CN102847194A - Stent type silk fibroin film insoluble in water, and preparation and application of stent type silk fibroin film - Google Patents
Stent type silk fibroin film insoluble in water, and preparation and application of stent type silk fibroin film Download PDFInfo
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Abstract
The invention discloses a stent type silk fibroin film insoluble in water, and preparation and application of the stent type silk fibroin film. A preparation method includes the steps of placing silk fibroin solution in a smooth, flat and horizontal mold, and obtaining a basilar film by humid and heat crosslinking; and laying the silk fibroin solution on the surface of the basilar film and forming the stent type silk fibroin film insoluble in water after freezing, drying and humid and heat crosslinking. Silk fibroin is used as a raw material, and biocompatibility is good; a preparation process is non-toxic and environment-friendly, and the thickness of the film and the aperture of a stent layer are easy to control; the film is soft in a moist environment, the basilar film is compact, and cells, microorganisms and biomacromolecules cannot penetrate through the basilar film; the stent layer is in the shape of a porous sponge, and is beneficial to fixation of the film, hemostasis of a wound and regeneration of transmitting tissues; and the film has a wide market in the field of anti-adhesive films and artificial film patches.
Description
(1) technical field
The present invention relates to a kind of fibroin protein film and preparation method thereof, particularly a kind of fibroin protein film that is insoluble in water and preparation method thereof and application.
(2) background technology
Silkworm silk is one of best natural fiber of intensity, and the fibroin albumen by 70 ~ 80% and 20 ~ 30% sericin form.The aminoacid sequence of fibroin albumen mainly is made of " Gly-Ala-Gly-Ala--glycine-serine " repetitive (GAGAGS), and wherein glycine, alanine and serine account for more than 85% of total amino acids content.This repetitive sequence overwhelming majority is positioned at the crystal region of fibroin albumen, and is self-assembled into the antiparallel folded conformation (structure of anti-parallel β-sheet).These structures are given the characteristic that fibroin albumen has good mechanics and slow degradation rate.
The sericin reaction that can cause inflammation in vivo needs to carry out degumming process (degummed) before use.Fibroin albumen is that 325KD and two kinds of albumen of 25KD form by molecular weight, and its catabolite is aminoacid.A large amount of fibroin albumens that studies show that have good biocompatibility, and inflammatory reaction (inflammatory responses) will be far below biomaterials commonly used such as collagen and polylactic acid in its body.Fibroin fiber is used as artificial ligament and tissue patch after braiding in the field of study as the existing long history of surgical sutures.But using in present biomaterial research field more is the regenerative fibroin albumen.Be fibroin albumen through after coming unstuck, dissolve, purifying, pass through again physics, chemical method, form the forms such as spongy, membranaceous, gel, microspheroidal.
Aspect the fibroin albumen film preparation, as Chinese invention patent " a kind of solvable-preparation method of high elasticity fibroin protein film " (CN101234212A) in, it is 30 ~ 50um dissolvable film that silk fibroin protein solution after purifying is formed thickness at polystyrene culture dish (40 ~ 70 ℃ of temperature, relative humidity 60 ~ 80%) convection drying in climatic chamber.In the Chinese invention patent " regenerated silk fibroin film and preparation method thereof " (publication number CN 101760027A), with the silk fibroin protein solution convection drying film forming (drying condition not being claimed) in the PS ware after purifying, use again polar solvent (C1-C5 alcohol and water mixture, water or water vapour) in conjunction with mechanical stretch, carry out the swelling degenerative treatments, to improve the fragility of fibroin protein film, namely increase tensile elongation.In the Chinese invention patent " a kind of transparent fibroin protein film that is insoluble in water and preparation method thereof " (CN101967282 A), add polyhydric alcohol in the silk fibroin protein solution after purification as cross-linking agent, with both mixture in mould through the forced air drying film forming, in relative humidity is 30 ~ 98% environment, process 1 ~ 48 hour (treatment temperature not being claimed) again, obtain needed film.Chinese invention patent " flexible silk fibroin membrane and preparation method thereof " (CN1316465) in, prepare water-fast fibroin protein film by adding the crosslinked fibroin albumen of epoxy resin.
Aspect silk fibroin porous scaffold prepares, in the Chinese invention patent " the spongy three-dimensional porous material preparation method of fibroin albumen " (CN 1262579C), in silk fibroin protein solution, add alcohol organic solvent as denaturant, through the freezing and acquisition milky porous material that thaws, material embathes through alcohol solution, more freezing, the dry material requested that obtains.In the Chinese invention patent " a kind of preparation method of silk fibroin micropore bracket " (CN 102133432 A), silk fibroin protein solution is mixed with organic solvent, inject in the mould, obtain Silk fibroin gel, with the organic solvent in the hot water flush away gel, lyophilization obtains described support again.In the Chinese invention patent " a kind of preparation method of porous silk fibroin material " (CN 101864177 A), silk fibroin protein solution is mixed with coagulant colloid (carboxylic acid, Polyethylene Glycol), obtain gel, obtain support in the heated drying method.In the Chinese invention patent " a kind of silk fibroin porous material that is insoluble in water and preparation method thereof " (CN 101857729 A), with silk fibroin protein solution and salt solution mix, obtain required support through steps such as lyophilization, wet-hot steam processing, washings.In the Chinese invention patent " a kind of medicinal microporous sponge and preparation method thereof " (CN 101967254A), silk fibroin protein solution is mixed with PVA solution, slowly adding formalin in the heating process, curing molding, demoulding washing, lyophilization get described support.In the Chinese invention patent " a kind of preparation method of three-dimensional silk fibroin porous scaffold material " (CN101596327 A), in the silk fibroin protein solution of stirring, add sodium chloride particle, get the precipitation part after leaving standstill, shorten type into, dry, embathe step and obtain described support through mould inner pressure.
The preparation method of existing fibroin protein film all is that silk fibroin protein solution or its mixture are poured in the mould, and is dry by solvent evaporates, and deposition of solute is in mold bottom, and then forms approximately 10 ~ 500um solvable or be insoluble in the film of water of thickness.In the film forming procedure of this step, the planarization of mold bottom upper surface and horizontality, whether mould is in vibration environment, blows the wind speed in solution surface, all will affect the homogeneity of film thickness and the microstructure on film surface; In this step film forming procedure, dry temperature and humidity conditions also will affect the transparency and the film homogeneity (such as excess Temperature, understanding forming microbubble in film) of film; The degree of drying of film in this step also affects the performance of film; Approximately several hours to several days drying time of this step film, the clean level of mould environment of living in will affect microorganism and the endotoxin content of film; The film that this step forms, the processing such as the polar solvent by subsequent step or wet heat condition are processed humiture and also can the crystallization degree of film be impacted again.
The preparation method of the existing spongy support of fibroin albumen has been used degeneration, coagulant, cross-linking agent etc. substantially, and these additives flush away in follow-up step perhaps becomes the part of support.Take sodium chloride particle as degeneration and pore-forming mode as example, the Silk fibroin gel that forms is deposited on the gap of sodium chloride particle, because the inhomogeneity in gap, the inhomogeneity that causes the porous support pore wall thickness, do not wait from several microns to the hundreds of micron, the fibroin albumen support mechanics that forms is higher, and degradation rate is slow, but complete a little sodium chloride microgranule that is wrapped up fully by fibroin albumen and being not easy in the flush away support.The fibroin albumen sponge that prepare so thickness and be nanoscale hole wall, micron order aperture, is evenly distributed will there be good application prospect.
For the problems referred to above, patent expectation of the present invention, obtains a kind of support rack type fibroin protein film that is insoluble in water, and stablizes its preparation technology on basement membrane and shelf layer preparation method by simple and the most most economical method.
(3) summary of the invention
The object of the invention provides a kind of support rack type fibroin protein film that is insoluble in water and preparation method thereof, do not contact noxious substance in the preparation process, preparation technology is simple, be beneficial to a large amount of productions, the thickness of easy controlling diaphragm and porosity, the aperture of shelf layer, and then produce different physics and biology performance, satisfy the needs of different purposes; This film one-sided smooth and densification is unfavorable for that the Cell and organism macromolecular substances penetrates, the another side shelf layer is porous spongy to be affixed on site of injury, is beneficial to hemostasis and film the fixing of this position, and the porous spongy shelf layer also has and is the guide tissue regeneration effect.
The technical solution used in the present invention is:
A kind of support rack type fibroin protein film that is insoluble in water, described support rack type fibroin protein film prepares as follows: (1) is take Bombyxmori Linnaeus silkworm silk as raw material, through coming unstuck, dissolve, dialysing, obtain trapped fluid a, the concentrated solution of trapped fluid a or trapped fluid a filtered or centrifugal, get filtrate or upper strata centrifugal liquid obtains silk fibroin protein solution; The mass concentration water of the silk fibroin protein solution that (2) step (1) is obtained is adjusted to 0.3 ~ 30%(preferred 0.5 ~ 20%) (silk fibroin protein solution that the step that at first takes a morsel (1) obtains is dried to constant weight at 60 ℃, calculate the silk fibroin protein solution water content, and then the consumption of definite water water when adjusting concentration), (the smooth smooth bottom of mould of the present invention is that float glass is made to pour mould into, the square mould of mould length of side 20cm) in, the silk fibroin protein solution addition is counted 2 ~ 50mg/cm with the fibroin albumen quality in the described mould
2With mould at 10 ~ 60 ℃ of temperature (preferred 35 ~ 55 ℃), relative humidity 10 ~ 70%(preferred 40%) be dried to without macroscopic moisture under the condition, mold weight increased add 1.2 ~ 1.8 times of fibroin albumen solute weight, in mould, form fibroin protein film; Then with mould (together with fibroin protein film) in 50 ~ 100 ℃ of temperature (preferred 65 ~ 70 ℃), relative humidity 70 ~ 100%(preferred 88 ~ 92%) place the preferred 120min of 20 ~ 200min(under the condition) carry out damp and hot crosslinked, in mould, add purified water (surface that covers the mould inner membrance gets final product), be cooled to room temperature, the purified water of inclining, form the fibroin protein film that is insoluble in water in mould, namely thickness is the basement membrane of 20 ~ 500 μ m; (3) add mass concentration 0.3 ~ 30%(preferred 0.5 ~ 20% that step (1) obtains on the described basement membrane of step (2) surface) silk fibroin protein solution, described silk fibroin protein solution volume addition is 100 ~ 10000ml/m
2Mould, mould is descended freezing 8 ~ 20h) in preferred-40 ℃ of-10 ~-80 ℃ of freezing 2 ~ 30h(under level, again vacuum lyophilization under-40 ~-110 ℃ of conditions (preferred-80 ℃ of lower vacuum dryings 16 ~ 48h), in mould, form duplicature and (namely form one deck support film on the basement membrane surface, described basement membrane is smooth film, the support film is spongy film), remove mould, with duplicature at 50 ~ 100 ℃ of temperature (preferred 65 ~ 70 ℃), relative humidity 70 ~ 100%(preferred 88 ~ 92%) place 20 ~ 200min(preferred 100 ~ 120min) under the condition, and then place volumetric concentration 50 ~ 90%(preferred 75%) ethanol water soak 10 ~ 60min(preferred 20 ~ 30min), fully embathe with purified water again, obtain to be insoluble in the support rack type fibroin protein film of water.
Further, the described silk fibroin protein solution addition of step (2) is preferably 3 ~ 20mg/cm in the fibroin albumen quality
2Mould, making base film thickness is 20 ~ 500 μ m.
Further, the preferred 200 ~ 4000ml/m of volumetric usage of the described silk fibroin protein solution of step (3)
2Mould, the thickness that makes shelf layer are 100～10000 μ m.
Further, the described mould of step (3) is at-20 ~-80 ℃ of lower freezing 5 ~ 30h, and again vacuum lyophilization under-60 ~-110 ℃ of conditions forms the fibroin protein film shelf layer.
Further, the concentrated solution preparation method of the described trapped fluid a of step (1) is: the bag filter after will dialysing is put into the Aqueous Solutions of Polyethylene Glycol of mass concentration 20 ~ 60%, leaves standstill 1 ~ 10h and concentrates, and gets the concentrated solution that trapped fluid b is trapped fluid a; Described Polyethylene Glycol mean molecule quantity 1000～10000.
Further, the concentrated solution of the described trapped fluid a of step (1) or trapped fluid a filters or centrifugal method is one of following: a) with the concentrated solution of trapped fluid a or trapped fluid a centrifugal 5 ~ 20min under 4 ℃, 3000 ~ 6000g condition, discard precipitation, get upper solution and be described silk fibroin protein solution; B) concentrated solution with trapped fluid a or trapped fluid a is the filter filtration of 2 ~ 20 μ m with the aperture, removes insoluble granule, and filtrate is described silk fibroin protein solution.
Further, step (1) is described comes unstuck, dissolving, dialysis process are: a) come unstuck: the 2M aqueous sodium carbonate of 100g Bombyxmori Linnaeus silkworm silk being put into 4 ~ 8L, 90 ~ 100 ℃ of water-bath 20 ~ 60min, pure water cleans, this process repeats 3 times, sloughs sericin, stays fibroin albumen, fibroin albumen 20 ~ 60 ℃ of oven dry, is obtained dried fibroin albumen; B) dissolving: above-mentioned dried fibroin albumen is dissolved in the lithium bromide water solution of 9 ~ 11M, and 55 ~ 65 ℃ of water-bath 30 ~ 300min fully dissolve to fibroin albumen, and acquisition contains the mixed liquor of fibroin albumen; The mass volume ratio of described dried fibroin albumen and lithium bromide water solution is 0.1 ~ 0.2:1; C) dialysis: the mixed liquor that will contain fibroin albumen is dialysed with molecular cut off 1000 ~ 20000 daltonian bag filters, sterile deionized water with 10 times of mixeding liquid volumes is dialysed 10 ~ 12 times at 3 days as dialysis solution, remove the lithium bromide composition in the solution, obtain trapped fluid a.
The present invention also provides the described application of fibroin protein film in preparation Antiadhesive film, wound-protecting film or synthetic membrane that is insoluble in water.
Further, described synthetic membrane is periodontal membrane, Guided Bone Regeneration film, pleura, cerebral dura mater or spinal dura mater.
Plate or box body that institute of the present invention mould can make various materials make, because the bottom requirement is extremely smooth, preferred float glass is as the bottom surface of mould.
Support rack type fibroin protein film of the present invention is duplicature, the shiny surface of film is basal layer, in its preparation process, the quality that in the fixed-area mould, adds fibroin albumen in the silk fibroin protein solution, determined the thickness of final fibroin protein film, be described thickness relevant with the area of mould and silk fibroin protein solution mass concentration and addition, when die area one regularly, adjust the silk fibroin protein solution mass concentration or add the fibroin albumen basement membrane that is insoluble in water that volume obtains different-thickness.Then add again silk fibroin protein solution on the surface of basement membrane, process forming the porous support layer through lyophilization.The concentration of the aperture of porous support layer and the parameters such as porosity and used silk fibroin protein solution and freezing temperature thereof are relevant, generally are that concentration is larger, and cryogenic temperature is lower, and the aperture is less.The thickness of shelf layer is relevant with the volume and the concentration that add silk fibroin protein solution, generally is that the sample path length above the basement membrane is close with shelf layer thickness.The concrete thickness of basement membrane and shelf layer is adjusted according to actual needs, and more common thickness can be basement membrane 30 ~ 200 μ m, and the thickness of shelf layer is 200 ~ 4000 μ m.Smooth layer is dense film, can stop cell, microorganism and biomacromolecule to pass through, and the shelf layer pore-size distribution is 20-500 μ m, is beneficial to growing into of cell and blood vessel.
The concentrated solution of trapped fluid a of the present invention and trapped fluid a is the same solution of fibroin albumen of variable concentrations, names for ease of statement, and letter itself does not have implication.
Compared with prior art, beneficial effect of the present invention is mainly reflected in: (1) the object of the invention provides a kind of preparation method that is insoluble in the support rack type fibroin protein film of water, do not contact noxious substance among the preparation technology, do not use degeneration, coagulant and the additive such as crosslinked, preparation technology is simple, is beneficial to a large amount of productions; (2) the extremely smooth float glass in surface is adopted in the mould bottom surface, but and with the rustless steel platform of mold storing in the level intense adjustment; (3) this film is take fibroin albumen as raw material, and existing fibroin albumen and the catabolite (aminoacid) thereof of studies show that has good biocompatibility; (4) this film is under moist environment, and quality is soft, can be obedient to the curvature in body surface and the body thereof, effectively reduces the foreign body sensation in the body; (5) this film is easily controlled the thickness of basement membrane and shelf layer, and then produces different physics and biology performance, satisfies the needs of different purposes; (6) the smooth densification of substrate face can stop cell, microorganism and biomacromolecule to pass, and has every bacterium and anti effect; Shelf layer is porous spongy, is affixed on site of injury and is beneficial to the fixing of hemostasis, film, also is conducive to the cell blood vessel and grows into, and transmitting tissue grows in shelf layer; (7) this film has wide market in preparation Antiadhesive film, wound-protecting film or synthetic membrane field.
(4) description of drawings
The electron-microscope scanning figure of the support rack type fibroin protein film substrate face that is insoluble in water of Fig. 1 embodiment 1 preparation;
The electron-microscope scanning figure of the support rack type fibroin protein film support aspect that is insoluble in water of Fig. 2 embodiment 1 preparation.
(5) specific embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1(basement membrane 300 μ m, shelf layer 4000 μ m)
(1) preparation of silk fibroin protein solution: a) come unstuck: with 100g Bombyxmori Linnaeus silkworm silk (silk Co., Ltd of Zhejiang Huajing-Toshiba, the 5A level) puts into the 2M aqueous sodium carbonate of 5L, 98 ℃ of water-bath 30min, pure water cleans, and this process repeats 3 times, sloughs sericin, stay fibroin albumen, fibroin albumen 50 ℃ of oven dry, is obtained the dried fibroin albumen of 70g, for subsequent use; B) dissolving: above-mentioned dried fibroin albumen is dissolved in mass volume ratio 0.2:1 in lithium bromide (LiBr) aqueous solution of 9.3M, and 60 ℃ of water-bath 90min fully dissolve to fibroin albumen, and acquisition contains the mixed liquor of fibroin albumen and a small amount of insoluble granule composition; C) dialysis: mixed liquor with regenerated cellulose bag filter (molecular cut off 4000 dalton) dialysis, is dialysed 12 times at 3d with the sterile pure water of 10 times of mixeding liquid volumes, remove the LiBr ion in the solution, obtain trapped fluid a; D) concentrated: the bag filter after will dialysing is put into mass concentration 50% Polyethylene Glycol (PEG, the molecular weight 7000) aqueous solution of 4 times of volumes, and room temperature leaves standstill concentrated 5h, gets trapped fluid b, namely obtains the concentrated solution of trapped fluid a; E) with trapped fluid b at horizontal rotor 5000g, 4 ℃, centrifugal 10min removes in the not dissolving fibroin albumen of bottom and the lithium bromide and may have insoluble granule, gets supernatant, the silk fibroin protein solution 300ml after namely purifying; F) concentration determination: get the silk fibroin protein solution 10ml after the purification, dry in plate, it is 20% that method of poor quality records the silk fibroin protein solution mass concentration.4 ℃ of silk fibroin protein solutions save backup.
(2) with mould (bottom is float glass, the square ware of length of side 20cm, other embodiment mold material is identical) fine setting level, add the silk fibroin protein solution 60ml(30mg/cm of mass concentration 20%
2), 35 ℃ of temperature, relative humidity 50% environment leave standstill dry to naked eyes without visible liquid, increase 18g than empty mold weight, form fibroin protein film at mold bottom;
(3) mould is placed temperature 70 C, place 120min in the damp and hot crosslinked case of relative humidity 92%, mould is placed room temperature (25 ℃), in the situation that film surface coverage one deck purified water, be cooled to room temperature, the purified water of inclining forms the fibroin protein film that is insoluble in water in mould, namely obtain basement membrane;
(4) be level with above-mentioned die adjustment, mass concentration 20% silk fibroin protein solution that step (1) is obtained is 15% with purified water adjustment mass concentration, getting 160ml adds in the mould, die horizontal is placed-80 ℃ of freezing 30h, vacuum lyophilization 48h under the condition of-80 ℃ of cold-traps, in mould, form duplicature, namely form shelf layer on the surface of basement membrane.
(5) with duplicature at temperature 70 C, place 120min under relative humidity 92% condition, and then place the ethanol water of volumetric concentration 75% to soak 20min, fully embathe with pure water again, acquisition is insoluble in the support rack type fibroin protein film of water, the substrate thickness is 300 μ m approximately, and the support bed thickness is 4000 μ m approximately.The electron-microscope scanning of the described support rack type fibroin protein film basement membrane aspect that is insoluble in water as shown in Figure 1, the electron-microscope scanning of support aspect is as shown in Figure 2.
Embodiment 2(basement membrane 200 μ m, shelf layer 1000 μ m)
(1) preparation of silk fibroin protein solution: a) come unstuck: with 100g Bombyxmori Linnaeus silkworm silk (silk Co., Ltd of Zhejiang Huajing-Toshiba, the 5A level) puts into the 2M aqueous sodium carbonate of 5L, 98 ℃ of water-bath 30min, washed with de-ionized water, this process repeats 3 times, sloughs sericin, stay fibroin albumen, fibroin albumen 50 ℃ of oven dry, is obtained the dried fibroin albumen of 70g, for subsequent use; B) dissolving: above-mentioned dried fibroin albumen is dissolved in mass volume ratio 0.2:1 in lithium bromide (LiBr) aqueous solution of 9.3M, and 60 ℃ of water-bath 90min fully dissolve to fibroin albumen, and acquisition contains the mixed liquor of fibroin albumen and a small amount of insoluble granule composition; C) dialysis: above-mentioned mixed liquor with regenerated cellulose bag filter (molecular cut off 8000 dalton) dialysis, is dialysed 12 times at 3d with the sterile deionized water of 10 times of mixeding liquid volumes, remove the LiBr ion in the solution, obtain trapped fluid a; D) with trapped fluid a at horizontal rotor 5100g, 4 ℃, centrifugal 10min, remove the bottom not dissolving fibroin albumen and lithium bromide in may have insoluble granule, get the silk fibroin protein solution 1000ml after supernatant namely obtains to purify; E) concentration determination: get the silk fibroin protein solution 10ml after the purification, dry in plate, the mass concentration that method of poor quality records silk fibroin protein solution is 5%.4 ℃ of silk protein liquid save backup.
(2) with mould (with embodiment 1) adjustment level, add the mass concentration 5% silk protein liquid 140ml of above-mentioned preparation, remove the inner bubble that produces.10 ℃ of temperature, relative humidity 40% environment leave standstill dry to naked eyes without visible liquid, increase 10.7g than empty mold weight, form fibroin protein film at mold bottom.
(3) mould is placed temperature 70 C, place 100min in the damp and hot crosslinked case of relative humidity 90%, mould is placed room temperature, in the situation that film surface coverage one deck purified water is cooled to room temperature, the purified water of inclining, in mould, form the fibroin protein film that is insoluble in water, namely obtain basement membrane;
(4) in mould, add the mass concentration 5% silk fibroin protein solution 40ml that step (1) obtains, the horizontal positioned mould, die horizontal is placed-40 ℃ of freezing 10h, vacuum lyophilization 20h under the condition of-80 ℃ of cold-traps, in mould, form duplicature, remove mould, obtain duplicature, namely form shelf layer on the basement membrane surface.
(5) with duplicature 65 ℃ of temperature, place 100min under relative humidity 90% condition, and then place the ethanol water of volumetric concentration 75% to soak 20min, fully embathe with pure water again, acquisition is insoluble in the support rack type fibroin protein film of water, the substrate thickness is 200 μ m approximately, and the support bed thickness is 1000 μ m approximately.
Embodiment 3(basement membrane 100 μ m, shelf layer 2000 μ m)
(1) preparation of silk fibroin protein solution: a) come unstuck: with 100g Bombyxmori Linnaeus silkworm silk (silk Co., Ltd of Zhejiang Huajing-Toshiba, the 5A level) puts into the 2M aqueous sodium carbonate of 5L, 98 ℃ of water-bath 30min, washed with de-ionized water, this process repeats 3 times, sloughs sericin, stay fibroin albumen, fibroin albumen 50 ℃ of oven dry, is obtained the dried fibroin albumen of 70g, for subsequent use; B) dissolving: fibroin albumen is dissolved in mass volume ratio 0.20:1 in lithium bromide (LiBr) aqueous solution of 9.3M, and 60 ℃ of water-bath 90min fully dissolve to fibroin albumen, and acquisition contains the mixed liquor of fibroin albumen and a small amount of insoluble granule composition; C) dialysis: mixed liquor with regenerated cellulose bag filter (molecular cut off 2000 dalton) dialysis, is dialysed 12 times at 3d with the sterile deionized water of 10 times of mixeding liquid volumes, remove the LiBr ion in the solution, obtain trapped fluid a; D) with the filter of trapped fluid a with aperture 10 μ m, remove the insoluble granule of bottom, get the silk fibroin protein solution 1200ml after filtrate namely obtains to purify; E) concentration determination: get the silk fibroin protein solution 10ml after the purification, dry in plate, the mass concentration that method of poor quality records silk fibroin protein solution is 4%.4 ℃ of silk protein liquid save backup.
(2) with mould (with embodiment 1) adjustment level, the mass concentration that step (1) is obtained is that 4% silk fibroin protein solution 88ml adds in the mould, removes the inner bubble that produces.At temperature 45 C, relative humidity 50% environment leave standstill dry to naked eyes without visible liquid, increase 5.4g than empty mold weight, form fibroin protein film at mold bottom.
(3) mould is placed 65 ℃ of temperature, place 100min in the damp and hot crosslinked case of relative humidity 90%, mould is placed room temperature, in the situation that film surface coverage one deck purified water is cooled to room temperature, the purified water of inclining, in mould, form the fibroin protein film that is insoluble in water, namely obtain basement membrane;
(4) in mould, add the mass concentration 4% silk fibroin protein solution 80ml that step (1) obtains, the horizontal positioned mould, die horizontal is placed-40 ℃ of freezing 10h, vacuum lyophilization 20h under the condition of-80 ℃ of cold-traps, in mould, form duplicature, remove mould, obtain duplicature, namely form shelf layer on the basement membrane surface.
(5) with duplicature 65 ℃ of temperature, place 120min under relative humidity 88% condition, and then place the ethanol water of volumetric concentration 75% to soak 30min, fully embathe with pure water again, acquisition is insoluble in the support rack type fibroin protein film of water, the substrate thickness is 100 μ m approximately, and the support bed thickness is 2000 μ m approximately.
Embodiment 4(basal layer 30 μ m, shelf layer 100 μ m)
(1) preparation of silk fibroin protein solution: a) come unstuck: with 100g Bombyxmori Linnaeus silkworm silk (silk Co., Ltd of Zhejiang Huajing-Toshiba, the 5A level) puts into the 2M aqueous sodium carbonate of 5L, 98 ℃ of water-bath 30min, washed with de-ionized water, this process repeats 3 times, sloughs sericin, stay fibroin albumen, 50 ℃ of oven dry of fibroin albumen obtain the 70g fibroin albumen, and are for subsequent use; B) dissolving: fibroin albumen is dissolved in mass volume ratio 0.2:1% in lithium bromide (LiBr) aqueous solution of 9.3M, and 60 ℃ of water-bath 90min fully dissolve to fibroin albumen, and acquisition contains the mixed liquor of fibroin albumen and a small amount of insoluble granule composition; C) dialysis: mixed liquor with regenerated cellulose bag filter (molecular cut off 8000 dalton) dialysis, is dialysed 12 times at 3d with the sterile deionized water of 10 times of mixeding liquid volumes, remove the LiBr ion in the solution, obtain trapped fluid a; D) with the filter of trapped fluid a with aperture 10 μ m, remove the insoluble granule of bottom, the silk fibroin protein solution 1200ml after obtaining to purify; E) concentration determination: get the silk fibroin protein solution 10ml after the purification, dry in plate, the mass concentration that method of poor quality records silk fibroin protein solution concentration is 4%.4 ℃ of silk protein liquid save backup.
(2) silk fibroin protein solution being diluted to mass concentration 0.5% with purified water, with mould (with embodiment 1) adjustment level, is that 0.5% silk fibroin protein solution 210ml adds in the mould with mass concentration, removes the inner bubble that produces.25 ℃ of temperature, relative humidity 50% environment leave standstill dry to naked eyes without visible liquid, increase 1.6g than empty mold weight, form fibroin protein film at mold bottom.
(3) mould is placed 63 ℃ of temperature, place 100min in the damp and hot crosslinked case of relative humidity 90%, mould is placed room temperature, in the situation that film surface coverage one deck purified water is cooled to room temperature, the purified water of inclining, in mould, form the fibroin protein film that is insoluble in water, namely obtain basement membrane;
(4) in mould, add the mass concentration 4% silk fibroin protein solution 30ml that step (1) obtains, again the silk fibroin protein solution in the mould is outwelled behind the tiling die surface, at basement membrane surface adhesion skim silk fibroin protein solution, die horizontal is placed-40 ℃ of freezing 8h, vacuum lyophilization 16h under the condition of-80 ℃ of cold-traps forms duplicature in mould, remove mould, obtain duplicature, namely form shelf layer on the basement membrane surface.
(5) with duplicature 65 ℃ of temperature, place 100min under relative humidity 88% condition, and then place the ethanol water of volumetric concentration 75% to soak 20min, fully embathe with pure water again, acquisition is insoluble in the support rack type fibroin protein film of water, and the substrate thickness is 30 μ m approximately, and the support bed thickness is 100 μ m approximately.
Claims (9)
1. support rack type fibroin protein film that is insoluble in water, it is characterized in that described support rack type fibroin protein film prepares as follows: (1) is take Bombyxmori Linnaeus silkworm silk as raw material, through coming unstuck, dissolve, dialysing, obtain trapped fluid a, the concentrated solution of trapped fluid a or trapped fluid a filtered or centrifugal, get filtrate or upper strata centrifugal liquid obtains silk fibroin protein solution; The mass concentration water of the silk fibroin protein solution that (2) step (1) is obtained is adjusted to 0.3 ~ 30%, pours in the mould, and the silk fibroin protein solution addition is counted 2 ~ 50mg/cm with the fibroin albumen quality in the described mould
2Mould 10 ~ 60 ℃ of temperature, under relative humidity 10 ~ 70% conditions is dried to mould without macroscopic moisture, forms fibroin protein film in mould; Then with mould in 50 ~ 100 ℃ of temperature, place 20 ~ 200min under relative humidity 70 ~ 100% conditions, in mould, add purified water, be cooled to room temperature, the purified water of inclining forms the fibroin protein film be insoluble in water, i.e. basement membrane in mould; (3) add mass concentration 0.3 ~ 30% silk fibroin protein solution that step (1) obtains on the described basement membrane of step (2) surface, described silk fibroin protein solution volume addition is 100 ~ 10000ml/m
2Mould, with mould under level in-10 ~-80 ℃ of freezing 2 ~ 30h, again vacuum lyophilization under-40 ~-110 ℃ of conditions, in mould, form duplicature, remove mould, with duplicature 50 ~ 100 ℃ of temperature, place 20 ~ 200min under relative humidity 70 ~ 100% conditions, and then place the ethanol water of volumetric concentration 50 ~ 90% to soak 10 ~ 60min, and fully embathe with pure water again, obtain to be insoluble in the support rack type fibroin protein film of water.
2. the support rack type fibroin protein film that is insoluble in water as claimed in claim 1 is characterized in that the described silk fibroin protein solution addition of step (2) counts 3 ~ 20mg/cm with the fibroin albumen quality
2Mould.
3. the support rack type fibroin protein film that is insoluble in water as claimed in claim 1 is characterized in that the volumetric usage of the described silk fibroin protein solution of step (3) is 200 ~ 4000ml/m
2Mould.
4. the support rack type fibroin protein film that is insoluble in water as claimed in claim 1 is characterized in that the described mould of step (3) at-20 ~-80 ℃ of lower freezing 5 ~ 30h, and again vacuum lyophilization under-60 ~-110 ℃ of conditions forms the fibroin protein film shelf layer.
5. the support rack type fibroin protein film that is insoluble in water as claimed in claim 1, the concentrated solution preparation method that it is characterized in that the described trapped fluid a of step (1) is: the bag filter after will dialysing is put into the Aqueous Solutions of Polyethylene Glycol of mass concentration 20 ~ 60%, leave standstill 1 ~ 10h, get the concentrated solution that trapped fluid b is trapped fluid a; Described Polyethylene Glycol mean molecule quantity 1000 ~ 10000.
6. the support rack type fibroin protein film that is insoluble in water as claimed in claim 1, the concentrated solution that it is characterized in that the described trapped fluid a of step (1) or trapped fluid a filters or centrifugal method is one of following: a) with the concentrated solution of trapped fluid a or trapped fluid a centrifugal 5 ~ 20min under 4 ℃, 3000 ~ 6000g condition, discard precipitation, get upper solution and be described silk fibroin protein solution; B) concentrated solution with trapped fluid a or trapped fluid a is the filter filtration of 2 ~ 20 μ m with the aperture, removes insoluble granule, and filtrate is described silk fibroin protein solution.
7. the support rack type fibroin protein film that is insoluble in water as claimed in claim 1, it is characterized in that step (1) is describedly come unstuck, dissolving, dialysis process be: a) come unstuck: the 2M aqueous sodium carbonate of 100g Bombyxmori Linnaeus silkworm silk being put into 4 ~ 8L, 90 ~ 100 ℃ of water-bath 20 ~ 60min, pure water cleans, this process repeats 3 times, sloughs sericin, stays fibroin albumen, fibroin albumen 20 ~ 60 ℃ of oven dry, is obtained dried fibroin albumen; B) dissolving: above-mentioned dried fibroin albumen is dissolved in the lithium bromide water solution of 9 ~ 11M, and 55 ~ 65 ℃ of water-bath 30 ~ 300min fully dissolve to fibroin albumen, and acquisition contains the mixed liquor of fibroin albumen; The mass volume ratio of described dried fibroin albumen and lithium bromide water solution is 0.1 ~ 0.2:1; C) dialysis: the mixed liquor that will contain fibroin albumen is dialysed with molecular cut off 1000 ~ 20000 daltonian bag filters, sterile deionized water with 10 times of mixeding liquid volumes is dialysed 10 ~ 12 times at 3 days as dialysis solution, remove the lithium bromide composition in the solution, obtain trapped fluid a.
8. such as the described application of support rack type fibroin protein film in preparation Antiadhesive film, wound-protecting film or synthetic membrane that is insoluble in water of one of claim 1 ~ 7.
9. application as claimed in claim 8 is characterized in that described synthetic membrane is periodontal membrane, Guided Bone Regeneration film, pleura, cerebral dura mater or spinal dura mater.
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| CN 201210344126 CN102847194A (en) | 2012-09-17 | 2012-09-17 | Stent type silk fibroin film insoluble in water, and preparation and application of stent type silk fibroin film |
| CN201310426437.7A CN103536963B (en) | 2012-09-17 | 2013-09-17 | A kind of support rack type fibroin protein film and preparation and application thereof being insoluble in water |
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| CN 201210344126 CN102847194A (en) | 2012-09-17 | 2012-09-17 | Stent type silk fibroin film insoluble in water, and preparation and application of stent type silk fibroin film |
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| CN103536963B (en) | 2015-09-16 |
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