CN103341175A - Preparation method of fibroin microsphere - Google Patents

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

A kind of preparation method of silk fibroin microsphere

technical field

The invention relates to a preparation method of silk fibroin microspheres, and the silk fibroin microspheres can be used as carriers for sustained release of medicines.

Background technique

Silk fibroin is composed of 18 kinds of amino acids. Materials made of silk fibroin have excellent biocompatibility and can be slowly degraded in vivo. Therefore, the application of silk fibroin microspheres in drug sustained-release carriers has attracted extensive attention. In the preparation of silk fibroin materials in the form of microspheres, there are currently emulsification (Yaowalak Srisuwanl), coagulation precipitation (Joydip Kundu), spray drying (Joo-Hong Yeo), spray freezing-freeze drying-crosslinking (Esther wenk) and other methods . For example, the publication date is August 20, 2008, and the Chinese patent publication number CN101244277 discloses a preparation method of silk fibroin drug-loaded microspheres. Assembly technology, after fully mixing the water-soluble drug and the regenerated silk fibroin solution, adding it to the oil phase containing a certain amount of emulsifier in the stirring to emulsify, and then adding an organic solvent to stir, so that the silk fibroin is denatured and its structure is changed to form a milky white crystal. At the same time as the silk fibroin particles, the drug is embedded, and the supernatant is removed by centrifugation, and then an organic solvent is added to further crystallize the silk fibroin. The preparation method of the drug-loaded microsphere belongs to the emulsification method.

Microspheres with different preparation methods have different effects when embedding different drugs, and the release mechanism and time are also different. The design of the microsphere preparation method needs to be based on the characteristics of the drug. At present, there is no high embedding efficiency. It can be degraded and released slowly and evenly, and has excellent biocompatibility, and is suitable for the preparation method of silk fibroin microspheres for embedding and slow-release nanoparticle medicines, polypeptide medicines and enzyme medicines.

Contents of the invention

The purpose of the present invention is to overcome the above-mentioned deficiencies in the prior art, and provide a method for preparing silk fibroin microspheres with high embedding efficiency, slow and uniform drug release along with the degradation of silk fibroin microspheres, and excellent biocompatibility .

The technical solution adopted by the present invention to solve the above problems is: the preparation method of the silk fibroin microspheres is characterized in that: the steps of the preparation method are as follows: add the required embedding and Slowly release the drug, then add 2-10 wt% coagulant dropwise, and slowly stir at a speed of 80-120 rpm to obtain a mixed solution; spray the mixed solution into a space of -20°C to -80°C to condense, Collect the ice particles, keep the ice particles in the space of -20°C ~ -80°C for more than 2 hours; then thaw the ice particles at 0°C ~ room temperature, wash off the coagulant at 0°C ~ room temperature, centrifuge or filter Collect silk fibroin microspheres. The silk fibroin microspheres in the present invention have the advantages of high embedding efficiency and excellent biocompatibility, and the drug can be released slowly and uniformly with the degradation of the silk fibroin microspheres. used in biomaterials.

Preferably, the drug required for embedding and sustained release in the present invention is a nanoparticle drug, a polypeptide drug or an enzyme drug.

Preferably, the regenerated silk fibroin solution of the present invention is a mixed regenerated silk fibroin solution of one or more silks of silkworms, tussah silkworms and other wild silkworms.

Preferably, the coagulant in the present invention is one of dimethyl sulfoxide, water-soluble epoxy compound, methanol, ethanol, propanol, isopropanol, n-butanol and tert-butanol.

Preferably, in the present invention, when the coagulant is washed out at 0° C. to room temperature, the coagulant is washed out by soaking in distilled water several times.

Preferably, the particle size of the silk fibroin microspheres in the present invention is related to the spray fineness, the particle size of the silk fibroin microspheres is discretely distributed, and the average particle size is 10-1000 microns.

Preferably, the silk fibroin microspheres of the present invention are stored at 0°C to 4°C until use, or after low-temperature vacuum drying, or after freeze-drying.

As a preference, when the coagulant used in the present invention is a water-soluble epoxy compound, the mixed solution is sprayed into a space of -20°C to -80°C to condense, and after collecting the ice particles, the ice particles continue to cool at -20°C to -80°C When the coagulant used is dimethyl sulfoxide, the mixed solution is sprayed into a space of -20°C to -80°C to condense, and after collecting the ice particles, the ice particles continue to cool at -20°C to -80°C. Store in a space of ℃ for more than 12 hours; when the coagulant used is propanol, isopropanol, n-butanol or tert-butanol, spray the mixed solution into a space of -20℃～-80℃ to condense, and collect ice particles , the ice particles continue to be stored in the space of -20°C to -80°C for more than 24 hours; when the coagulant used is methanol or ethanol, spray the mixed solution into the space of -20°C to -80°C to condense, and collect the ice particles , The ice particles continue to be stored in the space of -20℃～-80℃ for more than 48 hours.

Compared with the prior art, the present invention has the following advantages and effects: high embedding efficiency, slow drug release along with the degradation of silk fibroin microspheres, excellent biocompatibility; drug slow-release silk fibroin microspheres can be used directly, and It can be compounded into other materials and feed, etc.; the invention has formed water-insoluble silk fibroin particles in the process of spraying and freezing, without further freeze-drying and cross-linking; the internal and external solidification structure of silk fibroin microspheres is uniform, and the degradation is slow Uniform; because the silk fibroin microspheres are solidified at low temperature, the curing agent does not react with silk fibroin and drugs, and these water-soluble curing agents can be soaked and removed, while nanoparticle drugs, polypeptides, and enzyme drugs are weakly combined with silk fibroin. Fixed in the silk fibroin microspheres will be released with the degradation of the silk fibroin microspheres. The invention is especially suitable for embedding and slow-releasing nano particle medicine, polypeptide and enzyme medicine.

The difference between the present invention and the spray freezing-freeze drying-crosslinking method lies in the following aspects, (1) the present invention has formed water-insoluble silk fibroin particles during the spray freezing process, and no further freeze drying-crosslinking method is required. In the present invention, freeze-drying is only used for drying, so low-temperature vacuum drying can also be used, and it can also be used directly; and the spray freeze-freeze-drying-crosslinking method must go through freeze-drying-crosslinking. (2) The internal and external solidification structure of the silk fibroin microspheres of the present invention is uniform, so the degradation is slow and uniform; the spray freezing-freeze drying-crosslinking method is crosslinked and solidified after the microspheres are formed, and the internal and external solidification structures are easy to be uneven and fast degradation. (3) The present invention cures silk fibroin microspheres at low temperature, and the curing agent does not react with silk fibroin and drugs. These water-soluble curing agents can be soaked and removed, while nanoparticle drugs, polypeptides, enzyme drugs and silk fibroin are weak. Combined, it is fixed in the silk fibroin microspheres and will be released with the degradation of the silk fibroin microspheres; the spray freeze-freeze-drying-crosslinking method requires high concentrations of crosslinking agents and temperatures after the microspheres are formed and then crosslinked and solidified and other cross-linking reaction conditions, which may cause the cross-linking agent to react with the embedded drug.

Description of drawings

Fig. 1 is a schematic diagram of the composite state of nano-silver in silk fibroin microspheres in Example 13 of the present invention observed through a transmission electron microscope.

Fig. 2 is a schematic diagram of the degradation rate of the silk fibroin microspheres in Example 13 of the present invention in a protease solution at 37°C.

Detailed ways

The present invention will be further described in detail below in conjunction with the accompanying drawings and examples. The following examples are explanations of the present invention and the present invention is not limited to the following examples.

Example 1.

Prepared in the present embodiment are silk fibroin microspheres of slow-release nano-silver. The steps of _the preparation method of the silk fibroin microspheres are as follows: 1000 grams of silkworm cocoon shells are degummed twice with 0.5wt% _Na2CO3 aqueous solution. 30 minutes each time, the degumming temperature is 100°C, and the liquor ratio is 1:100. After degumming, it is fully washed with water and dried at 60°C to obtain silk fibers. Dissolve the silk fiber with CaCl ₂ aqueous solution, silk fiber: CaCl ₂ ·2H ₂ O:water=1g:25g:25ml, the dissolution temperature is 100°C, and the dissolution time is 5min. The solution obtained after dissolving is filtered and injected into a cellulose dialysis membrane for dialysis for 3 days. The molecular weight cut-off of the cellulose dialysis membrane used is 8000-14000. Concentrate the silk fibroin solution to 4wt%, add 0.3wt% nano-silver, dropwise add 5% diglycidyl ether, and mix slowly and uniformly at 100 rpm. Spray at -20°C to collect ice particles. Continue to store the ice particles at -20°C for 24 hours. Thaw the ice particles at room temperature, wash off the coagulant at room temperature, change the water every 2 hours, and change the water 3 times in total. The silk fibroin microspheres were collected with a filter, dried in vacuum at low temperature and stored for later use.

During the preparation process of silk fibroin dressing, 2-10wt% silk fibroin microspheres were evenly mixed into the silk fibroin solution to make a silk fibroin dressing with sustained release of nano-silver. The preparation method of silk dressing can refer to the Chinese invention patent whose patent number is ZL 200510060655.9.

In the preparation process of collagen biological dressings, 5wt% nano-silver fibroin microspheres are compounded, and nano-silver and silk fibroin can be slowly released with the degradation of silk fibroin microspheres, which can play a long-term antibacterial role and promote wound healing. effect.

Example 2.

Prepared in the present embodiment are silk fibroin microspheres of slow-release growth factor, and the steps of _the preparation method of the silk fibroin microspheres are as follows: 1000 grams of silkworm cocoon shells are degummed with 0.5wt% _Na2CO3 aqueous solution twice, each 30 minutes each time, the degumming temperature is 100°C, and the bath ratio is 1:100. After degumming, it is fully washed with water and dried at 60°C to obtain silk fibers. Dissolve silk fiber with CaCl ₂ aqueous solution, silk fiber: CaCl ₂ ·2H ₂ O:water=1g:25g:25ml, dissolution temperature 100°C, dissolution time 5min. The solution obtained after dissolving is filtered, poured into a cellulose dialysis membrane for dialysis for 3 days, and the molecular weight cut-off of the cellulose dialysis membrane used is 8000-14000. The silk fibroin solution was concentrated to 4 wt%. Cool the sterilized silk fibroin solution to about 0°C, add 0.5wt% human epidermal growth factor (hEGF) at a low temperature of about 0°C, and add 3wt% dimethyl sulfoxide dropwise, at about 100 rpm Mix slowly and evenly. Spray at -40°C to collect ice particles. Continue to store the ice particles at -40°C for 48 hours. Thaw the ice particles at 0-4°C. Wash off the coagulant at 0-4°C, change the water once every 0.5 hours, and change the water 5 times in total. The silk fibroin microspheres were collected by centrifugation, freeze-dried and stored for later use.

In the silver sulfadiazine ointment, add 10-20wt% hEGF silk fibroin microspheres, mix well and apply directly on the burn and scald wound after debridement. With the degradation of silk fibroin microspheres, hEGF and silk fibroin can be slowly released to promote wound healing.

Example 3.

Prepared in the present embodiment are silk fibroin microspheres of slow-release antimicrobial peptides, and the steps of the preparation method of the silk fibroin microspheres are as follows: 500 grams of silkworm cocoon shells are degummed with 0.5wt% _Na CO _aqueous solution for 2 times, each time 30min, degumming temperature 100℃, bath ratio 1:100. After degumming, it is fully washed with water and dried at 60°C to obtain silk fibers. Dissolve silk fiber with calcium chloride, water and ethanol solution with a molar ratio of 1:8:2, the dissolution temperature is 70°C, the bath ratio is 1:25, and the time is 10 minutes. The solution obtained after dissolving is filtered and poured into a cellulose dialysis membrane for dialysis for 3 days. The molecular weight cut-off of the cellulose dialysis membrane used is 8000-14000. Concentrate the silk fibroin solution to 5wt%, add 1wt% antibacterial peptide, add 5wt% ethanol dropwise, and mix slowly and uniformly at about 100 rpm. Spray at -20°C to collect ice particles. Continue to store the ice particles at -20°C for 96 hours. Thaw the ice particles at 4°C. Wash off the coagulant at 4°C, change the water every 2 hours, and change the water 5 times in total. The silk fibroin microspheres were collected by a filter, freeze-dried and stored for later use.

The slow-release silk fibroin microspheres can be compounded into various ointment coating drugs, and the antimicrobial peptides can be slowly released along with the degradation of the silk fibroin microspheres, which can inhibit bacteria and promote the healing of skin diseases.

Example 4.

In this example, silk fibroin microspheres embedded with enzymes added to feed were prepared. The steps of the preparation method of the silk microspheres were as follows: dissolve silk fiber with CaCl ₂ aqueous solution, silk fiber: CaCl ₂ 2H ₂ O: Water=1g:25g:25ml, dissolution temperature 100°C, time 5min. The solution obtained after dissolving is filtered and poured into a cellulose dialysis membrane for dialysis for 3 days. The molecular weight cut-off of the cellulose dialysis membrane used is 8000-14000. Concentrate the silk fibroin solution to 6wt%, add enzyme, add 5wt% ethanol dropwise, and mix slowly and uniformly at 100 rpm. Spray at -40°C to collect ice particles. Continue to store the ice particles at -40°C for 96 hours. The ice particles were thawed at 4°C. Wash off the coagulant at 4°C, change the water every 2 hours, and change the water 3 times in total. The silk fibroin microspheres were collected by a filter, freeze-dried and stored for later use.

Mixing the enzyme-embedded silk fibroin microspheres into animal feed can prolong the activity of the enzyme, increase the activity temperature of the enzyme, and widen the pH range of the enzyme activity.

Example 5.

The steps of the preparation method of silk fibroin microspheres in this example are as follows: Add the required embedding and sustained-release drug to the 3wt% regenerated silk fibroin solution, and the required embedded and sustained-release drug is a nanoparticle drug , the regenerated silk fibroin solution used is the regenerated silk fibroin solution of silkworm. Then dropwise add the coagulant of 2 wt%, stir slowly under the rotating speed of 100 rev/mins, obtain mixed solution, used coagulant is dimethyl sulfoxide. Spray the mixed solution into the -50°C space to condense, collect the ice particles, and keep the ice particles in the -50°C space for more than 48 hours. Then the ice particles were thawed at 15°C, the coagulant was washed off at 15°C, and the silk fibroin microspheres were collected by a filter. When washing off the coagulant at 15°C, the water can be changed every 2 hours for a total of 5 times to wash off the coagulant.

The present invention can add the drug required for embedding and sustained release into the regenerated silk fibroin solution of 3-10 wt%. The drug required for embedding and sustained release can be nanoparticle drug, polypeptide drug or enzyme drug. The regenerated silk fibroin solution can be the regenerated silk fibroin solution of silkworm, tussah silkworm and/or other wild silkworms. Then the coagulant of 2-10 wt% can be added dropwise, and can be slowly stirred at a rotating speed of 80-120 rpm to obtain a mixed solution. The coagulant used can be dimethyl sulfoxide, water-soluble epoxy compound, methyl alcohol, One of ethanol, propanol, isopropanol, n-butanol and tert-butanol.

The present invention can spray the mixed solution into the space of -20°C to -80°C to condense, collect the ice particles, and keep the ice particles in the space of -20°C to -80°C for more than 24 hours. When the coagulant used is water-soluble For epoxy compounds, spray the mixed solution into the space of -20℃～-80℃ to condense. After collecting the ice particles, the ice particles can continue to be stored in the space of -20℃～-80℃ for more than 2 hours; when the coagulant used When it is dimethyl sulfoxide, spray the mixed solution into the space of -20℃～-80℃ to condense, and after collecting the ice particles, the ice particles can continue to be stored in the space of -20℃～-80℃ for more than 12 hours; when the used When the coagulant is propanol, isopropanol, n-butanol or tert-butanol, spray the mixed solution into the space of -20℃～-80℃ to condense. After collecting the ice particles, the ice particles can continue to cool at -20℃～- Store at 80°C for more than 24 hours; when the coagulant used is methanol or ethanol, spray the mixed solution into a space at -20°C to -80°C to condense, and after collecting ice particles, the ice particles can continue to cool at -20°C to Store at -80°C for more than 48 hours.

The invention can thaw the ice particles at 0°C to room temperature, wash off the coagulant at 0°C to room temperature, and collect the silk fibroin microspheres through a filter screen. When washing off the coagulant at 0°C to room temperature, you can wash off the coagulant by changing the water every few hours and changing the water several times. The room temperature mentioned in the present invention usually refers to about 25°C.

The particle size of the silk fibroin microspheres in the present invention is related to the spray fineness, and the particle size of the silk fibroin microspheres is discretely distributed, with an average particle size of 10-1000 microns. The silk fibroin microspheres prepared by the present invention can be stored at 0° C. to 4° C. for use, can also be stored for use after low-temperature vacuum drying, and can also be stored for use after being freeze-dried.

A variety of drugs can be compounded in the silk fibroin microspheres of the present invention, and the silk fibroin microspheres can be compounded into various biological dressings and various ointment coating drugs, and the silk fibroin microspheres can be slowly degraded. Release the drug and play a therapeutic role.

Example 6.

The steps of the preparation method of the silk fibroin microspheres in this embodiment are as follows: Add the required embedding and slow-release drug to the 10 wt% regenerated silk fibroin solution, the required embedding and slow-release drug is a polypeptide drug , the regenerated silk fibroin solution used is the regenerated silk fibroin solution of tussah. Then dropwise add the coagulant of 10 wt%, stir slowly under the rotating speed of 80 rev/mins, obtain mixed solution, used coagulant is methanol. Spray the mixed solution into the -20°C space to condense, collect the ice particles, and keep the ice particles in the -20°C space for more than 96 hours. Then the ice particles were thawed at 0°C, the coagulant was washed off at 0°C, and the silk fibroin microspheres were collected by a filter.

Example 7.

The steps of the preparation method of the silk fibroin microspheres in this example are as follows: Add the required embedding and slow-release drug to the 5 wt% regenerated silk fibroin solution, and the required embedding and slow-release drug is an enzyme Medicine, the regenerated silk fibroin solution used is the regenerated silk fibroin solution of tussah. Then dropwise add the coagulant of 9 wt%, stir slowly under the rotating speed of 120 revs/min, obtain mixed solution, used coagulant is propanol. Spray the mixed solution into the -80°C space to condense, collect the ice particles, and keep the ice particles in the -80°C space for more than 72 hours. Then the ice particles were thawed at 25°C, the coagulant was washed off at 25°C, and the silk fibroin microspheres were collected by a filter.

Example 8.

The steps of the preparation method of the silk fibroin microspheres in this embodiment are as follows: Add the required embedding and slow-release drug to the 8 wt% regenerated silk fibroin solution, and the required embedding and slow-release drug is a polypeptide drug , the regenerated silk fibroin solution used is the regenerated silk fibroin solution of silkworm. Add dropwise the coagulant of 6 wt% then, stir slowly under the rotating speed of 100 rev/mins, obtain mixed solution, used coagulant is n-butanol. Spray the mixed solution into the -60°C space to condense, collect the ice particles, and keep the ice particles in the -60°C space for more than 72 hours. Then the ice particles were thawed at 10°C, the coagulant was washed off at 10°C, and the silk fibroin microspheres were collected by a filter.

Example 9.

The steps of the preparation method of the silk fibroin microspheres in this embodiment are as follows: Add the required embedding and slow-release drug to the 6 wt% regenerated silk fibroin solution, and the required embedding and slow-release drug is a nanoparticle Medicine, the regenerated silk fibroin solution used is the regenerated silk fibroin solution of tussah. Then dropwise add the coagulant of 3 wt%, stir slowly under the rotating speed of 100 rev/mins, obtain mixed solution, used coagulant is isopropanol. Spray the mixed solution into the -30°C space to condense, collect the ice particles, and keep the ice particles in the -30°C space for more than 72 hours. Then the ice particles were thawed at 20°C, the coagulant was washed off at 20°C, and the silk fibroin microspheres were collected by a filter. When washing off the coagulant at 20°C, the water can be changed every 2 hours for a total of 5 times to wash off the coagulant.

Example 10.

The steps of the preparation method of the silk fibroin microspheres in this embodiment are as follows: Add the required embedding and slow-release drug to the 4 wt% regenerated silk fibroin solution, and the required embedded and slow-release drug is an enzyme Medicine, the regenerated silk fibroin solution used is the regenerated silk fibroin solution of tussah. Then dropwise add the coagulant of 7wt%, stir slowly under the rotating speed of 100 rev/mins, obtain mixed solution, used coagulant is tert-butanol. Spray the mixed solution into the -40°C space to condense, collect the ice particles, and keep the ice particles in the -40°C space for more than 72 hours. Then the ice particles were thawed at 5°C, the coagulant was washed off at 5°C, and the silk fibroin microspheres were collected by a filter.

Example 11.

The steps of the preparation method of the silk fibroin microspheres in this embodiment are as follows: Add the required embedding and slow-release drug to the 7 wt% regenerated silk fibroin solution, and the required embedding and slow-release drug is an enzyme Medicine, the regenerated silk fibroin solution used is the regenerated silk fibroin solution of silkworm. Then dropwise add the coagulating agent of 4 wt%, slowly stir under the rotating speed of 105 rev/mins, obtain mixed solution, used coagulating agent is water-soluble epoxy compound. Spray the mixed solution into the -70°C space to condense, collect the ice particles, and keep the ice particles in the -70°C space for more than 24 hours. Then the ice particles were thawed at 18°C, the coagulant was washed off at 18°C, and the silk fibroin microspheres were collected by a filter.

Example 12.

The steps of the preparation method of the silk fibroin microspheres in this example are as follows: Add the required embedding and slow-release drug to the 9 wt% regenerated silk fibroin solution, the required embedding and slow-release drug is a polypeptide drug , the regenerated silk solution used is tussah. Then dropwise add the coagulant of 5 wt%, stir slowly under the rotating speed of 95 rev/mins, obtain mixed solution, used coagulant is ethanol. Spray the mixed solution into the -55°C space to condense, collect the ice particles, and keep the ice particles in the -55°C space for more than 96 hours. Then the ice particles were thawed at 22°C, the coagulant was washed off at 22°C, and the silk fibroin microspheres were collected by a filter. When washing off the coagulant at 22°C, the water can be changed every 2 hours for a total of 5 times to wash off the coagulant.

Example 13.

Prepared in the present embodiment are silk fibroin microspheres of slow-release nano-silver. The steps of _the preparation method of the silk fibroin microspheres are as follows: 1000 grams of silkworm cocoon shells are degummed twice with 0.5wt% _Na2CO3 aqueous solution. 30 minutes each time, the degumming temperature is 100°C, and the liquor ratio is 1:100. After degumming, it is fully washed with water and dried at 60°C to obtain silk fibers. Dissolve the silk fiber with CaCl ₂ aqueous solution, silk fiber: CaCl ₂ ·2H ₂ O:water=1g:25g:25ml, the dissolution temperature is 100°C, and the dissolution time is 5min. The solution obtained after dissolving is filtered and injected into a cellulose dialysis membrane for dialysis for 3 days. The molecular weight cut-off of the cellulose dialysis membrane used is 8000-14000. Concentrate the silk fibroin solution to 4wt%, add 0.05wt% nano-silver, dropwise add 3% diglycidyl ether, and mix slowly and uniformly at 100 rpm. Spray at -20°C to collect ice particles. Continue to store the ice particles at -40°C for 24 hours. Thaw the ice particles at room temperature, wash off the coagulant at room temperature, change the water every 2 hours, and change the water 6 times in total. The silk fibroin microspheres were collected with a filter, dried in vacuum at low temperature and stored for later use.

As shown in accompanying drawing 1, it shows the composite state of nano-silver in silk fibroin microspheres under a transmission electron microscope, and accompanying drawing 2 shows the degradation rate of silk fibroin microspheres in a protease solution at 37°C.

Example 14.

The steps of the preparation method of the silk fibroin microspheres in this example are as follows: Add the required embedding and slow-release drug to the 5.5wt% regenerated silk fibroin solution, and the required embedding and slow-release drug is a polypeptide drug , the regenerated silk fibroin solution used is the regenerated silk fibroin solution of tussah. Then 6.5wt% coagulant was added dropwise and stirred slowly at a speed of 98 rpm to obtain a mixed solution. The coagulant used was methanol. Spray the mixed solution into the -20°C space to condense, collect the ice particles, and keep the ice particles in the -20°C space for more than 96 hours. Then the ice particles were thawed at 0°C, the coagulant was washed off at 0°C, and the silk fibroin microspheres were collected by a filter.

Example 15.

The steps of the preparation method of the silk fibroin microspheres in this example are as follows: Add the required embedding and slow-release drug to the 10 wt% regenerated silk fibroin solution, and the required embedding and slow-release drug is a polypeptide drug , the regenerated silk fibroin solution used is the regenerated silk fibroin solution of tussah. Then dropwise add the coagulant of 10 wt%, slowly stir under the rotating speed of 102 rev/mins, obtain mixed solution, used coagulant is methyl alcohol. Spray the mixed solution into a space of -35°C to condense, collect ice particles, and keep the ice particles in a space of -20°C for more than 96 hours. Then the ice particles were thawed at 8°C, the coagulant was washed off at 8°C, and the silk fibroin microspheres were collected by a filter.

Although the present invention has been disclosed as above with the embodiments, it is not intended to limit the scope of protection of the present invention. Any changes and modifications made by those skilled in the art without departing from the concept and scope of the present invention shall be Belong to the 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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