Uniform-sized silk fibroin microspheres, their preparation method and applications

Abstract

This invention discloses a uniform-size silk fibroin microsphere, its preparation method, and its application, comprising the following steps: taking a regenerated silk fibroin solution, digesting it with trypsin to obtain a modified silk fibroin precipitate, dissolving it in an aqueous solution of a β-sheet inhibitor to obtain a silk fibroin solution as the dispersed phase; emulsifying it using an external pressure membrane emulsifier to obtain an emulsion containing silk fibroin droplets; adding a cross-linking agent to the emulsion to carry out a cross-linking reaction, and then adding a crystallization inducing agent to induce crystallization to obtain primary silk fibroin microspheres; and freeze-drying under vacuum to obtain silk fibroin microspheres. This invention modifies silk fibroin by enzymatic digestion to retain silk fibroin fragments with high β-sheet content, exhibiting high structural stability; and uses membrane emulsification combined with a multiple crystallization induction method to improve the crystallinity of the microspheres, obtaining silk fibroin microspheres with uniform particle size and smooth surface.

Description

Technical Field

[0001] This invention relates to the field of silk fibroin microsphere preparation technology, specifically to a uniform-size silk fibroin microsphere, its preparation method, and its application. Background Technology

[0002] Silk fibroin is a natural polymer extracted from silkworm silk. It possesses excellent mechanical properties, biodegradability, low immunogenicity, and high biocompatibility, making it widely applicable in regenerative medical materials, cosmetics, and pharmaceuticals. The molecular structure of silk fibroin consists of alternating highly ordered β-sheet crystalline regions and loosely ordered amorphous regions. The crystalline regions are primarily composed of repeating hydrophobic small-molecule amino acid residues such as glycine, alanine, and serine, forming a tightly packed antiparallel β-sheet sheet structure, which endows silk fibroin with excellent mechanical strength and stability. The amorphous regions, on the other hand, have a higher content of hydrophilic amino acids, which can regulate the hydrophobicity and hydrophilicity of silk fibroin and inhibit the assembly of the β-sheet crystalline regions.

[0003] Silk fibroin microspheres are biodegradable, and their degradation products are amino acids, which are non-irritating and can be absorbed by the body, making them a promising candidate material for filler-type medical and cosmetic products. The particle size, sphericity, and surface smoothness of the microspheres significantly affect their efficacy. Microspheres that are too small (typically <20μm) can be phagocytosed by macrophages, causing severe inflammation; microspheres that are too large result in a noticeable granular texture after implantation and a high risk of vascular embolism. The commonly used microsphere particle size in dermal fillers is between 20-100μm. The more uniform the microsphere size, the higher the sphericity, and the smoother the surface, the less inflammation and the higher the safety after implantation into local tissue.

[0004] Common methods for preparing silk fibroin microspheres include cryogenic crystallization, electrospray ionization, emulsification, and microfluidics. Silk fibroin microspheres prepared by cryogenic crystallization and electrospray ionization typically have low particle size and high surface roughness due to ice crystal formation or rapid liquid evaporation. While conventional mechanical emulsification can produce silk fibroin microspheres with smooth surfaces, it results in low particle size uniformity and low yield after microsphere screening, leading to high production costs. Microfluidics can produce microspheres with uniform particle size, but its throughput is low, making large-scale production difficult.

[0005] Patent CN114874466A discloses a silk fibroin microsphere, its preparation method, and its application. The method involves preparing a silk fibroin microsphere solution by low-temperature coagulation of a silk fibroin solution containing an ionic dispersant and a spheroidizing agent, followed by dilution, hydrothermal crystallization, and spray drying to prepare solid silk fibroin microspheres. However, this method can only produce microspheres with a relatively small particle size, approximately 2 μm.

[0006] Patent CN119331274A discloses a method for preparing silk fibroin nanospheres. First, a crystallization inducer is directly added to a silk fibroin solution and mixed thoroughly. Then, a gelatin solution is added as a dispersant, and the mixture is further mixed to obtain a pretreated liquid. The pretreated liquid is then subjected to low-temperature coagulation and ultrasonic dispersion to obtain a silk fibroin nanosphere liquid. Finally, the silk fibroin nanosphere liquid is centrifuged and freeze-dried to obtain solid silk fibroin nanospheres. This method has two limitations: firstly, it can only produce silk fibroin microspheres with an average diameter of approximately 260 nm, limiting its application range; secondly, electron microscopy reveals that most of the microspheres are aggregated, and the surface of the microspheres is very rough and the particle size is uneven.

[0007] Patent CN117304519A discloses a method for preparing silk fibroin microspheres. The method involves adding a silk fibroin solution dropwise to a mixed oil phase containing liquid paraffin, Span 80, and Tween 80 under stirring at 35-60°C. After stirring for 30-90 minutes, silk fibroin microspheres with the desired particle size of 20-80 μm can be obtained by separation. However, the silk fibroin microspheres prepared by this method have poor sphericity, and rapid curing after ethanol washing leads to severe shrinkage of most of the microspheres.

[0008] Patent CN118420936A discloses a silk fibroin microsphere and its preparation method. The method involves dissolving silk fibroin in a solvent and then chemically modifying it with a modifier to obtain a silk fibroin stock solution. An excipient is then added to the silk fibroin stock solution to obtain a silk fibroin pre-solution. The silk fibroin pre-solution is then used to prepare size-tunable silk fibroin microspheres using microfluidics. Finally, a reinforcing agent is added to the size-tunable silk fibroin microspheres to obtain silk fibroin microspheres with excellent mechanical properties. While this method utilizes microfluidics to prepare microspheres with a particle size of approximately 60 μm, the throughput of microfluidics for microsphere preparation is relatively low, making it difficult to scale up for production. Furthermore, some of the prepared microspheres adhere together, and some microspheres exhibit wrinkles on their surface.

[0009] In summary, there remains a significant challenge in preparing silk fibroin microspheres with a particle size between 20 and 100 μm, high uniformity in particle size distribution, good sphericity, and smooth surface. Summary of the Invention

[0010] To address the shortcomings of existing technologies, this invention provides a uniform-size silk fibroin microsphere, its preparation method, and its application. Modified silk fibroin raw materials are used to prepare silk fibroin microspheres with uniform particle size (20-100 μm) and smooth surface through membrane emulsification and multiple microsphere crystallization.

[0011] To address the aforementioned technical problems, this invention provides a method for preparing silk fibroin microspheres with uniform particle size, comprising the following steps:

[0012] S1. Take the regenerated silk fibroin solution, digest it with trypsin to obtain modified silk fibroin precipitate, dissolve it in an aqueous solution of β-sheet inhibitor to obtain silk fibroin solution, and use it as the dispersed phase;

[0013] S2. Emulsification is performed using an external pressure membrane emulsifier to disperse the dispersed phase in a continuous phase, resulting in an emulsion containing silk fibroin droplets.

[0014] S3. Add a cross-linking agent to the emulsion to carry out the cross-linking reaction of silk fibroin, and then add a crystallization inducer to induce the crystallization of silk fibroin to obtain primary silk fibroin microspheres.

[0015] S4. The primary silk fibroin microspheres are freeze-dried under vacuum to obtain silk fibroin microspheres.

[0016] The presence of hydrophilic amorphous regions in silk fibroin can affect the crystallization process during the preparation of silk fibroin microspheres, resulting in poor sphericity and an uneven surface. This invention modifies silk fibroin by enzymatic digestion, removing the hydrophilic sites in the amorphous regions. The raw material mainly retains silk fibroin fragments with high β-sheet content of repetitive GAGAS sequences, exhibiting high structural stability. Compared to unmodified silk fibroin, it is easier to induce crystallization and form microspheres. The resulting microspheres have better sphericity, higher crystallinity, smoother surface, and are more resistant to enzymatic hydrolysis.

[0017] This invention employs membrane emulsification, enabling the preparation of microspheres with more uniform particle size and higher yield, which is more conducive to scale-up production. Simultaneously, it combines a multiple crystallization induction method to enhance the crystallinity of the microspheres: firstly, cross-linking of the emulsified droplets is achieved through a cross-linking agent, bringing the silk fibroin molecular chains closer together to form crystal nuclei; then, treatment with a crystallization inducing agent and vacuum freeze-drying further enhance the crystallinity of the microspheres. During vacuum freeze-drying, a rapid freezing-low temperature sublimation process is used to control the rate of water loss from the microspheres, resulting in a smoother surface and a denser structure compared to traditional freeze-drying processes.

[0018] Furthermore, in S3, the crosslinking agent is one or more of butanediol glycidyl ether (BDDE), divinyl sulfone (DVS), and epoxy-polyethylene glycol-epoxy (Epoxide-PEG-Epoxide, with a molecular weight of 1kDa-20kDa), preferably butanediol glycidyl ether;

[0019] And / or, the volume ratio of the crosslinking agent to the dispersed phase is 0.5%-5%;

[0020] And / or, the conditions for the crosslinking reaction are: temperature 30-80℃, preferably 40-60℃, stirring speed 150-500rpm, preferably 250-400rpm, and time 20-90min, preferably 30-60min.

[0021] Furthermore, in S3, the crystallization inducer is one or more of methanol, ethanol, isopropanol, n-butanol, tert-butanol, acetone, formic acid, N,N-dimethylformamide, dimethyl sulfoxide, chloroform, and ethylene oxide, preferably methanol;

[0022] And / or, the volume ratio of the crystallization inducer to the dispersed phase is 100%-800%;

[0023] And / or, the conditions for inducing silk fibroin crystallization are: temperature 30-80℃, preferably 40-60℃, stirring speed 150-500rpm, preferably 250-400rpm, and time 2-10h, preferably 5-8h.

[0024] Furthermore, in S3, after inducing silk fibroin crystallization, the upper liquid is discarded, and the lower layer of silk fibroin primary microspheres is washed: 3 times with water, 3 times with ethyl acetate, 3 times with petroleum ether, 3 times with ethyl acetate, and 3 times with water, while ultrasonic treatment is performed during the washing process.

[0025] Furthermore, in S2, the membrane tube of the external pressure membrane emulsifier is an SPG membrane with a pore size of 5-40 μm, preferably 10-20 μm;

[0026] And / or, the emulsification parameters are: temperature 5-30℃, preferably 10-20℃, continuous phase flow rate 0.5-1.5m / s, preferably 0.8-1.2m / s, membrane pressure 0.5kPa-5kPa, preferably 1.0kPa-2.5kPa, and the volume-to-mass ratio of dispersed phase to continuous phase is 10%-50%, in mL / g.

[0027] Furthermore, in S2, the mass-to-volume ratio of silk fibroin to aqueous solution in the dispersed phase is 2%-25%, preferably 5%-15%, in g / mL;

[0028] And / or, the continuous phase is one or more of light liquid paraffin, heavy liquid paraffin, soybean oil, castor oil, palm oil, olive oil, sesame oil, corn oil, lecithin, petroleum ether, n-hexane, n-heptane, petrolatum, silicone oil, and ceresin, preferably light liquid paraffin;

[0029] And / or, the continuous phase further includes a surfactant at a mass concentration of 0.5%-3%, preferably 1%-1.5%.

[0030] Furthermore, the surfactant is one or more of the following: Tween 20, Tween 40, Tween 60, Tween 80, Tween 85, 3-[(3-cholesterol aminopropyl)dimethylamino]-1-propanesulfonic acid (CHAPS), 3-sulfopropyltetradecyl dimethylammonium, Span 20, Span 40, Span 60, Span 80, Span 85, sodium dodecyl sulfonate, sodium dodecyl sulfate (SDS), sodium dodecylbenzene sulfonate, polyvinyl alcohol, polyvinylpyrrolidone, polyoxyethylene stearyl alcohol ether, polyglycerol fatty acid ester, calcium rosinate, magnesium stearate, and sorbitan palmitate.

[0031] Furthermore, in S4, the vacuum freeze-drying includes the following steps:

[0032] Pre-cool to 0-4℃ at a rate of 0.4-1℃ / min for 20-40 min; pre-freeze to -50℃ ~ -40℃ at a rate of 0.9-1.2℃ / min for 60-120 min; heat to -20℃ ~ 0℃ and sublimate under a vacuum of 0-30Pa for 10-24 h; heat to 0-30℃ and dry under a vacuum of 0-30Pa for 5-30 h.

[0033] Rapid freezing during vacuum freeze drying can form more uniform and smaller ice crystals in the microspheres, preventing large ice crystals from forming on the surface of the microspheres due to slow crystallization; low-temperature sublimation can make the ice crystals sublimate slowly, thereby reducing the shrinkage of the microspheres during the sublimation process.

[0034] Furthermore, in S1, the weight-average molecular weight of the regenerated silk fibroin is 50kDa-250kDa.

[0035] Furthermore, in S1, the β-sheet inhibitor is one or more of LiBr, NaSCN, ZnCl2, and CaCl2, preferably LiBr;

[0036] And / or, the concentration of the β-sheet inhibitor in the aqueous solution of the β-sheet inhibitor is 4-9.3M, preferably 6-9.3M.

[0037] The second aspect of the present invention provides a method for preparing silk fibroin microspheres as described in the first aspect.

[0038] The third aspect of this invention provides the application of the silk fibroin microspheres described in the second aspect in medical products and cosmetic products.

[0039] The beneficial effects of this invention are:

[0040] This invention modifies silk fibroin by enzymatic digestion, removing hydrophilic sites in the non-crystalline region. The raw material mainly retains silk fibroin fragments with high β-sheet content of repetitive GAGAS sequences, which have high structural stability. Compared with unmodified silk fibroin, it is easier to induce crystallization to form microspheres. The prepared microspheres have better sphericity, higher crystallinity, smoother surface, and are more resistant to enzymatic hydrolysis.

[0041] This invention employs a membrane emulsification method, which can prepare microspheres with more uniform particle size, higher microsphere yield, and is more conducive to scale-up production. At the same time, it combines a multiple crystallization induction method to improve the crystallinity of microspheres: first, the droplets are cross-linked after emulsification by a cross-linking agent, which brings the distance between silk fibroin molecular chains closer to form crystal nuclei. Then, the crystallinity of microspheres is improved by treatment with a crystallization inducing agent and vacuum freeze-drying to remove water.

[0042] In this invention, a freeze-drying process using rapid freezing and cooling followed by low-temperature sublimation is employed to control the rate of moisture loss from the microspheres during vacuum freeze-drying. Compared to traditional freeze-drying processes, this results in a smoother surface and a denser structure for the microspheres. Attached Figure Description

[0043] To more clearly illustrate the technical solution of the present invention, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0044] Figure 1 This is a SEM image of the silk fibroin microspheres obtained in Example 1 of the present invention;

[0045] Figure 2 This is a SEM image of the silk fibroin microspheres obtained in Example 5 of the present invention;

[0046] Figure 3 This is a SEM image of the silk fibroin microspheres obtained in Comparative Example 1 of the present invention;

[0047] Figure 4 This is a SEM image of the silk fibroin microspheres obtained in Comparative Example 2 of the present invention;

[0048] Figure 5 This is a SEM image of the silk fibroin microspheres obtained in Comparative Example 3 of the present invention;

[0049] Figure 6 This is a SEM image of the silk fibroin microspheres obtained in Comparative Example 4 of the present invention;

[0050] Figure 7 These are the degradation curves of the silk fibroin microspheres obtained in Example 1 and Comparative Examples 1-2 of the present invention. Detailed Implementation

[0051] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0052] Example 1

[0053] This embodiment relates to a method for preparing silk fibroin microspheres with uniform particle size, comprising the following steps:

[0054] (1) Dissolve 150g of anhydrous sodium carbonate in 15kg of water, and after boiling, add 150g of silk and boil for another 45min to obtain degummed silk. Take out the degummed silk, wash it three times with water and dry it; dissolve 50g of the dried degummed silk in 500mL of 9.3M LiBr solution, filter, and purify it by molecular weight screening to obtain a regenerated silk fibroin aqueous solution with a weight average molecular weight of 100 kDa-250 kDa. Mix it with an equal volume of 3mg / g trypsin solution, react at 37℃ for 20h, heat at 100℃ for 10min to inactivate the enzyme, centrifuge at 5000rpm for 10min to remove the supernatant, and dry to obtain modified silk fibroin precipitate raw material.

[0055] (2) Dissolve 9.0g of silk fibroin precipitate in 60mL of 9.3M LiBr aqueous solution until completely dissolved to prepare a 15% (w / v) silk fibroin solution as the dispersed phase.

[0056] (3) An external pressure SPG membrane emulsifier is used. The dispersed phase is located outside the hydrophobic SPG membrane tube, and the continuous phase is located inside and flows continuously through a pump. It is pressurized by external nitrogen and acts directly on the outside of the membrane tube to automatically and continuously process and prepare silk fibroin emulsion droplets. Specifically, 60 mL of the silk fibroin solution prepared in step (2) is taken as the dispersed phase and cooled to 20°C in an ice-water bath and then added to the dispersed phase storage tank. 4.5 g of Tween 80 is weighed and added to 300 g of light liquid paraffin continuous phase and stirred evenly. The temperature of the continuous phase is kept at 20°C by using a cooling circulation pump. The magnetic pump is turned on to fill the membrane tube with the continuous phase. The membrane pressure is set to 1.0 kPa. The nitrogen valve is turned on and the dispersed phase solution passes through the SPG membrane under the action of nitrogen pressure to prepare a light liquid paraffin emulsion containing silk fibroin emulsion droplets. The emulsion is collected in the post-processing tank.

[0057] The membrane tube specifications are: Φ10mm x L125mm (effective length 105mm); membrane tube pore size 10μm; continuous phase flow rate: 1.2m / s; and the ratio of dispersed phase to continuous phase is 20% (v / w).

[0058] (4) Under the conditions of heating at 60℃ and stirring at 250rpm, add 1.2mL of BDDE to the emulsion prepared in step (3) and continue heating and stirring for 60min.

[0059] (5) Add 328 mL of methanol to the emulsion prepared in step (4) and continue stirring at 250 rpm for 8 h under heating conditions at 60 °C to induce microsphere crystallization and solidification.

[0060] (6) After stopping stirring and allowing the emulsion to stand and separate into layers in step (5), pour off the upper layer of liquid and collect the lower layer of microspheres. Add 100 mL of deionized water and wash for 15 min under ultrasonic and stirring conditions at 250 rpm. Repeat the washing process 3 times. Wash the microspheres 3 times with ethyl acetate, 3 times with petroleum ether, 3 times with ethyl acetate, and 3 times with deionized water using the same operation. After washing, the primary silk fibroin microspheres are obtained.

[0061] (7) Vacuum freeze drying: cool to 0℃ at 0.5℃ / min for 30 min; cool to -45℃ at 1℃ / min for 90 min; heat to -15℃ and sublimate under vacuum of 5Pa for 12 h; heat to 20℃ and dry under vacuum of 5Pa for 26 h to obtain silk fibroin microspheres.

[0062] Example 2

[0063] The difference between this embodiment and embodiment 1 is that the ratio of dispersed phase to continuous phase in step (3) is 33% (v / w), while other steps and parameters remain unchanged.

[0064] Example 3

[0065] The difference between this embodiment and embodiment 1 is that the membrane tube pore size in step (3) is 20 μm, while the other steps and parameters remain unchanged.

[0066] Example 4

[0067] The difference between this embodiment and embodiment 1 is that the continuous phase flow velocity in step (3) is 1.0 m / s, while the other steps and parameters remain unchanged.

[0068] Example 5

[0069] The difference between this embodiment and embodiment 1 is that the membrane pressure in step (3) is 2.5 kPa, while the other steps and parameters remain unchanged.

[0070] Example 6

[0071] The difference between this embodiment and embodiment 1 is that the amount of BDDE added in step (4) is 0.6 mL, while the other steps and parameters remain unchanged.

[0072] Example 7

[0073] The difference between this embodiment and embodiment 1 is that the amount of methanol added in step (4) is 164 mL, while the other steps and parameters remain unchanged.

[0074] Comparative Example 1

[0075] The difference between this comparative example and Example 1 is that the enzymatic digestion process is omitted in step (1), and the undigested silk fibroin raw material is obtained by vacuum freeze-drying after purification. Other steps and parameters remain unchanged.

[0076] Comparative Example 2

[0077] This comparative example uses a mechanical emulsification method to prepare microspheres, specifically:

[0078] (1) Dissolve 150g of anhydrous sodium carbonate in 15kg of water, and after boiling, add 150g of silk and boil for another 45min to obtain degummed silk. Take out the degummed silk, wash it three times with water and dry it; dissolve 50g of the dried degummed silk in 500mL of 9.3M LiBr solution, filter, and purify it by molecular weight screening to obtain a regenerated silk fibroin aqueous solution with a weight average molecular weight of 100 kDa-250 kDa. Mix it with an equal volume of 3mg / g trypsin solution, react at 37℃ for 20h, heat at 100℃ for 10min to inactivate the enzyme, centrifuge at 5000rpm for 10min to remove the supernatant, and dry to obtain modified silk fibroin precipitate raw material.

[0079] (2) Dissolve 9.0g of silk fibroin precipitate in 60mL of 9.3M LiBr aqueous solution until completely dissolved to prepare a 15% (w / v) silk fibroin solution as the dispersed phase.

[0080] (3) At 45°C, liquid paraffin, Span 80 and Tween 80 were mixed evenly in a mass ratio of 100:7.5:2.5 to obtain a mixed oil phase.

[0081] (4) Add 2 mL of the dispersed phase at 45°C dropwise to 10 mL of the mixed oil phase under stirring using a syringe. After the addition is complete, continue stirring for 40 min. The stirring speed is 300 rpm.

[0082] (5) After washing with ethanol and pure water in sequence, the silk fibroin microspheres were obtained by vacuum freeze drying using the same process as in Example 1.

[0083] Comparative Example 3

[0084] The difference between this comparative example and Example 1 is that in step (7), the temperature was lowered to -45°C at a rate of 0.3°C / min during the pre-freezing process, while other steps and parameters remained unchanged.

[0085] Comparative Example 4

[0086] This comparative example uses SPG film emulsification combined with a traditional crystallization induction method, specifically:

[0087] Steps (1)-(3) are the same as in Example 1;

[0088] (4) Add 10% methanol to the emulsion prepared in step (3) to obtain a pretreatment solution. Add 1% gelatin solution at a volume ratio of 2:1 to the pretreatment solution and mix well. Place the mixture in a -80℃ refrigerator and take it out after 48 hours. Dissolve it in a 37℃ water bath. After sonication, centrifugation, and washing, freeze dry it under vacuum with the parameters of step (7) in Example 1 to obtain silk fibroin microsphere samples.

[0089] Test Example 1: Microsphere Size Detection

[0090] Take an appropriate amount of silk fibroin microspheres and place them in a 5 mL test tube. Add 2 mL of 0.5% Tween 80 solution and shake well to ensure that the microspheres are fully wetted by the dispersion medium. Turn on the stirrer and the ultrasonic device. Add the sample to the particle size analysis sample cell containing 150 mL of deionized water until the transmittance is between 70% and 90%. Turn on the stirrer and the ultrasonic device. After the transmittance stabilizes, measure D50, D10, and D90, and calculate the particle size span of the microspheres according to the formula: Span = (D90 - D10) / D50.

[0091] Wherein, D50 is the median particle size, representing that 50% of the microspheres are smaller than this value; D90 represents that 90% of the microspheres are smaller than this value; D10 represents that 10% of the microspheres are smaller than this value; the Span value represents the span of the microsphere particle sizes, and the smaller the Span value, the narrower the microsphere particle size distribution. The particle size parameters of the microspheres obtained in the examples and comparative examples are shown in Table 1.

[0092] Table 1

[0093]

[0094] As shown in Table 1, the microspheres prepared in Examples 1-7 of this invention meet the required size range and have low Span values, indicating good microsphere particle size uniformity. Specifically, the microspheres prepared in Example 1 have a D50 of approximately 35.8 μm and a Span value of 1.26. In Example 2, compared to Example 1, the ratio of dispersed phase to continuous phase was increased from 20% (v / w) to 33.3% (v / w), resulting in an increase in the D50 of the microspheres to 40.3 μm. This is because the increased proportion of dispersed phase leads to the fusion of some silk fibroin droplets after emulsification. In Example 3, compared to Example 1, the pore size of the SPG membrane was increased to 20 μm, significantly increasing the D50 of the microspheres to 62.3 μm. This demonstrates that the membrane pore size is the most significant factor affecting the microsphere particle size, directly determining the size of the emulsified silk fibroin droplets, and the microsphere particle size can be adjusted by regulating the pore size. In Example 4, compared to Example 1, the continuous phase flow rate was reduced to 1.0 m / s, and the D50 of the microspheres increased to 37.2 μm. The flow rate mainly affected the fluid drag force of the continuous phase relative to the emulsion droplets, thus affecting the microsphere size. A lower continuous phase flow rate reduced the fluid drag force, making it more difficult for the dispersed phase droplets to be sheared away from the membrane pores, resulting in larger microsphere sizes. In Example 5, compared to Example 1, the permeabilization pressure was increased to 2.5 kPa, and the microsphere D50 increased to 43.2 μm. This is because the increased permeabilization pressure increased the velocity at which the dispersed phase was squeezed through the membrane pores, resulting in larger droplet volumes at the membrane pore outlet, thus producing larger microspheres. In Examples 6-7, compared to Example 1, the amounts of crosslinking agent and crystallization inducing agent were adjusted within appropriate ranges, and the results showed that the D50 of the prepared microspheres was similar to that of Example 1.

[0095] Comparative Example 1 used silk fibroin raw material containing hydrophilic amorphous regions that had not undergone enzymatic digestion to prepare microspheres. The results showed that the D50 of the prepared microspheres was approximately 48.3 μm, significantly larger than the microspheres prepared from the enzymatically digested silk fibroin raw material in Example 1. This may be because silk fibroin molecules containing hydrophilic amorphous regions cannot form a dense structure during induced crystallization, resulting in a loose molecular arrangement, leading to larger microsphere size and more uneven particle size distribution, with a Span value as high as 1.48. Comparative Example 2 used a mechanical emulsification method, resulting in microspheres with uneven particle size and a Span value exceeding 1.5. Comparative Example 4 used silk fibroin microspheres prepared using the membrane emulsification method disclosed in this invention combined with a traditional induced crystallization method. The detected particle size reached 118.3 μm, significantly higher than in the examples. This is because the traditional induced crystallization method cannot completely induce silk fibroin crystallization, causing the silk fibroin microspheres to adhere and fuse when 1% gelatin solution was subsequently added as a dispersant, ultimately forming large-sized microsphere clusters.

[0096] Test Example 2: Microsphere Morphology Detection

[0097] Figure 1 and Figure 2The images show SEM images of the microspheres prepared in Examples 1 and 5, respectively. It can be seen that the microspheres have uniform particle size and smooth surface. Figure 3 The SEM image of the microspheres prepared for Comparative Example 1 shows that the surface of the microspheres is uneven and there are some large pores. It is speculated that this is because the silk fibroin molecules containing hydrophilic non-crystalline regions cannot form a dense structure during induced crystallization, and the uneven molecular crystallization leads to the shrinkage of the surface of the final microspheres, resulting in wrinkles and even large pores. Figure 4 The image shows the SEM image of the microspheres prepared in Comparative Example 2. It can be seen that the microspheres prepared by the pure mechanical emulsification method have a large difference in the largest and smallest particle sizes, and need to be further sieved before they can be used. The yield of qualified microspheres is significantly reduced. Figure 5 The image shows a SEM image of the microspheres prepared in Comparative Example 3. It can be seen that there are a large number of cracks and wrinkles on the surface of the microspheres, suggesting that the large ice crystals formed by the slow freezing process have damaged the surface morphology of the microspheres. Figure 6 The image shows the SEM image of the microspheres prepared in Comparative Example 4. As can be seen from the image, the particle size of the prepared microspheres is generally greater than 100 μm, and there is obvious aggregation of microspheres of different sizes. It is speculated that this is because the traditional induced crystallization method cannot completely induce the crystallization of silk fibroin, which leads to the adhesion and fusion of silk fibroin microspheres when 1% gelatin solution is added as a dispersant, ultimately forming large-sized microsphere agglomerates, consistent with the particle size test results.

[0098] Test Example 3: Microsphere Crystallinity Detection

[0099] The crystallinity of microsphere samples from different embodiments and comparative examples was analyzed using the ATR mode of an infrared spectrometer. The crystallinity was determined using OMNIC software via the amide I band (1585-1710 cm⁻¹). -1 The proportion of β structure in the secondary structure of silk fibroin molecules was determined by Fourier self-deconvolution, and the crystallinity was obtained by the proportion of β structure. The results are shown in Table 2.

[0100] Table 2

[0101]

[0102] As shown in Table 2, the microspheres obtained in Examples 1-7 of this invention have good crystallinity. The crystallinity of the microspheres obtained in Examples 1-5 ranged from 49.3% to 55.8%, showing no significant difference. In Examples 6-7, after appropriately reducing the crosslinking agent and crystallization inducer, the crystallinity decreased appropriately. This is because the use of the crosslinking agent can significantly shorten the distance between the molecular chains in the crystallization region, promoting intermolecular crystallization of silk fibroin molecules; the crystallization inducer mainly induces crystallization by disrupting the hydration layer on the surface of silk fibroin molecules, promoting the formation of β-sheet structures with hydrogen bond interactions between silk fibroin molecules.

[0103] The crystallinity of the microspheres in Comparative Example 1 was significantly reduced to 28.3%, which may be because the non-enzymatically cleaved silk fibroin molecules containing hydrophilic amorphous regions hindered the formation of β-sheet structures during crystallization induction, ultimately leading to low crystallinity. Microspheres prepared using a purely mechanical emulsification method in Comparative Example 2, without cross-linking by a cross-linking agent or crystallization induction by a crystallization inducing agent, showed a crystallinity reduction to 22.3%. Microspheres dried using a different freeze-drying process than in Example 1 in Comparative Example 3 showed a slight decrease in crystallinity to 46.3%. Silk fibroin microspheres prepared using the membrane emulsification method disclosed in this invention combined with a traditional crystallization induction method in Comparative Example 4 had a measured crystallinity of 31.2%, significantly lower than the crystallinity in the examples, indicating that traditional crystallization induction methods are insufficient to induce sufficient crystallization of silk fibroin.

[0104] Test Example 4: Microsphere Swelling Rate Detection

[0105] The swelling rate of the microsphere samples in the examples and comparative examples was detected, and the results are shown in Table 3. The specific detection process is as follows: Take a 2.5 mL EP tube, weigh it, and record the weight as W0. Take about 0.5 g of dried silk fibroin microsphere sample and add it to the EP tube, weigh it, and record the weight as W1. Add 1.5 mL of physiological saline to the centrifuge tube to resuspend the microspheres, and incubate overnight at 37°C. After the incubation, centrifuge at 10000 rpm for 5 min, discard the supernatant, and then accurately weigh the entire centrifuge tube, recording the weight as W2. The formula for calculating the swelling rate of the microspheres is as follows:

[0106] Microsphere swelling rate % = (W2-W0) / (W1-W0)×100%.

[0107] Table 3

[0108]

[0109] As shown in Table 3, the swelling degree of the microspheres in Examples 1-7 was relatively low, ranging from 8.6% to 20.4%. The swelling degree of the microspheres prepared using non-enzymatically digested silk fibroin raw material containing hydrophilic non-crystalline regions in Comparative Example 1 was 25.8%; the highest swelling degree was found in Comparative Example 2, reaching 37.2%.

[0110] Test Example 5: Microsphere Degradability Detection

[0111] Take a 2.5 mL EP tube (weight M0) and fill it with approximately 50 mg of microspheres prepared in Example 1, Comparative Example 1, and Comparative Example 2. Weigh the total weight of the microspheres and EP tube (M1). Set up three replicates for each group, and take five aliquots of each sample. Add 1 mL of PBS buffer (pH=7.4) containing 1 U / mL proteinase K, and incubate at 37°C and 60 rpm for 21 days. Every 3 days, remove the EP tube, centrifuge at 10000 rpm for 5 min, discard the supernatant, and add freshly prepared proteinase K hydrolysate. For each replicate in each group, take one EP tube on days 3, 6, 9, 15, and 21, centrifuge at 4500 rpm for 5 min, discard the supernatant, wash twice with purified water, centrifuging at 4500 rpm for 5 min after each wash, and then lyophilize. Weigh the total weight of the lyophilized microspheres and EP tube (M2), and calculate the degradation rate of the silk fibroin microspheres using the following formula:

[0112] Microsphere degradation rate % = (M2-M1) / (M1-M0)×100%.

[0113] Figure 7 The figures show the degradation curves of the microspheres in Example 1 and Comparative Examples 1-2. It can be seen that the degradation rate in Example 1 is significantly lower than that in Comparative Examples 1-2. This is because the microspheres obtained in Example 1 have better crystallinity. The degradation experiment results indicate that the microspheres prepared in this invention have a longer degradation time and higher resistance to enzymatic degradation.

[0114] The present invention has been described in detail above with reference to specific embodiments and exemplary examples; however, these descriptions should not be construed as limiting the present invention. Those skilled in the art will understand that various equivalent substitutions, modifications, or improvements can be made to the technical solutions and embodiments of the present invention without departing from the spirit and scope of the invention, and all such modifications and improvements fall within the scope of the present invention. The scope of protection of the present invention is defined by the appended claims.

Claims

1. A method for preparing uniform size silk fibroin microspheres, characterized by, It comprises the following steps: S1, taking regenerated silk fibroin solution, trypsin enzyme cutting, the hydrophilic site in the non-crystalline region is cut off, and the modified silk fibroin precipitate is obtained, which is dissolved in the aqueous solution of beta-sheet inhibitor to obtain silk fibroin solution as the dispersed phase; S2, emulsification is carried out by an external pressure type membrane emulsifier, and the dispersed phase is dispersed in the continuous phase to obtain an emulsion containing silk fibroin emulsion droplets; S3, a crosslinking agent is added to the emulsion to carry out a crosslinking reaction of silk fibroin, and then a crystallization inducer is added to induce silk fibroin crystallization to obtain silk fibroin primary microspheres; S4, vacuum freeze-drying the silk fibroin primary microspheres to obtain silk fibroin microspheres; The vacuum freeze-drying comprises the following steps: pre-cooling at a rate of 0.4-1℃ / min to 0-4℃ for 20-40min; pre-freezing at a rate of 0.9-1.2℃ / min to-50℃ ~ -40℃ for 60-120min; warming up to-20℃ ~ 0℃, sublimation under the condition of vacuum degree 0-30Pa for 10-24h; warming up to 0-30℃, drying under the condition of vacuum degree 0-30Pa for 5-30h; The crosslinking agent is one or several of butanediol glycidyl ether, divinyl sulfone, epoxy-polyethylene glycol-epoxy; The crystallization inducer is one or several of methanol, ethanol, isopropanol, n-butanol, tert-butanol, acetone, formic acid, N,N-dimethylformamide, dimethyl sulfoxide, chloroform, and oxirane.

2. The method of claim 1, wherein the uniform size of the silk fibroin microspheres is 1-10 μm. In S3, the volume ratio of the crosslinking agent to the dispersed phase is 0.5%-5%; And / or, the conditions of the crosslinking reaction are: temperature 30-80℃, stirring speed 150-500rpm, and time 20-90min.

3. The method for preparing uniform-sized silk fibroin microspheres as described in claim 1, characterized in that, In S3, the volume ratio of the crystallization inducer to the dispersed phase is 100%-800%; And / or, the conditions of inducing silk fibroin crystallization are: temperature 30-80℃, stirring speed 150-500rpm, and time 2-10h.

4. The method for preparing uniform-sized silk fibroin microspheres as described in claim 1, characterized in that, In S2, the membrane tube of the external pressure type membrane emulsifier is SPG membrane, and the membrane pore size is 5-40μm; And / or, the parameters of the emulsification are: temperature 5-30℃, continuous phase flow rate 0.5-1.5m / s, membrane pressure 0.5kPa-5kPa, and volume mass ratio of the dispersed phase to the continuous phase 10%-50%, unit mL / g.

5. The method for preparing uniform-sized silk fibroin microspheres as described in claim 1, characterized in that, In S2, the mass volume ratio of silk fibroin to the aqueous solution in the dispersed phase is 2%-25%, unit g / mL; And / or, the continuous phase is one or more of light liquid paraffin, heavy liquid paraffin, soybean oil, castor oil, palm oil, olive oil, sesame oil, corn oil, lecithin, petroleum ether, n-hexane, n-heptane, vaseline, silicone oil, and ozokerite; And / or, the continuous phase further comprises a surfactant with a mass concentration of 0.5%-3%.

6. The method for preparing uniform-sized silk fibroin microspheres as described in claim 1, characterized in that, In S1, the weight average molecular weight of the regenerated silk fibroin is 50kDa-250kDa.

7. The method for preparing uniform-sized silk fibroin microspheres as described in claim 1, characterized in that, In S1, the beta-sheet inhibitor is one or several of LiBr, NaSCN, ZnCl2, and CaCl2. and / or the concentration of the beta-sheet breaker in the aqueous solution of the beta-sheet breaker is 4-9.3 M.

8. The silk fibroin microspheres obtained by the preparation method of any one of claims 1-7.

9. The silk fibroin microspheres of claim 8 for use in medical products, cosmetic products.

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