CN106730052A - A kind of anticoagulant fimbrin material and preparation method thereof - Google Patents
A kind of anticoagulant fimbrin material and preparation method thereof Download PDFInfo
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- CN106730052A CN106730052A CN201710108408.4A CN201710108408A CN106730052A CN 106730052 A CN106730052 A CN 106730052A CN 201710108408 A CN201710108408 A CN 201710108408A CN 106730052 A CN106730052 A CN 106730052A
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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
- A61L33/12—Polypeptides, proteins or derivatives thereof, e.g. degradation products thereof
- A61L33/128—Other specific proteins or polypeptides not covered by A61L33/122 - A61L33/126
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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
- A61L33/0023—Anticoagulant, e.g. heparin, platelet aggregation inhibitor, fibrinolytic agent, other than enzymes, attached to the substrate using a quaternized group or a protonated amine group of the substrate
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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
- A61L33/0076—Chemical modification of the substrate
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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
- A61L33/068—Use of macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
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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
Technical field
The invention belongs to anticoagulant material technical field, more particularly to a kind of anticoagulant fimbrin material and its preparation side
Method.
Background technology
The annual cardiovascular and cerebrovascular disease death toll of China has millions of, and increasing year by year, therefore, contacting blood material is
Biomaterial (medicine equipment) clinically the most in short supply at present, especially synthetic vascular grafts, even obtain clinical practice
In, heavy caliber artificial blood vessel, also very rare in China's product, the annual use ratio of homemade goodses only has 20% or so,
And the transplanting of small-caliber artificial blood vessel or clinical blank, its maximum problem is exactly easily to form thrombus, long-term patency rate compared with
Difference.
Artificial blood vessel's product for medically applying is mainly and is made up of synthetic materials such as terylene, expanded PTFEs at present
, there is clinical practice in terms of medium-large caliber artificial blood vessel, but these synthetic material cell compatibilities are poor, are unfavorable for endothelialization,
Thrombus is easily formed, organization healing is influenceed.Silkworm silk be by silkworm synthesize with secretion natural animal albumen, wide material sources,
Its fibroin albumen has good biocompatibility, is made up of 20 kinds of absorbable amino acid of human body, and final catabolite is ammonia
Base acid or small peptide, are easily absorbed or are swallowed by cell, will not cause obvious immune response.Existing substantial amounts of literature research shows silk
Fibroin material can support the growth of various kinds of cell, more and more deep in terms of tissue engineering material research, and achieve prominent
The progress of broken property, recently, is applied to also increasingly be attracted attention on intravascular tissue engineering.
Although fibroin material has the advantage of cell compatibility and histocompatbility in itself, as exogenous material,
Blood coagulation system can all be stimulated when with contacting blood, haemolysis or blood coagulation is induced.In order to improve the anticoagulation function of silk fibroin material, state
Inside and outside some researchers have also been concerned about improves the anticoagulation function research of fibroin material.
At present, to the modified main macromolecule for having reported grafting with anticoagulation of anticoagulation and sulphation of fibroin albumen
Or Heparinizing method.Such as She adds heparin in fibroin/chitosan stent under mild conditions, enhances anticoagulant
(Polymer International,2010,59(1):55-61)；Liu etc. is treated chlorosulfonic acid using electrostatic spinning technique
Fibroin albumen be made fibroin nano-bracket, the anticoagulant property of sulphation nanometer fibroin support significantly increase (Biomaterials,
2011,32(15):3784-3793)；Wang etc. is prepared for heparin modified fibroin nano material using electrostatic spinning technique, in vitro
Blood coagulation test result shows that the anticoagulant property of modified fibroin nano material is far above pure silk element (International
Journal of Biological Macromolecules, 2011,48 (2):345-353)；The sulphation prepared with chlorosulfonic acid
The anticoagulant active of fibroin albumen greatly improves (Biomaterials, 2004,25 (3) than sulphation fibroin prepared by sulfuric acid：
377-383), but much not as good as heparin.Heparin modified fibroin protein material anticoagulant property studies existing intellectual property (such as anti-freezing
The preparation of blood dermis scaffold, the Chinese patent of Application No. CN200910223207.4；Nano fiber artificial blood vessel and preparation side
Method, the Chinese patent of Application No. CN200910228843.6), so it is that current silk fibroin material anticoagulant property is modified to introduce heparin
Main method, but heparin belongs to a kind of fibrin ferment indirect inhibitor, anticoagulation to rely on antithrombase and it is specific it is auxiliary because
Son, even if so the heparin for being blended in material or being bonded differs surely or can play anticoagulation.
Hirudin is a kind of thrombin inhibitor, can direct inhibition thrombosis, can also be to established
Thrombus has certain thrombolytic effect.We have researched and developed a kind of a kind of hirudin/anticoagulant fimbrin material (anti-freezing
Trace of blood cellulosic material and preparation method thereof, the Chinese patent of Application No. ZL201310250951.X；A kind of anticoagulation fibroin membrane and
Its preparation method, the Chinese patent of Application No. ZL201310251819.0；Journal of Biomedical Materials
Research Part B,2015:103B:556-562) result of study and hirudin, but further investigation finds, are suppressed into blood coagulation
The mechanism of enzymatic activity combines analysis, and the activity of hirudin can be reduced using the method for modifying of Covalent bonding together.
The content of the invention
In view of this, the technical problem to be solved in the present invention is to provide a kind of anticoagulant fimbrin material and its preparation
Method, anticoagulant fimbrin material prepared by the method can efficiently suppress thrombin activity, persistently have anticoagulant functions.
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 poly- second two
The fibroin material of hydro diamine cationization；
S2 during the fibroin material that the polyethylene glycol diamines are cationized) impregnated in into hirudin solution, silk is obtained
Fibroin anticoagulant material.
Preferably, the mass concentration of the silk fibroin protein solution is 3%~20%.
Preferably, the quality of the polyethylene glycol diamines and the mass ratio of fibroin albumen in silk fibroin protein solution are A, 0 ＜ A
≤0.5。
Preferably, the crosslinking agent is selected from 1- (3- dimethylamino-propyls) -3- ethyl carbodiimides, N- hydroxysuccinimidyls acyl Asia
One or more in amine, MES, carbodiimides, Geniposide and polyethylene glycol glycerol ether.
Preferably, the quality of the crosslinking agent is the B% of fibroin albumen quality in silk fibroin protein solution, B >=20.
Preferably, the step S1) in reaction time be 10~30min.
Preferably, the time of the dialysis is 12~48h.
Preferably, the concentration of hirudin is C U/ml, 0 ＜ C≤500 in the hirudin solution.
Preferably, the step S2) in dipping time be 2~10h.
Preferably, including through polyethylene glycol diamines the fibroin albumen and hirudin for ionizing.
The invention provides a kind of preparation method of anticoagulant fimbrin material, comprise the following steps：S1) by fibroin egg
White solution, polyethylene glycol diamines mix with crosslinking agent, are dialysed after reaction, dry, and obtain the silk of polyethylene glycol diamines cationization
Fibroin material；S2 during the fibroin material that the polyethylene glycol diamines are cationized) impregnated in into hirudin solution, obtain
Anticoagulant fimbrin material.Compared with prior art, the preparation method that the present invention is provided is protected and fibrin ferment calmodulin binding domain CaM
Functional group (- COOH ,-NH2,-OH) not because the domain for being influenceed to be combined with fibrin ferment by reaction can make again hirudin with compared with
The ionic bond of strong adhesion is incorporated on the fibroin albumen of polyethylene glycol diamine cationization, reaches the work that stabilization plays anti-freezing
With, the function of thrombin activity is significantly inhibited so as to have the anticoagulant fimbrin material for obtaining, it is particularly applicable to prevent
The only such as formation of the neointimal hyperplasia and postoperative thrombus of artificial blood vessel.
Specific embodiment
Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described,
Obviously, described embodiment is only a part of embodiment of the invention, rather than whole embodiments.Based in the present invention
Embodiment, the every other embodiment that those of ordinary skill in the art are obtained under the premise of creative work is not made, all
Belong to the scope of protection of the invention.
The invention provides a kind of anticoagulant fimbrin material, including it is cationized through polyethylene glycol diamines and crosslinking agent
Fibroin albumen and hirudin.
Wherein, the polyethylene glycol diamines are preferably A, 0 ＜ A≤0.5, more preferably 0.01 with the mass ratio of fibroin albumen
~0.5, it is further preferably 0.01~0.2, most preferably 0.05~0.1；In some embodiments that the present invention is provided, the poly- second
Two hydro diamines are preferably 0.01 with the mass ratio of fibroin albumen；In some embodiments that the present invention is provided, the polyethylene glycol
Diamines is preferably 0.05 with the mass ratio of fibroin albumen；In other embodiments that the present invention is provided, the polyethylene glycol two
Amine is preferably 0.1 with the mass ratio of fibroin albumen.
The crosslinking agent is crosslinking agent well known to those skilled in the art, has no special limitation, excellent in the present invention
Elect as-(3- dimethylamino-propyls) -3- ethyl carbodiimides, N-hydroxy-succinamide, MES, carbonization two be sub-
One or more in amine, Geniposide and polyethylene glycol glycerol ether；The quality of the crosslinking agent is preferably fibroin protein amount
B%, B >=20, more preferably 20%~50%.
Present invention also offers a kind of preparation method of above-mentioned anticoagulant fimbrin material, comprise the following steps：S1) will
Silk fibroin protein solution, polyethylene glycol diamines mix with crosslinking agent, are dialysed after reaction, dry, and obtain polyethylene glycol diamines cation
The fibroin material of change；S2 the fibroin material that the polyethylene glycol diamines are cationized) be impregnated in into hirudin solution
In, obtain anticoagulant fimbrin material.
The present invention is not particularly limited to the source of all raw materials, is commercially available or self-control.
Wherein, the mass concentration of fibroin albumen is preferably 3%~20% in the silk fibroin protein solution；The fibroin albumen
It is fibroin albumen well known to those skilled in the art, has no special limitation, silk fibroin protein is preferably in the present invention；
The silk fibroin protein solution is preferably prepared in accordance with the following methods：After silk or silkworm shell are carried out into degumming, dissolving, bag filter is poured into
It is interior, dialysed with deionized water, obtain silk fibroin protein solution.
Wherein, the method for the degumming is method well known to those skilled in the art, has no special limitation, this hair
Preferably silk or silkworm shell are heated in aqueous sodium carbonate in bright, washed, after pulling loose, obtain the fibroin fiber of degumming；
The aqueous sodium carbonate is the aqueous solution well known to those skilled in the art, has no special limitation, in the present invention preferably
It is the aqueous sodium carbonate of concentration 0.1%~1%, more preferably 0.1%~0.5%, it is further preferably 0.2%~0.3%；It is described
The ratio of silk or silkworm shell and aqueous sodium carbonate is preferably (0.1~10) g：50ml, more preferably (0.5~5) g：50ml, then
Preferably (0.5~2) g：50ml, most preferably 1g：50ml；The temperature of the heating is preferably 98 DEG C~100 DEG C；It is described
The time of heating is preferably 20~40min；The number of times of the heating is preferably 2~4 times.
The fibroin fiber of degumming is dissolved, fibroin lysate is obtained；The method of the dissolving is that those skilled in the art are ripe
The method known, has no special limitation, is preferably the fibroin fiber of degumming and the water in calcium chloride-ethanol in the present invention
Solution mixes, and after heating for dissolving, obtains fibroin lysate；The fibroin fiber of the degumming and the aqueous solution of calcium chloride-ethanol
Ratio is preferably (0.1~5) g：10ml, more preferably (0.5~3) g：10ml, is further preferably (1~2) g：10ml；The chlorination
Calcium is preferably 1 with the mol ratio of ethanol：2；The temperature of the heating for dissolving is preferably 60 DEG C~80 DEG C, more preferably 65 DEG C~75
DEG C, most preferably 70 DEG C；The time of the heating for dissolving is preferably 1~3h, more preferably 2~3h.
Fibroin lysate is poured into bag filter, is dialysed with deionized water, obtain silk fibroin protein solution；The bag filter
It is pellicle, its molecular cut off is preferably 12.0~16.0kDa；Preferably every 1~3h during dialysis, more preferably every 2h with newly
Deionized water or pure water change dialysis used by deionized water；The time of the dialysis is preferably 3 days.
Silk fibroin protein solution, polyethylene glycol diamines are mixed with crosslinking agent, preferably first by silk fibroin protein solution and poly- second two
After hydro diamine mixing, crosslinking agent is added；The matter of fibroin albumen in the quality and silk fibroin protein solution of the polyethylene glycol diamines
Amount ratio preferably A, 0 ＜ A≤0.5, more preferably 0.01~0.5, it is further preferably 0.01~0.2, most preferably 0.05~0.1；
The crosslinking agent is crosslinking agent well known to those skilled in the art, has no special limitation, and 1- (3- are preferably in the present invention
Dimethylamino-propyl) -3- ethyl carbodiimides, N-hydroxy-succinamide, MES, carbodiimides, Geniposide
With one or more in polyethylene glycol glycerol ether, more preferably 1- (3- dimethylamino-propyls) -3- ethyl carbodiimides, N- hydroxyls
Base succinimide and MES；1- (3- the dimethylamino-propyls) -3- ethyl carbodiimides, N- hydroxysuccinimidyl acyls
Imines is preferably 2 with the mass ratio of MES:1:2；The quality of the crosslinking agent is preferably silk in silk fibroin protein solution
The B% of fibroin quality, B >=20, more preferably 20%~50%.
Then reacted；The time of the reaction is preferably 10min~1h, more preferably 15~45min, further preferably for
20min。
Dialysed after reaction；The molecular cut off of the pellicle used by the dialysis is preferably 12.0~14.0kDa；It is described
The time of analysis is preferably 12~48h, obtains the silk fibroin protein solution of polyethylene glycol diamines cationization.
After the silk fibroin protein solution that polyethylene glycol diamines are cationized is dried, polyethylene glycol diamines cationization is obtained
Fibroin material；The drying can be heat drying, or freeze-drying；When drying, preferably by polyethylene glycol diamines sun
The silk fibroin protein solution of ionization is poured into flat board, then is thermally dried or freeze-drying；The flat board is preferably polyphenyl second
Alkene plate；The temperature of the heat drying is preferably 50 DEG C~70 DEG C.
During the fibroin material that the polyethylene glycol diamines are cationized impregnated in into hirudin solution；The hirudin
The concentration of hirudin is preferably C U/ml in solution, 0 ＜ C≤500, more preferably 10~500U/ml, further preferably for 10~
300U/ml, is further preferably 10~200U/ml, is further preferably 10~100U/ml, most preferably 10~50U/ml；The poly- second two
It is molten that the fibroin material of hydro diamine cationization and the ratio of hirudin solution are preferably every milligram of material 1~10ml hirudin
Liquid, more preferably every milligram material 1~8ml hirudin solution is further preferably every milligram of material 1~5ml hirudin solution, then excellent
Elect every milligram of material 2~5ml hirudin solution, most preferably every milligram material 3ml hirudin solution as；The time of the dipping
Preferably 2~10h, more preferably 2~8h, are further preferably 4~6h, most preferably 5h.
After dipping, preferably take out and air-dry, then with wash buffer, after air-drying, obtain anticoagulant fimbrin material；It is described
Buffer solution is preferably phosphate buffer (PBS)；The number of times of the flushing is preferably 3~4 times.
The preparation method that the present invention is provided protects functional group (- the COOH ,-NH with fibrin ferment calmodulin binding domain CaM2,-OH) no
Because again the domain for being influenceed to be combined with fibrin ferment by reaction can make hirudin be incorporated into poly- second with the ionic bond of stronger adhesion
On the fibroin albumen of glycol diamine cationization, the effect that stabilization plays anti-freezing is reached, so as to make the fibroin albumen anti-freezing for obtaining
Blood material has and significantly inhibits the function of thrombin activity, be particularly applicable to prevent as artificial blood vessel neointimal hyperplasia and
The formation of postoperative thrombus.
In order to further illustrate the present invention, a kind of anticoagulant fimbrin material for providing the present invention with reference to embodiments
Material and preparation method thereof is described in detail.
Reagent used is commercially available in following examples.
Embodiment 1
The cocoon shell of silkworm raw silk or drying layering is pressed 1 by 1.1:The bath raio of 50 (g/mL) is put into the carbonic acid that concentration is 0.2%
In sodium water solution, processed three times in 98 DEG C~100 DEG C, 30 minutes per treatment, silk is fully then cleaned dry with deionized water
Only, pull loose, be placed in 60 DEG C of baking ovens and dry, obtain the bombyx mori silk fibroin fiber after degumming.
1.2 weigh the bombyx mori silk fibroin after degumming by 1:The bath raio of 10 (g/mL) is dissolved in mol ratio 1:2 calcium chloride-ethanol
The aqueous solution (mol ratio 1 of ethanol and water:4) in, 70 DEG C of dissolvings obtain bombyx mori silk fibroin lysate in 2 hours.
1.3 are poured into bag filter bombyx mori silk fibroin lysate, and bag filter wall is pellicle, and molecular cut off is 12.0~
16.0kDa scopes, the bag filter that will be filled with bombyx mori silk fibroin lysate was placed in the container for filling deionized water, every 2 hours
The water in container is changed with new deionized water or pure water, is persistently dialysed 3 days, the silk fibroin protein for obtaining after purification is water-soluble
Liquid.Silk fibroin protein solution concentration after adjustment dialysis is 4%.
1.4 in mass ratio 100:The mixed solution of 1 configuration fibroin albumen and polyethylene glycol diamines, stir, to above-mentioned
Mass ratio is added in mixed solution for 1- (3- dimethylamino-propyls) -3- ethyl carbodiimides stirring of fibroin albumen 20% is equal
Even, 10% N-hydroxy-succinamide and 20% MES, reaction 20 minutes after with deionized water dialyse 12~
48 hours, the silk fibroin protein solution of polyethylene glycol diamines cationization is obtained, be subsequently poured into 60 in a smooth XPS
DEG C drying obtain polyethylene glycol diamines cationization fibroin material.
1.5 small pieces that the fibroin material that polyethylene glycol diamines are cationized is cut into certain area, according to every milligram
The ratio of material 3ml hirudin solution impregnated in the hirudin solution of 10U/mL, is taken out after 5 hours after standing is air-dried and is rushed with PBS
Wash 3~4 times, finally air-dry, obtain anticoagulant fimbrin material.
Anticoagulant fimbrin material prepared by embodiment 1 carries out antithrombin activity test, blood coagulation at detection 450nm
The absorbance of enzymatic activity is 80% or so of the absorbance of pure thrombin activity, i.e., anticoagulant fimbrin material is to blood coagulation
The activity of enzyme has certain inhibitory action, illustrates that hirudin preferably can be incorporated on fibroin material.
Embodiment 2
The cocoon shell of silkworm raw silk or drying layering is pressed 1 by 2.1:The bath raio of 50 (g/mL) is put into the carbonic acid that concentration is 0.2%
In sodium water solution, processed three times in 98 DEG C~100 DEG C, 30 minutes per treatment, silk is fully then cleaned dry with deionized water
Only, pull loose, be placed in 60 DEG C of baking ovens and dry, obtain the bombyx mori silk fibroin fiber after degumming.
2.2 weigh the bombyx mori silk fibroin after degumming by 1:The bath raio of 10 (g/mL) is dissolved in mol ratio 1:2 calcium chloride-ethanol
The aqueous solution (mol ratio 1 of ethanol and water:4) in, 70 DEG C of dissolvings obtain bombyx mori silk fibroin lysate in 2 hours.
2.3 are poured into bag filter bombyx mori silk fibroin lysate, and bag filter wall is pellicle, and molecular cut off is 12.0~
16.0kDa scopes, the bag filter that will be filled with bombyx mori silk fibroin lysate was placed in the container for filling deionized water, every 2 hours
The water in container is changed with new deionized water or pure water, is persistently dialysed 3 days, the silk fibroin protein for obtaining after purification is water-soluble
Liquid.Silk fibroin protein solution concentration after adjustment dialysis is 4%.
2.4 in mass ratio 100:The mixed solution of 5 configuration fibroin albumens and polyethylene glycol diamines, stir, to above-mentioned
Mass ratio is added in mixed solution for 1- (3- dimethylamino-propyls) -3- ethyl carbodiimides stirring of fibroin albumen 20% is equal
Even, 10% N-hydroxy-succinamide and 20% MES, with deionized water dialysis 12-48 after reacting 20 minutes
Hour, the silk fibroin protein solution of polyethylene glycol diamines cationization is obtained, it is subsequently poured into 60 DEG C in a smooth XPS
Drying obtains the fibroin material of polyethylene glycol diamines cationization.
2.5 small pieces that the fibroin material that polyethylene glycol diamines are cationized is cut into certain area, according to every milligram
The ratio of material 3ml hirudin solution impregnated in the hirudin solution of 10U/mL, is taken out after 5 hours after standing is air-dried and is rushed with PBS
Wash 3~4 times, finally air-dry, obtain anticoagulant fimbrin material.
Anticoagulant fimbrin material prepared by embodiment 2 carries out antithrombin activity test, blood coagulation at detection 450nm
The absorbance of enzymatic activity is 45% or so of the absorbance of pure thrombin activity, will embodiment 2 prepare fibroin albumen
Anticoagulant material can significantly suppress the activity of fibrin ferment, illustrate that hirudin is securely joined with fibroin material.
Embodiment 3
The cocoon shell of silkworm raw silk or drying layering is pressed 1 by 3.1:The bath raio of 50 (g/mL) is put into the carbonic acid that concentration is 0.2%
In sodium water solution, processed three times in 98~100 DEG C, 30 minutes per treatment, then fully cleaned up silk with deionized water,
Pull loose, be placed in 60 DEG C of baking ovens and dry, obtain the bombyx mori silk fibroin fiber after degumming.
3.2 weigh the bombyx mori silk fibroin after degumming by 1:The bath raio of 10 (g/mL) is dissolved in mol ratio 1:2 calcium chloride-ethanol
The aqueous solution (mol ratio 1 of ethanol and water:4) in, 70 DEG C of dissolvings obtain bombyx mori silk fibroin lysate in 2 hours.
3.3 are poured into bag filter bombyx mori silk fibroin lysate, and bag filter wall is pellicle, and molecular cut off is 12.0~
16.0kDa scopes, the bag filter that will be filled with bombyx mori silk fibroin lysate was placed in the container for filling deionized water, every 2 hours
The water in container is changed with new deionized water or pure water, is persistently dialysed 3 days, the silk fibroin protein for obtaining after purification is water-soluble
Liquid.Silk fibroin protein solution concentration after adjustment dialysis is 5%.
3.4 in mass ratio 100:The mixed solution of 5 configuration fibroin albumens and polyethylene glycol diamines, stir, to above-mentioned
Mass ratio is added in mixed solution for 1- (3- dimethylamino-propyls) -3- ethyl carbodiimides stirring of fibroin albumen 20% is equal
Even, 10% N-hydroxy-succinamide and 20% MES, reaction 20 minutes after with deionized water dialyse 12~
48 hours, the silk fibroin protein solution of polyethylene glycol diamines cationization is obtained, be subsequently poured into cold in a smooth XPS
Jelly is dried to obtain the fibroin material of polyethylene glycol diamines cationization.
3.5 small pieces that the regenerated silk material of cationization is cut into certain area, according to every milligram of material 3ml hirudin
The ratio of solution impregnated in the hirudin solution of 20U/mL, with PBS flushings 3~4 times after taking-up stands and air-dries after 5 hours, finally
Air-dry, obtain anticoagulant fimbrin material.
Anticoagulant fimbrin material prepared by embodiment 3 carries out antithrombin activity test, blood coagulation at detection 450nm
The absorbance of enzymatic activity is 33% or so of the absorbance of pure thrombin activity, i.e., plus anticoagulant fimbrin material can
Significantly suppress the activity of fibrin ferment, illustrate that hirudin is securely joined with fibroin material.
Embodiment 4
The cocoon shell of silkworm raw silk or drying layering is pressed 1 by 4.1:The bath raio of 50 (g/mL) is put into the carbonic acid that concentration is 0.2%
In sodium water solution, processed three times in 98~100 DEG C, 30 minutes per treatment, then fully cleaned up silk with deionized water,
Pull loose, be placed in 60 DEG C of baking ovens and dry, obtain the bombyx mori silk fibroin fiber after degumming.
4.2 weigh the bombyx mori silk fibroin after degumming by 1:The bath raio of 10 (g/mL) is dissolved in mol ratio 1:2 calcium chloride-ethanol
The aqueous solution (mol ratio 1 of ethanol and water:4) in, 70 DEG C of dissolvings obtain bombyx mori silk fibroin lysate in 2 hours.
4.3 are poured into bag filter bombyx mori silk fibroin lysate, and bag filter wall is pellicle, and molecular cut off is 12.0~
16.0kDa scopes, the bag filter that will be filled with bombyx mori silk fibroin lysate was placed in the container for filling deionized water, every 2 hours
The water in container is changed with new deionized water or pure water, is persistently dialysed 3 days, the silk fibroin protein for obtaining after purification is water-soluble
Liquid.Silk fibroin protein solution concentration after adjustment dialysis is 5%.
4.4 in mass ratio 100:The mixed solution of 10 configuration fibroin albumens and polyethylene glycol diamines, stir, to above-mentioned
Mass ratio is added in mixed solution for 1- (3- dimethylamino-propyls) -3- ethyl carbodiimides stirring of fibroin albumen 20% is equal
Even, 10% N-hydroxy-succinamide and 20% MES, reaction 20 minutes after with deionized water dialyse 12~
48 hours, the silk fibroin protein solution of polyethylene glycol diamines cationization is obtained, be subsequently poured into cold in a smooth XPS
Jelly is dried to obtain the fibroin material of polyethylene glycol diamines cationization.
4.5 small pieces that the fibroin material that polyethylene glycol diamines are cationized is cut into certain area, according to every milligram
The ratio of material 3ml hirudin solution impregnated in the hirudin solution of 40U/mL, is taken out after 5 hours after standing is air-dried and is rushed with PBS
Wash 3~4 times, finally air-dry, obtain anticoagulant fimbrin material.
Anticoagulant fimbrin material prepared by embodiment 4 carries out antithrombin activity test, blood coagulation at detection 450nm
The absorbance of enzymatic activity is less than the 10% of the absorbance of pure thrombin activity, i.e., the fibroin albumen that embodiment 4 is obtained resists
Coagulant material can significantly suppress the activity of fibrin ferment, illustrate that more hirudins are securely joined with fibroin material
.
Comparative example 1
The cocoon shell of silkworm raw silk or drying layering is pressed 1 by 1.1:The bath raio of 50 (g/mL) is put into the carbonic acid that concentration is 0.2%
In sodium water solution, processed three times in 98~100 DEG C, 30 minutes per treatment, then fully cleaned up silk with deionized water,
Pull loose, be placed in 60 DEG C of baking ovens and dry, obtain the bombyx mori silk fibroin fiber after degumming.
1.2 weigh the bombyx mori silk fibroin after degumming by 1:The bath raio of 10 (g/mL) is dissolved in mol ratio 1:2 calcium chloride-ethanol
The aqueous solution (mol ratio 1 of ethanol and water:4) in, 70 DEG C of dissolvings obtain bombyx mori silk fibroin lysate in 2 hours.
1.3 are poured into bag filter bombyx mori silk fibroin lysate, and bag filter wall is pellicle, and molecular cut off is 12.0~
16.0kDa scopes, the bag filter that will be filled with bombyx mori silk fibroin lysate was placed in the container for filling deionized water, every 2 hours
The water in container is changed with new deionized water or pure water, is persistently dialysed 3 days, the silk fibroin protein for obtaining after purification is water-soluble
Liquid.Silk fibroin protein solution concentration after adjustment dialysis is 5%.
1.4 is 1- (3- dimethylamino-propyls) -3- of fibroin albumen 20% to mass ratio is added in silk fibroin water solution
Ethyl carbodiimide stirs, 10% N-hydroxy-succinamide and 20% MES, after stirring 20 minutes
Dialysed 12~48 hours with deionized water, be subsequently poured into freeze-drying in a smooth XPS and obtain regenerated silk material.
1.5 small pieces that pure regenerated silk material is cut into certain area, according to every milligram of material 3ml hirudin solution
Ratio impregnated in the hirudin solution of 20U/mL, is taken out after 5 hours and rinsed 3~4 times with PBS after standing is air-dried, and finally air-dry, and obtain
To the silk fibroin material of loading hirudin.
The silk fibroin material of the loading hirudin that will be prepared carries out antithrombin activity test, fibrin ferment at detection 450nm
The absorbance of activity is more than the 96% of the absorbance of pure thrombin activity, that is, the fibroin material for loading hirudin does not have
There is the activity that can effectively suppress fibrin ferment, illustrate that hirudin is not loaded on fibroin material securely.
Comparative example 2
The cocoon shell of silkworm raw silk or drying layering is pressed 1 by 2.1:The bath raio of 50 (g/mL) is put into the carbonic acid that concentration is 0.2%
In sodium water solution, processed three times in 98~100 DEG C, 30 minutes per treatment, then fully cleaned up silk with deionized water,
Pull loose, be placed in 60 DEG C of baking ovens and dry, obtain the bombyx mori silk fibroin fiber after degumming.
2.2 weigh the bombyx mori silk fibroin after degumming by 1:The bath raio of 10 (g/mL) is dissolved in mol ratio 1:2 calcium chloride-ethanol
The aqueous solution (mol ratio 1 of ethanol and water:4) in, 70 DEG C of dissolvings obtain bombyx mori silk fibroin lysate in 2 hours.
2.3 are poured into bag filter bombyx mori silk fibroin lysate, and bag filter wall is pellicle, and molecular cut off is 12.0~
16.0kDa scopes, the bag filter that will be filled with bombyx mori silk fibroin lysate was placed in the container for filling deionized water, every 2 hours
The water in container is changed with new deionized water or pure water, is persistently dialysed 3 days, the silk fibroin protein for obtaining after purification is water-soluble
Liquid.Silk fibroin protein solution concentration after adjustment dialysis is 5%.
2.4 is 1- (3- dimethylamino-propyls) -3- of fibroin albumen 20% to mass ratio is added in silk fibroin water solution
Ethyl carbodiimide stirs, 10% N-hydroxy-succinamide and 20% MES, after stirring 20 minutes
Dialysed 12~48 hours with deionized water, be subsequently poured into freeze-drying in a smooth XPS and obtain regenerated silk material.
The regenerated silk material that will be prepared carries out antithrombin activity test, the suction of thrombin activity at detection 450nm
Shading value is the 98% of the absorbance of pure thrombin activity, i.e. regenerated silk material does not suppress the ability of thrombin activity.
Claims (10)
1. a kind of preparation method of anticoagulant fimbrin material, it is characterised in that comprise the following steps：
S1) silk fibroin protein solution, polyethylene glycol diamines are mixed with crosslinking agent, is dialysed after reaction, dried, obtain polyethylene glycol two
The fibroin material of amine cation；
S2 during the fibroin material that the polyethylene glycol diamines are cationized) impregnated in into hirudin solution, fibroin egg is obtained
White anticoagulant material.
2. preparation method according to claim 1, it is characterised in that the mass concentration of the silk fibroin protein solution is 3%
~20%.
3. preparation method according to claim 1, it is characterised in that the quality and fibroin albumen of the polyethylene glycol diamines
The mass ratio of fibroin albumen is A, 0 ＜ A≤0.5 in solution.
4. preparation method according to claim 1, it is characterised in that the crosslinking agent is selected from 1- (3- dimethylaminos third
Base) -3- ethyl carbodiimides, N-hydroxy-succinamide, MES, carbodiimides, Geniposide and polyethylene glycol
One or more in glycerin ether.
5. preparation method according to claim 1, it is characterised in that during the quality of the crosslinking agent is silk fibroin protein solution
The B% of fibroin albumen quality, B >=20.
6. preparation method according to claim 1, it is characterised in that the step S1) in reaction time for 10~
30min。
7. preparation method according to claim 1, it is characterised in that the time of the dialysis is 12~48h.
8. preparation method according to claim 1, it is characterised in that the concentration of hirudin is C in the hirudin solution
U/ml, 0 ＜ C≤500.
9. preparation method according to claim 1, it is characterised in that the step S2) in time of dipping be 2~10h.
10. a kind of anticoagulant fimbrin material, it is characterised in that including the fibroin albumen being cationized through polyethylene glycol diamines
With hirudin.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110038162A (en) * | 2019-04-16 | 2019-07-23 | 苏州大学 | It is a kind of to have the function of modulating vascular cell growth effect silk fibroin material and preparation method thereof |
| CN112043878A (en) * | 2020-08-06 | 2020-12-08 | 苏州大学 | Anticoagulation blood vessel stent covering film and preparation method thereof |
| CN112043877A (en) * | 2020-08-06 | 2020-12-08 | 苏州大学 | Silk anticoagulant tube stent tectorial membrane and preparation method thereof |
| CN114887123A (en) * | 2022-04-18 | 2022-08-12 | 南通大学 | Hirudin grafted nanofiber vascular stent material, preparation method and application |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 thereof |
| CN103301506A (en) * | 2013-06-21 | 2013-09-18 | 苏州大学 | Anticoagulation fibroin membrane and preparation method thereof |
| US20140315828A1 (en) * | 2013-04-22 | 2014-10-23 | Allergan, Inc. | Cross-linked silk-hyaluronic acid compositions |
| 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 |
-
2017
- 2017-02-27 CN CN201710108408.4A patent/CN106730052B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6139433B2 (en) * | 1978-08-21 | 1986-09-03 | Kanebo Kenshi Kyobijin Kk | |
| US20140315828A1 (en) * | 2013-04-22 | 2014-10-23 | Allergan, Inc. | Cross-linked silk-hyaluronic acid compositions |
| CN103285431A (en) * | 2013-06-21 | 2013-09-11 | 苏州大学 | Anticoagulation fibroin material and preparation method thereof |
| 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 |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110038162A (en) * | 2019-04-16 | 2019-07-23 | 苏州大学 | It is a kind of to have the function of modulating vascular cell growth effect silk fibroin material and preparation method thereof |
| CN110038162B (en) * | 2019-04-16 | 2021-08-31 | 苏州大学 | Functional silk fibroin material with function of regulating and controlling growth of vascular cells and preparation method thereof |
| CN112043878A (en) * | 2020-08-06 | 2020-12-08 | 苏州大学 | Anticoagulation blood vessel stent covering film and preparation method thereof |
| CN112043877A (en) * | 2020-08-06 | 2020-12-08 | 苏州大学 | Silk anticoagulant tube stent tectorial membrane and preparation method thereof |
| WO2022028395A1 (en) * | 2020-08-06 | 2022-02-10 | 苏州大学 | Anticoagulant intravascular stent cover film and preparation method therefor |
| WO2022028396A1 (en) * | 2020-08-06 | 2022-02-10 | 苏州大学 | Silk anticoagulant vascular stent cover film and preparation method therefor |
| CN114887123A (en) * | 2022-04-18 | 2022-08-12 | 南通大学 | Hirudin grafted nanofiber vascular stent material, preparation method and application |
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