CN103341175B - A kind of preparation method of fibroin microsphere - Google Patents
A kind of preparation method of fibroin microsphere Download PDFInfo
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- CN103341175B CN103341175B CN201310228078.4A CN201310228078A CN103341175B CN 103341175 B CN103341175 B CN 103341175B CN 201310228078 A CN201310228078 A CN 201310228078A CN 103341175 B CN103341175 B CN 103341175B
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- 108010022355 Fibroins Proteins 0.000 title claims abstract description 137
- 239000004005 microsphere Substances 0.000 title claims abstract description 108
- 238000002360 preparation method Methods 0.000 title claims abstract description 38
- 239000004531 microgranule Substances 0.000 claims abstract description 84
- 239000003814 drug Substances 0.000 claims abstract description 82
- 239000000701 coagulant Substances 0.000 claims abstract description 70
- 239000000243 solution Substances 0.000 claims abstract description 60
- 239000011259 mixed solution Substances 0.000 claims abstract description 40
- 238000009833 condensation Methods 0.000 claims abstract description 26
- 230000005494 condensation Effects 0.000 claims abstract description 26
- 238000003756 stirring Methods 0.000 claims abstract description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 114
- 229940079593 drug Drugs 0.000 claims description 35
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 27
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 21
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 21
- DKGAVHZHDRPRBM-UHFFFAOYSA-N Tert-Butanol Chemical compound CC(C)(C)O DKGAVHZHDRPRBM-UHFFFAOYSA-N 0.000 claims description 21
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 20
- 108090000790 Enzymes Proteins 0.000 claims description 18
- 102000004190 Enzymes Human genes 0.000 claims description 18
- -1 dimethyl sulfoxine Chemical compound 0.000 claims description 15
- 229920001184 polypeptide Polymers 0.000 claims description 13
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 13
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 13
- 238000004108 freeze drying Methods 0.000 claims description 7
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 claims description 7
- 241000255789 Bombyx mori Species 0.000 claims description 6
- 239000002245 particle Substances 0.000 claims description 6
- 238000001291 vacuum drying Methods 0.000 claims description 6
- 238000005507 spraying Methods 0.000 claims description 4
- 238000004321 preservation Methods 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 2
- 238000002791 soaking Methods 0.000 claims description 2
- 239000000835 fiber Substances 0.000 description 13
- 239000007921 spray Substances 0.000 description 11
- 239000001913 cellulose Substances 0.000 description 10
- 229920002678 cellulose Polymers 0.000 description 10
- 238000000502 dialysis Methods 0.000 description 10
- 238000000034 method Methods 0.000 description 10
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 9
- 229910052709 silver Inorganic materials 0.000 description 9
- 239000004332 silver Substances 0.000 description 9
- 238000004132 cross linking Methods 0.000 description 8
- 150000001875 compounds Chemical class 0.000 description 7
- 238000004090 dissolution Methods 0.000 description 7
- 238000002156 mixing Methods 0.000 description 6
- 238000001035 drying Methods 0.000 description 5
- 206010013786 Dry skin Diseases 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 230000015556 catabolic process Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000003910 polypeptide antibiotic agent Substances 0.000 description 3
- 206010052428 Wound Diseases 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 239000003431 cross linking reagent Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- GYZLOYUZLJXAJU-UHFFFAOYSA-N diglycidyl ether Chemical compound C1OC1COCC1CO1 GYZLOYUZLJXAJU-UHFFFAOYSA-N 0.000 description 2
- 230000001804 emulsifying effect Effects 0.000 description 2
- 230000035876 healing Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 238000013268 sustained release Methods 0.000 description 2
- 239000012730 sustained-release form Substances 0.000 description 2
- 102000044503 Antimicrobial Peptides Human genes 0.000 description 1
- 108700042778 Antimicrobial Peptides Proteins 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000003385 bacteriostatic effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 238000001804 debridement Methods 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 229960003600 silver sulfadiazine Drugs 0.000 description 1
- UEJSSZHHYBHCEL-UHFFFAOYSA-N silver(1+) sulfadiazinate Chemical compound [Ag+].C1=CC(N)=CC=C1S(=O)(=O)[N-]C1=NC=CC=N1 UEJSSZHHYBHCEL-UHFFFAOYSA-N 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Processes Of Treating Macromolecular Substances (AREA)
- Medicinal Preparation (AREA)
Abstract
The present invention relates to a kind of preparation method of fibroin microsphere, fibroin microsphere can as the carrier of medicament slow release.Also high less than embedding efficiency at present, medicine is the preparation method of the fibroin microsphere of slowly uniform release with fibroin microsphere degraded.Step of the present invention is as follows: the medicine adding required embedding and slow release in the regenerated silk solution of 3-10wt%, is then added dropwise to the coagulant of 2-10wt%, slowly stirs, obtain mixed solution under the rotating speed of 80-120 rev/min; Mixed solution is sprayed into the space condensation of-20 DEG C ~-80 DEG C, collect ice microgranule, ice microgranule is continued to preserve more than 2 hours in the space of-20 DEG C ~-80 DEG C; Then by ice microgranule at 0 DEG C ~ thawed at room temperature, eccysis coagulant under 0 DEG C ~ room temperature, centrifugal or filter screen is collected and is obtained fibroin microsphere.Embedding efficiency of the present invention is high, and medicine slowly uniform release with fibroin microsphere degraded, biocompatibility is excellent.
Description
Technical field
The present invention relates to a kind of preparation method of fibroin microsphere, fibroin microsphere can as the carrier of medicament slow release.
Background technology
Natural silk is made up of 18 seed amino acids, and the Biocompatibility prepared by fibroin is excellent, can slowly degrade in vivo, and therefore the application of fibroin microsphere in slow releasing carrier of medication is paid close attention to widely.In prepared by the silk fibroin material of microsphere form, there is emulsifying (Yaowalak Srisuwanl) at present, solidified precipitation (Joydip Kundu), the method such as spraying dry (Joo-Hong Yeo), spray chilling-lyophilization-crosslinked (Esther wenk).If publication date is on 08 20th, 2008, publication number is in the Chinese patent of CN101244277, disclose a kind of preparation method of medicine carrying fibroin microsphere, it adopts the emulsion technology of water/oil/water type and the self-assembling technique of protein, after water soluble drug is fully mixed with regenerated silk solution, join in stirring containing emulsifying in the oil phase of a certain amount of emulsifying agent, add organic solvent again to stir, make fibroin degeneration and structure Βization and while forming creamy white crystals sex pilus element microgranule, medicine is embedded, organic solvent is added again through centrifugal removing upper liquid, allow the further crystallization of fibroin albumen, medicine continues to be embedded, centrifugally again collect microsphere except desolventizing and residual oil phase, the preparation method of this medicine carrying fibroin microsphere belongs to emulsification method.
The effect of microsphere when embedding different pharmaceutical of different preparation methoies is different, releasing mechanism and time are also different, the design of method for preparing microsphere needs the characteristic for medicine, also high less than embedding efficiency at present, medicine slowly uniform release with fibroin microsphere degraded, biocompatibility is excellent, is suitable for the preparation method of the fibroin microsphere of embedding and slow release nanoparticulate drug, polypeptide drugs and enzyme drug.
Summary of the invention
The object of the invention is to overcome above shortcomings in prior art, and provide a kind of embedding efficiency high, medicine slowly uniform release with fibroin microsphere degraded, the preparation method of the fibroin microsphere that biocompatibility is excellent.
The present invention's adopted technical scheme that solves the problem is: the feature of the preparation method of this fibroin microsphere is: the step of described preparation method is as follows: the medicine adding required embedding and slow release in the regenerated silk solution of 3-10 wt%, then the coagulant of 2-10 wt% is added dropwise to, slowly stir under the rotating speed of 80-120 rev/min, obtain mixed solution; Mixed solution is sprayed into the space condensation of-20 DEG C ~-80 DEG C, collect ice microgranule, ice microgranule is continued to preserve more than 2 hours in the space of-20 DEG C ~-80 DEG C; Then by ice microgranule at 0 DEG C ~ thawed at room temperature, eccysis coagulant under 0 DEG C ~ room temperature, centrifugal or filter screen is collected and is obtained fibroin microsphere.It is high that fibroin microsphere in the present invention has embedding efficiency, the advantage that biocompatibility is excellent, and medicine slowly uniform release with fibroin microsphere degraded, outside medicament slow release fibroin microsphere can directly use, can also compound enter in biomaterial to use.
As preferably, the medicine of required embedding of the present invention and slow release is nanoparticulate drug, polypeptide drugs or enzyme drug.
As preferably, regenerated silk solution of the present invention is one in silkworm, Antherea pernyi Guerin-Meneville and other tussahs or two or more mixed regenerated silk solution.
As preferably, coagulant of the present invention is the one in dimethyl sulfoxine, soluble epoxide compound, methanol, ethanol, propanol, isopropyl alcohol, n-butyl alcohol and the tert-butyl alcohol.
As preferably, the present invention during eccysis coagulant, adopts distilled water repeatedly to change the mode eccysis coagulant of water soaking under 0 DEG C ~ room temperature.
As preferably, the particle diameter of fibroin microsphere of the present invention is relevant with 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 DEG C ~ 4 DEG C preservations, or preserves stand-by after low-temperature vacuum drying, or preserves stand-by after lyophilization.
As preferably, the present invention is when coagulant used is soluble epoxide compound, and sprayed by mixed solution into the space condensation of-20 DEG C ~-80 DEG C, after collecting ice microgranule, ice microgranule continues to preserve more than 2 hours in the space of-20 DEG C ~-80 DEG C; When coagulant used is dimethyl sulfoxine, sprayed by mixed solution into the space condensation of-20 DEG C ~-80 DEG C, after collecting ice microgranule, ice microgranule continues to preserve more than 12 hours in the space of-20 DEG C ~-80 DEG C; When coagulant used is propanol, isopropyl alcohol, n-butyl alcohol or the tert-butyl alcohol, sprayed by mixed solution into the space condensation of-20 DEG C ~-80 DEG C, after collecting ice microgranule, ice microgranule continues to preserve more than 24 hours in the space of-20 DEG C ~-80 DEG C; When coagulant used be methanol or ethanol time, sprayed by mixed solution into the space condensation of-20 DEG C ~-80 DEG C, after collecting ice microgranule, ice microgranule continues to preserve more than 48 hours in the space of-20 DEG C ~-80 DEG C.
The present invention compared with prior art, has the following advantages and effect: embedding efficiency is high, and medicine is slow releasing with fibroin microsphere degraded, and biocompatibility is excellent; Outside medicament slow release fibroin microsphere can directly use, can also compound enter in other materials and feedstuff etc. to use; The present invention has defined water-fast fibroin microgranule in spraying-refrigerating process, without the need to again through lyophilization-crosslinked; Inside and outside fibroin microsphere, consolidated structures is even, and degraded slowly evenly; Because solidifying fibroin microsphere at low temperatures, firming agent not with fibroin and drug reaction, these water miscible firming agent can soak removing, and nanoparticulate drug, polypeptide, enzyme drug and fibroin albumen have weak binding, are fixed and will discharge with fibroin microsphere degraded in fibroin microsphere.The present invention is particularly useful for embedding and slow release nanoparticulate drug, 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 defined water-fast fibroin microgranule in spray chilling process, without the need to again through lyophilization-crosslinked, in the present invention with lyophilization just in order to drying, therefore also can use low-temperature vacuum drying, also can directly use; And spray chilling-lyophilization-cross-linking method must through lyophilization-crosslinked.(2) inside and outside fibroin microsphere of the present invention, consolidated structures is even, and therefore degraded slowly evenly; Spray chilling-lyophilization-cross-linking method crosslinking curing again after defining microsphere, easily inside and outside crosslinking curing structure is uneven, fast degradation.(3) the present invention solidifies fibroin microsphere at low temperatures, firming agent not with fibroin and drug reaction, these water miscible firming agent can soak removing, and nanoparticulate drug, polypeptide, enzyme drug and fibroin albumen have weak binding, be fixed and will discharge with fibroin microsphere degraded in fibroin microsphere; Spray chilling-lyophilization-cross-linking method crosslinking curing again after defining microsphere, need the cross-linking reaction condition such as cross-linking agent and temperature of high concentration, these likely can make the medicine of cross-linking agent and embedding react.
Accompanying drawing explanation
Fig. 1 be by transmission electron microscope observing to the embodiment of the present invention 13 in the combined state schematic diagram of nanometer silver in fibroin microsphere.
Fig. 2 is the degradation speed schematic diagram of fibroin microsphere at 37 DEG C in protein enzyme solution in the embodiment of the present invention 13.
Detailed description of the invention
Below in conjunction with accompanying drawing, also by embodiment, the present invention is described in further detail, 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: by the Na of 1000 grams of Cocoon shell 0.5wt %
2cO
3aqueous solution comes unstuck 2 times, each 30min, temperature of coming unstuck 100 DEG C, bath raio 1:100.Fully clean with water after coming unstuck, 60 DEG C of dryings, obtain fibroin fiber.Use CaCl
2aqueous dissolution fibroin fiber, fibroin fiber: CaCl
22H
2o: water=1g:25g:25ml, solution temperature is 100 DEG C, and dissolution time is 5min.Dialyse injection cellulose dialysis film after the solution obtained after dissolving filtration 3d, and the molecular cut off of cellulose dialysis film used is 8000-14000.Silk fibroin solution is concentrated into 4wt%, adds 0.3wt% nanometer silver, is added dropwise to the diglycidyl ether of 5%, 100 revs/min of slow Homogeneous phase mixing.Spray at-20 DEG C, collect ice microgranule.Continue at-20 DEG C, to preserve ice microgranule 24 hours.By ice microgranule thawed at room temperature, eccysis coagulant under room temperature, 2 hours change water once, altogether change water 3 times.Collect fibroin microsphere with filter screen, preserve stand-by after low-temperature vacuum drying.
In fibroin dressing preparation process, the fibroin microsphere Homogeneous phase mixing of 2 ~ 10wt % is entered 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 see the patent No..
In collagenous biological dressing preparation process, compound enters the nanometer silver fibroin microsphere of 5wt %, along with the degraded of fibroin microsphere can slowly release nanometer silver and fibroin albumen, plays long-acting bacteriostatic, the effect of wound healing.
Embodiment 2.
What prepare in the present embodiment is the fibroin microsphere of the sustained release growth factor, and the step of the preparation method of this fibroin microsphere is as follows: by the Na of 1000 grams of Cocoon shell 0.5wt %
2cO
3aqueous solution comes unstuck 2 times, each 30min, temperature of coming unstuck 100 DEG C, bath raio 1:100.Fully clean with water after coming unstuck, 60 DEG C of dryings, obtain fibroin fiber.Use CaCl
2aqueous dissolution fibroin fiber, fibroin fiber: CaCl
22H
2o: water=1g:25g:25ml, solution temperature 100 DEG C, dissolution time 5min.Filtered by the solution that obtains after dissolving, inject cellulose dialysis film and to dialyse 3d, cellulose dialysis retaining molecular weight used is 8000-14000.Silk fibroin solution is concentrated into 4wt%.Silk fibroin solution after sterilizing is cooled to about 0 DEG C, under the cryogenic conditions of about 0 DEG C, adds the hEGF (hEGF) of 0.5wt %, is added dropwise to 3wt % dimethyl sulfoxine, at about 100 revs/min slow Homogeneous phase mixing.Spray at-40 DEG C, collect ice microgranule.Continue at-40 DEG C, to preserve ice microgranule 48 hours.Ice microgranule is thawed at 0 ~ 4 DEG C.Eccysis coagulant at 0 ~ 4 DEG C, 0.5 hours changes water once, altogether changes water 5 times.Use collected by centrifugation fibroin microsphere, preserve stand-by after lyophilization.
In silver sulfadiazine ointment, add 10-20wt % hEGF fibroin microsphere, be directly coated in after mix homogeneously on the empyrosis wound surface after 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: by 500 grams of Cocoon shell 0.5wt % Na
2cO
3aqueous solution comes unstuck 2 times, each 30min, temperature of coming unstuck 100 DEG C, bath raio 1:100.Fully clean with water after coming unstuck, 60 DEG C of dryings, obtain fibroin fiber.With calcium chloride, water, dissolve with ethanol solution fibroin fiber that mol ratio is 1:8:2, solution temperature 70 DEG C, bath raio 1:25,10 minutes time.Dialyse injection cellulose dialysis film after the solution obtained after dissolving filtration 3d, and cellulose dialysis retaining molecular weight used is 8000-14000.Silk fibroin solution is concentrated into 5wt %, adds antibacterial peptide 1wt %, is added dropwise to 5wt % ethanol, about 100 revs/min slow Homogeneous phase mixing.Spray at-20 DEG C, collect ice microgranule.Continue at-20 DEG C, to preserve ice microgranule 96 hours.Thaw at ice microgranule 4 DEG C.Eccysis coagulant at 4 DEG C, 2 hours change water once, altogether change water 5 times.Filter screen collects fibroin microsphere, preserves stand-by after lyophilization.
Slow release fibroin microsphere compound can be entered in various paste coating medicine, along with the degraded of fibroin microsphere can slowly release antibacterial peptide, play antibacterial, to promote dermopathic healing effect.
Embodiment 4.
What prepare in the present embodiment is the fibroin microsphere that embedding feedstuff adds enzyme, and the step of the preparation method of this fibroin microsphere is as follows: use CaCl
2aqueous dissolution fibroin fiber, fibroin fiber: CaCl
22H
2o: water=1g:25g:25ml, solution temperature 100 DEG C, time 5min.Dialyse injection cellulose dialysis film after the solution obtained after dissolving filtration 3d, and cellulose dialysis retaining molecular weight used is 8000-14000.Silk fibroin solution is concentrated into 6wt %, adds enzyme, is added dropwise to 5wt % ethanol, 100 revs/min of slow Homogeneous phase mixing.Spray at-40 DEG C, collect ice microgranule.Continue at-40 DEG C, to preserve ice microgranule 96 hours.Ice microgranule is thawed at 4 DEG C.Eccysis coagulant at 4 DEG C, 2 hours change water once, altogether change water 3 times.Filter screen collects fibroin microsphere, preserves stand-by after lyophilization.
The fibroin microsphere embedding enzyme is mixed in animal feed, can the activity of extending enzyme, improve the active temperature of enzyme, widen enzymatic activity pH scope.
Embodiment 5.
The step of the preparation method of the fibroin microsphere in the present embodiment is as follows: the medicine adding required embedding and slow release in the regenerated silk solution of 3wt%, the medicine of this required embedding and slow release is nanoparticulate drug, and regenerated silk solution used is the regenerated silk solution of silkworm.Then be added dropwise to the coagulant of 2 wt%, slowly stir, obtain mixed solution under the rotating speed of 100 revs/min, coagulant used is dimethyl sulfoxine.Mixed solution is sprayed into the space condensation of-50 DEG C, collect ice microgranule, ice microgranule is continued to preserve more than 48 hours in the space of-50 DEG C.Then thawed at 15 DEG C by ice microgranule, eccysis coagulant at 15 DEG C, filter screen is collected and is obtained fibroin microsphere.At 15 DEG C during eccysis coagulant, can adopt within every 2 hours, change water once, altogether change the mode eccysis coagulant of water 5 times.
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 nanoparticulate drug, polypeptide drugs or enzyme drug, and regenerated silk solution used can be the regenerated silk solution of silkworm, Antherea pernyi Guerin-Meneville and/or other tussahs.Then the coagulant of 2-10 wt% can be added dropwise to, slowly can stir under the rotating speed of 80-120 rev/min, obtain mixed solution, coagulant used can be the one in dimethyl sulfoxine, soluble epoxide compound, methanol, ethanol, propanol, isopropyl alcohol, n-butyl alcohol and the tert-butyl alcohol.
The present invention mixed solution can be sprayed into-20 DEG C ~-80 DEG C space condensation, collect ice microgranule, ice microgranule is continued to preserve more than 24 hours in the space of-20 DEG C ~-80 DEG C, when coagulant used is soluble epoxide compound, mixed solution is sprayed into-20 DEG C ~-80 DEG C space condensation, after collecting ice microgranule, ice microgranule can continue to preserve more than 2 hours in the space of-20 DEG C ~-80 DEG C; When coagulant used is dimethyl sulfoxine, sprayed by mixed solution into the space condensation of-20 DEG C ~-80 DEG C, after collecting ice microgranule, ice microgranule can continue to preserve more than 12 hours in the space of-20 DEG C ~-80 DEG C; When coagulant used is propanol, isopropyl alcohol, n-butyl alcohol or the tert-butyl alcohol, sprayed by mixed solution into the space condensation of-20 DEG C ~-80 DEG C, after collecting ice microgranule, ice microgranule can continue to preserve more than 24 hours in the space of-20 DEG C ~-80 DEG C; When coagulant used be methanol or ethanol time, sprayed by mixed solution into the space condensation of-20 DEG C ~-80 DEG C, after collecting ice microgranule, ice microgranule can continue to preserve more than 48 hours in the space of-20 DEG C ~-80 DEG C.
The present invention can by ice microgranule at 0 DEG C ~ thawed at room temperature, can under 0 DEG C ~ room temperature eccysis coagulant, filter screen is collected and is obtained fibroin microsphere.Under 0 DEG C ~ room temperature during eccysis coagulant, can adopt and change water once every several hours, change water mode eccysis coagulant repeatedly.Said room temperature in the present invention, normal conditions refer to about 25 DEG C.
The particle diameter of the fibroin microsphere in the present invention is relevant with 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 DEG C ~ 4 DEG C preservations, also can preserve stand-by after low-temperature vacuum drying, can also preserve stand-by after lyophilization.
Can compound multi-medicament in fibroin microsphere of the present invention, fibroin microsphere can compound enter in various biological dressing again, and compound enters in various paste coating medicine, 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 adding 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 regenerated silk solution used is the regenerated silk solution of Antherea pernyi Guerin-Meneville.Then be added dropwise to the coagulant of 10 wt%, slowly stir, obtain mixed solution under the rotating speed of 80 revs/min, coagulant used is methanol.Mixed solution is sprayed into the space condensation of-20 DEG C, collect ice microgranule, ice microgranule is continued to preserve more than 96 hours in the space of-20 DEG C.Then thawed at 0 DEG C by ice microgranule, eccysis coagulant at 0 DEG C, filter screen is collected and is obtained fibroin microsphere.
Embodiment 7.
The step of the preparation method of the fibroin microsphere in the present embodiment is as follows: the medicine adding 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 regenerated silk solution used is the regenerated silk solution of Antherea pernyi Guerin-Meneville.Then be added dropwise to the coagulant of 9 wt%, slowly stir, obtain mixed solution under the rotating speed of 120 revs/min, coagulant used is propanol.Mixed solution is sprayed into the space condensation of-80 DEG C, collect ice microgranule, ice microgranule is continued to preserve more than 72 hours in the space of-80 DEG C.Then thawed at 25 DEG C by ice microgranule, eccysis coagulant at 25 DEG C, filter screen is collected and is obtained fibroin microsphere.
Embodiment 8.
The step of the preparation method of the fibroin microsphere in the present embodiment is as follows: the medicine adding 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 regenerated silk solution used is the regenerated silk solution of silkworm.Then be added dropwise to the coagulant of 6 wt%, slowly stir, obtain mixed solution under the rotating speed of 100 revs/min, coagulant used is n-butyl alcohol.Mixed solution is sprayed into the space condensation of-60 DEG C, collect ice microgranule, ice microgranule is continued to preserve more than 72 hours in the space of-60 DEG C.Then thawed at 10 DEG C by ice microgranule, eccysis coagulant at 10 DEG C, filter screen is collected and is obtained fibroin microsphere.
Embodiment 9.
The step of the preparation method of the fibroin microsphere in the present embodiment is as follows: the medicine adding required embedding and slow release in the regenerated silk solution of 6 wt%, the medicine of this required embedding and slow release is nanoparticulate drug, and regenerated silk solution used is the regenerated silk solution of Antherea pernyi Guerin-Meneville.Then be added dropwise to the coagulant of 3 wt%, slowly stir, obtain mixed solution under the rotating speed of 100 revs/min, coagulant used is isopropyl alcohol.Mixed solution is sprayed into the space condensation of-30 DEG C, collect ice microgranule, ice microgranule is continued to preserve more than 72 hours in the space of-30 DEG C.Then thawed at 20 DEG C by ice microgranule, eccysis coagulant at 20 DEG C, filter screen is collected and is obtained fibroin microsphere.At 20 DEG C during eccysis coagulant, can adopt within every 2 hours, change water once, altogether change the mode eccysis coagulant of water 5 times.
Embodiment 10.
The step of the preparation method of the fibroin microsphere in the present embodiment is as follows: the medicine adding 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 regenerated silk solution used is the regenerated silk solution of Antherea pernyi Guerin-Meneville.Then be added dropwise to the coagulant of 7 wt%, slowly stir, obtain mixed solution under the rotating speed of 100 revs/min, coagulant used is the tert-butyl alcohol.Mixed solution is sprayed into the space condensation of-40 DEG C, collect ice microgranule, ice microgranule is continued to preserve more than 72 hours in the space of-40 DEG C.Then thawed at 5 DEG C by ice microgranule, eccysis coagulant at 5 DEG C, filter screen is collected and is obtained fibroin microsphere.
Embodiment 11.
The step of the preparation method of the fibroin microsphere in the present embodiment is as follows: the medicine adding 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 regenerated silk solution used is the regenerated silk solution of silkworm.Then be added dropwise to the coagulant of 4 wt%, slowly stir, obtain mixed solution under the rotating speed of 105 revs/min, coagulant used is soluble epoxide compound.Mixed solution is sprayed into the space condensation of-70 DEG C, collect ice microgranule, ice microgranule is continued to preserve more than 24 hours in the space of-70 DEG C.Then thawed at 18 DEG C by ice microgranule, eccysis coagulant at 18 DEG C, filter screen is collected and is obtained fibroin microsphere.
Embodiment 12.
The step of the preparation method of the fibroin microsphere in the present embodiment is as follows: the medicine adding 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 regenerated silk solution used is Antherea pernyi Guerin-Meneville.Then be added dropwise to the coagulant of 5 wt%, slowly stir, obtain mixed solution under the rotating speed of 95 revs/min, coagulant used is ethanol.Mixed solution is sprayed into the space condensation of-55 DEG C, collect ice microgranule, ice microgranule is continued to preserve more than 96 hours in the space of-55 DEG C.Then thawed at 22 DEG C by ice microgranule, eccysis coagulant at 22 DEG C, filter screen is collected and is obtained fibroin microsphere.At 22 DEG C during eccysis coagulant, can adopt within every 2 hours, change water once, altogether change the mode eccysis coagulant of water 5 times.
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: by the Na of 1000 grams of Cocoon shell 0.5wt %
2cO
3aqueous solution comes unstuck 2 times, each 30min, temperature of coming unstuck 100 DEG C, bath raio 1:100.Fully clean with water after coming unstuck, 60 DEG C of dryings, obtain fibroin fiber.Use CaCl
2aqueous dissolution fibroin fiber, fibroin fiber: CaCl
22H
2o: water=1g:25g:25ml, solution temperature is 100 DEG C, and dissolution time is 5min.Dialyse injection cellulose dialysis film after the solution obtained after dissolving filtration 3d, and the molecular cut off of cellulose dialysis film used is 8000-14000.Silk fibroin solution is concentrated into 4wt%, adds 0.05wt% nanometer silver, is added dropwise to the diglycidyl ether of 3%, 100 revs/min of slow Homogeneous phase mixing.Spray at-20 DEG C, collect ice microgranule.Continue at-40 DEG C, to preserve ice microgranule 24 hours.By ice microgranule thawed at room temperature, eccysis coagulant under room temperature, 2 hours change water once, altogether change water 6 times.Collect fibroin microsphere with filter screen, preserve stand-by after low-temperature vacuum drying.
As shown in Figure 1, display be under transmission electron microscope, the combined state of nanometer silver in fibroin microsphere, accompanying drawing 2 show be the degradation speed of fibroin microsphere at 37 DEG C in protein enzyme solution.
Embodiment 14.
The step of the preparation method of the fibroin microsphere in the present embodiment is as follows: the medicine adding 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 regenerated silk solution used is the regenerated silk solution of Antherea pernyi Guerin-Meneville.Then be added dropwise to the coagulant of 6.5wt%, slowly stir, obtain mixed solution under the rotating speed of 98 revs/min, coagulant used is methanol.Mixed solution is sprayed into the space condensation of-20 DEG C, collect ice microgranule, ice microgranule is continued to preserve more than 96 hours in the space of-20 DEG C.Then thawed at 0 DEG C by ice microgranule, eccysis coagulant at 0 DEG C, filter screen is collected and is obtained fibroin microsphere.
Embodiment 15.
The step of the preparation method of the fibroin microsphere in the present embodiment is as follows: the medicine adding 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 regenerated silk solution used is the regenerated silk solution of Antherea pernyi Guerin-Meneville.Then be added dropwise to the coagulant of 10 wt%, slowly stir, obtain mixed solution under the rotating speed of 102 revs/min, coagulant used is methanol.Mixed solution is sprayed into the space condensation of-35 DEG C, collect ice microgranule, ice microgranule is continued to preserve more than 96 hours in the space of-20 DEG C.Then thawed at 8 DEG C by ice microgranule, eccysis coagulant at 8 DEG C, filter screen is collected and is obtained fibroin microsphere.
Although the present invention with embodiment openly as above; but it is also not used to limit protection scope of the present invention; any technical staff being familiar with this technology, not departing from the change and retouching done in the spirit and scope of the present invention, all should belong to protection scope of the present invention.
Claims (7)
1. the preparation method of a fibroin microsphere, it is characterized in that: the step of described preparation method is as follows: the medicine adding required embedding and slow release in the regenerated silk solution of 3-10 wt%, then the coagulant of 2-10 wt% is added dropwise to, slowly stir under the rotating speed of 80-120 rev/min, obtain mixed solution; Mixed solution is sprayed into the space condensation of-20 DEG C ~-80 DEG C, collect ice microgranule, ice microgranule is continued to preserve more than 2 hours in the space of-20 DEG C ~-80 DEG C; Then by ice microgranule at 0 DEG C ~ thawed at room temperature, eccysis coagulant under 0 DEG C ~ room temperature, centrifugal or filter screen is collected and is obtained fibroin microsphere, the medicine of described required embedding and slow release is nanoparticulate drug, polypeptide drugs or enzyme drug, and being fixed on the nanoparticulate drug in fibroin microsphere, polypeptide drugs or enzyme drug will discharge with fibroin microsphere degraded.
2. the preparation method of fibroin microsphere according to claim 1, is characterized in that: described regenerated silk solution is one in silkworm, Antherea pernyi Guerin-Meneville and other tussahs or two or more mixed regenerated silk solution.
3. the preparation method of fibroin microsphere according to claim 1, is characterized in that: described coagulant is the one in dimethyl sulfoxine, soluble epoxide compound, methanol, ethanol, propanol, isopropyl alcohol, n-butyl alcohol and the tert-butyl alcohol.
4. the preparation method of fibroin microsphere according to claim 1, is characterized in that: under 0 DEG C ~ room temperature during eccysis coagulant, adopts distilled water repeatedly to change the mode eccysis coagulant of water soaking.
5. the preparation method of fibroin microsphere according to claim 1, is characterized in that: the particle diameter of described fibroin microsphere is relevant with spraying fineness, and the particle diameter of fibroin microsphere is in a discrete distribution, and mean diameter is at 10 ~ 1000 microns.
6. the preparation method of fibroin microsphere according to claim 1, is characterized in that: described fibroin microsphere is stand-by 0 DEG C ~ 4 DEG C preservations, or preserves stand-by after low-temperature vacuum drying, or preserves stand-by after lyophilization.
7. the preparation method of fibroin microsphere according to claim 3, it is characterized in that: when coagulant used is soluble epoxide compound, mixed solution is sprayed into-20 DEG C ~-80 DEG C space condensation, after collecting ice microgranule, ice microgranule continues to preserve more than 2 hours in the space of-20 DEG C ~-80 DEG C; When coagulant used is dimethyl sulfoxine, sprayed by mixed solution into the space condensation of-20 DEG C ~-80 DEG C, after collecting ice microgranule, ice microgranule continues to preserve more than 12 hours in the space of-20 DEG C ~-80 DEG C; When coagulant used is propanol, isopropyl alcohol, n-butyl alcohol or the tert-butyl alcohol, sprayed by mixed solution into the space condensation of-20 DEG C ~-80 DEG C, after collecting ice microgranule, ice microgranule continues to preserve more than 24 hours in the space of-20 DEG C ~-80 DEG C; When coagulant used be methanol or ethanol time, sprayed by mixed solution into the space condensation of-20 DEG C ~-80 DEG C, after collecting ice microgranule, ice microgranule continues to preserve more than 48 hours in the space of-20 DEG C ~-80 DEG C.
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