CN106902398A - Cationization fibroin material, its preparation method and application - Google Patents
Cationization fibroin material, its preparation method and application Download PDFInfo
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- CN106902398A CN106902398A CN201710108832.9A CN201710108832A CN106902398A CN 106902398 A CN106902398 A CN 106902398A CN 201710108832 A CN201710108832 A CN 201710108832A CN 106902398 A CN106902398 A CN 106902398A
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- fibroin
- hirudin
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- A—HUMAN NECESSITIES
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- 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
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- 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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- 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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- 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
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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
- A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
- A61L2300/602—Type of release, e.g. controlled, sustained, slow
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Abstract
The invention provides a kind of preparation method of the fibroin material that is cationized, comprise the following steps：Silk fibroin protein solution, polyethylene glycol diamines are mixed with crosslinking agent, is dialysed after reaction, obtain the fibroin material that is cationized.Compared with prior art; cationization fibroin material prepared by the present invention can prepare anticoagulant material with hirudin; mix with hirudin after especially dialysing, obtain anticoagulant material, both protected functional group (COOH, NH with fibrin ferment calmodulin binding domain CaM2, OH) not because the domain for being influenceed to be combined with fibrin ferment by reaction can make again hirudin be incorporated into the ionic bond of stronger adhesion polyethylene glycol diamine cationization fibroin albumen on, and part hirudin is wrapped in inside fibroin albumen, gradually discharged with the degraded of fibroin albumen, so as to make the anticoagulant material for obtaining also reach stabilization while there is the function of significantly inhibiting thrombin activity and play the effect of anti-freezing for a long time.
Description
Technical field
The invention belongs to anticoagulant material technical field, more particularly to a kind of cationization fibroin material, its preparation
Method and application.
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 cationization fibroin material, its system
Preparation Method and its application, the method prepare cationization fibroin material can prepare with efficiently suppress thrombin activity,
Persistently there is the anticoagulant material of anticoagulant functions.
The invention provides a kind of preparation method of the fibroin material that is cationized, comprise the following steps：
Silk fibroin protein solution, polyethylene glycol diamines are mixed with crosslinking agent, is dialysed after reaction, obtain the fibroin egg that is cationized
White 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 time of the reaction is 10~30min；The time of the dialysis is 12~48h.
Present invention also offers one kind cationization fibroin material, including the silk being cationized through polyethylene glycol diamines
Fibroin.
Present invention also offers application of the above-mentioned cationization fibroin material in anticoagulant material.
Preferably, the anticoagulant material is prepared according to following steps：
Silk fibroin protein solution, polyethylene glycol diamines are mixed with crosslinking agent, is dialysed after reaction, obtain the fibroin egg that is cationized
White solution, is subsequently adding hirudin, obtains anticoagulant material.
Preferably, the hirudin addition is to make the concentration of hirudin in reaction solution for C U/ml, 0 ＜ C≤500.
The invention provides a kind of preparation method of the fibroin material that is cationized, comprise the following steps：By fibroin egg
White solution, polyethylene glycol diamines mix with crosslinking agent, are dialysed after reaction, obtain the fibroin material that is cationized.With existing skill
Art is compared, and cationization fibroin material prepared by the present invention can prepare anticoagulant material with hirudin, protect and blood coagulation
Functional group (- the COOH ,-NH of enzyme calmodulin binding domain CaM2,-OH) not because again the domain for being influenceed to be combined with fibrin ferment by reaction can make
Hirudin is incorporated on the fibroin albumen of polyethylene glycol diamine cationization with the ionic bond of stronger adhesion, reaches stabilization performance
The effect of anti-freezing, so as to make the anticoagulant material for obtaining that there is the function of significantly inhibiting thrombin activity.
Present invention also offers application of the above-mentioned cationization fibroin material in anticoagulant material, the anticoagulation
Material is prepared according to following steps：Silk fibroin protein solution, polyethylene glycol diamines are mixed with crosslinking agent, is dialysed after reaction, obtained
Cationization silk fibroin protein solution, is subsequently adding hirudin, obtains anticoagulant material.Compared with prior art, prepared by the present invention
Anticoagulant material protect functional group (- COOH ,-NH with fibrin ferment calmodulin binding domain CaM2,-OH) not because influenceed by reaction with
The domain that fibrin ferment is combined can make hirudin be incorporated into the cationization of polyethylene glycol diamine with the ionic bond of stronger adhesion again
Fibroin albumen on, part hirudin is wrapped in inside fibroin albumen, is gradually discharged with the degraded of fibroin albumen so that
The anticoagulant material for obtaining also reaches stabilization and plays anti-freezing for a long time while having the function of significantly inhibiting thrombin activity
Effect.
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 one kind cationization fibroin material, including the fibroin being cationized through polyethylene glycol diamines
Albumen.
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 0.05 with the mass ratio of fibroin albumen；In some embodiments that the present invention is provided, the polyethylene glycol
Diamines is preferably 0.01 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.
In the present invention, it is preferred to also include crosslinking agent；The crosslinking agent is for crosslinking agent well known to those skilled in the art
Can, special limitation is had no, preferably-(3- dimethylamino-propyls) -3- ethyl carbodiimides, N- hydroxysuccinimidyl acyls in the present invention
One or more in imines, MES, carbodiimides, Geniposide and polyethylene glycol glycerol ether；The crosslinking agent
Quality be preferably the B% of fibroin protein amount, B >=20, more preferably 20%~50%.
The cationization fibroin material that the present invention is provided can be used to prepare anticoagulant material.
Present invention also offers a kind of preparation method of above-mentioned cationization fibroin material, comprise the following steps：Will
Silk fibroin protein solution, polyethylene glycol diamines mix with crosslinking agent, are dialysed after reaction, obtain the fibroin material that is cationized.
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 time of the dialysis is preferably 12~48h, obtains the fibroin material that is cationized.
It can be molded by drying or freeze-drying.
Cationization fibroin material prepared by the present invention can prepare anticoagulant material with hirudin, protect and blood coagulation
Functional group (- the COOH ,-NH of enzyme calmodulin binding domain CaM2,-OH) not because again the domain for being influenceed to be combined with fibrin ferment by reaction can make
Hirudin is incorporated on the fibroin albumen of polyethylene glycol diamine cationization with the ionic bond of stronger adhesion, reaches stabilization performance
The effect of anti-freezing, so as to make the anticoagulant material for obtaining that there is the function of significantly inhibiting thrombin activity.
Present invention also offers a kind of application of the cationization fibroin material in anticoagulant material of above-mentioned preparation.
According to the present invention, the anticoagulant material is preferably prepared according to following steps：By silk fibroin protein solution, polyethylene glycol
Diamines mixes with crosslinking agent, is dialysed after reaction, obtains the silk fibroin protein solution that is cationized, and is subsequently adding hirudin, obtains anti-freezing
Blood material.
Wherein, the preparing for fibroin material that be cationized is same as above, will not be repeated here.
Hirudin is added after dialysis；The addition of the hirudin be preferably make hirudin in reaction solution concentration be C U/
Ml, 0 ＜ C≤500, more preferably 10~500U/ml is further preferably 10~300U/ml, is further preferably 10~200U/ml, then excellent
Elect 10~100U/ml as, be further preferably 10~50U/ml.
Add hirudin after, preferably stir, then room temperature air-dry or freeze-drying after, obtain anticoagulant material.
In the present invention, preferably pour into room temperature in the vessel of certain area and air-dry or freeze-drying, more preferably in flat board, further preferably pour into poly-
Styrene plate interior room warm air is done or freeze-drying.
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, and part hirudin is wrapped in inside fibroin albumen, with fibroin albumen
Degrade and gradually discharge, so as to make the anticoagulant material for obtaining also be reached while there is the function of significantly inhibiting thrombin activity
Stabilization and the effect of performance anti-freezing for a long time.
In order to further illustrate the present invention, a kind of cationization fibroin albumen for providing the present invention with reference to embodiments
Material, its preparation method and its application are 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 5%.
1.4 in mass ratio 100:The mixed solution of 1 configuration fibroin albumen and polyethylene glycol diamine, 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, obtain the silk fibroin protein solution that is cationized, be subsequently poured into a smooth XPS interior room warm air it is dry obtain sun from
Sonization fibroin material.
The cationization fibroin material obtained in embodiment 1 is analyzed using zeta potential instrument, measures its table
Surface charge is positive charge, Zeta potential>11.
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 5%.
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, reaction 20 minutes after with deionized water dialyse 12~
48 hours, obtain the silk fibroin protein solution that is cationized, be subsequently poured into a smooth XPS interior room warm air it is dry obtain sun from
Sonization fibroin material.
The cationization fibroin material obtained in embodiment 2 is analyzed using zeta potential instrument, measures its table
Surface charge is positive charge, Zeta potential>15.
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 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.
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, obtain the silk fibroin protein solution that is cationized.
3.5, to hirudin is added in cationization silk fibroin protein solution, make the concentration of hirudin in solution for 10U/mL, stir
The XPS interior room warm air that certain area is poured into after mixing uniformly is done, and obtains anticoagulant material.
The anticoagulation fibroin material that will be obtained in embodiment 3 carries out antithrombin activity test, is coagulated at detection 450nm
The absorbance OD of hemase activity450Being worth can be notable than absorbance decline 65%, the i.e. anticoagulant material of pure thrombin activity
Ground suppresses the activity of fibrin ferment.
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 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.
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 1 configuration fibroin albumen and polyethylene glycol diamine, 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, obtain the silk fibroin protein solution that is cationized.
4.5, to hirudin is added in cationization silk fibroin protein solution, make the concentration of hirudin in solution for 20U/mL, stir
The XPS interior room warm air that certain area is poured into after mixing uniformly is done, and obtains anticoagulant material.
The anti-freezing material that will be obtained in embodiment 4 carries out antithrombin activity test, thrombin activity at detection 450nm
Absorbance OD450Be worth has certain suppression blood coagulation than absorbance decline 25%, the i.e. anticoagulant material of pure thrombin activity
The ability of enzymatic activity.
Embodiment 5
The cocoon shell of silkworm raw silk or drying layering is pressed 1 by 5.1: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.
5.2 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.
5.3 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%.
5.4 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, obtain the silk fibroin protein solution that is cationized.
5.5, to hirudin is added in cationization silk fibroin protein solution, make the concentration of hirudin in solution for 20U/mL, stir
Uniform rear freeze-drying is mixed, anticoagulant material is obtained.
The anticoagulant material that will be obtained in embodiment 5 carries out antithrombin activity test, thrombin activity at detection 450nm
Absorbance OD450Being worth can significantly suppress solidifying than absorbance decline 80%, the i.e. anticoagulant material of pure thrombin activity
The activity of hemase.
Embodiment 6
The cocoon shell of silkworm raw silk or drying layering is pressed 1 by 6.1: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.
6.2 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.
6.3 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%.
6.4 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, obtain the silk fibroin protein solution that is cationized.
6.5, to hirudin is added in cationization silk fibroin protein solution, make the concentration of hirudin in solution for 40U/mL, stir
Uniform rear freeze-drying is mixed, anticoagulant material is obtained.
The anticoagulant material that will be obtained in embodiment 6 carries out antithrombin activity test, thrombin activity at detection 450nm
Absorbance OD450Being worth can significantly press down than absorbance decline more than 90%, the i.e. anticoagulant material of pure thrombin activity
The activity of fibrin ferment processed.
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 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 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 reaction 20 minutes
Dialysed 12~48 hours with deionized water, be subsequently poured into a smooth XPS interior room warm air and do to obtain regenerated silk material.
The regenerated silk material obtained in comparative example 1 is analyzed using zeta potential instrument, measuring its surface charge is
Negative electrical charge.
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 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 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 reaction 20 minutes
Dialysed 12~48 hours with deionized water；Being subsequently adding hirudin makes the concentration of hirudin in solution be 10U/mL, stirs
The XPS interior room warm air for pouring into certain area afterwards is done or freeze-drying, obtains anticoagulation fibroin material.
The anticoagulation fibroin material that will be obtained in comparative example 2 carries out antithrombin activity test, is coagulated at detection 450nm
The absorbance OD of hemase activity450Decline 6% than the absorbance of pure thrombin activity, material suppresses the energy of thrombin activity
Power very little, illustrating that hirudin is no with reference to comparative example 2 firmly can be loaded on fibroin material.
Comparative example 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 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 reaction 20 minutes
Dialysed 12~48 hours with deionized water, the XPS interior room warm air for being subsequently poured into certain area is done, obtain pure silk element egg
Tunica albuginea.
The pure fibroin protein film obtained in comparative example 3 is carried out into antithrombin activity test, blood coagulation enzyme activity at detection 450nm
The absorbance OD of property450It is worth and declines 2% than the absorbance of pure thrombin activity, illustrates that pure fibroin material does not suppress
The ability of thrombin activity.
Claims (10)
1. it is a kind of be cationized fibroin material preparation method, it is characterised in that comprise the following steps：
Silk fibroin protein solution, polyethylene glycol diamines are mixed with crosslinking agent, is dialysed after reaction, obtain the fibroin albumen material that is cationized
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 time of the reaction is 10~30min；It is described
The time of analysis is 12~48h.
7. the cationization fibroin material prepared by a kind of claim 1~6 any one, it is characterised in that including warp
The fibroin albumen of polyethylene glycol diamines cationization.
8. prepared by claim 1~6 any one cationization fibroin material or claim 7 described in cation
Change application of the fibroin material in anticoagulant material.
9. application according to claim 8, it is characterised in that the anticoagulant material is prepared according to following steps：
Silk fibroin protein solution, polyethylene glycol diamines are mixed with crosslinking agent, is dialysed after reaction, obtain cationization fibroin albumen molten
Liquid, is subsequently adding hirudin, obtains anticoagulant material.
10. application according to claim 9, it is characterised in that the hirudin addition is to make hirudin in reaction solution
Concentration be C U/ml, 0 ＜ C≤500.
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