CN1255189C - Prepn of anticoagulant insoluble silk protein material - Google Patents
Prepn of anticoagulant insoluble silk protein material Download PDFInfo
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- CN1255189C CN1255189C CNB2003101019844A CN200310101984A CN1255189C CN 1255189 C CN1255189 C CN 1255189C CN B2003101019844 A CNB2003101019844 A CN B2003101019844A CN 200310101984 A CN200310101984 A CN 200310101984A CN 1255189 C CN1255189 C CN 1255189C
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- fibroin
- heparin
- silk fibroin
- water
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Abstract
The present invention provides a method for preparing silk fibroin material with favorable anticoagulant properties, which relate to biological polymer material. Ternary solution of CaCl2/water/ethanol(the molar ratio is 1: 8: 2) is used as solvent, and water solution of silk fibroin with the concentration of 2 to 100 mg/ml is prepared by dialysis. 0.1 to 10% of heparin is dissolved in the solution of silk fibroin, and the solution is concentrated to 100 to 500 mg/ml by slight stirring at the temperature lower than 90 DEG C. The solution is spread on a polystyrene board at 20 to 200 DEG C to form a film or prepare a thin film or a silk fibroin tube by extrusion. The method has the advantages of simple operation and long residence time of heparin. The prepared material has the advantages of favorable mechanical properties, favorable biocompatibility and favorable blood compatibility, and the material can be used as blood contact material and has a favorable application prospect.
Description
Technical field
The present invention has a good biocompatibility for preparation, and the new method of blood compatibility and mechanical performance and fibroin material that can stable existence in water relates to biomaterial.
Background technology
Fibroin albumen has excellent biocompatibility as natural biological albumen, and low inflammatory reaction and favorable mechanical and physical and chemical performance are subjected to biomaterial circle extensive studies in recent years.At artificial skin, prosthetic ligament, the artificial bone, there is good prospects for application the artificial blood vessel aspect, and can be used as the medicament slow release material, enzyme immobilization material etc.In organizational project, its excellent cell growth property and slow degradability also make it be with a wide range of applications.Though, in actual applications, then need to be prepared into two-dimensional film or three-dimensional tubing form, so will dissolve regeneration meets instructions for use with preparation material to fibroin because fibroin albumen exists with the thread attitude of one dimension at nature.
After the fibroin albumen dissolving regeneration, generally be water-soluble material, cause loss in vivo easily, be difficult to use as biomaterial.The existing water-fast processing mode of fibroin albumen that makes is that fibroin is immersed in the methanol solution, and this method causes the harm problem of residual methanol to human body easily.For avoiding the harm of methanol to human body, we have studied the new method that directly prepares insoluble fibroin material under appropraite condition, and have obtained success.
But on the other hand, because the blood compatibility of fibroin albumen is relatively poor, though its cell growth property is good, thereby can produce new endothelial layer at film surface within a certain period of time, but the back that implants is not easy to generate blood coagulation under cell has the situation of covering surfaces, uses thereby be not suitable as blood contact class material.Be of the application of expansion wire fibroin, must carry out modification with the anticoagulation function that the improves material especially anticoagulation function of short-term fibroin albumen in technical field of biological material.The existing method of improving the fibroin anticoagulant property mainly contains grafting phosphate compounds and plasma modification etc., the reaction condition complexity, and poor repeatability, and the improvement of anticoagulant property has certain restriction, is not suitable for extensive use.
The present invention is for solving anticoagulant fimbrin, especially the straightforward procedure of short-term anticoagulant property, by a certain amount of heparin of blend, make good mechanical performance, water-fast have a thrombogenic material of good resistance, its excellent anticoagulant property can keep in water considering the excellent biological compatibility that fibroin albumen has more than 28 days that the material of the method preparation is with a wide range of applications in blood contact class biomaterial.
Fibroin albumen generally has two kinds of crystalline states, and wherein alpha-helix is soluble in water, and beta sheet is water-fast crystalline state.Fibroin albumen mainly exists with beta sheet under the naturalness, and water insoluble and other common solvent can be dissolved in CaCl
2In/water/ethanol (mol ratio 1: 8: 2) ternary solution or other dense saline solution, but can destroy the original beta sheet structure of fibroin albumen after the fibroin dissolving, and behind drying and forming-film, mainly exist with alpha-helix or indefinite form form, soluble in water, this has limited the application of this material at biological field greatly.Originally the method for the insoluble fibroin thin film of preparation mainly is to handle by methanol to make its crystal conversion, and we studies show that insoluble fibroin material can directly make under conditions such as suitable concentration, temperature and evaporation rate, and do not need methanol to handle.This method makes the preparation of insoluble fibroin albumen thin film safer and simple.On the other hand, the anticoagulation function of fibroin albumen is difficult to satisfy the instructions for use of blood contact class material.Studies show that fibroin material can cause blood coagulation immediately after implanting, but because fibroin has the good cell compatibility, prolongation along with the time of implantation, material surface has endothelial layer to generate gradually, thereby its blood compatibility also improves gradually, after implanting 28 days, endothelial layer will cover the fibroin surface fully.Therefore, if improve the blood compatibility of blood compatibility, the especially incipient stage of fibroin albumen, fibroin albumen will be had wide practical use in blood class technical field of biological material.
Heparin is the most frequently used anticoagulant, has good anticoagulant property, is widely used in the improvement of anticoagulant property of biomaterials such as polyurethane.But do not appear in the newspapers about the material of fibroin with the heparin blend always.Consider that heparin is a polysaccharide, the ability that has good hydrogen bond to form can be dissolved in the aqueous solution with fibroin, thus with heparin with the fibroin blend, by the bleed rate of hydrogen bond action reduction heparin, it is feasible making intermingling material have excellent anticoagulant property in a long time.
The present invention seeks to utilize heparin as anticoagulant for overcoming the shortcoming of fibroin albumen blood compatibility difference, the preparation heparin is with the new method of the insoluble fibroin material of the satisfactory mechanical property of the even blend of fibroin.
Summary of the invention:
After being dissolved in an amount of heparin in the silk fibroin solution, stir, making it has the transparent silk fibroin water solution of high concentration of heparin being lower than 90 ℃ of blend that are concentrated into 100～500mg/ml, aqueous solution is poured on polystyrene board or the glass plate, and 20～200 ℃ of following volatile dries become water-fast fibroin thin film in drying baker.
With the solution-cast of high concentration in the mould of different-diameter, under 20～200 ℃, the control rate of drying, volatile dry becomes silk fibroin tubular material.Make the water-fast fibroin albumen tubing of different-diameter with very good mechanical properties with blood compatibility.
After utilizing silk fibroin protein solution and heparin blend, obtain water-fastly have the fibroin thin film of good blood compatibility or under further spissated condition at the spontaneous crystal conversion under uniform temperature and the high concentration, directly by extruding, the method of blowing is made tubular structure, the reaction condition gentleness, film forming is even, heparin does not have harmful effect to the crystallization of fibroin albumen, and can form hydrogen bond with fibroin, the method is fit to the mass preparation of material, and on the basis that keeps material good mechanical properties and biocompatibility, make material improve blood compatibility.
Specific implementation of the present invention is: with silkworm silk at 0.5% Na
2CO
3100 ℃ are boiled 60min in the solution, repeatedly wash after drying with the sericin of removing the silkworm silk outside and the fibroin albumen that will boil with distilled water, obtain pure fibroin albumen.A certain amount of fibroin albumen is dissolved in CaCl
2In/water/ethanol (mol ratio 1: 8: 2) ternary solution or other dense saline solution, and with dialysis band dialysis three days, removing calcium chloride and the ethanol in the solution, thereby obtain purified silk fibroin water solution.Heparin with 0.1～10% joins in this silk fibroin solution and dissolves, with this aqueous solution through stirring, in being lower than 90 ℃ scope, this solution is concentrated, obtaining concentration is the transparent fibroin heparin mixed aqueous solution of 100～500mg/ml, the purpose that stirs is to prevent that silk fibroin solution from solidifying in concentration process, and give fibroin with dipole-dipole force, promote the transformation of fibroin to beta sheet.The clear solution of this high concentration is poured on polystyrene or the glass plate, in temperature is dry in 20～200 ℃ the drying baker, the control rate of drying, make transparent water-fast fibroin albumen thin film, after clear solution can also being poured on polystyrene or the glass plate, by its part is covered, the evaporation rate of control solution prepares transparent fibroin heparin blend film.
This clear solution can be by casting through further concentrating, and extrudes and blowing obtains the tubing of the water-fast fibroin heparin intermingling material of mechanical property excellence.
The present invention is on the basis of the direct insoluble fibroin thin film of preparation, the method by the blend heparin improves the anticoagulant property of material, has avoided the complexity of preparation method and method of modifying in the past, and preparation process is simplified greatly.
The present invention is directly preparing on the basis of insoluble fibroin thin film, improve the anticoagulant property of material by the method for blend heparin, key technology is: heparin is blended into silk fibroin solution 1., the biological activity that has kept heparin, 2. before solution concentration, add heparin, to avoid heparin in material, to assemble, and help fibroin and form with the hydrogen bond between the heparin. heparin is widely used at medical field as well behaved anticoagulant.In technical field of biological material, be to improve the anticoagulant property of material, the grafting of heparin and blend also are fundamental method.After the heparin grafting, can make material have long lasting anticoagulant property, but the grafting meeting destroys the active group of heparin, thereby the anticoagulation function of heparin is reduced.Heparin is blended into the highly blood coagulation resistant that material can keep heparin, but heparin can run off fully over time.Consider that fibroin has excellent biological compatibility, after a period of time that implants, generate new endothelial layer on the surface, thereby has good blood compatibility, simultaneously heparin and fibroin may form hydrogen bond and make heparin can be in the fibroin thin film existence of long period, so from taking all factors into consideration, using the method for heparin blend is more effectively to improve the method for fibroin anticoagulant property.Consider heparin active reduction easily when temperature is too high, and concentrating the gathering that heparin can take place in the back adding, heparin was added before concentrating, and thickening temperature is controlled at below 90 ℃, like this can be on the basis that keeps heparin activity as far as possible, fibroin fully interacts with heparin, makes heparin and fibroin have enough hydrogen bonds to form, and uniform distribution.
Fibroin thin film of preparing or tubing infrared spectrum, the x x ray diffraction, and under 37 ℃ of the fibroin heparin blend films in water 24 hours turnover rate measure the structure of thin film and the dissolubility in water.And study the wastage of heparin in material by amino toluene Fructus polygoni tinctorii test decide heparin in water 24 hours turnover rate and thin film soak the anticoagulant property after 28 days in water variation.The result shows that the fibroin thin film behind the blend heparin is still water insoluble fully, and main crystalline state is a beta sheet, and crystallization does not have harmful effect to fibroin in the adding of heparin.There is hydrogen bond to generate between fibroin and the heparin.The amino toluene Fructus polygoni tinctorii test show heparin bleed rate in time growth and reduce, 24 hours turnover rate is near 13%.Soak after 28 days in water, material still has good anticoagulant property.The mechanical property of materials is measured the ultimate tensile strength of finding its hygrometric state be not less than 10MPa, the maximum tension rate is not less than 40%, and mechanical performance is good.The anticoagulant property of material is measured and is shown, three index times of expression material anticoagulant property are thrombin time (TT), prothrombin time (PT), and partial thromboplastin time (APTT) surpasses the setting value of gauge respectively, i.e. TT＞200s, APTT＞150s, PT＞50s. and before the blend heparin, three indexs of fibroin anticoagulant property are respectively: TT=14.4s, this material had very excellent anticoagulation function after APTT=25.4s, PT=13.0s. showed the blend heparin.Afterwards still kept good anticoagulant property in 28 days through soaking in the water, still greater than the setting value of instrument, PT then has certain reduction (13.3s) for TT and APTT.This makes this material become good fugitive and middle effect anticoagulant material, consider that this material with good anticoagulant property can grow the endothelial layer that covers fully in vivo within 28 days, so this material can be applied to the blood contact material fully.
Advantage of the present invention has: the heparin blend can keep its good anticoagulant property, and the crystallization of fibroin is not had harmful effect, and preparation is simple, is easy to control, and good reproducibility obtains uniform thin film of quality or tubing easily.After heparin and the fibroin blend, the diffusion rate of heparin is slowed down, grow with adequate time to cell thereby can form hydrogen bond between the two.Be solvent with water in the whole process, safe.Therefore the application in blood contact class material is significant and real value to fibroin albumen in the present invention.
Embodiment
Embodiment 1:
With silkworm silk at 0.5% Na
2CO
3100 ℃ are boiled 60min in the solution, carefully wash after drying with the sericin of removing the silkworm silk outside and the fibroin albumen that will boil with distilled water, obtain pure fibroin albumen.1 gram fibroin albumen is dissolved in 10 milliliters CaCl
2In/water/ethanol (mol ratio 1: 8: the 2) ternary solution, this silk fibroin solution was dialysed three days in the dialysis band, remove calcium chloride and ethanol in the solution, thereby obtain transparent silk fibroin solution.The heparin that takes by weighing 0～30mg joins in the silk fibroin solution and dissolves.Pour solution into beaker, being lower than under 90 ℃ of heating in water bath situations, use the agitator gentle agitation, volatilization concentrates.With solution concentration to 150～300mg/ml, be clear solution.Subsequently this concentrated solution is sprawled on polystyrene board, 50～200 ℃ of following drying and forming-films in drying baker, its thickness can be controlled by solution quality.Become the thin film homogeneous transparent, anticoagulant property is good.
Embodiment 2:
Prepare pure silk fibroin water solution shown in embodiment 1.
The heparin that takes by weighing 20～50mg dissolves in silk fibroin solution.Pour solution into beaker, being lower than under 90 ℃ of heating in water bath situations, use the agitator gentle agitation, volatilization concentrates.With solution concentration to 200～350mg/ml, be clear solution.Subsequently this concentrated solution is sprawled on polystyrene board, 50～200 ℃ of following drying and forming-films in drying baker, its thickness can be controlled by solution quality.Become the thin film homogeneous transparent, anticoagulant property is good.
Embodiment 3:
Prepare pure silk fibroin water solution shown in embodiment 1.
The heparin that takes by weighing 0～30mg joins in the silk fibroin solution and dissolves.Pour solution into beaker, be lower than under 90 ℃ of heating in water bath situations, use the agitator gentle agitation, volatilization is the clear solution of 150～300mg/ml, with this solution under static state, be dried to half curdled appearance in drying baker under 50～200 ℃, the method for extruding with machinery is prepared the fibroin albumen pipe that is mixed with heparin then.
Embodiment 4:
Prepare pure silk fibroin water solution shown in embodiment 1.
The heparin that takes by weighing 0～30mg joins in the silk fibroin solution and dissolves.Pour solution into beaker, being lower than under 90 ℃ of heating in water bath situations, use the agitator gentle agitation, volatilization is the clear solution of 150～500mg/ml, solution is poured in the mould of different-diameter, prepares tubular material 50～200 ℃ of following dryings.
Embodiment 5
The heparin that takes by weighing 0～30mg joins in the silk fibroin solution and dissolves.Pour solution into beaker, being lower than under 90 ℃ of heating in water bath situations, use the agitator gentle agitation, volatilization concentrates.With solution concentration to 150～300mg/ml, be clear solution.Subsequently this concentrated solution is sprawled on polystyrene board, 50～200 ℃ of following drying and forming-films in drying baker become thin film to carry out surface treatment with the sulfur dioxide plasma institute, obtain the thin film that cell growth property is further improved.
Embodiment 6
The heparin that takes by weighing 0～30mg joins in the silk fibroin solution and dissolves.Pour solution into beaker, being lower than under 90 ℃ of heating in water bath situations, use the agitator gentle agitation, volatilization concentrates.With solution concentration to 150～300mg/ml, be clear solution.Subsequently this concentrated solution is sprawled on polystyrene board, 50～200 ℃ of following drying and forming-films in drying baker become thin film to carry out surface treatment with the carbon dioxide plasma institute, obtain the thin film that cell growth property is further improved.
Claims (2)
1. the preparation method of the insoluble silk fibroin material of anticoagulant property, it is characterized in that: will greater than 0 and be dissolved in the solution that the 1g fibroin albumen forms smaller or equal to the heparin of 50mg amount after, stir, making it has the transparent silk fibroin water solution of high concentration of heparin being lower than 90 ℃ of blend that are concentrated into 100～500mg/ml, aqueous solution is poured on polystyrene board or the glass plate, and 20～200 ℃ of following volatile dries become water-fast fibroin thin film in drying baker.
2. the preparation method of the insoluble silk fibroin material of anticoagulant property as claimed in claim 1, it is characterized in that: have the transparent silk fibroin water solution of heparin to cast in the mould of different-diameter the blend of 100-500mg/ml, under 20～200 ℃, the control rate of drying, volatile dry becomes silk fibroin tubular material, or be dried to half curdled appearance and extrude or be blow molded into tubulose through machinery, make the water-fast fibroin albumen tubing of different-diameter with very good mechanical properties with blood compatibility.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2003101019844A CN1255189C (en) | 2003-10-20 | 2003-10-20 | Prepn of anticoagulant insoluble silk protein material |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2003101019844A CN1255189C (en) | 2003-10-20 | 2003-10-20 | Prepn of anticoagulant insoluble silk protein material |
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| Publication Number | Publication Date |
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| CN1608683A CN1608683A (en) | 2005-04-27 |
| CN1255189C true CN1255189C (en) | 2006-05-10 |
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| CNB2003101019844A Expired - Fee Related CN1255189C (en) | 2003-10-20 | 2003-10-20 | Prepn of anticoagulant insoluble silk protein material |
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Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8591932B2 (en) * | 2009-09-17 | 2013-11-26 | W. L. Gore & Associates, Inc. | Heparin entities and methods of use |
| CN103301506B (en) * | 2013-06-21 | 2015-03-04 | 苏州大学 | Anticoagulation fibroin membrane and preparation method thereof |
| CN104922728A (en) * | 2015-06-01 | 2015-09-23 | 北京理工大学 | Preparation method for small-diameter artificial silk fibroin blood vessel with high unblocked rate |
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Granted publication date: 20060510 Termination date: 20091120 |