Silk fibroin microspheres with surface microporous structure, their preparation methods and applications

Abstract

This invention provides silk fibroin microspheres with a surface microporous structure, their preparation method, and applications. The method involves spraying a silk fibroin solution or a mixture of silk fibroin solution and a water-soluble polymer solution into liquid nitrogen to form microspheres, followed by annealing and crystallization at elevated temperatures, and finally thawing or freeze-drying to form Silk I silk fibroin microspheres. The silk fibroin microspheres obtained by this method are entirely based on an aqueous system, requiring no chemical reagents or post-treatment, and remain stable in water. The microspheres prepared by this method have a highly porous surface, a nanoscale hierarchical structure internally, and are rapidly biodegradable. Based on the advantages of green preparation, numerous micropores on the surface, high internal specific surface area, and rapid degradability, these silk fibroin microspheres can be applied in cell expansion, drug delivery, cell microscaffolds, hemostatic powders, and other fields.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, and in particular to a silk fibroin microsphere with a surface microporous structure, its preparation method, and its application. Background Technology

[0002] Silk fibroin is a natural protein extracted from silkworm cocoons. In recent years, silk fibroin has attracted increasing attention in numerous biomedical applications, such as drug delivery, tissue engineering, and cell carriers, due to its biocompatibility, biodegradability, and ability to stabilize other biomolecules. Currently, the preparation of silk fibroin microspheres widely employs methods such as emulsification solvent evaporation, phase separation, high-voltage electrostatic methods, and spray drying.

[0003] Since regenerated silk fibroin is water-soluble, organic solvents such as methanol and ethanol are typically used to induce β-sheet formation to obtain a stable silk II structure in order to improve the water stability of silk fibroin microspheres. However, the use of organic solvents reduces the biocompatibility of the material and can even lead to drug denaturation and changes in microsphere morphology. Secondly, silk II microspheres obtained by induction with organic solvents such as ethanol usually have a dense surface structure, limiting the application range of silk fibroin microspheres. In addition, silk II microspheres contain a large number of β-sheet structures, resulting in a slow biodegradation rate.

[0004] In the prior art, patent CN108553690B discloses a strontium-doped porous silk fibroin microsphere and its preparation method. This patent provides a method for preparing strontium-doped silk fibroin-based porous microspheres using silk fibroin and strontium chloride as the main materials, employing an emulsion method combined with freeze-drying. The patent involves pouring a silk fibroin and strontium chloride mixture into a pre-cooled petroleum ether solution containing an emulsifier, then allowing it to settle in a frozen environment; the petroleum ether is then removed under a freezing atmosphere to obtain strontium-doped silk fibroin microspheres containing ice crystals. These microspheres are then frozen and freeze-dried to obtain dried strontium-doped silk fibroin microspheres. Finally, alcohol treatment renders them insoluble in water. While this microsphere preparation method utilizes an emulsion method and leverages alcohol to induce the transformation of silk fibroin into a silk II crystalline structure, the residual organic solvent significantly reduces the biocompatibility of the material, and the slow degradation rate of silk II may affect the effective utilization rate of drugs.

[0005] In the prior art, patent CN 106637969 B discloses a method for preparing tussah silk fibroin microspheres with a three-dimensional porous structure. This patent involves adding a foaming agent and a cross-linking agent to a tussah silk fibroin solution, allowing it to stand, stirring, and centrifuging to form tussah silk fibroin particles. The tussah silk fibroin particles are then added to a sodium chloride solution, heated and stirred, centrifuged to separate the precipitate, washed with ethanol, removed, dried, and then freeze-dried at low temperature to obtain tussah silk fibroin microspheres with a three-dimensional porous structure. This method obtains a porous structure by adding a foaming agent and a cross-linking agent, and uses ethanol treatment to make the silk fibroin microspheres water-stable. However, the use of organic solvents and cross-linking agents significantly reduces the biocompatibility of the material and affects the degradation rate of the silk fibroin microspheres. In addition, a literature (Materials, 2018, 11(8):1280.) reports the preparation of porous silk fibroin microspheres by adding glycerol to a silk fibroin solution and using an electrostatic differentiation method. Although the silk fibroin microspheres prepared in this article also have water stability, the method involves adding glycerol to the silk fibroin solution in the initial stage, resulting in microspheres with a silk II structure and rich in β-sheets. This leads to a slow degradation rate of the silk fibroin microspheres, and the presence of glycerol also limits their application.

[0006] In view of this, it is necessary to design a silk fibroin microsphere with a multi-microporous structure, rapid degradation capability, preparation process without other chemical reagents, and water stability to solve the above problems. Summary of the Invention

[0007] To address the shortcomings of the existing technology, the present invention aims to provide silk fibroin microspheres with a surface microporous structure, their preparation method, and applications. This preparation method requires no chemical reagents, and the prepared microspheres possess a highly porous surface, an internal nanoscale hierarchical structure, water stability, and rapid biodegradability. Other polymers and small molecule drugs can be added to the prepared silk fibroin microspheres. Because these silk fibroin microspheres simultaneously possess advantages such as green preparation, numerous micropores on the surface, high internal specific surface area, and rapid degradability, they can be applied in fields such as cell expansion, hemostatic powders, cell microscaffolds, and drug carriers.

[0008] To achieve the above objectives, the present invention provides a method for preparing silk fibroin microspheres with a surface microporous structure, comprising the following steps:

[0009] S1. Prepare a silk fibroin solution for later use;

[0010] S21. The silk fibroin solution obtained in step S1 is sprayed into liquid nitrogen to form microspheres; then the microspheres are subjected to heating and annealing treatment, and finally freeze-dried or directly thawed to obtain silk fibroin microspheres with a surface microporous structure; or

[0011] S22. The silk fibroin solution obtained in step S1 is mixed with a water-soluble polymer solution to obtain a mixture. The mixture is then sprayed into liquid nitrogen to form microspheres. Subsequently, the microspheres are subjected to heating and annealing treatment, and finally freeze-dried or directly thawed to obtain silk fibroin microspheres with a surface microporous structure.

[0012] Furthermore, in steps S21 and S22, the spraying method is electrostatic differentiation, ultrasonic atomization, high-pressure airflow spraying, or microfluidic technology, etc.

[0013] Furthermore, the heating annealing treatment refers to freezing the microspheres removed from liquid nitrogen at a temperature of -20℃ to 0℃ for 12h to 96h. The thawing includes melting at a temperature of 4 to 60℃.

[0014] Furthermore, the freeze-drying refers to drying under reduced pressure at -50°C.

[0015] Further, in steps S21 and S22, the mass percentage of the silk fibroin solution is 0.05–20 wt%.

[0016] Further, in step S22, the concentration of the water-soluble polymer solution is 0.05–5.0 wt%; the water-soluble polymer is one or more of hyaluronic acid and its sodium salt, alginate, gelatin, chondroitin sulfate, cellulose derivatives, starch and its derivatives.

[0017] Furthermore, in step S22, the amount of the water-soluble polymer used is 1 to 50% of the silk mass.

[0018] Furthermore, in step S1, the silk fibroin solution is a regenerated silk fibroin solution obtained by degumming, dissolving, and dialysis of silk or cocoons. The silk or cocoons originate from mulberry silkworms or tussah silkworms.

[0019] Furthermore, the silk fibroin microspheres obtained after freeze-drying or direct thawing can be further screened to obtain silk fibroin microspheres with relatively uniform size; the mesh size used for screening is 15 to 40 mesh.

[0020] To achieve the above objectives, the present invention also provides a silk fibroin microsphere with a surface microporous structure, which is prepared by the preparation method described in any of the foregoing technical solutions.

[0021] The aforementioned silk fibroin microspheres with surface microporous structures can be used in cell expansion, drug carriers, cell microscaffolds, hemostatic powders, and other fields.

[0022] The beneficial effects of this invention are:

[0023] 1. The present invention provides a method for preparing silk fibroin microspheres with a surface microporous structure. This involves spraying a silk fibroin solution or a mixture of a silk fibroin solution and a water-soluble polymer solution into liquid nitrogen to form microspheres, followed by annealing and crystallization at elevated temperatures. Finally, the microspheres are directly thawed or freeze-dried to form water-stable silk I-structured silk fibroin microspheres. This invention provides a method entirely based on an aqueous solution system, eliminating the need for chemical additives or organic solvents.

[0024] 2. The silk fibroin microspheres prepared by this invention have micron-sized macropores on their surface and nanoscale hierarchical structures inside, and also have water stability; moreover, compared with silk II silk fibroin microspheres, the silk fibroin microspheres prepared by this invention can achieve rapid biodegradation.

[0025] 3. The silk fibroin microspheres prepared by this invention can be supplemented with other macromolecular substances and small molecule drugs. Based on the advantages of these silk fibroin microspheres—including green preparation, numerous micropores on the surface, high internal specific surface area, and rapid degradation—they can be applied in cell expansion, drug carriers, cell microscaffolds, hemostatic powders, and other fields. Attached Figure Description

[0026] Figure 1 The image shows the morphology of the silk I microspheres with a surface microporous structure provided in Example 1 under an electron microscope at a scale bar of 500 μm.

[0027] Figure 2 The image shows the morphology of the silk I microspheres with surface microporous structure provided in Example 1 under an electron microscope with a scale bar of 150 μm. The image shows that the microspheres have a nanoscale hierarchical structure inside.

[0028] Figure 3 Electron microscope image of the material provided for Comparative Example 1, with a scale bar of 150 μm.

[0029] Figure 4 The images show the stability of the materials provided in Example 1 and Comparative Example 1 in water; where a is an image of Example 1 and b is an image of Comparative Example 1.

[0030] Figure 5 The image shows the morphology of silk fibroin microspheres with a silk II structure obtained by ethanol treatment as provided in Comparative Example 2, with a scale bar of 100 μm. The image shows that there are no microporous structures on the surface of the microspheres.

[0031] Figure 6 The XRD curves of the silk fibroin microspheres provided in Example 1 and Comparative Example 2 are shown.

[0032] Figure 7The degradation curves of the silk fibroin microspheres provided in Example 1 and Comparative Example 2 after 14 days are shown.

[0033] Figure 8 The images show the morphology of endothelial cells proliferating on microporous silk I microspheres after one day and seven days, as provided in Example 1, under laser confocal microscopy at 200x magnification.

[0034] Figure 9 This is a quantitative schematic diagram of the proliferation of endothelial cells on microporous silk fibroin microspheres provided in Example 1 using CCK-8.

[0035] Figure 10 Electron microscope morphology of the material provided for Comparative Example 3, with a scale bar of 1 μm. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0037] Additionally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0038] This invention provides a method for preparing silk fibroin microspheres with a surface microporous structure, comprising the following steps:

[0039] S1. Prepare a silk fibroin solution for later use;

[0040] S21. The silk fibroin solution obtained in step S1 is sprayed into liquid nitrogen to form microspheres; then the microspheres are subjected to heating and annealing treatment, and finally freeze-dried or directly thawed to obtain silk fibroin microspheres with a surface microporous structure; or

[0041] S22. The silk fibroin solution obtained in step S1 is mixed with a water-soluble polymer solution to obtain a mixture. The mixture is then sprayed into liquid nitrogen to form microspheres. Subsequently, the microspheres are subjected to heating and annealing treatment, and finally freeze-dried or directly thawed to obtain silk fibroin microspheres with a surface microporous structure.

[0042] In step S1, the silk fibroin solution is a regenerated silk fibroin solution obtained by degumming, dissolving, and dialysis of silk or cocoons. The silk or cocoons are derived from mulberry silkworms or tussah silkworms. In steps S21 and S22, the mass percentage of the silk fibroin solution is 0.05–20 wt%.

[0043] In steps S21 and S22, the spraying method is electrostatic differentiation, ultrasonic atomization, high-pressure airflow spraying, or microfluidic technology, etc.

[0044] The heating annealing process refers to freezing the microspheres removed from liquid nitrogen at a temperature of -20℃ to 0℃ for 12h to 96h. The thawing process includes melting at a temperature of 4 to 60℃.

[0045] The freeze-drying refers to drying under reduced pressure at -50°C.

[0046] In step S22, the concentration of the water-soluble polymer solution is 0.05 to 5.0 wt%; the water-soluble polymer is one or more of hyaluronic acid and its sodium salt, alginate, gelatin, chondroitin sulfate, cellulose derivatives, starch and its derivatives.

[0047] In step S22, the amount of the water-soluble polymer used is 1 to 50% of the silk mass.

[0048] Furthermore, the silk fibroin microspheres obtained after freeze-drying or direct thawing can be further screened to obtain silk fibroin microspheres with relatively uniform size; the mesh size used for screening is 15 to 40 mesh.

[0049] The preparation method of silk fibroin microspheres with surface microporous structure provided by the present invention will be described below with reference to specific embodiments.

[0050] Example 1

[0051] This embodiment provides a method for preparing silk fibroin microspheres with a surface microporous structure, including the following steps:

[0052] S1. Prepare a silk fibroin solution for later use;

[0053] Specifically, the following process can be used: 100g of silkworm silk is immersed in 5L of 0.1% sodium carbonate solution and boiled at 98-100℃ for 30 minutes, repeated three times to degumm the silkworm cocoons. After thorough washing and drying, pure fibroin fibers are obtained. The treated fibroin fibers are then immersed in a 9.3M lithium bromide solution and dissolved at 60℃ for one hour. After cooling, the dissolved solution is poured into a dialysis bag with a molecular weight cutoff of 9-14kDa and dialyzed with deionized water. After dialysis, the solution is filtered through gauze to obtain a fibroin solution, and its concentration is determined by the dry weight method. To obtain a higher concentration solution, the fibroin solution contained in the dialysis membrane is placed in a PEG solution and reverse dialysis is performed. The prepared fibroin solution is poured into centrifuge tubes, centrifuged to remove aggregates and air bubbles, and stored in a 4℃ refrigerator for later use.

[0054] S2. The silk fibroin solution was diluted to 1% by mass, and the solution was sprayed into liquid nitrogen by electrostatic differentiation to obtain microspheres. The microspheres were then transferred to a -4°C freezer for annealing for 48 hours to induce a transformation in their crystalline structure. Finally, freeze-drying (i.e., drying under reduced pressure at -50°C for 48 hours) was performed to obtain silk I microspheres with a surface microporous structure. These microspheres were then sieved sequentially through 18-mesh and 25-mesh sieves to obtain relatively uniform-sized silk I microspheres with a surface microporous structure.

[0055] The final prepared silk I microspheres with surface microporous structure were visualized using scanning electron microscopy at scale bars of 500 μm and 150 μm, as shown in the following figures. Figure 1 and Figure 2 As shown, compared to Silk II silk fibroin microspheres, this microsphere has a large number of porous structures on its surface and a nanoscale hierarchical structure inside, which can provide a better three-dimensional growth environment for subsequent cell growth.

[0056] The stability test results in water (after immersion in water for 14 days) are as follows: Figure 4 As shown in Figure a, the microspheres can be seen to retain their shape in water and do not dissolve even after being soaked for 14 days.

[0057] After endothelial cells were placed on the porous silk I microspheres obtained in step S2 and proliferated for one day, their condition under a laser confocal microscope was as follows. Figure 8 As shown in Figure a, the results indicate good cell growth (green represents the cytoskeleton; more green indicates better cell growth). Endothelial cells, after proliferating for seven days on these microporous silk I microspheres, are shown in the following image under a laser confocal microscope. Figure 8 As shown in Figure b, the results indicate that cell growth remained in good condition over time.

[0058] In addition, the proliferation of CCK-8 cells on the microspheres prepared in Example 1 was statistically analyzed, and the results are as follows: Figure 9 As shown, the proliferation of endothelial cells on the microporous silk I microspheres prepared in Example 1 was relatively stable and steady at 1, 3, and 7 days, further confirming that the microporous silk I microspheres prepared in Example 1 can serve as an excellent carrier for cell growth and proliferation.

[0059] Example 2

[0060] The only difference between Example 2 and Example 1 is that in step S2, the microspheres after the heating and annealing treatment are thawed at room temperature (25°C) to obtain a well-dispersed silk fibroin microsphere suspension. The rest is roughly the same as in Example 1 and will not be repeated here.

[0061] Example 3

[0062] The only difference between Example 3 and Example 1 is that in step S2, the microspheres are heated and annealed for 72 hours. The rest is roughly the same as in Example 1 and will not be repeated here.

[0063] Example 4

[0064] The only difference between Example 4 and Example 1 is that in step S2, the silk fibroin solution is sprayed into liquid nitrogen by ultrasonic atomization to obtain microspheres. The rest is roughly the same as in Example 1, and will not be repeated here.

[0065] Examples 5-7

[0066] The difference between Examples 5-7 and Example 1 is that the mass fraction of the silk fibroin solution in step S2 is changed; otherwise, they are largely the same as in Example 1 and will not be repeated here. The mass fractions of the silk fibroin solution in Examples 5-7 are shown in the table below.

[0067] project mass fraction of silk fibroin solution Example 1 1wt% Example 5 0.05wt% Example 6 10wt% Example 7 20wt%

[0068] Example 8

[0069] The main difference between Example 8 and Example 1 lies in the solvent system used to prepare the silk fibroin solution, as detailed below:

[0070] S1. Prepare a silk fibroin solution for later use;

[0071] Specifically, the following process can be used: 100g of silkworm silk is immersed in 5L of 0.1% sodium carbonate solution and boiled at 98-100℃ for 30 minutes, repeated three times to degumm the silkworm cocoons. After thorough washing and drying, pure fibroin fibers are obtained. The treated fibroin fibers are then immersed in a prepared ternary solution of CaCl2 / CH3CH2OH / H2O with a molar ratio of 1:2:8 and dissolved at 72℃ for one hour. After cooling, the dissolved solution is poured into a dialysis bag with a molecular weight cutoff of 9-14kDa and dialyzed with deionized water. After dialysis, the solution is filtered through gauze to obtain a fibroin solution, and its concentration is determined by the dry weight method. To obtain a higher concentration solution, the fibroin solution contained in the dialysis membrane is placed in a PEG solution and subjected to reverse dialysis. The prepared fibroin solution is poured into centrifuge tubes, and aggregates and air bubbles are removed by centrifugation. The solution is then stored in a refrigerator at 4℃ for later use.

[0072] S2 is largely the same as step S2 in Example 1, and will not be repeated here.

[0073] Example 9

[0074] The only difference between Example 9 and Example 8 is that in step S2, the microspheres after the heating and annealing treatment are thawed at room temperature (25°C) to obtain a well-dispersed silk fibroin microsphere suspension. The rest is roughly the same as in Example 8 and will not be repeated here.

[0075] Example 10

[0076] The only difference between Example 10 and Example 8 is that in step S2, the microspheres are heated and annealed for 72 hours. The rest is roughly the same as in Example 8, and will not be repeated here.

[0077] Examples 11-13

[0078] The difference between Examples 11-13 and Example 8 is that the mass fraction of the silk fibroin solution in step S2 was changed; otherwise, they are largely the same as in Example 8 and will not be repeated here. The mass fractions of the silk fibroin solution in Examples 11-13 are shown in the table below.

[0079] project mass fraction of silk fibroin solution Example 8 1wt% Example 11 0.05wt% Example 12 10wt% Example 13 20wt%

[0080] Example 14

[0081] The main difference between Example 14 and Example 1 is that the source of the silkworm cocoons used to prepare the silk fibroin solution is different. Specifically, the cocoons used in step S1 are tussah silkworm cocoons. The rest is roughly the same as in Example 1, and will not be repeated here.

[0082] Example 15

[0083] The only difference between Example 15 and Example 14 is that in step S2, the silk fibroin solution is sprayed into liquid nitrogen by ultrasonic atomization to obtain microspheres. The rest is roughly the same as in Example 14, and will not be repeated here.

[0084] Example 16

[0085] The only difference between Example 16 and Example 14 is that in step S2, the microspheres are heated and annealed for 72 hours. The rest is roughly the same as in Example 14 and will not be repeated here.

[0086] Examples 17-19

[0087] The difference between Examples 17-19 and Example 14 is that the mass fraction of the silk fibroin solution in step S2 is changed; otherwise, they are largely the same as in Example 14 and will not be repeated here. The mass fractions of the silk fibroin solution in Examples 17-19 are shown in the table below.

[0088] project mass fraction of silk fibroin solution Example 14 1wt% Example 17 0.05wt% Example 18 10wt% Example 19 20wt%

[0089] Example 20

[0090] The main difference between Example 20 and Example 14 lies in the solvent system used to prepare the silk fibroin solution, as detailed below:

[0091] S1. Prepare a silk fibroin solution for later use;

[0092] Specifically, the following process can be used: 100g of tussah silkworm cocoons are immersed in 5L of 0.1% sodium carbonate solution and boiled at 98-100℃ for 30 minutes, repeated three times to degumm the cocoons. After thorough washing and drying, pure silk fibroin fibers are obtained. The treated silk fibroin fibers are then immersed in a prepared ternary solution of CaCl2 / CH3CH2OH / H2O with a molar ratio of 1:2:8 and dissolved at 72℃ for one hour. After cooling, the dissolved solution is poured into a dialysis bag with a molecular weight cutoff of 9-14kDa and dialyzed with deionized water. After dialysis, the solution is filtered through gauze to obtain a silk fibroin solution, which is stored in a refrigerator at 4℃ for later use, and its concentration is determined by the dry weight method. The prepared silk fibroin solution is poured into centrifuge tubes and centrifuged to remove aggregates and air bubbles.

[0093] S2 is largely the same as step S2 in Example 14, and will not be described again here.

[0094] Example 21

[0095] The only difference between Example 21 and Example 20 is that in step 2, the silk fibroin solution is sprayed into liquid nitrogen by ultrasonic atomization to obtain microspheres. The rest is roughly the same as in Example 20, and will not be repeated here.

[0096] Example 22

[0097] The only difference between Example 22 and Example 21 is that in step 2, the microspheres are heated and annealed for 72 hours. The rest is roughly the same as Example 21 and will not be repeated here.

[0098] Examples 23-25

[0099] The difference between Examples 23-25 ​​and Example 20 is that the mass fraction of the silk fibroin solution in step S2 is changed; otherwise, they are largely the same as in Example 20 and will not be repeated here. The mass fractions of the silk fibroin solution in Examples 23-25 ​​are shown in the table below.

[0100] project mass fraction of silk fibroin solution Example 20 1wt% Example 23 0.05wt% Example 24 10wt% Example 25 20wt%

[0101] Example 26

[0102] This embodiment provides a method for preparing silk fibroin-based composite microspheres with a surface microporous structure, including the following steps:

[0103] S1 is the same as step S1 in Example 1, and will not be repeated here;

[0104] S2. Add hyaluronic acid solution (hyaluronic acid content accounts for 1% of the silk fibroin protein content) to the solution in step S1, and stir thoroughly to obtain a reaction solution; obtain microspheres in liquid nitrogen by electrostatic differentiation of the reaction solution; then transfer the microspheres to a -4℃ freezer for heating and annealing for 48 hours to induce a transformation of their crystal structure. Finally, freeze-dry (i.e., dry under reduced pressure at -50℃ for 48 hours) to obtain silk I silk fibroin-based composite microspheres with a multi-microporous structure.

[0105] Example 27

[0106] The difference between Examples 27-28 and Example 26 is that the mass fraction of the silk fibroin solution, the type of water-soluble polymer, and the percentage of water-soluble polymer used relative to the silk mass in step S2 were changed, as shown in the table below. Other aspects are largely the same as in Example 26 and will not be repeated here.

[0107]

[0108] The water-soluble polymer may also be one or more of chondroitin sulfate, cellulose derivatives, starch and its derivatives.

[0109] Experiments show that silk fibroin microspheres with surface microporous structures can be obtained in Examples 2-28.

[0110] Comparative Example 1

[0111] The difference between Comparative Example 1 and Example 1 is that the silk fibroin solution was placed in liquid nitrogen, not sprayed into liquid nitrogen using an electrostatic differentiation method, and no heating annealing treatment was performed. Specifically:

[0112] S1 is the same as step S1 in Example 1, and will not be repeated here;

[0113] S2. Dilute the silk fibroin solution obtained in step S1 to 1% by mass, then freeze the silk fibroin solution under liquid nitrogen conditions, and then freeze-dry it (i.e., dry it under reduced pressure at -50°C for 48 hours).

[0114] The final prepared material is shown in the scanning electron microscope morphology image at a scale bar of 300 μm as follows. Figure 3 As shown in the figure, this method cannot produce silk fibroin microspheres with a surface microporous structure; it results in porous sponges. The stability test results in water (immersion in water for 5 minutes) are as follows: Figure 4As shown in b, the material dissolves immediately in water and cannot maintain its shape.

[0115] Comparative Example 2

[0116] The difference between Comparative Example 2 and Example 1 is that no annealing treatment was performed. Specifically:

[0117] S1 is the same as step S1 in Example 1, and will not be repeated here;

[0118] S2. The solution is diluted to 1%, and microspheres are obtained in liquid nitrogen by electrostatic differentiation. The microspheres are then freeze-dried (i.e., dried under reduced pressure at -50°C for 48 hours), and the dried silk fibroin microspheres are treated with 75% ethanol for 2 hours, followed by freeze-drying again to obtain silk fibroin microspheres with a silk II structure. Finally, they are sieved sequentially through 18-mesh and 25-mesh sieves to obtain relatively uniform silk II silk fibroin microspheres.

[0119] The final morphology of the prepared silk II microspheres under a scanning electron microscope with a scale bar of 100 μm is shown in the following figure. Figure 5 As shown, the material surface is dense and has no pore structure.

[0120] Figure 6 The XRD curves of the silk fibroin microspheres provided in Example 1 and Comparative Example 2 are shown. It can be seen that the material obtained in Example 1 has a silk I structure, while the material obtained in Comparative Example 2 has a silk II structure.

[0121] Figure 7 The degradation curves of the silk fibroin microspheres provided in Example 1 and Comparative Example 2 after 14 days are shown. It can be seen that the material obtained in Example 1 degraded by approximately 80% after 14 days, while the material obtained in Comparative Example 2 degraded by only approximately 30%, further demonstrating that the material prepared by the method provided in this patent can achieve rapid degradation.

[0122] Comparative Example 3

[0123] The difference between Comparative Example 3 and Example 1 is that the silk fibroin solution was placed in liquid nitrogen, rather than being sprayed into liquid nitrogen using an electrostatic differentiation method. Specifically:

[0124] S1 is the same as step S1 in Example 1, and will not be repeated here;

[0125] S2. Dilute the silk fibroin solution obtained in step S1 to 1% by mass, then freeze the silk fibroin solution under liquid nitrogen conditions, followed by annealing at -4°C for 48 hours, and finally freeze-dry (i.e., freeze-dry at -50°C under reduced pressure for 48 hours). The morphology of the finally prepared material under a scanning electron microscope with a scale bar of 1 μm is shown in the following figures. Figure 10 As shown, this method cannot prepare silk fibroin microspheres with surface microporous structures.

[0126] In summary, this invention provides silk I fibroin microspheres with a multi-microporous structure, their preparation method, and applications. The water-stable silk I fibroin microspheres obtained by this method are entirely based on an aqueous solution system, requiring no chemical reagents or post-treatment. Furthermore, the microspheres possess numerous porous structures on their surface and nanoscale hierarchical structures internally, enabling rapid biodegradation. In addition, other polymers and small molecule drugs can be added to the fibroin microspheres. Because these fibroin microspheres simultaneously possess advantages such as green preparation, numerous micropores on their surface, high internal specific surface area, and rapid degradability, they can be applied in cell expansion, drug delivery, cell microscaffolds, hemostatic powders, and other fields.

[0127] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing silk fibroin microspheres with a surface microporous structure, characterized in that, Includes the following steps: S1. Prepare a silk fibroin solution for later use; S21. The silk fibroin solution obtained in step S1 is sprayed into liquid nitrogen to form microspheres; then the microspheres are subjected to heating and annealing treatment, and finally freeze-dried or directly thawed to obtain silk fibroin microspheres with surface microporous structure. or S22. The silk fibroin solution obtained in step S1 is mixed with a water-soluble polymer solution to obtain a mixture, and then the mixture is sprayed into liquid nitrogen to form microspheres; subsequently, the microspheres are subjected to a heating annealing treatment, and finally freeze-dried or directly thawed to obtain silk fibroin microspheres with a surface microporous structure; the heating annealing treatment refers to freezing the microspheres taken out of the liquid nitrogen at a temperature of -20℃ to 0℃ for 12 h to 96 h; The silk fibroin solution has a mass percentage of 0.05~20 wt%. The concentration of the water-soluble polymer solution is 0.05~5.0wt%; the amount of the water-soluble polymer is 1~50% of the silk mass; the water-soluble polymer is one or more of hyaluronic acid and its sodium salt, alginate, gelatin, chondroitin sulfate, cellulose derivatives, starch and its derivatives.

2. The method for preparing silk fibroin microspheres with a surface microporous structure according to claim 1, characterized in that: In steps S21 and S22, the spraying method is electrostatic differentiation, ultrasonic atomization, high-pressure airflow spraying, or microfluidic technology.

3. The method for preparing silk fibroin microspheres with a surface microporous structure according to claim 1, characterized in that: The freeze-drying refers to drying under reduced pressure at -50°C.

4. The method for preparing silk fibroin microspheres with a surface microporous structure according to claim 1, characterized in that: In step S1, the silk fibroin solution is a regenerated silk fibroin solution obtained by degumming, dissolving, and dialysis of silk or cocoon.

5. A silk fibroin microsphere with a surface microporous structure, characterized in that: The silk fibroin microspheres with surface microporous structures are prepared by the preparation method according to any one of claims 1 to 4.

6. The application of a silk fibroin microsphere with a surface microporous structure prepared by the preparation method according to any one of claims 1 to 4, or the silk fibroin microsphere with a surface microporous structure according to claim 5, characterized in that: The silk fibroin microspheres with surface microporous structures are used to prepare drug carriers, cell microscaffolds, and hemostatic powders.

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