysuccinimide and 20% 2-morpholineethanesulfonic acid, stirred for 20 minutes, dialyzed with deionized water for 12 to 48 hours, then poured into a flat polystyrene plate and freeze-dried to obtain the regenerated silk fibroin material.

1.5 Cut the pure regenerated silk fibroin material into small pieces of a certain area, dip in the hirudin solution of 20U/mL according to the ratio of 3ml hirudin solution per milligram of material, take it out after 5 hours, let it stand and air-dry, and rinse it with PBS for 3-4 times , and finally air-dried to obtain hirudin-loaded silk fibroin material.

The prepared hirudin-loaded silk fibroin material was tested for antithrombin activity, and the absorbance value of the thrombin activity at 450 nm was more than 96% of that of pure thrombin activity, that is, the hirudin-loaded silk fibroin material There was no effective inhibition of thrombin activity, indicating that hirudin was not firmly loaded onto the silk fibroin material.

Comparative example 2

2.1 Put silkworm raw silk or cocoon shells dried and layered into 0.2% sodium carbonate aqueous solution at a bath ratio of 1:50 (g/mL), and treat them three times at 98-100°C for 30 minutes each time. Then, the silk is fully cleaned with deionized water, loosened, and dried in an oven at 60° C. to obtain degummed silkworm silk fiber.

2.2 Weigh the degummed silkworm silk fibroin and dissolve it in an aqueous solution of calcium chloride-ethanol with a molar ratio of 1:2 (the molar ratio of ethanol to water is 1:4) at a bath ratio of 1:10 (g/mL), 70 ℃ for 2 hours to obtain silkworm fibroin solution.

2.3 Pour the silkworm fibroin solution into the dialysis bag. The wall of the dialysis bag is a semi-permeable membrane with a molecular weight cut-off of 12.0-16.0kDa. Place the dialysis bag filled with the silkworm silk solution into a container filled with deionized water Inside, the water in the container was replaced with new deionized water or pure water every 2 hours, and the dialysis was continued for 3 days to obtain the purified aqueous silk fibroin protein solution. Adjust the concentration of silk fibroin solution after dialysis to 5%.

2.4 Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide with a mass ratio of 20% silk fibroin to the silk fibroin aqueous solution and stir evenly, 10% N-hydroxysuccinimide and 20% 2-morpholineethanesulfonic acid, stirred for 20 minutes, dialyzed with deionized water for 12 to 48 hours, then poured into a flat polystyrene plate and freeze-dried to obtain the regenerated silk fibroin material.

The prepared regenerated silk fibroin material was tested for antithrombin activity, and the absorbance value of thrombin activity at 450 nm was 98% of that of pure thrombin activity, that is, the regenerated silk fibroin material had no ability to inhibit thrombin activity.

Claims (10)

1. a preparation method of silk fibroin anticoagulant material, is characterized in that, comprises the following steps: S1) Mix the silk fibroin solution, polyethylene glycol diamine and a cross-linking agent, dialyze after the reaction, and dry to obtain a silk fibroin material cationized with polyethylene glycol diamine; S2) immersing the polyethylene glycol diamine cationized silk fibroin material in a hirudin solution to obtain a silk fibroin anticoagulant material. 2. The preparation method according to claim 1, characterized in that the mass concentration of the silk fibroin solution is 3%-20%. 3. The preparation method according to claim 1, characterized in that the mass ratio of the polyethylene glycol diamine to the silk fibroin in the silk fibroin solution is A, 0<A≤0.5. 4. preparation method according to claim 1 is characterized in that, described linking agent is selected from 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide One or more of amine, 2-morpholineethanesulfonic acid, carbodiimide, genipin and macroglyceryl ether. 5. The preparation method according to claim 1, characterized in that the mass of the cross-linking agent is B% of the silk fibroin in the silk fibroin solution, and B≧20. 6. The preparation method according to claim 1, characterized in that, the reaction time in the step S1) is 10-30 minutes. 7. The preparation method according to claim 1, characterized in that, the dialysis time is 12-48 hours. 8. The preparation method according to claim 1, characterized in that, the concentration of hirudin in the hirudin solution is CU/ml, 0<C≤500. 9. The preparation method according to claim 1, characterized in that the soaking time in the step S2) is 2-10 hours. 10. A silk fibroin anticoagulant material, characterized in that it comprises silk fibroin and hirudin cationized with polyethylene glycol diamine.