CN103341175A - Preparation method of fibroin microsphere - Google Patents
Preparation method of fibroin microsphere Download PDFInfo
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- CN103341175A CN103341175A CN2013102280784A CN201310228078A CN103341175A CN 103341175 A CN103341175 A CN 103341175A CN 2013102280784 A CN2013102280784 A CN 2013102280784A CN 201310228078 A CN201310228078 A CN 201310228078A CN 103341175 A CN103341175 A CN 103341175A
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Abstract
The invention relates to a preparation method of a fibroin microsphere, and the fibroin microsphere can be taken as a carrier of medicaments for slow release. At present, there is not a preparation method of a demand-satisfying fibroin microsphere; and wherein the demands are that the fibroin microsphere has high embedding efficiency, and the medicament can be released slowly and uniformly along with the degradation of the prepared fibroin microsphere. The preparation method comprises the following steps: adding the medicament needing to embed and release into a regenerated fibroin solution with a concentration of 3-10 wt%, then dropwise adding a coagulating agent with a concentration of 2-10 wt%, and slowly stirring at a rotary speed of 80-120 r/min to obtain a mixed solution; spraying the mixed solution into a space with a temperature of -20 DEG C to -80 DEG C and coagulating, collecting icy particles, continually preserving the icy particles in a space with a temperature of -20 DEG C to -80 DEG C for more than 2 hours, then unfreezing the icy particles at 0 DEGC to room temperature, washing and removing the coagulating agent at 0 DEG C to room temperature, collecting the fibroin microsphere by centrifuging or filtering. The fibroin microsphere prepared by the method is high in embedding efficiency and excellent in biological compatibility, and the medicament is released slowly and uniformly along with the degradation of the fibroin microsphere.
Description
Technical field
The present invention relates to a kind of preparation method of fibroin microsphere, the fibroin microsphere can be as the carrier of medicament slow release.
Background technology
Natural silk is made up of 18 seed amino acids, and is good by the material biocompatibility of fibroin preparation, can slowly degrade in vivo, so the application of fibroin microsphere aspect slow releasing carrier of medication paid close attention to widely.Aspect the silk fibroin material of microsphere form prepares, emulsifying (Yaowalak Srisuwanl) has been arranged at present, solidified precipitation (Joydip Kundu), spray drying (Joo-Hong Yeo), spray chilling-lyophilization-crosslinked methods such as (Esther wenk).As openly day is on 08 20th, 2008, publication number is in the Chinese patent of CN101244277, a kind of preparation method of medicine carrying fibroin microsphere is disclosed, it adopts the emulsion technology of water/oil/water type and the self-assembling technique of protein, with water soluble drug with after regenerated silk solution fully mixes, join emulsifying in the oil phase that contains a certain amount of emulsifying agent in the stirring, adding organic solvent again stirs, make fibroin degeneration and structure Βization and when forming milky crystallinity fibroin microgranule, medicine is by embedding, add organic solvent again through the centrifugal upper strata liquid of removing, allow the further crystallization of fibroin albumen, medicine continues by embedding, more centrifugal desolventizing and residual oil phase and collect microsphere; The preparation method of this medicine carrying fibroin microsphere belongs to emulsification method.
The effect difference of the microsphere of different preparation methoies when the embedding different pharmaceutical, releasing mechanism is also different with the time, the design of method for preparing microsphere need be at the characteristic of medicine, at present also less than the embedding efficiency height, medicine slowly discharges uniformly with the degraded of fibroin microsphere, biocompatibility is good, is suitable for the preparation method of the fibroin microsphere of embedding and sustained-release nano medicine, polypeptide drugs and enzyme drug.
Summary of the invention
The objective of the invention is to overcome above shortcomings in the prior art, and a kind of embedding efficiency height is provided, medicine slowly discharges the preparation method of the fibroin microsphere that biocompatibility is good uniformly with the degraded of fibroin microsphere.
The present invention addresses the above problem the technical scheme that adopts: the characteristics of the preparation method of this fibroin microsphere are: the step of described preparation method is as follows: the medicine that adds required embedding and slow release in the regenerated silk solution of 3-10 wt%, be added dropwise to the coagulant of 2-10 wt% then, under 80-120 rev/min rotating speed, slowly stir, obtain mixed solution; Mixed solution sprayed condense into-20 ℃～-80 ℃ space, collect the ice microgranule, the ice microgranule is continued to preserve more than 2 hours in-20 ℃～-80 ℃ space; To ice microgranule then and under 0 ℃～room temperature, thaw, eccysis coagulant under 0 ℃～room temperature, centrifugal or filter screen is collected and is obtained the fibroin microsphere.Fibroin microsphere among the present invention has the embedding efficiency height, the advantage that biocompatibility is good, and medicine is with fibroin microsphere degraded and slowly discharge uniformly, outside medicament slow release fibroin microsphere can directly use, can also be compound goes in the biomaterial and uses.
As preferably, the medicine of required embedding of the present invention and slow release is nano-particle medicine, polypeptide drugs or enzyme drug.
As preferably, regenerated silk solution of the present invention is a kind of or two or more mixed regenerated silk solution in silkworm, Antherea pernyi Guerin-Meneville and other tussahs.
As preferably, coagulant of the present invention is a kind of in dimethyl sulfoxine, soluble epoxide chemical compound, methanol, ethanol, propanol, isopropyl alcohol, n-butyl alcohol and the tert-butyl alcohol.
As preferably, the present invention during the eccysis coagulant, adopts distilled water repeatedly to change the mode eccysis coagulant of water logging bubble under 0 ℃～room temperature.
As preferably, the particle diameter of fibroin microsphere of the present invention is relevant with the spraying fineness, and the particle diameter of fibroin microsphere is in a discrete distribution, and mean diameter is at 10～1000 microns.
As preferably, fibroin microsphere of the present invention is stand-by 0 ℃～4 ℃ preservations, or stand-by through preserving behind the low-temperature vacuum drying, or stand-by through preserving after the lyophilization.
As preferably, when the present invention is the soluble epoxide chemical compound when used coagulant, mixed solution is sprayed condenses into-20 ℃～-80 ℃ space, collect the ice microgranule after, the ice microgranule continues to preserve more than 2 hours in-20 ℃～-80 ℃ space; When used coagulant is dimethyl sulfoxine, mixed solution sprayed condenses into-20 ℃～-80 ℃ space, collect the ice microgranule after, the ice microgranule continues to preserve more than 12 hours in-20 ℃～-80 ℃ space; When used coagulant is propanol, isopropyl alcohol, n-butyl alcohol or the tert-butyl alcohol, mixed solution sprayed condenses into-20 ℃～-80 ℃ space, collect the ice microgranule after, the ice microgranule continues to preserve more than 24 hours in-20 ℃～-80 ℃ space; When used coagulant is methanol or ethanol, mixed solution sprayed condenses into-20 ℃～-80 ℃ space, collect the ice microgranule after, the ice microgranule continues to preserve more than 48 hours in-20 ℃～-80 ℃ space.
The present invention compared with prior art has the following advantages and effect: the embedding efficiency height, and medicine slowly discharges with the degraded of fibroin microsphere, and biocompatibility is good; Outside medicament slow release fibroin microsphere can directly use, can also be compound go in other materials and the feedstuff etc. and use; The present invention has formed water-fast fibroin microgranule in spraying-refrigerating process, need not through lyophilization-crosslinked again; The inside and outside consolidated structures of fibroin microsphere is even, and degraded slowly evenly; Because solidifying the fibroin microsphere at low temperatures, firming agent not with fibroin and drug reaction, these water miscible firming agent can soak to be removed, and nano-particle medicine, polypeptide, enzyme drug and fibroin albumen have weak combination, is fixed in the fibroin microsphere to discharge with the degraded of fibroin microsphere.The present invention is particularly useful for embedding and sustained-release nano medicine, polypeptide and enzyme drug.
The difference of the present invention and spray chilling-lyophilization-cross-linking method is the following aspects, (1) the present invention has formed water-fast fibroin microgranule in the spray chilling process, need not again through lyophilization-crosslinked, among the present invention with lyophilization just for drying, therefore also can use low-temperature vacuum drying, also can directly use; And spray chilling-lyophilization-cross-linking method must pass through lyophilization-crosslinked.(2) the inside and outside consolidated structures of fibroin microsphere of the present invention is even, and therefore degraded slowly evenly; Spray chilling-lyophilization-cross-linking method crosslinking curing again after having formed microsphere, easily inside and outside crosslinking curing structure is inhomogeneous, fast degraded.(3) the fibroin microsphere is solidified in the present invention at low temperatures, firming agent not with fibroin and drug reaction, these water miscible firming agent can soak to be removed, and nano-particle medicine, polypeptide, enzyme drug and fibroin albumen have weak combination, are fixed in the fibroin microsphere to discharge with the degraded of fibroin microsphere; Spray chilling-lyophilization-cross-linking method crosslinking curing again after having formed microsphere needs cross-linking reaction conditions such as the cross-linking agent of high concentration and temperature, and these might make the medicine of cross-linking agent and embedding react.
Description of drawings
Fig. 1 be by transmission electron microscope observing to the embodiment of the invention 13 in the combined state sketch map of nanometer silver in the fibroin microsphere.
Fig. 2 is the degradation speed sketch map of fibroin microsphere in 37 ℃ of following protein enzyme solutions in the embodiment of the invention 13.
The specific embodiment
The present invention is described in further detail below in conjunction with accompanying drawing and by embodiment, and following examples are explanation of the invention and the present invention is not limited to following examples.
Embodiment 1.
What prepare in the present embodiment is the fibroin microsphere of slow release nanometer silver, and the step of the preparation method of this fibroin microsphere is as follows: with the Na of the man silkworm cocoon of 1000 grams with 0.5wt %
2CO
3Aqueous solution comes unstuck 2 times, each 30min, 100 ℃ of the temperature of coming unstuck, bath raio 1:100.The back water that comes unstuck is fully clean, and 60 ℃ of dryings obtain fibroin fiber.Use CaCl
2Aqueous solution dissolving fibroin fiber, fibroin fiber: CaCl
22H
2O: water=1g:25g:25ml, solution temperature is 100 ℃, dissolution time is 5min.The solution that obtains after the dissolving is filtered the back inject the cellulose dialyzer 3d that dialyses, the molecular cut off of used cellulose dialyzer is 8000-14000.Silk fibroin solution is concentrated into 4wt%, adds the 0.3wt% nanometer silver, is added dropwise to 5% diglycidyl ether, and 100 rev/mins are slowly evenly mixed.The ice microgranule is collected in-20 ℃ of sprayings down.Continuation was preserved the ice microgranule down 24 hours at-20 ℃.To ice under the microgranule room temperature and thaw, eccysis coagulant under the room temperature changed water once about 2 hours, changed water altogether 3 times.Collect the fibroin microsphere with filter screen, preserve stand-by behind the low-temperature vacuum drying.
In fibroin dressing preparation process, the fibroin microsphere of 2～10wt % evenly is mixed into silk fibroin solution, can be made into the fibroin dressing of slow release nanometer silver.The preparation method of fibroin dressing can be the Chinese invention patent of ZL 200510060655.9 referring to the patent No..
In collagen biological dressing preparation process, compound nanometer silver fibroin microsphere of going into 5wt % along with the degraded of fibroin microsphere can slowly release nanometer silver and fibroin albumen, plays long-acting bacteriostatic, promotes the effect of wound healing.
Embodiment 2.
What prepare in the present embodiment is the fibroin microsphere of slow release somatomedin, and the step of the preparation method of this fibroin microsphere is as follows: with the Na of the man silkworm cocoon of 1000 grams with 0.5wt %
2CO
3Aqueous solution comes unstuck 2 times, each 30min, 100 ℃ of the temperature of coming unstuck, bath raio 1:100.The back water that comes unstuck is fully clean, and 60 ℃ of dryings obtain fibroin fiber.Use CaCl
2Aqueous solution dissolving fibroin fiber, fibroin fiber: CaCl
22H
2O: water=1g:25g:25ml, 100 ℃ of solution temperatures, dissolution time 5min.The solution that obtains after the dissolving is filtered, inject the cellulose dialyzer 3d that dialyses, used cellulose dialyzer molecular cut off is 8000-14000.Silk fibroin solution is concentrated into 4wt%.Silk fibroin solution after the sterilization is cooled to about 0 ℃, under the cryogenic conditions about 0 ℃, adds the hEGF (hEGF) of 0.5wt %, is added dropwise to 3wt % dimethyl sulfoxine, slowly evenly mixes about 100 rev/mins.-40 ℃ of sprayings down, collect the ice microgranule.Continuation was preserved the ice microgranule down 48 hours at-40 ℃.To ice microgranule thaws under 0～4 ℃.0～4 ℃ of following eccysis coagulant changed water once about 0.5 hour, changed water altogether 5 times.With centrifugal collection fibroin microsphere, preserve stand-by after the lyophilization.
In the silver sulfadiazine ointment, add 10-20wt % hEGF fibroin microsphere, directly be coated in behind the mix homogeneously on the empyrosis wound surface after the debridement.Along with the degraded of fibroin microsphere can slowly release hEGF and fibroin albumen, promote the healing of wound surface.
Embodiment 3.
What prepare in the present embodiment is the fibroin microsphere of sustained-release antimicrobial peptide, and the step of the preparation method of this fibroin microsphere is as follows: with the man silkworm cocoon of 500 grams 0.5wt % Na
2CO
3Aqueous solution comes unstuck 2 times, each 30min, 100 ℃ of the temperature of coming unstuck, bath raio 1:100.The back water that comes unstuck is fully clean, and 60 ℃ of dryings obtain fibroin fiber.Be calcium chloride, water, the dissolve with ethanol solution fibroin fiber of 1:8:2 with mol ratio, 70 ℃ of solution temperatures, bath raio 1:25,10 minutes time.The solution that obtains after the dissolving is filtered the back inject the cellulose dialyzer 3d that dialyses, used cellulose dialyzer molecular cut off is 8000-14000.Silk fibroin solution is concentrated into 5wt %, adds antibacterial peptide 1wt %, is added dropwise to 5wt % ethanol, slowly evenly mixes about 100 rev/mins.-20 ℃ of sprayings down, collect the ice microgranule.Continuation was preserved the ice microgranule down 96 hours at-20 ℃.To ice under 4 ℃ of the microgranules and thaw.4 ℃ of following eccysis coagulant changed water once about 2 hours, changed water altogether 5 times.Filter screen is collected the fibroin microsphere, preserves stand-by after the lyophilization.
Can go in the various pastes coating medicines slow release fibroin microsphere is compound, along with the degraded of fibroin microsphere can slowly release antibacterial peptide, play antibacterial, as to promote dermopathic healing effect.
Embodiment 4.
What prepare in the present embodiment is the fibroin microsphere that the embedding feedstuff adds enzyme, and the step of the preparation method of this fibroin microsphere is as follows: use CaCl
2Aqueous solution dissolving fibroin fiber, fibroin fiber: CaCl
22H
2O: water=1g:25g:25ml, 100 ℃ of solution temperatures, time 5min.The solution that obtains after the dissolving is filtered the back inject the cellulose dialyzer 3d that dialyses, used cellulose dialyzer molecular cut off is 8000-14000.Silk fibroin solution is concentrated into 6wt %, adds enzyme, is added dropwise to 5wt % ethanol, and 100 rev/mins are slowly evenly mixed.-40 ℃ of sprayings down, collect the ice microgranule.Continuation was preserved the ice microgranule down 96 hours at-40 ℃.To ice microgranule thaws under 4 ℃.At 4 ℃ of following eccysis coagulant, change water once about 2 hours, change water altogether 3 times.Filter screen is collected the fibroin microsphere, preserves stand-by after the lyophilization.
With embedding the fibroin microsphere of enzyme sneak in the animal feed, can prolong the activity of enzyme, improve the active temperature of enzyme, widen enzymatic activity pH scope.
The step of the preparation method of the fibroin microsphere in the present embodiment is as follows: the medicine that adds required embedding and slow release in the regenerated silk solution of 3wt%, the medicine of this required embedding and slow release is the nano-particle medicine, and used regenerated silk solution is the regenerated silk solution of silkworm.Be added dropwise to the coagulant of 2 wt% then, slowly stir under 100 rev/mins rotating speed, obtain mixed solution, used coagulant is dimethyl sulfoxine.Mixed solution sprayed condense into-50 ℃ space, collect the ice microgranule, the ice microgranule is continued to preserve more than 48 hours in-50 ℃ space.To ice microgranule then and thaw under 15 ℃, at 15 ℃ of following eccysis coagulant, filter screen is collected and is obtained the fibroin microsphere.When 15 ℃ of following eccysis coagulant, can adopt and change water once in per 2 hours, change the mode eccysis coagulant of water 5 times altogether.
The present invention can add the medicine of required embedding and slow release in the regenerated silk solution of 3-10 wt%, the medicine of this required embedding and slow release can be nano-particle medicine, polypeptide drugs or enzyme drug, and used regenerated silk solution can be the regenerated silk solution of silkworm, Antherea pernyi Guerin-Meneville and/or other tussahs.Can be added dropwise to the coagulant of 2-10 wt% then, can under 80-120 rev/min rotating speed, slowly stir, obtain mixed solution, what used coagulant can be in dimethyl sulfoxine, soluble epoxide chemical compound, methanol, ethanol, propanol, isopropyl alcohol, n-butyl alcohol and the tert-butyl alcohol is a kind of.
The present invention can spray mixed solution and condense into-20 ℃～-80 ℃ space, collect the ice microgranule, the ice microgranule is continued to preserve more than 24 hours in-20 ℃～-80 ℃ space, when used coagulant is the soluble epoxide chemical compound, mixed solution sprayed condense into-20 ℃～-80 ℃ space, after collecting the ice microgranule, the ice microgranule can continue to preserve more than 2 hours in-20 ℃～-80 ℃ space; When used coagulant is dimethyl sulfoxine, mixed solution sprayed condenses into-20 ℃～-80 ℃ space, collect the ice microgranule after, the ice microgranule can continue to preserve more than 12 hours in-20 ℃～-80 ℃ space; When used coagulant is propanol, isopropyl alcohol, n-butyl alcohol or the tert-butyl alcohol, mixed solution sprayed condenses into-20 ℃～-80 ℃ space, collect the ice microgranule after, the ice microgranule can continue to preserve more than 24 hours in-20 ℃～-80 ℃ space; When used coagulant is methanol or ethanol, mixed solution sprayed condenses into-20 ℃～-80 ℃ space, collect the ice microgranule after, the ice microgranule can continue to preserve more than 48 hours in-20 ℃～-80 ℃ space.
The present invention can be thawed the ice microgranule under 0 ℃～room temperature, can be under 0 ℃～room temperature the eccysis coagulant, filter screen is collected and is obtained the fibroin microsphere.Under 0 ℃～room temperature, during the eccysis coagulant, can adopt every several hours and change water once, change water mode eccysis coagulant repeatedly.Said room temperature among the present invention, normal conditions refer to about 25 ℃.
The particle diameter of the fibroin microsphere among the present invention is relevant with the spraying fineness, and the particle diameter of fibroin microsphere is in a discrete distribution, and mean diameter is at 10～1000 microns.The fibroin microsphere that the present invention is prepared from can be stand-by 0 ℃～4 ℃ preservations, also can be stand-by through preserving behind the low-temperature vacuum drying, and can also be stand-by through preserving after the lyophilization.
Can compound multiple medicine in fibroin microsphere of the present invention, the fibroin microsphere again can be compound be gone in the various biological dressings, compoundly goes in the various pastes coating medicines, along with the degraded of fibroin microsphere can slowly release medicine, plays therapeutical effect.
Embodiment 6.
The step of the preparation method of the fibroin microsphere in the present embodiment is as follows: the medicine that adds required embedding and slow release in the regenerated silk solution of 10 wt%, the medicine of this required embedding and slow release is polypeptide drugs, and used regenerated silk solution is the regenerated silk solution of Antherea pernyi Guerin-Meneville.Be added dropwise to the coagulant of 10 wt% then, slowly stir under 80 rev/mins rotating speed, obtain mixed solution, used coagulant is methanol.Mixed solution sprayed condense into-20 ℃ space, collect the ice microgranule, the ice microgranule is continued to preserve more than 96 hours in-20 ℃ space.To ice microgranule then and thaw under 0 ℃, at 0 ℃ of following eccysis coagulant, filter screen is collected and is obtained the fibroin microsphere.
Embodiment 7.
The step of the preparation method of the fibroin microsphere in the present embodiment is as follows: the medicine that adds required embedding and slow release in the regenerated silk solution of 5 wt%, the medicine of this required embedding and slow release is enzyme drug, and used regenerated silk solution is the regenerated silk solution of Antherea pernyi Guerin-Meneville.Be added dropwise to the coagulant of 9 wt% then, slowly stir under 120 rev/mins rotating speed, obtain mixed solution, used coagulant is propanol.Mixed solution sprayed condense into-80 ℃ space, collect the ice microgranule, the ice microgranule is continued to preserve more than 72 hours in-80 ℃ space.To ice microgranule then and thaw under 25 ℃, at 25 ℃ of following eccysis coagulant, filter screen is collected and is obtained the fibroin microsphere.
Embodiment 8.
The step of the preparation method of the fibroin microsphere in the present embodiment is as follows: the medicine that adds required embedding and slow release in the regenerated silk solution of 8 wt%, the medicine of this required embedding and slow release is polypeptide drugs, and used regenerated silk solution is the regenerated silk solution of silkworm.Be added dropwise to the coagulant of 6 wt% then, slowly stir under 100 rev/mins rotating speed, obtain mixed solution, used coagulant is n-butyl alcohol.Mixed solution sprayed condense into-60 ℃ space, collect the ice microgranule, the ice microgranule is continued to preserve more than 72 hours in-60 ℃ space.To ice microgranule then and thaw under 10 ℃, at 10 ℃ of following eccysis coagulant, filter screen is collected and is obtained the fibroin microsphere.
Embodiment 9.
The step of the preparation method of the fibroin microsphere in the present embodiment is as follows: the medicine that adds required embedding and slow release in the regenerated silk solution of 6 wt%, the medicine of this required embedding and slow release is the nano-particle medicine, and used regenerated silk solution is the regenerated silk solution of Antherea pernyi Guerin-Meneville.Be added dropwise to the coagulant of 3 wt% then, slowly stir under 100 rev/mins rotating speed, obtain mixed solution, used coagulant is isopropyl alcohol.Mixed solution sprayed condense into-30 ℃ space, collect the ice microgranule, the ice microgranule is continued to preserve more than 72 hours in-30 ℃ space.To ice microgranule then and thaw under 20 ℃, at 20 ℃ of following eccysis coagulant, filter screen is collected and is obtained the fibroin microsphere.When 20 ℃ of following eccysis coagulant, can adopt and change water once in per 2 hours, change the mode eccysis coagulant of water 5 times altogether.
The step of the preparation method of the fibroin microsphere in the present embodiment is as follows: the medicine that adds required embedding and slow release in the regenerated silk solution of 4 wt%, the medicine of this required embedding and slow release is enzyme drug, and used regenerated silk solution is the regenerated silk solution of Antherea pernyi Guerin-Meneville.Be added dropwise to the coagulant of 7 wt% then, slowly stir under 100 rev/mins rotating speed, obtain mixed solution, used coagulant is the tert-butyl alcohol.Mixed solution sprayed condense into-40 ℃ space, collect the ice microgranule, the ice microgranule is continued to preserve more than 72 hours in-40 ℃ space.To ice microgranule then and thaw under 5 ℃, at 5 ℃ of following eccysis coagulant, filter screen is collected and is obtained the fibroin microsphere.
Embodiment 11.
The step of the preparation method of the fibroin microsphere in the present embodiment is as follows: the medicine that adds required embedding and slow release in the regenerated silk solution of 7 wt%, the medicine of this required embedding and slow release is enzyme drug, and used regenerated silk solution is the regenerated silk solution of silkworm.Be added dropwise to the coagulant of 4 wt% then, slowly stir under 105 rev/mins rotating speed, obtain mixed solution, used coagulant is the soluble epoxide chemical compound.Mixed solution sprayed condense into-70 ℃ space, collect the ice microgranule, the ice microgranule is continued to preserve more than 24 hours in-70 ℃ space.To ice microgranule then and thaw under 18 ℃, at 18 ℃ of following eccysis coagulant, filter screen is collected and is obtained the fibroin microsphere.
Embodiment 12.
The step of the preparation method of the fibroin microsphere in the present embodiment is as follows: add the medicine of required embedding and slow release in the regenerated silk solution of 9 wt%, the medicine of this required embedding and slow release is polypeptide drugs, and used regenerated silk solution is Antherea pernyi Guerin-Meneville.Be added dropwise to the coagulant of 5 wt% then, slowly stir under 95 rev/mins rotating speed, obtain mixed solution, used coagulant is ethanol.Mixed solution sprayed condense into-55 ℃ space, collect the ice microgranule, the ice microgranule is continued to preserve more than 96 hours in-55 ℃ space.To ice microgranule then and thaw under 22 ℃, at 22 ℃ of following eccysis coagulant, filter screen is collected and is obtained the fibroin microsphere.When 22 ℃ of following eccysis coagulant, can adopt and change water once in per 2 hours, change the mode eccysis coagulant of water 5 times altogether.
Embodiment 13.
What prepare in the present embodiment is the fibroin microsphere of slow release nanometer silver, and the step of the preparation method of this fibroin microsphere is as follows: with the Na of the man silkworm cocoon of 1000 grams with 0.5wt %
2CO
3Aqueous solution comes unstuck 2 times, each 30min, 100 ℃ of the temperature of coming unstuck, bath raio 1:100.The back water that comes unstuck is fully clean, and 60 ℃ of dryings obtain fibroin fiber.Use CaCl
2Aqueous solution dissolving fibroin fiber, fibroin fiber: CaCl
22H
2O: water=1g:25g:25ml, solution temperature is 100 ℃, dissolution time is 5min.The solution that obtains after the dissolving is filtered the back inject the cellulose dialyzer 3d that dialyses, the molecular cut off of used cellulose dialyzer is 8000-14000.Silk fibroin solution is concentrated into 4wt%, adds the 0.05wt% nanometer silver, is added dropwise to 3% diglycidyl ether, and 100 rev/mins are slowly evenly mixed.The ice microgranule is collected in-20 ℃ of sprayings down.Continuation was preserved the ice microgranule down 24 hours at-40 ℃.To ice under the microgranule room temperature and thaw, eccysis coagulant under the room temperature changed water once about 2 hours, changed water altogether 6 times.Collect the fibroin microsphere with filter screen, preserve stand-by behind the low-temperature vacuum drying.
As shown in Figure 1, demonstration be under transmission electron microscope, the combined state of nanometer silver in the fibroin microsphere, what accompanying drawing 2 showed is the degradation speed of fibroin microsphere in 37 ℃ of following protein enzyme solutions.
Embodiment 14.
The step of the preparation method of the fibroin microsphere in the present embodiment is as follows: the medicine that adds required embedding and slow release in the regenerated silk solution of 5.5wt%, the medicine of this required embedding and slow release is polypeptide drugs, and used regenerated silk solution is the regenerated silk solution of Antherea pernyi Guerin-Meneville.Be added dropwise to the coagulant of 6.5wt% then, slowly stir under 98 rev/mins rotating speed, obtain mixed solution, used coagulant is methanol.Mixed solution sprayed condense into-20 ℃ space, collect the ice microgranule, the ice microgranule is continued to preserve more than 96 hours in-20 ℃ space.To ice microgranule then and thaw under 0 ℃, at 0 ℃ of following eccysis coagulant, filter screen is collected and is obtained the fibroin microsphere.
The step of the preparation method of the fibroin microsphere in the present embodiment is as follows: the medicine that adds required embedding and slow release in the regenerated silk solution of 10 wt%, the medicine of this required embedding and slow release is polypeptide drugs, and used regenerated silk solution is the regenerated silk solution of Antherea pernyi Guerin-Meneville.Be added dropwise to the coagulant of 10 wt% then, slowly stir under 102 rev/mins rotating speed, obtain mixed solution, used coagulant is methanol.Mixed solution sprayed condense into-35 ℃ space, collect the ice microgranule, the ice microgranule is continued to preserve more than 96 hours in-20 ℃ space.To ice microgranule then and thaw under 8 ℃, at 8 ℃ of following eccysis coagulant, filter screen is collected and is obtained the fibroin microsphere.
Though the present invention with embodiment openly as above; but it is not in order to limit protection scope of the present invention; any technical staff who is familiar with this technology not breaking away from change and the retouching of doing in the spirit and scope of the present invention, all should belong to protection scope of the present invention.
Claims (8)
1. the preparation method of a fibroin microsphere, it is characterized in that: the step of described preparation method is as follows: the medicine that adds required embedding and slow release in the regenerated silk solution of 3-10 wt%, be added dropwise to the coagulant of 2-10 wt% then, under 80-120 rev/min rotating speed, slowly stir, obtain mixed solution; Mixed solution sprayed condense into-20 ℃～-80 ℃ space, collect the ice microgranule, the ice microgranule is continued to preserve more than 2 hours in-20 ℃～-80 ℃ space; To ice microgranule then and under 0 ℃～room temperature, thaw, eccysis coagulant under 0 ℃～room temperature, centrifugal or filter screen is collected and is obtained the fibroin microsphere.
2. the preparation method of fibroin microsphere according to claim 1, it is characterized in that: the medicine of described required embedding and slow release is nano-particle medicine, polypeptide drugs or enzyme drug.
3. the preparation method of fibroin microsphere according to claim 1, it is characterized in that: described regenerated silk solution is a kind of or two or more mixed regenerated silk solution in silkworm, Antherea pernyi Guerin-Meneville and other tussahs.
4. the preparation method of fibroin microsphere according to claim 1 is characterized in that: described coagulant is a kind of in dimethyl sulfoxine, soluble epoxide chemical compound, methanol, ethanol, propanol, isopropyl alcohol, n-butyl alcohol and the tert-butyl alcohol.
5. the preparation method of fibroin microsphere according to claim 1 is characterized in that: during the eccysis coagulant, adopt distilled water repeatedly to change the mode eccysis coagulant of water logging bubble under 0 ℃～room temperature.
6. the preparation method of fibroin microsphere according to claim 1 is characterized in that: the particle diameter of described fibroin microsphere is relevant with the spraying fineness, and the particle diameter of fibroin microsphere is in a discrete distribution, and mean diameter is at 10～1000 microns.
7. the preparation method of fibroin microsphere according to claim 1, it is characterized in that: described fibroin microsphere is stand-by 0 ℃～4 ℃ preservations, or stand-by through preserving behind the low-temperature vacuum drying, or stand-by through preserving after the lyophilization.
8. the preparation method of fibroin microsphere according to claim 4, it is characterized in that: when used coagulant is the soluble epoxide chemical compound, mixed solution sprayed condense into-20 ℃～-80 ℃ space, after collecting the ice microgranule, the ice microgranule continues to preserve more than 2 hours in-20 ℃～-80 ℃ space; When used coagulant is dimethyl sulfoxine, mixed solution sprayed condenses into-20 ℃～-80 ℃ space, collect the ice microgranule after, the ice microgranule continues to preserve more than 12 hours in-20 ℃～-80 ℃ space; When used coagulant is propanol, isopropyl alcohol, n-butyl alcohol or the tert-butyl alcohol, mixed solution sprayed condenses into-20 ℃～-80 ℃ space, collect the ice microgranule after, the ice microgranule continues to preserve more than 24 hours in-20 ℃～-80 ℃ space; When used coagulant is methanol or ethanol, mixed solution sprayed condenses into-20 ℃～-80 ℃ space, collect the ice microgranule after, the ice microgranule continues to preserve more than 48 hours in-20 ℃～-80 ℃ space.
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