CN102383267A - Natural polymer-based nano-fibrous membrane prepared by freeze-drying method - Google Patents
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Abstract
The invention relates a biodegradable and absorbable natural polymer-based nano-fibrous membrane prepared by a freeze-drying method, and the application thereof. The natural polymer-based nano-fibrous membrane is prepared through the following steps of: dissolving natural polymer powder into a corresponding solvent so as to prepare an extremely-dilute solution with the concentration of 0.001-0.1wt %; after the natural polymer powder is completely dissolved in the solvent, transferring the obtained natural polymer solution into a liquid nitrogen refrigerating device, so that the natural polymer solution is rapidly frozen in a liquid nitrogen environment; then, carrying out freeze-drying treatment on the obtained product in a freeze drier for 12-48 hours to obtain natural polymer-based nano fibers; and carrying out cross-linking on the obtained natural polymer-based nano fibers by a corresponding cross-linking agent to obtain a natural polymer-based nano-fibrous membrane, and then carrying out MTT (methyl thiazolyl tetrazolium) cytotoxicity test and cell vaccination experiments on the natural polymer-based nano-fibrous membrane, with the obtained results showing that the obtained fibrous membrane has no toxicity but has excellent cell adhesion and proliferation properties. The natural polymer-based nano-fibrous membrane disclosed by the invention is simple in the operation process, easy to control and low in cost; and by using the nano-fibrous membrane disclosed by the invention, ultra-fine natural polymer-based nano fibers can be prepared continuously on a large scale.
Description
Technical field
The present invention relates to prepare natural polymer based nano-fiber film and medical application thereof biodegradable and absorption with desivac.
Background technology
Vacuum Freezing & Drying Technology is to utilize ice crystal distillation principle, and under the environmental condition of high vacuum, the moisture in the material that will freeze directly makes the technology of dry materials for water vapour from the solid ice distillation without the thawing of ice.Can know by the Phase Equilibrium theory in the chemical thermodynamics: under certain pressure and temperature, reach certain balancing each other between three kinds of forms of water, obtain the phasor (Fig. 1) of water in view of the above.Three phase point has shown the pressure and temperature condition of the gas, liquid, solid three-phase coexistence of water.The OA line is the line of demarcation of solid phase and gas phase among the figure, is called the distillation line; The OB line is the line of demarcation of liquid phase and gas phase, is called the vaporization line; The OC line is the line of demarcation of solid phase and liquid phase, is called the thawing line; The O point is a three phase point.Reduce according to pressure, the basic physics principle that boiling point descends, as long as pressure P is positioned under the three phase point (diagram P<646.5Pa, T<0 ℃), the moisture in the material can from solid phase ice without liquid phase directly distillation be steam.
The operating process of vacuum freeze-drying technique comprises pre-freeze, distillation, parsing three phases, the phase I: pre-freeze stage.Water congeals into ice with free state in the material, for sublimation stage is prepared.Because material is in by frozen state, after the freeze-drying material form can keep with drying before basic identical, material bubbles, concentrates in the time of also can preventing to vacuumize drying, contraction and solute move etc., and irreversible change takes place.In this stage, pre-freeze speed has certain influence to the quality of material.The ice crystal that snap frozen forms is less, and is unfavorable to distilling, but the dissolving of dry back is fast, and the ice crystal that slow freezing forms is bigger, and is favourable to distilling, but the dissolving of dry back is slow.Second stage: sublimation stage.This stage mainly is that the water sublimate with free state in the material comes out.In this stage, temperature remains unchanged, and gets rid of frozen water content, and this stage is the constant speed process.Phase III: resolution phase.This stage mainly is sublimation stage to be failed dry bound water evaporate material.A part of bound water in the biological tissue is difficult to remove through desivac, need temperature be elevated to 30~35 ℃ through resolution phase and further remove.
Vacuum freeze drying has advantage:
(1) freeze drying is carried out at low temperatures, is applicable to the material of many thermal sensitivitys; (2) in freezing dry process, the effect of microbial growth and enzyme can't be carried out, and therefore can keep original proterties; (3) owing under the state that freezes, carry out drying, can keep the original structure of material, concentration phenomena can not take place; (4) dried matter is loose porous, is spongy, adds that dissolving almost recovers original proterties rapidly and fully immediately behind the water; (5) because freeze drying is carried out under vacuum, oxygen is few, can protect the material of some easy oxidations; (6) vacuum freeze drying can be got rid of the above moisture of 95-99%, makes the dry back product can long preservation and unlikely rotten etc.Therefore, freeze drying is at present at medical industry, food industry, and scientific research departments etc. are widely used.
Nanofiber has advantages such as specific area is big, porosity is high, draw ratio is big, and it is with a wide range of applications in organizational project, drug field.At present; Become nanofiber to attract the concern of a lot of seminars natural biological Process Technologies of Polymer such as shitosan, glucan, sodium alginate, gelatin, hyaluronic acid, cellulose ethanoate, collagen, silk, starch, nucleic acid, fibroin, mainly adopt the method for electrostatic spinning preparation.Wherein, application number is the preparation method that each application of 201010579804.3 and 200810100981.1 Chinese invention patent discloses a kind of sodium alginate nano fiber, prepares pure sodium alginate nano fiber through electrostatic spinning technique.Because its strand is rigidity, in solution, stretches, lack necessary chain entanglement effect, and be difficult to obtain a large amount of SA nanofibers through electrostatic spinning technique, make it on using, obtain certain restriction; Application number is that 200810202167.0 Chinese invention patent application discloses a kind of preparation method who is used for the stable gelatine nano fiber of bionic bracket material; Gelatin is dissolved in trifluoroethanol, hexafluoro ethanol, the trifluoroacetic acid equal solvent; Utilize electrostatic spinning technique that gelatin is processed into nanofiber; Because to make trifluoroethanol, hexafluoro ethanol, trifluoroacetic acid etc. be solvent, make the biocompatibility variation of gelatine nano fiber, its medical value reduction; Application number is that 201010137452.6 Chinese invention patent application discloses a kind of preparation method who prepares pure hyaluronic acid nano fiber, the pure hyaluronic acid of preparing through the electric field that changes in molecular chain structure and the electrostatic spinning process.Because the electrostatic spinning of natural polymer is had relatively high expectations to its process factors and environmental factor, preparation method's post processing trouble, repeatability is not strong, makes the natural polymer nanofiber can not well be used always.
Utilize the nanofiber of desivac preparation to have the following advantages:
1, desivac prepare the nanofiber operating process simple, be easy to control, with low cost;
2, utilize desivac can prepare superfine natural polymer nanofiber;
3, utilize desivac can prepare the natural polymer based nano-fiber in enormous quantities, continuously;
4, utilize the natural polymer nanofiber of desivac preparation to have the good cell adhesion property.
Summary of the invention
Natural polymer nanofiber existence to present preparation is had relatively high expectations to process factors and environmental factor; Preparation method's post processing trouble; Defectives such as repeatability is not strong the object of the present invention is to provide a kind of method for preparing the natural polymer nano fibrous membrane.
A kind of method biodegradable and the natural polymer based nano-fiber film that absorbs for preparing provided by the present invention may further comprise the steps:
(1) preparation of natural polymer solution: natural polymer is dissolved in the solvent, is made into the solution that percentage by weight is 0.001~0.1wt%, then solution is fully stirred, so that dissolving fully, promptly obtain natural polymer solution.Described natural polymer is sodium alginate, hyaluronic acid, shitosan, glucan, cellulose ethanoate, collagen, silk, gelatin, starch, nucleic acid and fibroin albumen.
(2) desivac prepares the natural polymer nanofiber: the natural polymer solution of preparation in the step (1) is transferred in the liquid nitrogen frozen device; Open refrigerating plant; Make the natural polymer solution of being prepared in liquid nitrogen environment, reach a certain specific freeze junction temperature rapidly; In certain cryogenic temperature scope and certain vacuum ranges, the natural polymer solution that will freeze carries out freeze-drying and handles 12~48h in freeze drier, obtain the natural polymer nanofiber then.
(3) chemical crosslink technique obtains the natural polymer nano fibrous membrane: with the natural polymer nanofiber that obtains in the step (2) under 20~40 ℃ of conditions; Carried out crosslinked 2 hours with crosslinking agent; Crosslinking agent unnecessary in the natural polymer nanofiber after crosslinked is fallen with deionized water or other solvent wash; The nano fiber non-woven fabric of handling at 40~60 ℃ of following vacuumize 1~12h, is obtained the natural polymer nano fibrous membrane.Wherein the concentration of crosslinking agent in mixed liquor is 1~10mmol/L.
Described crosslinking agent (corresponding natural polymer) is: calcium chloride solution (sodium alginate), glutaraldehyde (collagen, shitosan), 1-chloro-2,3-expoxy propane (glucan), Geniposide (shitosan, gelatin), Cyanuric Chloride (silk), carbodiimides (hyaluronic acid), dialdehyde starch (gelatin, shitosan), POCl3 (starch), ultraviolet light (nucleic acid), diglycidyl ether (fibroin).
(4) MTT cytotoxicity test: the PBS of the natural polymer nano fibrous membrane that obtains in the step (3) is embathed liquid handle the L929 cell respectively after 24 hours, 48 hours, 72 hours, abandon supernatant, with PBS washing 2 times.ELIASA is measured light absorption value, and the mensuration wavelength is 490nm, and calculates its P value.
(5) cell inoculation experiment: the natural polymer nano fibrous membrane that obtains in the step (3) is carried out cell (L929, l cell) inoculation experiments, and resulting natural polymer nano fibrous membrane is assessed.
Description of drawings
Fig. 1. the phasor of water
Fig. 2 is the sodium alginate nano fiber SEM pattern that obtains by the technical scheme that embodiment 1 is provided.
Fig. 3 is the nano fibrous membrane MTT test result with the hyaluronic acid preparation that obtains by the technical scheme that embodiment 2 is provided.
Fig. 4 is the SEM pattern of testing the cell growing state that obtains by the silk nano fibrous membrane cell inoculation that the technical scheme that embodiment 7 is provided obtains.
The specific embodiment
Embodiment 1:
(1) sodium alginate is dissolved in the deionized water, being made into percentage by weight is the solution 20mL of 0.001wt%, then solution is fully stirred, so that dissolving fully, sodium alginate soln.
(2) sodium alginate soln of preparation in the step (1) is transferred in the liquid nitrogen frozen device; Open refrigerating plant; The sodium alginate soln of being prepared is freezed in liquid nitrogen environment rapidly; Cryogenic temperature is under the condition of 600Pa for-80 ℃ with vacuum then, and the sodium alginate soln that will freeze carries out freeze-drying and handles 24h in freeze drier, obtain sodium alginate nano fiber.
(3) with the sodium alginate nano fiber that obtains in the step (2) under 25 ℃ of conditions; Calcium chloride with 10mmol/L carried out crosslinked 2 hours; Calcium chloride unnecessary in the sodium alginate nano fiber after crosslinked is spent deionised water to be fallen; The nano fiber non-woven fabric of handling at 60 ℃ of following vacuumize 1h, is obtained the sodium alginate nano fiber film, see Fig. 2.
(4) PBS of the sodium alginate nano fiber film that obtains in the step (3) is embathed liquid and handle the L929 cell respectively after 24 hours, 48 hours, 72 hours, abandon supernatant, with PBS washing 2 times.ELIASA is measured light absorption value, and the mensuration wavelength is 490nm, compares with blank, and its 24 hours, 48 hours, 72 hours P value does not have significant difference all greater than 0.05, shows that there is not toxicity in resulting nano fibrous membrane.
(5) the sodium alginate nano fiber film that obtains in the step (3) is carried out the cell inoculation experiment, have excellent cell and attach and multiplication characteristic.
Embodiment 2:
(1) hyaluronic acid is dissolved in the deionized water, being made into percentage by weight is the solution 20mL of 0.015wt%, then solution is fully stirred, so that dissolving fully, hyaluronic acid solution.
(2) hyaluronic acid solution of preparation in the step (1) is transferred in the liquid nitrogen frozen device; Open refrigerating plant; The hyaluronic acid solution of being prepared is freezed in liquid nitrogen environment rapidly; Cryogenic temperature is under the condition of 200Pa for-10 ℃ with vacuum then, and the hyaluronic acid solution that will freeze carries out freeze-drying and handles 12h in freeze drier, obtain hyaluronic acid nano fiber.
(3) with the hyaluronic acid nano fiber that obtains in the step (2) under 20 ℃ of conditions; Carbodiimides with 5mmol/L carried out crosslinked 2 hours; Carbodiimides unnecessary in the hyaluronic acid nano fiber after crosslinked is spent deionised water to be fallen; The nano fiber non-woven fabric of handling at 40 ℃ of following vacuumize 12h, is obtained the hyaluronic acid nano fiber film.
(4) PBS of the hyaluronic acid nano fiber film that obtains in the step (3) is embathed liquid and handle the L929 cell respectively after 24 hours, 48 hours, 72 hours, abandon supernatant, with PBS washing 2 times.ELIASA is measured light absorption value, and the mensuration wavelength is 490nm, compares with blank, and its 24 hours, 48 hours, 72 hours P value does not have significant difference all greater than 0.05, shows that there is not toxicity in resulting nano fibrous membrane.
(5) the hyaluronic acid nano fiber film that obtains in the step (3) is carried out the cell inoculation experiment, have excellent cell and attach and multiplication characteristic.
Embodiment 3:
(1) shitosan is dissolved in the 1wt% acetic acid solution, being made into percentage by weight is the solution 20mL of 0.1wt%, then solution is fully stirred, so that dissolving fully, chitosan solution.
(2) chitosan solution of preparation in the step (1) is transferred in the liquid nitrogen frozen device; Open refrigerating plant; The chitosan solution of being prepared is freezed in liquid nitrogen environment rapidly; Cryogenic temperature is under the condition of 1Pa for-40 ℃ with vacuum then, and the chitosan solution that will freeze carries out freeze-drying and handles 36h in freeze drier, obtain chitosan nano fiber.
(3) with the chitosan nano fiber that obtains in the step (2) under 40 ℃ of conditions; Geniposide with 2mmol/L carried out crosslinked 2 hours; Geniposide unnecessary in the chitosan nano fiber after crosslinked is fallen with absolute ethanol washing; The nano fiber non-woven fabric of handling at 50 times vacuumize 6h, is obtained chitosan nano fiber membrane.
(4) PBS of the chitosan nano fiber membrane that obtains in the step (3) is embathed liquid and handle the L929 cell respectively after 24 hours, 48 hours, 72 hours, abandon supernatant, with PBS washing 2 times.ELIASA is measured light absorption value, and the mensuration wavelength is 490nm, compares with blank, and its 24 hours, 48 hours, 72 hours P value does not have significant difference all greater than 0.05, shows that there is not toxicity in resulting nano fibrous membrane, sees Fig. 3.
(5) chitosan nano fiber membrane that obtains in the step (3) is carried out the cell inoculation experiment, have excellent cell and attach and multiplication characteristic.
Embodiment 4:
(1) glucan is dissolved in the deionized water, being made into percentage by weight is the solution 20mL of 0.05wt%, then solution is fully stirred, so that dissolving fully, dextran solution.
(2) dextran solution of preparation in the step (1) is transferred in the liquid nitrogen frozen device; Open refrigerating plant; The dextran solution of being prepared is freezed in liquid nitrogen environment rapidly; Cryogenic temperature is under the condition of 100Pa for-20 ℃ with vacuum then, and the dextran solution that will freeze carries out freeze-drying and handles 24h in freeze drier, obtain the glucan nanofiber.
(3) with the glucan nanofiber that obtains in the step (2) under 20 ℃ of conditions; 1-chloro-2 with 1mmol/L; The 3-expoxy propane carried out crosslinked 2 hours, and with 1-chloro-2 unnecessary in the glucan nanofiber after crosslinked, the 3-expoxy propane spends deionised water and falls; The nano fiber non-woven fabric of handling at 55 ℃ of following vacuumize 6h, is obtained the glucan nano fibrous membrane.
(4) PBS of the glucan nano fibrous membrane that obtains in the step (3) is embathed liquid and handle the L929 cell respectively after 24 hours, 48 hours, 72 hours, abandon supernatant, with PBS washing 2 times.ELIASA is measured light absorption value, and the mensuration wavelength is 490nm, compares with blank, and its 24 hours, 48 hours, 72 hours P value does not have significant difference all greater than 0.05, shows that there is not toxicity in resulting nano fibrous membrane.
(5) the glucan nano fibrous membrane that obtains in the step (3) is carried out the cell inoculation experiment, have excellent cell and attach and multiplication characteristic.
Embodiment 5:
(1) cellulose ethanoate is dissolved in the acetic acid aqueous solution of 2wt%, being made into percentage by weight is the solution 20mL of 0.1wt%, then solution is fully stirred, so that dissolving fully, the cellulose acetate ester solution.
(2) the cellulose acetate ester solution of preparation in the step (1) is transferred in the liquid nitrogen frozen device; Open refrigerating plant; The cellulose acetate ester solution of being prepared is freezed in liquid nitrogen environment rapidly; Cryogenic temperature is under the condition of 10Pa for-50 ℃ with vacuum then, and the cellulose acetate ester solution that will freeze carries out freeze-drying and handles 24h in freeze drier, obtain the cellulose ethanoate nanofiber.
(3) PBS of the cellulose ethanoate nano fibrous membrane that obtains in the step (2) is embathed liquid and handle the L929 cell respectively after 24 hours, 48 hours, 72 hours, abandon supernatant, with PBS washing 2 times.ELIASA is measured light absorption value, and the mensuration wavelength is 490nm, compares with blank, and its 24 hours, 48 hours, 72 hours P value does not have significant difference all greater than 0.05, shows that there is not toxicity in resulting nano fibrous membrane.
(4) the cellulose ethanoate nano fibrous membrane that obtains in the step (2) is carried out the cell inoculation experiment, have excellent cell and attach and multiplication characteristic.
Embodiment 6:
(1) collagen is dissolved in 40 ℃ the warm water, being made into percentage by weight is the solution 20mL of 0.001wt%, then solution is fully stirred, so that dissolving fully, collagen solution.
(2) collagen solution of preparation in the step (1) is transferred in the liquid nitrogen frozen device; Open refrigerating plant; The collagen solution of being prepared is freezed in liquid nitrogen environment rapidly; Cryogenic temperature is under the condition of 400Pa for-60 ℃ with vacuum then, and the collagen solution that will freeze carries out freeze-drying and handles 36h in freeze drier, obtain the collagen nanofiber.
(3) with the collagen nanofiber that obtains in the step (2) under 20 ℃ of conditions; Glutaraldehyde with 5mmol/L carried out crosslinked 2 hours; Unnecessary glutaraldehyde in the collagen nanofiber after crosslinked is spent deionised water to be fallen; The nano fiber non-woven fabric of handling at 45 ℃ of following vacuumize 12h, is obtained the collagen nano fibrous membrane.
(4) PBS of the collagen nano fibrous membrane that obtains in the step (3) is embathed liquid and handle the L929 cell respectively after 24 hours, 48 hours, 72 hours, abandon supernatant, with PBS washing 2 times.ELIASA is measured light absorption value, and the mensuration wavelength is 490nm, compares with blank, and its 24 hours, 48 hours, 72 hours P value does not have significant difference all greater than 0.05, shows that there is not toxicity in resulting nano fibrous membrane.
(5) the collagen nano fibrous membrane that obtains in the step (3) is carried out the cell inoculation experiment, have excellent cell and attach and multiplication characteristic.
Embodiment 7:
(1) silk is dissolved in the aqueous formic acid of 5wt%, being made into percentage by weight is the solution 20mL of 0.005wt%, then solution is fully stirred, so that dissolving fully, silk solution.
(2) the silk solution of preparation in the step (1) is transferred in the liquid nitrogen frozen device; Open refrigerating plant; The silk solution of being prepared is freezed in liquid nitrogen environment rapidly; Cryogenic temperature is under the condition of 80Pa for-80 ℃ with vacuum then, and the silk solution that will freeze carries out freeze-drying and handles 24h in freeze drier, obtain the silk nanofiber.
(3) with the silk nanofiber that obtains in the step (2) under 30 ℃ of conditions; Cyanuric Chloride with 1mmol/L carried out crosslinked 2 hours; Unnecessary Cyanuric Chloride in the silk nanofiber after crosslinked is spent deionised water to be fallen; The nano fiber non-woven fabric of handling at 40 ℃ of following vacuumize 12h, is obtained the silk nano fibrous membrane.
(4) PBS of the silk nano fibrous membrane that obtains in the step (3) is embathed liquid and handle the L929 cell respectively after 24 hours, 48 hours, 72 hours, abandon supernatant, with PBS washing 2 times.ELIASA is measured light absorption value, and the mensuration wavelength is 490nm, compares with blank, and its 24 hours, 48 hours, 72 hours P value does not have significant difference all greater than 0.05, shows that there is not toxicity in resulting nano fibrous membrane.
(5) the silk nano fibrous membrane that obtains in the step (3) is carried out the cell inoculation experiment, have excellent cell and attach and multiplication characteristic, see Fig. 4.
Embodiment 8:
(1) gelatin is dissolved in the aqueous formic acid of 5wt%, being made into percentage by weight is the solution 20mL of 0.008wt%, then solution is fully stirred, so that dissolving fully, gelatin solution.
(2) gelatin solution of preparation in the step (1) is transferred in the liquid nitrogen frozen device; Open refrigerating plant; The gelatin solution of being prepared is freezed in liquid nitrogen environment rapidly; Cryogenic temperature is under the condition of 100Pa for-80 ℃ with vacuum then, and the gelatin solution that will freeze carries out freeze-drying and handles 48h in freeze drier, obtain gelatine nano fiber.
(3) with the gelatine nano fiber that obtains in the step (2) under 40 ℃ of conditions; Dialdehyde starch with 8mmol/L carried out crosslinked 2 hours; Dialdehyde starch unnecessary in the gelatine nano fiber after crosslinked is spent deionised water to be fallen; The nano fiber non-woven fabric of handling at 60 ℃ of following vacuumize 6h, is obtained the gelatine nano fiber film.
(4) PBS of the gelatine nano fiber film that obtains in the step (3) is embathed liquid and handle the L929 cell respectively after 24 hours, 48 hours, 72 hours, abandon supernatant, with PBS washing 2 times.ELIASA is measured light absorption value, and the mensuration wavelength is 490nm, compares with blank, and its 24 hours, 48 hours, 72 hours P value does not have significant difference all greater than 0.05, shows that there is not toxicity in resulting nano fibrous membrane.
(5) the gelatine nano fiber film that obtains in the step (3) is carried out the cell inoculation experiment, have excellent cell and attach and multiplication characteristic.
Embodiment 9:
(1) with starch dissolution in 40 ℃ warm water, being made into percentage by weight is the solution 20mL of 0.1wt%, then solution is fully stirred so that fully the dissolving, starch solution.
(2) starch solution of preparation in the step (1) is transferred in the liquid nitrogen frozen device; Open refrigerating plant; The starch solution of being prepared is freezed in liquid nitrogen environment rapidly; Cryogenic temperature is under the condition of 300Pa for-30 ℃ with vacuum then, and the starch solution that will freeze carries out freeze-drying and handles 36h in freeze drier, obtain the starch nano fiber.
(3) with the starch nano fiber that obtains in the step (2) under 40 ℃ of conditions; POCl3 with 5mmol/L carried out crosslinked 2 hours; Unnecessary POCl3 in the starch nano fiber after crosslinked is spent deionised water to be fallen; The nano fiber non-woven fabric of handling at 40 ℃ of following vacuumize 12h, is obtained the starch nano tunica fibrosa.
(4) PBS of the starch nano tunica fibrosa that obtains in the step (3) is embathed liquid and handle the L929 cell respectively after 24 hours, 48 hours, 72 hours, abandon supernatant, with PBS washing 2 times.ELIASA is measured light absorption value, and the mensuration wavelength is 490nm, compares with blank, and its 24 hours, 48 hours, 72 hours P value does not have significant difference all greater than 0.05, shows that there is not toxicity in resulting nano fibrous membrane.
(5) the starch nano tunica fibrosa that obtains in the step (3) is carried out the cell inoculation experiment, have excellent cell and attach and multiplication characteristic.
Embodiment 10:
(1) nucleic acid is dissolved in 35 ℃ the warm water, being made into percentage by weight is the solution 20mL of 0.001wt%, then solution is fully stirred, so that dissolving fully, nucleic acid solution.
(2) nucleic acid solution of preparation in the step (1) is transferred in the liquid nitrogen frozen device; Open refrigerating plant; The nucleic acid solution of being prepared is freezed in liquid nitrogen environment rapidly; Cryogenic temperature is under the condition of 20Pa for-40 ℃ with vacuum then, and the nucleic acid solution that will freeze carries out freeze-drying and handles 48h in freeze drier, obtain the nucleic acid nano fiber.
(3) with the nucleic acid nano fiber that obtains in the step (2) under 35 ℃ of conditions, carried out crosslinked 2 hours with UV-irradiation, obtain the nucleic acid nano tunica fibrosa.
(4) PBS of the nucleic acid nano tunica fibrosa that obtains in the step (3) is embathed liquid and handle the L929 cell respectively after 24 hours, 48 hours, 72 hours, abandon supernatant, with PBS washing 2 times.ELIASA is measured light absorption value, and the mensuration wavelength is 490nm, compares with blank, and its 24 hours, 48 hours, 72 hours P value does not have significant difference all greater than 0.05, shows that there is not toxicity in resulting nano fibrous membrane.
(5) the nucleic acid nano tunica fibrosa that obtains in the step (3) is carried out the cell inoculation experiment, have excellent cell and attach and multiplication characteristic.
Embodiment 11:
(1) fibroin albumen is dissolved in the aqueous formic acid of 3wt%, being made into percentage by weight is the solution 20mL of 0.005wt%, then solution is fully stirred, so that dissolving fully, silk fibroin protein solution.
(2) silk fibroin protein solution of preparation in the step (1) is transferred in the liquid nitrogen frozen device; Open refrigerating plant; The silk fibroin protein solution of being prepared is freezed in liquid nitrogen environment rapidly; Cryogenic temperature is under the condition of 90Pa for-50 ℃ with vacuum then, and the silk fibroin protein solution that will freeze carries out freeze-drying and handles 24h in freeze drier, obtain silk fibroin nano-fiber.
(3) with the silk fibroin nano-fiber that obtains in the step (2) under 35 ℃ of conditions; Diglycidyl ether with 5mmol/L carried out crosslinked 2 hours; Diglycidyl ether unnecessary in the silk fibroin nano-fiber after crosslinked is spent deionised water to be fallen; The nano fiber non-woven fabric of handling at 50 ℃ of following vacuumize 12h, is obtained the silk fibroin nano-fiber film.
(4) PBS of the silk fibroin nano-fiber film that obtains in the step (3) is embathed liquid and handle the L929 cell respectively after 24 hours, 48 hours, 72 hours, abandon supernatant, with PBS washing 2 times.ELIASA is measured light absorption value, and the mensuration wavelength is 490nm, compares with blank, and its 24 hours, 48 hours, 72 hours P value does not have significant difference all greater than 0.05, shows that there is not toxicity in resulting nano fibrous membrane.
(5) the silk fibroin nano-fiber film that obtains in the step (3) is carried out the cell inoculation experiment, have excellent cell and attach and multiplication characteristic.
Claims (5)
1. one kind prepares method biodegradable and the natural polymer based nano-fiber film that absorbs, may further comprise the steps:
(1) preparation of natural polymer solution: natural polymer is dissolved in the solvent, is made into the solution that percentage by weight is 0.001~0.1wt%, then solution is fully stirred, so that dissolving fully, promptly obtain natural polymer solution.
(2) desivac prepares the natural polymer nanofiber: the natural polymer solution of preparation in the step (1) is transferred in the liquid nitrogen frozen device; Open refrigerating plant; The natural polymer solution of being prepared is freezed in liquid nitrogen environment rapidly; In certain cryogenic temperature scope and certain vacuum ranges, the natural polymer solution that will freeze carries out freeze-drying and handles 12~48h in freeze drier, obtain the natural polymer nanofiber then;
(3) chemical crosslink technique obtains the natural polymer nano fibrous membrane: with the natural polymer nanofiber that obtains in the step (2) under 20~40 ℃ of conditions; Carried out crosslinked 2 hours with crosslinking agent; Unnecessary crosslinking agent in the natural polymer nanofiber after crosslinked is spent deionised water to be fallen; The nano fiber non-woven fabric of handling at 40~60 ℃ of following vacuumize 1~12h, is obtained the natural polymer nano fibrous membrane.Wherein the concentration of crosslinking agent in mixed liquor is 1~10mmol/L;
2. according to the preparation method of claim 1, it is characterized in that the natural polymer described in the step (1) is a kind of of sodium alginate, hyaluronic acid, shitosan, glucan, cellulose ethanoate, collagen, silk, gelatin, starch, nucleic acid and fibroin albumen;
3. according to the preparation method of claim 1, the percentage by weight that it is characterized in that the natural polymer solution described in the step (1) is 0.001~0.1wt%;
4. according to the preparation method of claim 1, it is characterized in that the described vacuum ranges of step (2), the cryogenic temperature scope, the cooling time scope is respectively 1~600Pa, and-80~-10 ℃, 12~48h;
5. according to the preparation method of claim 1; It is characterized in that the crosslinking agent described in the step (3) is calcium chloride solution (sodium alginate), glutaraldehyde (collagen, shitosan), 1-chloro-2,3-expoxy propane (glucan), Geniposide (shitosan, gelatin), Cyanuric Chloride (silk), carbodiimides (hyaluronic acid), dialdehyde starch (gelatin, shitosan), POCl3 (starch), ultraviolet light (nucleic acid), diglycidyl ether (fibroin).
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Cited By (21)
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