CN106730052B - Silk fibroin anticoagulant material and preparation method thereof - Google Patents

Abstract

The present invention provides a method for preparing a silk fibroin anticoagulant material, which includes the following steps: S1) mixing a silk fibroin solution, polyethylene glycol diamine and a cross-linking agent, dialysis after the reaction, and drying to obtain polyethylene Diol diamine cationized silk fibroin material; S2) The polyethylene glycol diamine cationized silk fibroin material 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 group of the thrombin-binding region from being reacted to affect the thrombin-binding domain, and enables the hirudin to be bound by an ionic bond with a strong binding force. On the cationized silk fibroin of polyethylene glycol diamine, it can 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 preventing artificial Vascular neointimal hyperplasia and postoperative thrombosis.

Description

Silk fibroin anticoagulant material and preparation method thereof

Technical Field

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

Background

The number of deaths caused by cardiovascular and cerebrovascular diseases in China is millions and increases year by year, so blood contact materials are the most scarce biological materials (medical instruments) in clinical practice at present, especially for artificial blood vessel transplantation, even if the medium and large-caliber artificial blood vessels are clinically applied, products are rare in China, the annual use ratio of domestic products is only about 20%, the transplantation of the small-caliber artificial blood vessels is a clinical blank, and the biggest problem is that thrombus is easily formed and the long-term patency rate is poor.

At present, the artificial blood vessel products applied in medicine are mainly made of synthetic materials such as terylene, expanded polytetrafluoroethylene and the like, and have clinical application in the aspect of large and medium caliber artificial blood vessels, but the synthetic materials have poor cell compatibility, are not beneficial to endothelialization, are easy to form thrombus and influence tissue healing. Silkworm silk is natural animal protein synthesized and secreted by silkworm, has wide source, and the silk fibroin has good biocompatibility and consists of 20 amino acids absorbable by human body, and the final degradation product is amino acid or small peptide which is easily absorbed or phagocytized by cells and can not cause obvious immune reaction. A great deal of literature research shows that the silk fibroin material can support the growth of various cells, and has more and more deepened research on tissue engineering materials and made breakthrough progress, and recently, the silk fibroin material also has more and more attention to the application to blood vessel tissue engineering.

Although silk fibroin materials have the advantages of cell compatibility and histocompatibility, the silk fibroin materials used as foreign materials stimulate the blood coagulation system when contacting blood, and induce hemolysis or coagulation. In order to improve the anticoagulation performance of the silk fibroin material, some researchers at home and abroad pay attention to the research on improving the anticoagulation performance of the silk fibroin material.

At present, the anticoagulation modification of fibroin mainly reports grafting of high molecules with anticoagulation effect and sulfation or heparylation methods. Heparin, added to fibroin/chitosan scaffolds under mild conditions, such as She et al, enhances anticoagulation (Polymer International,2010,59(1): 55-61); liu and the like utilize an electrostatic spinning technology to prepare the silk fibroin nano-scaffold from the silk fibroin treated by chlorosulfonic acid, and the anticoagulant property of the sulfated nano-fibroin scaffold is obviously enhanced (Biomaterials,2011,32(15): 3784-3793); wang et al prepared heparin-modified fibroin nano-materials by using an electrostatic spinning technology, and the results of in vitro coagulation tests show that the anticoagulant property of the modified fibroin nano-materials is much higher than that of pure fibroin (International journal of Biological Macromolecules, 2011, 48(2): 345-; sulfated silk fibroin prepared with chlorosulfonic acid has greatly improved anticoagulant activity over sulfated silk fibroin prepared with sulfuric acid (Biomaterials,2004,25 (3): 377-383), but far less than heparin. The research on the anticoagulant property of the heparin modified silk fibroin material has the intellectual property (such as the preparation of an anticoagulant dermal scaffold, Chinese patent with the application number of CN 200910223207.4; nanofiber artificial blood vessel and a preparation method, Chinese patent with the application number of CN 200910228843.6), so that the introduction of heparin is the main method for the anticoagulant property modification of the silk fibroin material at present, but the heparin belongs to a thrombin indirect inhibitor, and the anticoagulant effect depends on antithrombin and specific cofactors, so that even the blended or bonded heparin in the material can not necessarily or can play the anticoagulant effect.

Hirudin is a thrombin specific inhibitor, can directly inhibit thrombosis, and has thrombolytic effect. We have studied and developed a hirudin/silk fibroin anticoagulant material (an anticoagulant fibroin material and a preparation method thereof, Chinese patent with application number ZL201310250951. X; an anticoagulant fibroin membrane and a preparation method thereof, Chinese patent with application number ZL 201310251819.0; Journal of biological materials research Part B2015: 103B:556 one 562), but in-depth research finds that the research result is combined with the mechanism of the hirudin for inhibiting the thrombin activity, and the modification method of covalent bond combination can reduce the activity of hirudin.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a silk fibroin anticoagulant material and a preparation method thereof, wherein the silk fibroin anticoagulant material prepared by the method can efficiently inhibit thrombin activity and continuously has an anticoagulant function.

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

s1) mixing the silk fibroin solution, polyethylene glycol diamine and a cross-linking agent, reacting, dialyzing and drying to obtain a polyethylene glycol diamine cationized silk fibroin material;

s2) soaking the silk fibroin material cationized by polyethylene glycol diamine in a hirudin solution to obtain the silk fibroin anticoagulation 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, wherein A is more than 0 and less than or equal to 0.5.

Preferably, the cross-linking agent is selected from one or more of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, N-hydroxysuccinimide, 2-morpholine ethanesulfonic acid, carbodiimide, genipin and polyethylene glycol glycerol ether.

Preferably, the mass of the cross-linking agent is B% of the mass of silk fibroin in the silk fibroin solution, and B is more than or equal to 20%.

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

Preferably, the dialysis time is 12-48 h.

Preferably, the concentration of hirudin in the hirudin solution is C U/ml, and C is more than 0 and less than or equal to 500.

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

Preferably, the composition comprises silk fibroin and hirudin ionized by polyethylene glycol diamine.

The invention provides a preparation method of a silk fibroin anticoagulant material, which comprises the following steps: s1) mixing the silk fibroin solution, polyethylene glycol diamine and a cross-linking agent, reacting, dialyzing and drying to obtain a polyethylene glycol diamine cationized silk fibroin material; s2) soaking the silk fibroin material cationized by polyethylene glycol diamine in a hirudin solution to obtain the silk fibroin anticoagulation material. Compared with the prior art, the preparation method provided by the invention protects the functional group (-COOH, -NH) of the thrombin binding region₂and-OH) is not influenced by the reaction, and hirudin can be bonded to the silk fibroin cationized by polyethylene glycol diamine through strong bonding force ionic bonds to stably exert the anticoagulation effect, so that the obtained silk fibroin anticoagulation material has the function of obviously inhibiting the thrombin activity, and can be particularly applied to preventing neointimal hyperplasia of artificial blood vessels and formation of postoperative thrombus.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

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

The mass ratio of the polyethylene glycol diamine to the silk fibroin is preferably A, A is more than 0 and less than or equal to 0.5, more preferably 0.01-0.5, still more preferably 0.01-0.2, and most preferably 0.05-0.1; in some embodiments provided herein, the mass ratio of polyethylene glycol diamine to silk fibroin is preferably 0.01; in some embodiments provided herein, the mass ratio of polyethylene glycol diamine to silk fibroin is preferably 0.05; in other embodiments provided herein, the mass ratio of polyethylene glycol diamine to silk fibroin is preferably 0.1.

The crosslinking agent is well known to those skilled in the art, and is not particularly limited, and in the present invention, one or more of- (3-dimethylaminopropyl) -3-ethylcarbodiimide, N-hydroxysuccinimide, 2-morpholinoethanesulfonic acid, carbodiimide, genipin and polyethylene glycol glycerol ether are preferred; the mass of the cross-linking agent is preferably B% of the mass of the silk fibroin, B is more than or equal to 20%, and more preferably 20% -50%.

The invention also provides a preparation method of the silk fibroin anticoagulation material, which comprises the following steps: s1) mixing the silk fibroin solution, polyethylene glycol diamine and a cross-linking agent, reacting, dialyzing and drying to obtain a polyethylene glycol diamine cationized silk fibroin material; s2) soaking the silk fibroin material cationized by polyethylene glycol diamine in a hirudin solution to obtain the silk fibroin anticoagulation material.

The sources of all raw materials are not particularly limited in the invention, and the raw materials can be either commercially available or self-made.

Wherein the mass concentration of silk fibroin in the silk fibroin solution is preferably 3% -20%; the silk fibroin is silk fibroin well known to those skilled in the art, and is not particularly limited, and in the present invention, silkworm silk fibroin is preferred; the silk fibroin solution is preferably prepared according to the following method: degumming and dissolving silk or silkworm shell, filling into a dialysis bag, and dialyzing with deionized water to obtain silk fibroin solution.

Wherein, the degumming method is the method known by the technicians in the field, and is not limited in particular, in the invention, silk or silkworm shell is preferably heated and treated in sodium carbonate aqueous solution, washed by water and loosened by pulling to obtain degummed fibroin fiber; the sodium carbonate aqueous solution is known to those skilled in the art, and is not particularly limited, and in the present invention, the sodium carbonate aqueous solution having a concentration of 0.1% to 1% is preferred, more preferably 0.1% to 0.5%, and still more preferably 0.2% to 0.3%; the preferable proportion of the silk or the silkworm shell to the sodium carbonate aqueous solution is (0.1-10) g: 50ml, more preferably (0.5 to 5) g: 50ml, more preferably (0.5 to 2) g: 50ml, most preferably 1 g: 50 ml; the temperature of the heating treatment is preferably 98-100 ℃; the time of the heating treatment is preferably 20-40 min; the number of times of the heat treatment is preferably 2 to 4.

Dissolving the degummed silk fibroin to obtain silk fibroin dissolving solution; the dissolving method is a method well known to those skilled in the art, and is not particularly limited, in the present invention, preferably, the degummed silk fibroin fiber is mixed with an aqueous solution of calcium chloride-ethanol, and after being heated and dissolved, the silk fibroin dissolving solution is obtained; the ratio of the degummed silk fibroin fibers to the calcium chloride-ethanol aqueous solution is preferably (0.1-5) g: 10ml, more preferably (0.5 to 3) g: 10ml, more preferably (1-2) g: 10 ml; the molar ratio of calcium chloride to ethanol is preferably 1: 2; the heating and dissolving temperature is preferably 60-80 ℃, more preferably 65-75 ℃, and most preferably 70 ℃; the heating and dissolving time is preferably 1-3 h, and more preferably 2-3 h.

Filling the silk fibroin dissolving solution into a dialysis bag, and dialyzing with deionized water to obtain a silk fibroin solution; the dialysis bag is a semipermeable membrane, and the preferred molecular weight cut-off is 12.0-16.0 kDa; during dialysis, preferably replacing the deionized water used for dialysis with new deionized water or purified water every 1-3 hours, more preferably every 2 hours; the dialysis time is preferably 3 days.

Mixing the silk fibroin solution, polyethylene glycol diamine and a cross-linking agent, preferably mixing the silk fibroin solution and the polyethylene glycol diamine, and then adding the cross-linking agent; the mass ratio of the polyethylene glycol diamine to the silk fibroin in the silk fibroin solution is preferably A, A is more than 0 and less than or equal to 0.5, more preferably 0.01-0.5, still more preferably 0.01-0.2, and most preferably 0.05-0.1; the crosslinking agent is well known to those skilled in the art, and is not particularly limited, and in the present invention, one or more of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, N-hydroxysuccinimide, 2-morpholine ethanesulfonic acid, carbodiimide, genipin and polyethylene glycol glyceryl ether are preferable, and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, N-hydroxysuccinimide and 2-morpholine ethanesulfonic acid are more preferable; the mass ratio of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide to the N-hydroxysuccinimide to the 2-morpholine ethanesulfonic acid is preferably 2:1: 2; the mass of the cross-linking agent is preferably B% of the mass of the silk fibroin in the silk fibroin solution, wherein B is more than or equal to 20%, and more preferably 20% -50%.

Then carrying out reaction; the reaction time is preferably 10min to 1h, more preferably 15 min to 45min, and still more preferably 20 min.

Dialyzing after reaction; the molecular weight cut-off of the semipermeable membrane used for dialysis is preferably 12.0-14.0 kDa; the dialysis time is preferably 12-48 h, and the polyethylene glycol diamine cationized silk fibroin solution is obtained.

Drying the silk fibroin solution cationized by the polyethylene glycol diamine to obtain a silk fibroin material cationized by the polyethylene glycol diamine; the drying can be heating drying or freeze drying; when drying, preferably pouring the silk fibroin solution cationized by polyethylene glycol diamine into a flat plate, and then heating and drying or freeze drying; the flat plate is preferably a polystyrene plate; the temperature for heating and drying is preferably 50 ℃ to 70 ℃.

Soaking the silk fibroin material cationized by polyethylene glycol diamine in a hirudin solution; the concentration of hirudin in the hirudin solution is preferably C U/ml, C is more than 0 and less than or equal to 500, more preferably 10-500U/ml, still more preferably 10-300U/ml, still more preferably 10-200U/ml, still more preferably 10-100U/ml, and most preferably 10-50U/ml; the ratio of the polyethylene glycol diamine cationized silk fibroin material to the hirudin solution is preferably 1-10 ml of hirudin solution per mg of material, more preferably 1-8 ml of hirudin solution per mg of material, further preferably 1-5 ml of hirudin solution per mg of material, further preferably 2-5 ml of hirudin solution per mg of material, and most preferably 3ml of hirudin solution per mg of material; the soaking time is preferably 2-10 h, more preferably 2-8 h, still more preferably 4-6 h, and most preferably 5 h.

After dipping, preferably taking out and air-drying, washing with a buffer solution, and air-drying to obtain the silk fibroin anticoagulation material; the buffer is preferably Phosphate Buffered Saline (PBS); the number of times of washing is preferably 3-4.

The preparation method provided by the invention protects the functional group (-COOH, -NH) of the thrombin binding region₂and-OH) is not influenced by the reaction, and hirudin can be bonded to the silk fibroin cationized by polyethylene glycol diamine through strong bonding force ionic bonds to stably exert the anticoagulation effect, so that the obtained silk fibroin anticoagulation material has the function of obviously inhibiting the thrombin activity, and can be particularly applied to preventing neointimal hyperplasia of artificial blood vessels and formation of postoperative thrombus.

In order to further illustrate the present invention, the following describes a silk fibroin anticoagulant material and a preparation method thereof in detail with reference to the following examples.

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

Example 1

1.1, putting the raw silk or the dried and layered cocoon shells of the silkworms into a sodium carbonate aqueous solution with the concentration of 0.2 percent according to the bath ratio of 1:50(g/mL), treating for three times at the temperature of 98-100 ℃ for 30 minutes each time, then fully cleaning the silk with deionized water, loosening, and drying in an oven at the temperature of 60 ℃ to obtain the degummed silk fibroin fibers of the silkworms.

1.2 weighing degummed bombyx mori silk fibroin, dissolving in a calcium chloride-ethanol aqueous solution (molar ratio of ethanol to water is 1:4) with a molar ratio of 1:2 according to a bath ratio of 1:10(g/mL), and dissolving at 70 ℃ for 2 hours to obtain bombyx mori silk fibroin solution.

1.3, the bombyx mori silk fibroin solution is filled into a dialysis bag, the wall of the dialysis bag is a semipermeable membrane, the molecular weight cutoff is within the range of 12.0-16.0 kDa, the dialysis bag filled with the bombyx mori silk fibroin solution is placed into a container filled with deionized water, the water in the container is replaced by new deionized water or pure water every 2 hours, and dialysis is continued for 3 days, so that the purified bombyx mori silk fibroin aqueous solution is obtained. The concentration of the silk fibroin solution after dialysis was adjusted to 4%.

1.4, preparing a mixed solution of silk fibroin and polyethylene glycol diamine according to a mass ratio of 100:1, uniformly stirring, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide with a mass ratio of 20% of silk fibroin into the mixed solution, uniformly stirring, adding 10% of N-hydroxysuccinimide and 20% of 2-morpholine ethanesulfonic acid, reacting for 20 minutes, dialyzing for 12-48 hours by using deionized water to obtain a silk fibroin solution with cationization of polyethylene glycol diamine, and pouring into a flat polystyrene board, and drying at 60 ℃ to obtain the silk fibroin material with cationization of polyethylene glycol diamine.

1.5 cutting the silk fibroin material cationized by polyethylene glycol diamine into small pieces with a certain area, soaking the small pieces in a hirudin solution of 10U/mL according to the proportion of 3mL of the hirudin solution per milligram of the material, taking out the small pieces after 5 hours, standing, air-drying, washing the small pieces for 3-4 times by PBS, and finally air-drying to obtain the silk fibroin anticoagulant material.

The silk fibroin anticoagulant material prepared in the embodiment 1 is subjected to antithrombin activity test, and the absorbance value of thrombin activity detected at 450nm is about 80% of that of pure thrombin activity, namely the silk fibroin anticoagulant material has a certain inhibition effect on the activity of thrombin, which shows that hirudin can be well combined with the silk fibroin material.

Example 2

2.1, putting the raw silk or the dried and layered cocoon shells of the silkworms into a sodium carbonate aqueous solution with the concentration of 0.2 percent according to the bath ratio of 1:50(g/mL), treating for three times at the temperature of 98-100 ℃ for 30 minutes each time, then fully cleaning the silk with deionized water, loosening, and drying in an oven at the temperature of 60 ℃ to obtain the degummed silk fibroin fibers of the silkworms.

2.2 weighing degummed bombyx mori silk fibroin, dissolving in a calcium chloride-ethanol aqueous solution (molar ratio of ethanol to water is 1:4) with a molar ratio of 1:2 according to a bath ratio of 1:10(g/mL), and dissolving at 70 ℃ for 2 hours to obtain bombyx mori silk fibroin solution.

And 2.3, filling the bombyx mori silk fibroin solution into a dialysis bag, wherein the wall of the dialysis bag is a semipermeable membrane, the molecular weight cutoff is in the range of 12.0-16.0 kDa, placing the dialysis bag filled with the bombyx mori silk fibroin solution into a container filled with deionized water, replacing the water in the container with new deionized water or pure water every 2 hours, and continuously dialyzing for 3 days to obtain the purified bombyx mori silk fibroin aqueous solution. The concentration of the silk fibroin solution after dialysis was adjusted to 4%.

2.4 preparing a mixed solution of silk fibroin and polyethylene glycol diamine according to the mass ratio of 100:5, uniformly stirring, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide with the mass ratio of 20% of silk fibroin into the mixed solution, uniformly stirring, adding 10% of N-hydroxysuccinimide and 20% of 2-morpholine ethanesulfonic acid, reacting for 20 minutes, dialyzing for 12-48 hours by using deionized water to obtain a silk fibroin solution with cationization of polyethylene glycol diamine, and pouring into a flat polystyrene board and drying at 60 ℃ to obtain the silk fibroin material with cationization of polyethylene glycol diamine.

2.5 cutting the silk fibroin material cationized by polyethylene glycol diamine into small pieces with a certain area, soaking the small pieces in a hirudin solution of 10U/mL according to the proportion of 3mL of the hirudin solution per milligram of the material, taking out the small pieces after 5 hours, standing, air-drying, washing the small pieces for 3-4 times by PBS, and finally air-drying to obtain the silk fibroin anticoagulant material.

The silk fibroin anticoagulant material prepared in the example 2 is subjected to antithrombin activity test, and the absorbance value of thrombin activity detected at 450nm is about 45% of that of pure thrombin activity, namely, the silk fibroin anticoagulant material prepared in the example 2 can obviously inhibit the activity of thrombin, which shows that hirudin is firmly combined on the silk fibroin material.

Example 3

3.1, putting the raw silk or the dried and layered cocoon shells of the silkworms into a sodium carbonate aqueous solution with the concentration of 0.2% according to the bath ratio of 1:50(g/mL), treating for three times at 98-100 ℃ for 30 minutes each time, then fully cleaning the silk with deionized water, loosening, and drying in an oven at 60 ℃ to obtain the degummed silk fibroin fibers of the silkworms.

3.2 weighing degummed bombyx mori silk fibroin, dissolving in a calcium chloride-ethanol aqueous solution (molar ratio of ethanol to water is 1:4) with a molar ratio of 1:2 according to a bath ratio of 1:10(g/mL), and dissolving at 70 ℃ for 2 hours to obtain bombyx mori silk fibroin solution.

3.3, the bombyx mori silk fibroin solution is filled into a dialysis bag, the wall of the dialysis bag is a semipermeable membrane, the molecular weight cutoff is within the range of 12.0-16.0 kDa, the dialysis bag filled with the bombyx mori silk fibroin solution is placed into a container filled with deionized water, the water in the container is replaced by new deionized water or pure water every 2 hours, and dialysis is continued for 3 days, so that the purified bombyx mori silk fibroin aqueous solution is obtained. The concentration of the dialyzed silk fibroin solution was adjusted to 5%.

3.4, preparing a mixed solution of silk fibroin and polyethylene glycol diamine according to the mass ratio of 100:5, uniformly stirring, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide with the mass ratio of 20% of silk fibroin into the mixed solution, uniformly stirring, adding 10% of N-hydroxysuccinimide and 20% of 2-morpholine ethanesulfonic acid, reacting for 20 minutes, dialyzing for 12-48 hours by using deionized water to obtain a silk fibroin solution with cationization of polyethylene glycol diamine, and pouring the silk fibroin solution into a flat polystyrene plate for cooling and freeze drying to obtain the silk fibroin material with cationization of polyethylene glycol diamine.

3.5 cutting the cationized regenerated silk fibroin material into small pieces with a certain area, soaking the small pieces in 20U/mL hirudin solution according to the proportion of 3mL hirudin solution per milligram of the material, taking out the small pieces after 5 hours, standing, air-drying, washing the small pieces with PBS for 3-4 times, and finally air-drying to obtain the silk fibroin anticoagulation material.

The silk fibroin anticoagulant material prepared in the embodiment 3 is subjected to antithrombin activity test, and the absorbance value of thrombin activity at 450nm is detected to be about 33% of the absorbance value of pure thrombin activity, namely, the silk fibroin anticoagulant material can obviously inhibit the activity of thrombin, so that hirudin is firmly combined on the silk fibroin material.

Example 4

4.1, putting the raw silk or the dried and layered cocoon shells of the silkworms into a sodium carbonate aqueous solution with the concentration of 0.2% according to the bath ratio of 1:50(g/mL), treating for three times at 98-100 ℃ for 30 minutes each time, then fully cleaning the silk with deionized water, loosening, and drying in an oven at 60 ℃ to obtain the degummed silk fibroin fibers of the silkworms.

4.2 weighing degummed bombyx mori silk fibroin, dissolving in a calcium chloride-ethanol aqueous solution (molar ratio of ethanol to water is 1:4) with a molar ratio of 1:2 according to a bath ratio of 1:10(g/mL), and dissolving at 70 ℃ for 2 hours to obtain bombyx mori silk fibroin solution.

And 4.3, filling the bombyx mori silk fibroin solution into a dialysis bag, wherein the wall of the dialysis bag is a semipermeable membrane, the molecular weight cutoff is 12.0-16.0 kDa, placing the dialysis bag filled with the bombyx mori silk fibroin solution into a container filled with deionized water, replacing the water in the container with new deionized water or pure water every 2 hours, and continuously dialyzing for 3 days to obtain the purified bombyx mori silk fibroin aqueous solution. The concentration of the dialyzed silk fibroin solution was adjusted to 5%.

4.4, preparing a mixed solution of silk fibroin and polyethylene glycol diamine according to a mass ratio of 100:10, uniformly stirring, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide with a mass ratio of 20% of silk fibroin into the mixed solution, uniformly stirring, adding 10% of N-hydroxysuccinimide and 20% of 2-morpholine ethanesulfonic acid, reacting for 20 minutes, dialyzing for 12-48 hours by using deionized water to obtain a silk fibroin solution with cationization of polyethylene glycol diamine, and pouring into a flat polystyrene board for cooling and freeze-drying to obtain the silk fibroin material with cationization of polyethylene glycol diamine.

4.5 cutting the silk fibroin material cationized by polyethylene glycol diamine into small pieces with a certain area, soaking the small pieces in a hirudin solution of 40U/mL according to the proportion of 3mL of the hirudin solution per milligram of the material, taking out the small pieces after 5 hours, standing, air-drying, washing the small pieces for 3-4 times by PBS, and finally air-drying to obtain the silk fibroin anticoagulant material.

The silk fibroin anticoagulant material prepared in the example 4 is subjected to antithrombin activity test, and the absorbance value of thrombin activity detected at 450nm is less than 10% of the absorbance value of pure thrombin activity, namely, the silk fibroin anticoagulant material obtained in the example 4 can obviously inhibit the activity of thrombin, which shows that more hirudin is firmly combined on the silk fibroin material.

Comparative example 1

1.1, putting the raw silk or the dried and layered cocoon shells of the silkworms into a sodium carbonate aqueous solution with the concentration of 0.2% according to the bath ratio of 1:50(g/mL), treating for three times at 98-100 ℃ for 30 minutes each time, then fully cleaning the silk with deionized water, loosening, and drying in an oven at 60 ℃ to obtain the degummed silk fibroin fibers of the silkworms.

1.2 weighing degummed bombyx mori silk fibroin, dissolving in a calcium chloride-ethanol aqueous solution (molar ratio of ethanol to water is 1:4) with a molar ratio of 1:2 according to a bath ratio of 1:10(g/mL), and dissolving at 70 ℃ for 2 hours to obtain bombyx mori silk fibroin solution.

1.3, the bombyx mori silk fibroin solution is filled into a dialysis bag, the wall of the dialysis bag is a semipermeable membrane, the molecular weight cutoff is within the range of 12.0-16.0 kDa, the dialysis bag filled with the bombyx mori silk fibroin solution is placed into a container filled with deionized water, the water in the container is replaced by new deionized water or pure water every 2 hours, and dialysis is continued for 3 days, so that the purified bombyx mori silk fibroin aqueous solution is obtained. The concentration of the dialyzed silk fibroin solution was adjusted to 5%.

1.4 adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide with the mass ratio of 20% of silk fibroin into the silk fibroin aqueous solution, uniformly stirring, adding 10% of N-hydroxysuccinimide and 20% of 2-morpholine ethanesulfonic acid, stirring for 20 minutes, dialyzing with deionized water for 12-48 hours, and pouring into a flat polystyrene plate for cooling, freeze-drying and drying to obtain the regenerated silk fibroin material.

1.5 cutting the pure regenerated silk fibroin material into small pieces with a certain area, soaking the small pieces in a hirudin solution with the volume of 20U/mL according to the proportion of 3mL of the hirudin solution per milligram of the material, taking out the small pieces after 5 hours, standing, air-drying, washing with PBS for 3-4 times, and finally air-drying to obtain the hirudin-loaded silk fibroin material.

The prepared silk fibroin material loaded with hirudin is subjected to antithrombin activity test, and the absorbance value of thrombin activity detected at 450nm is more than 96% of the absorbance value of pure thrombin activity, namely the silk fibroin material loaded with hirudin can not effectively inhibit thrombin activity, which indicates that hirudin is not firmly loaded on the silk fibroin material.

Comparative example 2

2.1, putting the raw silk or the dried and layered cocoon shells of the silkworms into a sodium carbonate aqueous solution with the concentration of 0.2% according to the bath ratio of 1:50(g/mL), treating for three times at 98-100 ℃ for 30 minutes each time, then fully cleaning the silk with deionized water, loosening, and drying in an oven at 60 ℃ to obtain the degummed silk fibroin fibers of the silkworms.

2.2 weighing degummed bombyx mori silk fibroin, dissolving in a calcium chloride-ethanol aqueous solution (molar ratio of ethanol to water is 1:4) with a molar ratio of 1:2 according to a bath ratio of 1:10(g/mL), and dissolving at 70 ℃ for 2 hours to obtain bombyx mori silk fibroin solution.

And 2.3, filling the bombyx mori silk fibroin solution into a dialysis bag, wherein the wall of the dialysis bag is a semipermeable membrane, the molecular weight cutoff is in the range of 12.0-16.0 kDa, placing the dialysis bag filled with the bombyx mori silk fibroin solution into a container filled with deionized water, replacing the water in the container with new deionized water or pure water every 2 hours, and continuously dialyzing for 3 days to obtain the purified bombyx mori silk fibroin aqueous solution. The concentration of the dialyzed silk fibroin solution was adjusted to 5%.

2.4 adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide with the mass ratio of 20% of silk fibroin into the silk fibroin aqueous solution, uniformly stirring, adding 10% of N-hydroxysuccinimide and 20% of 2-morpholine ethanesulfonic acid, stirring for 20 minutes, dialyzing with deionized water for 12-48 hours, and pouring into a flat polystyrene plate for cooling, freeze-drying and drying to obtain the regenerated silk fibroin material.

And (3) performing an antithrombin activity test on the prepared regenerated silk material, and detecting that the absorbance value of the thrombin activity at 450nm is 98% of the absorbance value of the pure thrombin activity, namely that the regenerated silk material has no capability of inhibiting the thrombin activity.

Claims (6)

1. a preparation method of silk fibroin anticoagulant material, is characterized in that, comprises the following steps: S1) mixing silk fibroin solution, polyethylene glycol diamine and a cross-linking agent, dialysis after the reaction, and drying to obtain a cationized silk fibroin material of polyethylene glycol diamine; The mass ratio of the mass to the silk fibroin in the silk fibroin solution is 0.01 to 0.5; S2) immersing the polyethylene glycol diamine cationized silk fibroin material in a hirudin solution to obtain a silk fibroin anticoagulant material; The crosslinking agent is selected from 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, 2-morpholineethanesulfonic acid, carbodiimide, One or more of nipin and polyethylene glycol glyceryl ether; The quality of the cross-linking agent is B% of the amount of silk fibroin in the silk fibroin solution, and B ≥ 20; The reaction time in the step S1) is 10-30 min. 2 . The preparation method according to claim 1 , wherein the mass concentration of the silk fibroin solution is 3% to 20%. 3 . 3 . The preparation method according to claim 1 , wherein the dialysis time is 12-48 h. 4 . 4. The preparation method according to claim 1, wherein the concentration of hirudin in the hirudin solution is CU/ml, and 0<C≤500. 5 . The preparation method according to claim 1 , wherein the immersion time in the step S2) is 2-10 h. 6 . 6. A silk fibroin anticoagulant material prepared according to claim 1, characterized in that it comprises cationized silk fibroin and hirudin with polyethylene glycol diamine.