Silk fibroin microspheres, and preparation method and application thereof

Abstract

The application provides a silk fibroin microsphere and a preparation method and application thereof, and belongs to the technical field of biopolymer microsphere preparation. The preparation method comprises the following steps: 1) adding an ionic dispersing agent into a silk fibroin solution, uniformly mixing, then adding a sphere forming agent into the solution, and obtaining a pretreated liquid after mixing; 2) performing low-temperature coagulation treatment on the pretreated liquid, and obtaining a silk fibroin microsphere solution after thawing; 3) diluting the silk fibroin microsphere solution, then performing wet heat crystallization treatment, and obtaining a high-crystallinity silk fibroin microsphere solution; and 4) performing spray drying on the high-crystallinity silk fibroin microsphere solution, and obtaining a solid silk fibroin microsphere. The silk fibroin microsphere obtained by the application has excellent biocompatibility, degradability and good mechanical properties. The silk fibroin microsphere prepared by the method of the application is regular spherical, has high crystallinity, slow degradation speed, is not easy to absorb moisture and adhere, and can be stored for a long time; and the preparation method is simple and has high yield.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomacromolecule microsphere preparation, and particularly relates to a silk fibroin microsphere and a preparation method and application thereof. BACKGROUND

[0002] Related researches show that in clinical trials, injectable micro / nanoparticles, hydrogels or implantable devices and other polymers can be used as embedding matrices of various therapeutic drugs to treat various diseases, and such materials have advantages of mild processing, high biocompatibility, slow degradation and controllable release. Among various polymers used, silk fibroin (SF) as a natural macromolecular structural protein has been widely used for drug sustained release, especially for drugs susceptible to environmental changes. Silk fibroin materials are also widely used in the fields of biological medicine and tissue engineering.

[0003] Silk fibroin contains 18 kinds of amino acids, about 80% of which are composed of glycine (Gly), alanine (Ala) and serine (Ser). The preparation of silk fibroin microspheres can be carried out under mild conditions and without the use of toxic chemicals. In the preparation process, SF in the solution is transformed from a random coil dominant structure to a beta-sheet dominant structure (i.e. Silk II crystal structure), so that a water-insoluble, thermodynamically stable biomaterial can be obtained.

[0004] At present, the preparation methods of silk fibroin microspheres mainly include enzyme method, spray drying method, emulsification-solvent evaporation method, template method, salting-out method, solvent-non-solvent method and electrospinning method.

[0005] In Chinese patent CN106474482B, "Silk fibroin nanoparticles and its preparation method and application", it is recorded that "the silk fibroin solution is quickly frozen in liquid nitrogen, then freeze-dried, followed by alcohol treatment, after centrifugation, the upper liquid is taken for washing, centrifugation and ultrasonic dispersion, and finally the silk fibroin nanoparticles with good dispersibility are obtained". This method must use low concentration of silk fibroin solution for liquid nitrogen freezing, which will cause a large amount of liquid nitrogen to be used, increasing the cost; and the microspheres treated by alcoholization must be centrifuged to take the upper liquid, and the lower precipitate is discarded, which will cause more waste and cost increase; moreover, the particle size range of the microspheres fluctuates greatly, which is uncontrollable, and the production is difficult, and the practicality is low; in addition, after ethanol treatment, simple water washing cannot effectively remove it, and its application in human body has certain harmfulness. Chinese patent CN102417733B, "Preparation method of silk fibroin nanospheres", discloses a method for preparing silk fibroin nanospheres by using ultrasonic reverse phase microemulsion in-situ drying method; the cyclohexane used in this method may remain in the microspheres after simple centrifugation, which has certain physiological toxicity, and cyclohexane is highly flammable and explosive, so the industrial mass production is greatly limited. Chinese patent CN103739691B, "Green and environmentally friendly method for preparing silk fibroin powder", discloses a method for preparing silk fibroin powder by using superfine crushing; the powder prepared by this method has large particle size and irregular shape. Yeo et al. reported in the literature "Yeo J H, Lee K G, Lee YW, et al. Simple preparation and characteristics of silk fibroin microsphere [J]. European Polymer Journal, 2003, 39(6) 1195-1199" that a spray drying method was used to prepare microspheres; the microspheres have irregular shape and are easy to absorb moisture in air. Wang et al. reported in the literature "Wang L, Xia ZR, Lv L L, et al. Preparation of silk fibroin microspheres [J]. Advanced Materials Research, 2011, 236-238: 1902-1905." that silk fibroin microspheres with particle size of about 100-400 μm and porous shape were prepared by using electrostatic spraying and freeze-drying method; this method has high requirements for equipment and low yield.

[0006] The existing silk fibroin microsphere preparation methods in the prior art have the problems of low yield, poor safety, and uneven particle size. SUMMARY

[0007] Therefore, the present application aims to provide a kind of silk fibroin microspheres with spherical shape, uniform particle size, simple preparation method, high yield, long-term storage and less moisture absorption and agglomeration, and a preparation method and application thereof.

[0008] The present application provides a kind of silk fibroin microspheres preparation method, comprising the following steps:

[0009] 1) after mixing ion dispersing agent with silk fibroin solution, add ball forming agent, mix to obtain pretreatment liquid;

[0010] 2) the pretreatment liquid is treated by low temperature coagulation, and silk fibroin microspheres solution is obtained after thawing;

[0011] 3) the silk fibroin microspheres solution is diluted, and then treated by wet heat crystallization to obtain high crystalline silk fibroin microspheres solution;

[0012] 4) the high crystalline silk fibroin microspheres solution is spray dried to obtain solid silk fibroin microspheres.

[0013] Preferably, in step 1), the ion dispersing agent is one or more of sodium chloride, potassium chloride, disodium hydrogen phosphate and dipotassium hydrogen phosphate;The mass of the ion dispersing agent is 0.1-2wt% of the mass of the silk fibroin solution;The ball forming agent is one or more of volatile organic solvents, including ethanol, isopropanol, acetone, acetonitrile, methyl ethyl ketone, ethyl acetate, methanol, 2-methyl-2-propanol and acetaldehyde.

[0014] Preferably, in step 1), the volume ratio of the ball forming agent to the silk fibroin solution is (1-18):20, and the ball forming agent is added to the silk fibroin solution within 3-5 minutes. The concentration of the silk fibroin solution is 1-60 mg / mL, and the average molecular weight of the silk fibroin in the silk fibroin solution is 5000 Da-20 kDa.

[0015] Preferably, in step 1), the mixing process is accompanied by stirring, the stirring speed is 60-120 rpm, and the mixing time is 5-25 min.

[0016] Preferably, in step 2), the low temperature coagulation treatment includes spray condensation, low temperature freezing or liquid nitrogen freezing;The temperature of the low temperature coagulation treatment is-80--20 ℃, and the time of the low temperature coagulation treatment is 12-48 h.

[0017] Preferably, in step 2), the thawing temperature is 10-30 ℃, and the step of dialysis is further included after thawing;The molecular weight cut-off of the dialysis material used in dialysis is 3500 Da;The dialysis time is 24-48 h;Wherein the dialysis water is replaced every 1-2 h.

[0018] Preferably, the dilution fold of the dilution in step 3) is 1-20; the temperature of the wet heat crystallization treatment is 100-134℃, and the time of the wet heat crystallization treatment is 5-30 min.

[0019] Preferably, the temperature of the spray drying in step 4) is 85-200℃, and the flow rate of the feed liquid in the spray drying process is 5-25 mL / min; after obtaining the solid silk fibroin microspheres in step 4), the solid silk fibroin microspheres are added into water for injection, a silk fibroin solution or normal saline to obtain a silk fibroin microsphere suspension.

[0020] The application also provides the silk fibroin microspheres obtained by the preparation method.

[0021] The application also provides the use of the silk fibroin microspheres in the preparation of sustained-release drugs.

[0022] Compared with the prior art, the application has the following beneficial effects: the preparation method of the silk fibroin microspheres provided by the application adopts the methods of balling with a balling agent, low-temperature coagulation, wet heat crystallization and spray drying to form silk fibroin microspheres. The silk fibroin microspheres obtained by the application are made of pure silk fibroin and have excellent biocompatibility, degradability and good mechanical properties. The microspheres prepared by the method of the application are regular spherical, have a particle size of about 100-5000 nm, a uniform particle size, a small difference, and a large specific surface area. The microspheres have a high crystallinity, a slow degradation speed, and are not easy to absorb moisture and adhere. The preparation method of the application can effectively improve the utilization of silk fibroin, has little waste, can make the high-volatility balling agent volatilize quickly through spray drying, has almost no residual organic matter, is suitable for industrial large-scale production, has very low production cost, and is green and harmless.

[0023] In the application, a volatile organic solvent that cannot dissolve silk fibroin is added to a silk fibroin solution to cause phase separation, induce silk fibroin to form a microcapsule structure, promote silk fibroin to change from a random coil structure to a β-sheet structure, and form a microgel state. Further, the solution is subjected to low-temperature coagulation treatment before each silk fibroin microsphere is completely formed into a gel to stop the movement of silk fibroin molecular chains and fix the silk fibroin molecular chains into a microsphere state. After the wet heat crystallization treatment, the sik II crystalline structure of the silk fibroin microspheres gradually increases, and the microsphere system is more stable.

[0024] Due to the great uncontrollability of the coagulation time point, although more silk fibroin microsphere particles can be formed, part of the silk fibroin is not formed into microspheres, resulting in a decrease in material utilization. The present application further adopts the method of water dialysis to remove the ball forming agent, and then performs spray drying, which can greatly increase the crystallinity of the formed silk fibroin microspheres, and at the same time can promote the silk fibroin that has not formed into microspheres to form microspheres, greatly improving the utilization of raw materials, and the high temperature can completely remove the volatile ball forming agent. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 is an electron microscope photo of the silk fibroin microspheres prepared in Example 2 of the present application;

[0026] Figure 2 is an electron microscope photo of the silk fibroin microspheres prepared in Example 2 of the present application, magnified by 20,000 times;

[0027] Figure 3 is an infrared spectrum of the silk fibroin microspheres prepared in Example 2;

[0028] Figure 4 is an infrared spectrum of the silk fibroin microspheres prepared in Example 4;

[0029] Figure 5 is an X-ray diffraction of the silk fibroin microspheres prepared in Example 5. DETAILED DESCRIPTION

[0030] The present application provides a preparation method of silk fibroin microspheres, comprising the following steps: 1) mixing an ionic dispersing agent with a silk fibroin solution, adding a ball forming agent, mixing to obtain a pretreated liquid; 2) performing low-temperature coagulation treatment on the pretreated liquid, and thawing to obtain a silk fibroin microsphere liquid; 3) diluting the silk fibroin microsphere liquid, and then performing wet heat crystallization treatment to obtain a high-crystallinity silk fibroin microsphere liquid; 4) performing spray drying on the high-crystallinity silk fibroin microsphere liquid to obtain solid silk fibroin microspheres.

[0031] In the present application, the concentration of the silk fibroin solution is preferably 1-60 mg / mL, further preferably 5-50 mg / mL, and more preferably 10-40 mg / mL; in the present application, the average molecular weight of the silk fibroin in the silk fibroin solution is 5000 Da-20 kDa. In the present application, the silk fibroin solution is preferably stored at 2-8℃, and further preferably at 4℃; in the present application, the silk fibroin solution is preferably prepared from mulberry silk, and the present application does not have special limitations on the preparation method of the silk fibroin solution, and a conventional preparation method of silk fibroin solution in the art can be used; see Example 1 of the present application for details.

[0032] After obtaining the silk fibroin solution, the ion dispersing agent is added into the silk fibroin solution and mixed uniformly, and then the ball forming agent is added into the solution to obtain a pretreatment liquid. In the present application, the ion dispersing agent is one or several of sodium chloride, potassium chloride, disodium hydrogen phosphate and dipotassium hydrogen phosphate; the mass of the ion dispersing agent is 0.1-2% of the mass of the silk fibroin solution, preferably 0.4-1.6%. The ball forming agent includes one or several of ethanol, isopropyl alcohol, acetone, acetonitrile, methyl ethyl ketone, ethyl acetate, methanol, 2-methyl-2-propanol and acetaldehyde. When the ball forming agent is two, in a specific implementation process of the present application, the ball forming agent is preferably isopropyl alcohol and methanol, and the volume ratio of the isopropyl alcohol and methanol is preferably 1:(8-10), more preferably 1:9; in another specific implementation process of the present application, the ball forming agent is preferably acetone and acetonitrile, and the volume ratio of the acetone and acetonitrile is preferably 1:(18-22), further preferably 1:20. In the present application, the volume ratio of the ball forming agent to the silk fibroin solution is preferably (1-18):20, further preferably (5-15):20; the ball forming agent is preferably added into the silk fibroin solution dropwise within 3-5 min. After the ball forming agent is added into the silk fibroin solution dropwise, mixing is performed, and the mixing is accompanied by stirring, the stirring speed is preferably 60-120 rpm, further preferably 70-90 rpm, more further preferably 80 rpm; the mixing time is preferably 5-25 min, further preferably 18-22 min, more further preferably 20 min.

[0033] In the present application, the pretreatment liquid is subjected to low-temperature coagulation treatment, and silk fibroin microsphere solution is obtained after thawing. In the present application, the low-temperature coagulation treatment preferably includes spray condensation, low-temperature freezing or liquid nitrogen freezing. The low-temperature coagulation temperature is -80 to -20℃, and the low-temperature coagulation treatment time is 12-48 h.

[0034] In the present application, the spray condensation is that the pretreatment liquid is stirred at a speed of 50-70 rpm for 5-30 min, then the pretreatment liquid is sprayed into ice particles by instantaneous condensation at a flow rate of 2-20 mL / min under a vacuum environment at -20℃ to -80℃ or in liquid nitrogen, and stored at -20℃ to -80℃ for 12-48 h. In the present application, the liquid nitrogen freezing is that the pretreatment liquid is stirred at a speed of 50-70 rpm for 15-30 min, then the container containing the pretreatment liquid is placed in liquid nitrogen until the silk fibroin solution is completely frozen, and then stored at -20℃ to -80℃ for 12-48 h. In the present application, the low-temperature freezing is that the pretreatment liquid is stirred at a low speed for 3-5 min, then the solution is directly frozen at -20℃ to -80℃, and stored for 12-48 h.

[0035] In this invention, the thawing temperature is preferably 10–30°C. The thawing time is not specifically limited, but is determined by the complete thawing of the solution, which is a milky white solution. Preferably, after thawing, the invention also includes a dialysis step. The molecular weight cutoff of the dialysis material is preferably 3500 Da. The dialysis material is preferably a dialysis tube or dialysis bag. The dialysis time is preferably 24–48 hours, more preferably 28–40 hours. The water is changed every 1–2 hours during the dialysis process.

[0036] In this invention, the silk fibroin microsphere solution is diluted and then subjected to hydrothermal crystallization to obtain a highly crystalline silk fibroin microsphere solution. In this invention, the temperature of the hydrothermal crystallization treatment is 100–134°C, preferably 105–130°C; the time of the hydrothermal crystallization treatment is preferably 5–30 min, more preferably 10–20 min.

[0037] In this invention, the highly crystalline silk fibroin microsphere liquid is spray-dried to obtain solid silk fibroin microspheres. In this invention, the spray-drying temperature is 85℃~200℃, more preferably 100~190℃; the flow rate of the liquid during the spray-drying process is preferably 5~25mL / min, more preferably 10~20mL / min.

[0038] In this invention, after obtaining solid silk fibroin microspheres, the solid silk fibroin microspheres are added to water for injection, silk fibroin solution, or physiological saline to obtain a silk fibroin microsphere suspension. In this invention, the dissolution is preferably assisted by ultrasound, stirring, or oscillation, the frequency of the ultrasound is 20-40 kHz, and the dissolution time is 3-5 minutes.

[0039] The present invention also provides silk fibroin microspheres prepared by the aforementioned preparation method. In the present invention, the silk fibroin microspheres exist in the form of powder or silk fibroin microsphere suspension.

[0040] This invention also provides the application of the aforementioned silk fibroin microspheres in the preparation of sustained-release drugs. In this invention, the silk fibroin microspheres are non-cytotoxic and can be used as a matrix or carrier in the preparation of sustained-release drugs.

[0041] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0042] Example 1

[0043] The preparation steps for silk fibroin solution are as follows:

[0044] Accurately weigh 500g of fresh, clean mulberry silk using a balance. Prepare 50L of 0.02M sodium carbonate aqueous solution. Heat the 50L of water to boiling, add 106g of sodium carbonate, stir well, then add the mulberry silk and simmer for 30 minutes. Next, wash the silk with purified water to remove the sericin from the surface. Repeat the degumming process three times. Dry the degummed, moistened silk in an oven at 60℃ for 12 hours to obtain dried, degummed silk.

[0045] Degummed silk was dissolved in a 9.3M lithium bromide solution at a mass ratio of 1:4 at 65°C. The silk was stirred at 60 rpm for 2 hours to fully dissolve, yielding a silk fibroin-lithium bromide mixture. After cooling, the mixture was placed in an ultrafiltration unit (molecular weight cutoff of 3000 Da–5000 Da) and desalted with purified water to obtain a pure silk fibroin solution, which was then stored at 4°C for later use.

[0046] In this embodiment, the concentration of the pure silk fibroin solution was calculated by the drying and weighing method, and the concentration was approximately 60 mg / mL, wherein the average molecular weight of the silk fibroin was 5000 Da to 20 kDa.

[0047] Example 2

[0048] Take the silk fibroin solution from Example 1, and then perform the following steps:

[0049] (1) Preparation of initial solution: Prepare a 500mL solution (containing 15g pure silk fibroin) by mixing silk fibroin solution and water for injection. Stir at 60rpm and let stand until the solution is clear and transparent.

[0050] (2) Addition of ionic dispersant: Add 0.9wt% NaCl to the initial silk fibroin solution.

[0051] (3) Addition of spheroidizing agent: Add 50 mL of anhydrous ethanol dropwise to the silk fibroin solution within 5 min, and stir with a glass rod at 80 rpm for 20 min.

[0052] (4) Formation of silk fibroin microspheres: The homogenized solution was sprayed in liquid nitrogen at a flow rate of 2 mL / min to obtain ice particles. The ice particles were collected and stored at -20℃ for 20 h. Then, the ice particles were thawed at 30℃ to obtain a milky white solution. The milky white solution was then placed in a dialysis bag (molecular weight cutoff of 3500 Da) and dialyzed for 24 h, with the water changed every 2 h to remove anhydrous ethanol.

[0053] (5) Crystallization treatment of silk fibroin microspheres: Dilute the dialyzed microsphere solution by 1 time, and then treat it with wet heat at 110℃ for 15 min to improve the crystallinity of the microspheres.

[0054] (6) Enhanced treatment of silk fibroin microspheres: The crystallized microsphere solution was spray-dried at 120°C at a flow rate of 10 mL / min to remove residual anhydrous ethanol, improve the crystallinity of the microspheres, increase their water insolubility, and finally obtain pure and dry silk fibroin microsphere powder.

[0055] (7) Packaging: The silk fibroin microsphere powder is packaged in 10g / vial and stored in a lyophilized bottle.

[0056] (8) Swelling and Loss Performance: 1g of silk fibroin microsphere powder was dried to constant weight in a 60℃ oven. Three parallel samples were weighed and recorded as M1. PBS buffer solution was added at a bath ratio of 1:100, and the mixture was kept at a constant temperature and shaken in a 37℃ water bath for 24 hours. The microspheres were then removed, placed on an ultrafiltration membrane, rinsed three times with purified water, and the remaining moisture on the surface of the microspheres was removed with absorbent kitchen paper. The weight of the microspheres was recorded as M2. The microspheres were then dried to constant weight in a 60℃ oven and the weight was recorded as M3. The swelling rate of the microspheres was calculated to be 18.9% ± 2.2%, and the loss rate was 17.2% ± 1.3%. Compared with pure silk fibroin material, both the swelling rate and loss rate were relatively low.

[0057] Dissolution rate = (M1 - M3) / M1 × 100%

[0058] Swelling rate = (M2 - M1) / M1 × 100%

[0059] Table 1. Dissolution rate and swelling rate of silk fibroin microspheres and pure silk fibroin in Example 2.

[0060]

[0061] (9) Infrared Spectrum: A small amount of silk fibroin microsphere powder was compressed into a potassium bromide tablet and subjected to infrared spectroscopy analysis, such as... Figure 3 As shown, silk fibroin microspheres at 1618 cm⁻¹ -1 1513cm -1 and 1230cm -1 Characteristic peaks are present at these locations, corresponding to the amide I, amide II, and amide III bands of silk fibroin, respectively, indicating a β-sheet structure.

[0062] (10) Microstructure: 10 mg of silk fibroin microsphere powder was ultrasonically dispersed in 15 mL of purified water, dried, and adhered to conductive adhesive. Scanning electron microscope images were taken, such as... Figure 1 and Figure 2 As shown, the microspheres have a particle size of approximately 2 μm.

[0063] Example 3

[0064] (1) Preparation of initial solution: Prepare a 500mL solution (containing 10g pure silk fibroin) by mixing silk fibroin solution and water for injection. Stir at 60rpm and let stand until the solution is clear and transparent.

[0065] (2) Addition of ionic dispersant: Add 0.8wt% NaCl and 0.2wt% Na2HPO4 to the initial silk fibroin solution.

[0066] (3) Addition of spheroidizing agent: Add 30 mL of a mixture of isopropanol and methanol (volume ratio of 1:9) to the silk fibroin solution, complete the addition within 3 min, and stir with a glass rod at 200 rpm for 3 min.

[0067] (4) Formation of silk fibroin microspheres: The homogeneous solution was directly frozen at -80°C for 18 hours. Then it was thawed at 30°C to obtain a milky white solution. The milky white solution was then placed in a dialysis bag (molecular weight cutoff of 3500 Da) and dialyzed for 48 hours to remove isopropanol and methanol.

[0068] (5) Crystallization treatment of silk fibroin microspheres: The dialyzed microsphere solution was diluted by 2 times and then subjected to wet heat crystallization at 121℃ for 8 minutes to improve the crystallinity of the microspheres.

[0069] (6) Enhanced treatment of silk fibroin microspheres: The dialyzed microsphere solution was spray-dried at 150°C at a flow rate of 20 mL / min to remove residual isopropanol and methanol, and to improve the crystallinity of the microspheres and increase their water insolubility, finally obtaining pure and dry silk fibroin microsphere powder.

[0070] (7) Preparation of microsphere solution: Silk fibroin microsphere powder was ultrasonically dispersed in a 2 wt% silk fibroin solution, and the final concentration of silk fibroin microspheres was 1%. The silk fibroin solution was taken from Example 1.

[0071] (8) Packaging: The microsphere solution is packaged in lyophilized bottles at a dose of 3 mL / vial and stored.

[0072] (9) Sterilization: The silk fibroin microsphere solution was sterilized by high-temperature steam at 121°C for 20 minutes.

[0073] (10) Cytotoxicity: The silk fibroin microsphere solution was diluted 10 times and directly seeded into L-929 cells (purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.) in 96-well plates. Cells seeded in MEM culture medium containing 10% fetal bovine serum and antibiotics (penicillin 100U / mL, streptomycin 100μg / mL) served as the control group. The cytotoxicity test was performed according to GB / T 16886.10. The results showed that the cytotoxicity of both the sample group and the control group was grade 0, with no cytotoxicity.

[0074] Example 4

[0075] (1) Preparation of initial solution: Prepare 500 mL of solution (containing 30 g pure silk fibroin) by mixing silk fibroin solution and water for injection, stir slowly and let stand until the solution is clear and transparent.

[0076] (2) Addition of ionic dispersant: Add 0.5wt% NaCl and 0.5wt% Na2HPO4 to the initial silk fibroin solution.

[0077] (3) Addition of spheroidizing agent: Add 100 mL of acetonitrile to the silk fibroin solution within 5 min and stir with a glass rod at 120 rpm for 30 min.

[0078] (4) Formation of silk fibroin microspheres: The homogeneous solution was placed in a stainless steel container, and then the container was placed in liquid nitrogen until the silk fibroin solution was completely frozen. It was then stored at -20°C for 24 hours. It was then thawed at 25°C to obtain a milky white solution. The milky white solution was then placed in a dialysis bag (molecular weight cutoff of 3500 Da) and dialyzed for 48 hours to remove acetonitrile.

[0079] (5) Crystallization treatment of silk fibroin microspheres: The dialyzed microsphere solution was diluted 5 times and then subjected to wet heat crystallization at 120℃ for 12 min to improve the crystallinity of the microspheres.

[0080] (6) Enhanced treatment of silk fibroin microspheres: The dialyzed microsphere solution was spray-dried at 135°C at a flow rate of 5 mL / min to remove residual acetonitrile, improve the crystallinity of the microspheres, increase their water insolubility, and finally obtain pure and dry silk fibroin microsphere powder.

[0081] (7) Packaging: The silk fibroin microsphere powder is packaged in 10g / vial and stored in a lyophilized bottle.

[0082] (8) Solubility and swelling properties: 10g of silk fibroin microsphere powder was dried in an oven at 60℃ to constant weight. Three parallel samples were weighed and PBS buffer solution was added at a bath ratio of 1:100. The samples were then kept at a constant temperature and shaken in a water bath at 37℃ for 24h. The microspheres were then removed, placed on an ultrafiltration membrane, rinsed three times with purified water, and the remaining moisture on the surface of the microspheres was removed with absorbent kitchen paper. The samples were weighed and then dried in an oven at 60℃ to constant weight. The swelling rate of the microspheres was calculated to be 23.2% ± 3.4%, and the solubility loss rate was 18.5% ± 1.3%. Compared with pure silk fibroin material, both the swelling rate and the solubility loss rate were relatively low.

[0083] Table 2 shows the solubility and swelling rates of silk fibroin microspheres and pure silk fibroin in Example 4.

[0084]

[0085] (9) Infrared Spectrum: A small amount of silk fibroin microsphere powder was compressed into a potassium bromide tablet and subjected to infrared spectroscopy analysis, such as... Figure 4 As shown, silk fibroin microspheres at 1618 cm⁻¹ -1 1513cm -1 and 1230cm -1 Characteristic peaks are present at these locations, corresponding to the amide I, amide II, and amide III bands of silk fibroin, respectively, indicating a β-sheet structure.

[0086] Example 5

[0087] (1) Preparation of initial solution: Prepare 500 mL of solution (containing 10 g pure silk fibroin) by mixing silk fibroin solution and water for injection, stir slowly and let stand until the solution is clear and transparent.

[0088] (2) Addition of ionic dispersant: Add 0.5wt% NaCl to the initial silk fibroin solution.

[0089] (3) Addition of spheroidizing agent: Add 25 mL of acetone and acetonitrile (volume ratio of 1:20) mixture to the silk fibroin solution within 3 min, and stir with a glass rod at 60 rpm for 3 min.

[0090] (4) Formation of silk fibroin microspheres: The homogeneous solution was frozen at -20°C for 24 hours. Then it was thawed at 25°C to obtain a milky white solution. The milky white solution was then placed in a dialysis bag (molecular weight cutoff of 3500 Da) and dialyzed for 48 hours to remove acetone and acetonitrile.

[0091] (5) Crystallization treatment of silk fibroin microspheres: Dilute the dialyzed microsphere solution by 1 time, and then treat it with wet heat at 100℃ for 15 minutes to improve the crystallinity of the microspheres.

[0092] (6) Enhanced treatment of silk fibroin microspheres: The dialyzed microsphere solution was spray-dried at 140°C at a flow rate of 15 mL / min to remove residual acetone and acetonitrile, improve the crystallinity of the microspheres, increase their water insolubility, and finally obtain pure and dry silk fibroin microsphere powder.

[0093] (7) Packaging: The silk fibroin microsphere powder is packaged in 10g / vial and stored in a lyophilized bottle.

[0094] (8) Solubility and swelling properties: 10g of silk fibroin microsphere powder was dried in an oven at 60℃ to constant weight. Three parallel samples were weighed and PBS buffer solution was added at a bath ratio of 1:100. The microspheres were then kept at a constant temperature and shaken in a water bath at 37℃ for 24h. The microspheres were then removed, placed on an ultrafiltration membrane, rinsed three times with purified water, and the remaining moisture on the surface of the microspheres was removed with absorbent kitchen paper. The microspheres were weighed and then dried in an oven at 60℃ to constant weight. The swelling rate of the microspheres was calculated to be 17.6% ± 1.1%, and the solubility rate was 13.5% ± 2.1%. Compared with pure silk fibroin material, the swelling rate and solubility rate were both lower.

[0095] Table 3 shows the solubility and swelling rates of silk fibroin microspheres and pure silk fibroin in Example 5.

[0096]

[0097] (9) X-ray diffraction test: Take 1g of silk fibroin microsphere powder for XRD analysis, such as... Figure 5 As shown, silk fibroin has distinct peaks at 9.1°, 20.7° and 24.3°, which is typical of the silk II crystal structure.

[0098] As demonstrated by the above embodiments, the silk fibroin microspheres provided by this invention possess excellent biocompatibility, biodegradability, and good mechanical properties. The microspheres prepared by the method of this invention are regular spheres with a particle size of approximately 100 nm to 5000 nm, exhibiting uniform particle size with minimal variation and a large specific surface area. The microspheres also possess high crystallinity, slow degradation rate, and are not prone to moisture absorption or aggregation. The preparation method of this invention effectively improves the utilization rate of silk fibroin with minimal waste. Furthermore, spray drying allows for the rapid volatilization of highly volatile granulating agents, leaving virtually no organic residue. This method is suitable for large-scale industrial production, with extremely low production costs and is environmentally friendly and harmless.

Claims

1. A method for preparing silk fibroin microspheres, characterized in that, Includes the following steps: 1) After mixing the ionic dispersant with the silk fibroin solution, add the pelletizing agent, mix, and obtain the pretreated liquid; 2) The pretreated liquid is subjected to low-temperature coagulation treatment, and thawed to obtain silk fibroin microsphere liquid; 3) Dilute the silk fibroin microsphere solution and then subject it to wet heat crystallization to obtain a highly crystalline silk fibroin microsphere solution; 4) The highly crystalline silk fibroin microsphere liquid is spray-dried to obtain solid silk fibroin microspheres; Step 1) The ionic dispersant is one or more of sodium chloride, potassium chloride, disodium hydrogen phosphate, and dipotassium hydrogen phosphate; the mass of the ionic dispersant is 0.1~2 wt% of the mass of the silk fibroin solution. The pelleting agent is a volatile organic solvent, and is one or more of ethanol, isopropanol, acetone, acetonitrile, methyl ethyl ketone, ethyl acetate, methanol and 2-methyl-2-propanol; Step 1) The volume ratio of the spheroidizing agent to the silk fibroin solution is (1~18):

20. The spheroidizing agent is completely added to the silk fibroin solution within 3 min~5 min. The concentration of the silk fibroin solution is 1~60 mg / mL. The average molecular weight of the silk fibroin in the silk fibroin solution is 5000 Da~20 kDa. Step 2) The thawing process also includes a dialysis step, wherein the molecular weight cutoff of the dialysis material used for dialysis is 3500 Da; the dialysis time is 24-48 hours; and the dialysis water is changed every 1-2 hours. Step 3) The dilution factor is 1 to 20 times; the temperature of the wet heat crystallization treatment is 100 to 134°C; and the time of the wet heat crystallization treatment is 5 to 30 minutes.

2. The preparation method according to claim 1, characterized in that, Step 1) The mixing process is accompanied by stirring, the stirring speed is 60~120 rpm, and the mixing time is 5~25 min.

3. The preparation method according to claim 1, characterized in that, Step 2) The low-temperature solidification treatment method includes spray condensation, low-temperature freezing or liquid nitrogen freezing; the temperature of the low-temperature solidification treatment is -80~-20℃, and the time of the low-temperature solidification treatment is 12~48h.

4. The preparation method according to claim 1, characterized in that, The thawing temperature described in step 2) is 10~30℃.

5. The preparation method according to claim 1, characterized in that, Step 4) The spray drying temperature is 85~200℃, and the flow rate of the liquid during the spray drying process is 5~25mL / min; Step 4) After obtaining solid silk fibroin microspheres, the solid silk fibroin microspheres are added to water for injection, silk fibroin solution or physiological saline to obtain a silk fibroin microsphere suspension.

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