CN106693082B - Anticoagulation material and preparation method thereof - Google Patents
Anticoagulation material and preparation method thereof Download PDFInfo
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- CN106693082B CN106693082B CN201710108825.9A CN201710108825A CN106693082B CN 106693082 B CN106693082 B CN 106693082B CN 201710108825 A CN201710108825 A CN 201710108825A CN 106693082 B CN106693082 B CN 106693082B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L33/00—Antithrombogenic treatment of surgical articles, e.g. sutures, catheters, prostheses, or of articles for the manipulation or conditioning of blood; Materials for such treatment
- A61L33/0005—Use of materials characterised by their function or physical properties
- A61L33/0011—Anticoagulant, e.g. heparin, platelet aggregation inhibitor, fibrinolytic agent, other than enzymes, attached to the substrate
- A61L33/0041—Anticoagulant, e.g. heparin, platelet aggregation inhibitor, fibrinolytic agent, other than enzymes, attached to the substrate characterised by the choice of an antithrombatic agent other than heparin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L33/00—Antithrombogenic treatment of surgical articles, e.g. sutures, catheters, prostheses, or of articles for the manipulation or conditioning of blood; Materials for such treatment
- A61L33/06—Use of macromolecular materials
- A61L33/062—Mixtures of macromolecular compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/42—Anti-thrombotic agents, anticoagulants, anti-platelet agents
Abstract
The invention provides a preparation method of an anticoagulant material, which comprises the following steps: s1) mixing the silk fibroin solution, polyethylene glycol diamine and a cross-linking agent, and reacting to obtain a polyethylene glycol diamine cationized silk fibroin material; s2) soaking the polyethylene glycol cationized silk fibroin material in water to obtain a soaked cationized silk fibroin material; s3) soaking the soaked cationized silk fibroin material in a hirudin solution to obtain the anticoagulant material. Compared with the prior art, the preparation method provided by the invention protects the functional group in the thrombin binding region from being affected by reaction to the thrombin binding domain, and enables hirudin to be bound to the polyethylene glycol diamine cationized silk fibroin by strong binding force ionic bonds, so as to stably exert the anticoagulation effect, and the obtained anticoagulation material has the function of obviously inhibiting the thrombin activity.
Description
Technical Field
The invention belongs to the technical field of anticoagulant materials, and particularly relates to an 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 an anticoagulant material and a preparation method thereof, wherein the anticoagulant material prepared by the method can inhibit thrombin activity and continuously has an anticoagulant function.
The invention provides a preparation method of an anticoagulant material, which comprises the following steps:
s1) mixing the silk fibroin solution, polyethylene glycol diamine and a cross-linking agent, and reacting to obtain a polyethylene glycol diamine cationized silk fibroin material;
s2) soaking the polyethylene glycol cationized silk fibroin material in water to obtain a soaked cationized silk fibroin material;
s3) soaking the soaked cationized silk fibroin material in a hirudin solution to obtain the 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, 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 step S2) is specifically:
and soaking the polyethylene glycol cationized silk fibroin material in water, replacing the water for soaking every 2-4 hours, and soaking for 1-3 days to obtain the soaked cationized silk fibroin material.
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 S3) is 2-10 h.
The invention also provides an anticoagulant material which comprises silk fibroin and hirudin which are cationized by polyethylene glycol diamine.
The invention provides a preparation method of an anticoagulant material, which comprises the following steps: s1) mixing the silk fibroin solution, polyethylene glycol diamine and a cross-linking agent, and reacting to obtain a polyethylene glycol diamine cationized silk fibroin material; s2) soaking the polyethylene glycol cationized silk fibroin material in water to obtain a soaked cationized silk fibroin material; s3) soaking the soaked cationized silk fibroin material in a hirudin solution to obtain the anticoagulant 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 affected by reaction, and hirudin can be bonded to the polyethylene glycol diamine cationized silk fibroin by strong bonding force ionic bond to stably exert anticoagulation effect, so that the obtained anticoagulation material has the function of obviously inhibiting thrombin activity, and can be particularly applied to preventing neointimal hyperplasia of artificial blood vessels and postoperative thrombosis.
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 0.005; 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, and reacting to obtain a polyethylene glycol diamine cationized silk fibroin material; s2) soaking the polyethylene glycol cationized silk fibroin material in water to obtain a soaked cationized silk fibroin material; s3) soaking the soaked cationized silk fibroin material 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.
After the reaction, preferably drying to obtain the polyethylene glycol diamine cationized silk fibroin material; the drying can be heating drying or freeze drying; preferably, the silk fibroin solution cationized by polyethylene glycol diamine is poured into a flat plate during drying, and then heating drying or freeze drying is carried out; 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 water; the water is preferably deionized water; the soaking time is preferably 1-3 days, more preferably 2-3 days, and further preferably 2 days; during soaking, preferably replacing water used for soaking every 2-4 hours; and after soaking, preferably taking out, air-drying or freeze-drying to obtain the soaked cationized silk fibroin material.
Soaking the soaked cationized silk fibroin material 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 5-500U/ml, still more preferably 5-300U/ml, still more preferably 5-200U/ml, still more preferably 5-100U/ml, still more preferably 5-50U/ml, and most preferably 5-20U/ml; the proportion of the soaked 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, more preferably 1-5 ml of hirudin solution per mg of material, more 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 an anticoagulant 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 affected by reaction, and hirudin can be bonded to the polyethylene glycol diamine cationized silk fibroin by strong bonding force ionic bond to stably exert anticoagulation effect, so that the obtained anticoagulation material has the function of obviously inhibiting thrombin activity, and can be particularly applied to preventing neointimal hyperplasia of artificial blood vessels and postoperative thrombosis.
In order to further illustrate the present invention, the following detailed description will be made of an anticoagulant material and a preparation method thereof.
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 100:5, 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, stirring for 10 minutes, and then pouring into a flat polystyrene plate to dry at 60 ℃ to obtain the silk fibroin material cationized by polyethylene glycol diamine.
1.5, placing the silk fibroin material cationized by polyethylene glycol diamine into a container containing deionized water, soaking for 2 days, replacing the deionized water in the container every 2-4 hours, taking out after 2 days, and air-drying to obtain the cationized silk fibroin material after soaking.
1.6 cutting the soaked cationized silk fibroin material into small pieces with certain mass, 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 anticoagulant material.
The anticoagulant material obtained in the example 1 is subjected to antithrombin activity test, and the absorbance value of thrombin activity detected at 450nm is 50% of the absorbance value of pure thrombin activity, namely, the anticoagulant material obtained in the example 1 can obviously inhibit the activity of thrombin, which shows that hirudin is firmly combined on 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 dialyzed silk fibroin solution was adjusted to 5%.
2.4 preparing a mixed solution of silk fibroin and polyethylene glycol diamine according to the mass ratio of 100:10, 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, stirring for 10 minutes, pouring into a flat polystyrene plate, and freeze-drying to obtain the polyethylene glycol diamine cationized silk fibroin material.
2.5, placing the silk fibroin material cationized by polyethylene glycol diamine into a container containing deionized water, soaking for 2 days, replacing the deionized water in the container every 2-4 hours, taking out after 2 days, and air-drying or freeze-drying to obtain the soaked cationized silk fibroin material.
2.6 cutting the soaked cationized silk fibroin material into small pieces with certain mass, soaking the small pieces in a hirudin solution of 5U/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 anticoagulant material.
And (3) performing an antithrombin activity test on the anticoagulant material obtained in the example 2, wherein the absorbance value of thrombin activity at 450nm is 52% of the absorbance value of pure thrombin activity, namely, the anticoagulant material obtained in the example 2 can obviously inhibit the activity of thrombin. Indicating that the hirudin is firmly bonded to 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 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.
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 100:10, 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, stirring for 10 minutes, pouring into a flat polystyrene plate, and freeze-drying to obtain the polyethylene glycol diamine cationized silk fibroin material.
3.5, placing the silk fibroin material cationized by polyethylene glycol diamine into a container containing deionized water, soaking for 2 days, replacing the deionized water in the container every 2-4 hours, taking out after 2 days, and air-drying or freeze-drying to obtain the soaked cationized silk fibroin material.
3.6 cutting the soaked cationized silk fibroin material into small pieces with certain mass, soaking the small pieces in a hirudin solution 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 the small pieces for 3-4 times by PBS, and finally air-drying to obtain the anticoagulant material.
The anticoagulant material obtained in the embodiment 3 is subjected to antithrombin activity test, and the absorbance value of thrombin activity detected at 450nm is 15% of the absorbance value of pure thrombin activity, namely, the anticoagulant material obtained in the embodiment 3 can obviously inhibit the activity of thrombin. Indicating that the hirudin is firmly bonded to 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 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 dialyzed silk fibroin solution was adjusted to 5%.
1.4 adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide with the mass ratio of 20 percent of silk fibroin into the silk fibroin aqueous solution, uniformly stirring, adding 10 percent of N-hydroxysuccinimide and 20 percent of 2-morpholine ethanesulfonic acid, stirring for 10 minutes, and pouring into a flat polystyrene plate to be dried or freeze-dried at 60 ℃. And (3) soaking the regenerated silk fibroin material in a container filled with deionized water for 2 days, replacing the deionized water in the container every 2-4 hours, and taking out and air-drying (drying film) or freeze-drying (porous silk fibroin) after 2 days.
1.5 shearing the pure regenerated fibroin porous material into small pieces with a certain area, soaking the small pieces in a hirudin solution 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 composite material.
The composite material obtained in comparative example 1 was subjected to an antithrombin activity test, and the absorbance value of thrombin activity at 450nm was measured to be 99% of that of pure thrombin activity, i.e., the composite material obtained in comparative example 1 failed to effectively inhibit thrombin activity, indicating that hirudin was 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 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 100:0.5, 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, stirring for 10 minutes, and pouring into a flat polystyrene plate to dry at 60 ℃. And (3) soaking the polyethylene glycol diamine modified cationized regenerated silk fibroin material in a container filled with deionized water for 2 days, replacing the deionized water in the container every 2-4 hours, and taking out and air-drying after 2 days.
2.5 cutting the cationized regenerated silk fibroin film into small pieces with a certain area, soaking the small pieces in 10U/mL hirudin solution, standing and air-drying the small pieces, washing the small pieces with PBS for 3-4 times, and finally air-drying the small pieces.
And (3) performing an antithrombin activity test on the prepared silk fibroin film loaded with hirudin, and detecting that the absorbance value of thrombin activity at 450nm is 95% of that of pure thrombin activity, namely the material does not obviously inhibit the activity of thrombin, which indicates that the hirudin is not firmly loaded on the silk fibroin material.
Due to the mass ratio of the silk fibroin to the polyethylene glycol diamine of 100:0.5, the silk fibroin is grafted with the cation NH3+The quantity is very small, so that the hirudin bound by ionic bonds is very small, and no obvious anticoagulant activity exists.
Claims (6)
1. The preparation method of the anticoagulant material is characterized by comprising the following steps:
s1) mixing the silk fibroin solution, polyethylene glycol diamine and a cross-linking agent, and reacting to obtain a polyethylene glycol diamine cationized silk fibroin material; the mass ratio of the polyethylene glycol diamine to the silk fibroin in the silk fibroin solution is 0.01-0.5;
s2) soaking the silk fibroin material cationized by polyethylene glycol diamine in water to obtain a soaked cationized silk fibroin material;
s3) soaking the soaked cationized silk fibroin material in a hirudin solution to obtain an anticoagulation material;
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;
the mass of the cross-linking agent is B% of the mass of the silk fibroin in the silk fibroin solution, and B is more than or equal to 20%;
the reaction time in the step S1) is 10-30 min.
2. The preparation method of claim 1, wherein the mass concentration of the silk fibroin solution is 3% to 20%.
3. The preparation method according to claim 1, wherein the step S2) is specifically:
and soaking the silk fibroin material cationized by the polyethylene glycol diamine in water, replacing the water for soaking every 2-4 hours, and soaking for 1-3 days to obtain the cationized silk fibroin material after soaking.
4. The process according to claim 1, wherein the hirudin solution has a hirudin concentration of CU/ml, 0 < C.ltoreq.500.
5. The method as claimed in claim 1, wherein the dipping time in the step S3) is 2-10 h.
6. An anticoagulant material as claimed in any one of claims 1 to 5 comprising silk fibroin cationized with polyethylene glycol diamine and hirudin.
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| CN103285431A (en) * | 2013-06-21 | 2013-09-11 | 苏州大学 | Anticoagulation fibroin material and preparation method |
| CN103301506A (en) * | 2013-06-21 | 2013-09-18 | 苏州大学 | Anticoagulation fibroin membrane and preparation method thereof |
| CN104524632A (en) * | 2015-01-21 | 2015-04-22 | 北京航空航天大学 | Preparation method of anti-coagulating composite tubular scaffold with good compliance |
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| JPS6139433B2 (en) * | 1978-08-21 | 1986-09-03 | Kanebo Kenshi Kyobijin Kk | |
| CN103285431A (en) * | 2013-06-21 | 2013-09-11 | 苏州大学 | Anticoagulation fibroin material and preparation method |
| CN103301506A (en) * | 2013-06-21 | 2013-09-18 | 苏州大学 | Anticoagulation fibroin membrane and preparation method thereof |
| CN104524632A (en) * | 2015-01-21 | 2015-04-22 | 北京航空航天大学 | Preparation method of anti-coagulating composite tubular scaffold with good compliance |
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