Silk fibroin microsphere with controllable size and structure and preparation method thereof

Abstract

The embodiment of the invention discloses silk fibroin microspheres with controllable size and structure and a preparation method of the silk fibroin microspheres. The method comprises the following steps: filling a silk fibroin solution into an injector provided with a needle, controlling the propelling speed of the injector by using an injection pump, so that the silk fibroin solution is dropped into liquid nitrogen in a liquid drop form for quick freezing, fishing out after forming, freezing and drying, and performing water vapor treatment on the obtained freeze-dried microspheres to reduce the particle size, thereby obtaining the silk fibroin microspheres. The size and the structure of the silk fibroin microspheres are controllable. According to the invention, the size regulation of the silk fibroin microspheres is realized by changing the concentration of silk fibroin or inducing the microspheres to be converted from flexibility to rigidity (random curling and alpha-helix are converted into beta-folding) to trigger volume reduction times; the structure regulation and control of the silk fibroin microspheres are realized by controlling the reduction multiple of the microspheres, adding a pore-foaming agent into the silk fibroin solution or changing the aggregation state (converting molecules into nanofibers) of the silk fibroin solution.

Description

Technical Field

[0001] The embodiments of the present invention relate to the technical field of microsphere preparation, and particularly to silk fibroin microspheres with controllable size and structure and a preparation method thereof. Background Art

[0002] Microspheres are tiny spherical or quasi-spherical particles made from polymers through physical or chemical methods, with particle sizes ranging from 1 to 1000 μm. Microspheres of varying sizes and structures have varying specific surface areas, porosities, and spatial structures, significantly impacting tissue regeneration, drug delivery, and microreactor efficiency. Most cells have a particle size greater than 20 μm. Microspheres that are too small (<20 μm) are easily recognized and phagocytosed by macrophages in the body. Different application scenarios require different microsphere size and structure. For example, microspheres used for tissue filling typically range in size from 20 to 70 μm. Depending on the intended tissue regeneration, the microsphere structure can be either solid (polycaprolactone microspheres) or porous (hydroxyapatite microspheres). For drug delivery, the size and structure of the microspheres can be flexibly controlled to achieve high drug loading and sustained, constant release. For microreactors for cells or active substances, the size of the microspheres can be controlled between 300 and 1000 μm, with a porous network structure that facilitates cell loading and proliferation. Furthermore, compared to irregular particles, well-rounded microspheres have a smoother surface, resulting in less friction with human tissue after entry, less likely to cause a foreign body reaction, and a more aesthetically pleasing appearance. Therefore, the development of microspheres with controllable size and structure has broad application prospects in medical aesthetics, drug delivery, tissue engineering, microreactor construction, water purification, and energy conversion.

[0003] Silk fibroin (molecular weight approximately 325,000 Da) is composed of light and heavy polypeptide chains, which are bound together by disulfide bonds to form a crystalline phase (approximately 2 / 3) and an amorphous phase (approximately 1 / 3). Within silk fibroin, the heavy chains can form various β-sheet structures, known as crystallites, which serve as the primary structural component and provide rigidity to the fiber material. The light chains, on the other hand, provide flexibility to the fiber material. The heavy chains are primarily composed of glycine (approximately 43-46%), alanine (approximately 25-30%), serine (approximately 12%), tyrosine (approximately 5%), and valine (approximately 2%). Silk fibroin exhibits two major forms: the amorphous phase Silk I and the crystalline phase Silk II. The amorphous phase Silk I consists of amorphous regions with randomly coiled chains and is readily soluble in water. The crystalline phase Silk II is composed of a large number of antiparallel β-sheets, resulting in an ordered structure that is insoluble in water (as well as weak acids, bases, and chaotropic solutions). The amorphous Silk I phase can be easily transformed into the crystalline Silk II phase under specific conditions (such as methanol, ethanol, salt, or pH). The crystallinity of silk fibroin is closely related to conformational changes (from random coil to β-sheet), which can be controlled by the extraction, dissolution, modification, reorganization, and post-processing conditions (humidity, temperature, pH, shear force, ultrasound, etc.) applied to the final material. These unique structures endow silk fibroin with excellent plasticity, controllable size and structure, and stability. Therefore, the development of microspheres with controllable size and structure using silk fibroin is feasible from a material perspective.

[0004] Traditional methods for preparing microspheres include emulsification, spray drying, phase separation, and spray freezing. Emulsification involves dispersing two immiscible liquids, one in the form of microparticles (droplets or liquid crystals), in the other to form an emulsion. The increased interfacial area between the two liquids during emulsion formation makes this system thermodynamically unstable. To stabilize the emulsion, a third component, such as an emulsifier, is added to reduce the interfacial energy. The microspheres produced by the emulsification method have a wide size distribution, requiring screening to achieve uniformity. This increases the number of steps and reduces yield. Furthermore, the development of implantable microspheres places high demands on the selection of emulsifiers, requiring the addition of additional additives during microsphere preparation and cleaning, which is complex and can lead to residual additives in the prepared microspheres, posing a safety risk.

[0005] Spray drying is a method that applies spray system technology to material drying. After the thin material is atomized in a drying chamber, the water rapidly vaporizes upon contact with hot air, resulting in a dry product. This method can directly dry solutions and emulsions into powdered or granular products, eliminating steps such as evaporation and pulverization. Marco et al. used spray drying at high temperatures to prepare silk fibroin microspheres, resulting in irregular morphology, a wide range of particle sizes, and poor uniformity. Furthermore, this method has a low yield (<50%).

[0006] Phase separation refers to the process by which a homogeneous mixture tends to separate into separate phases due to interactions between the different components, leading to thermodynamic equilibrium. This process is typically driven by a decrease in the system's free energy and involves kinetic processes such as nucleation and growth. Advantages: No toxic reagents or organic solvents are required. Disadvantages: A wide particle size distribution is achieved, and the yield of microspheres larger than 20 μm is low (<5%).

[0007] Spray freezing uses an atomizer to convert a liquid material into tiny droplets, which are then rapidly frozen by contact with a cold medium. Finally, vacuum freeze-drying is performed to produce a powder product. This process not only increases the surface area of ​​the liquid material but also improves heat and mass transfer rates. Compared to spray drying, this method produces a porous product with higher bioactivity and better quality. Ji et al. combined high-voltage electrostatic spray freezing with freeze-drying, using glucose as a pore-forming agent to control pore formation and produce silk fibroin microspheres with enhanced cell adhesion and proliferation. However, the particle size of the microspheres produced by this method is controlled by the needle size: the smaller the needle, the smaller the microsphere size. However, due to the inherent viscosity of silk fibroin solutions, the use of fine needles is prone to clogging, resulting in low production efficiency. Compared to fine needles, coarse needles are more convenient and reliable, maintaining a stable output and improving yield. However, the silk fibroin microspheres produced with coarse needles have a larger particle size, making them difficult to control. Therefore, how to use a thick needle to prepare silk fibroin microspheres with controllable particle size, and on this basis obtain microspheres with controllable structure (solid, porous) and good dispersibility is a key technical problem that needs to be solved in the present invention. Summary of the Invention

[0008] To this end, an embodiment of the present invention provides a silk fibroin microsphere with controllable size and structure and a preparation method thereof.

[0009] In order to achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0010] According to a first aspect of an embodiment of the present invention, the present invention provides a method for preparing silk fibroin microspheres with controllable size and structure as described above, the method comprising:

[0011] The silk fibroin solution is loaded into a syringe equipped with a needle, and the propulsion speed of the syringe is controlled by a syringe pump, so that the silk fibroin solution is dripped into liquid nitrogen in the form of droplets for quick freezing. After forming, the solution is fished out and freeze-dried. The resulting freeze-dried microspheres are treated with water vapor to reduce the particle size, thereby obtaining silk fibroin microspheres with controllable size and structure.

[0012] Furthermore, the concentration of the silk fibroin solution is 0.1% to 20%.

[0013] Furthermore, the preparation method of the silk fibroin solution is as follows:

[0014] (1) The silk was soaked in a sodium carbonate solution with a concentration of 0.02M~0.04M, treated at 90~100℃ for 10min~1.5h, washed and dried to obtain degummed silk;

[0015] (2) soaking the degummed silk in a lithium bromide solution having a concentration of 40% to 45% and treating the solution at 58 to 62° C. for 3 to 4 hours to obtain a silk solution;

[0016] (3) The silk solution was transferred to a dialysis bag with a molecular weight cutoff of 3500 Da, dialyzed in pure water for 48 h, and centrifuged to obtain the silk fibroin solution.

[0017] Furthermore, the needle size is 25~32G;

[0018] The advancement speed is 0.16 ml / min to 0.25 ml / min;

[0019] The freeze-drying conditions are: -80°C for at least 2 hours, or quick freezing using liquid nitrogen.

[0020] Furthermore, the conditions of the water vapor treatment are: temperature 15-100° C., humidity 70-100%, and time 1-10 hours.

[0021] Furthermore, during the process of dripping the silk fibroin solution into liquid nitrogen in the form of droplets, the needle is subjected to an oscillation treatment with an oscillation frequency of 0.1 Hz to 15 Hz.

[0022] Furthermore, before the step of loading the silk fibroin solution into a syringe equipped with a needle, the method further comprises: adding ammonium bicarbonate to the silk fibroin solution, wherein the amount of ammonium bicarbonate added is 0.1% to 250% of the mass of the silk fibroin in the silk fibroin solution.

[0023] Furthermore, a mixed solution containing water and ethanol is used for water vapor treatment, wherein the mass ratio of water to ethanol in the mixed solution is 8-12:1.

[0024] According to a second aspect of the embodiments of the present invention, the present invention provides a silk fibroin microsphere with controllable size and structure, which is prepared by the method described in any one of the above items.

[0025] Furthermore, the size of the silk fibroin microspheres ranges from 20 μm to 2000 μm, the structure of the microspheres includes solid or porous, the shape of the porous includes regular circles or irregular layers, the size of the pores ranges from 200 nm to 500 μm, and the reduction factor of the microsphere particle size is 1 to 20. The transformation from porous structure to dense structure can be achieved through the reduction factor of the microspheres themselves.

[0026] The present invention achieves size control of silk fibroin microspheres by changing the concentration of silk fibroin or inducing the transformation of microspheres from flexibility to rigidity (from random coil, α-helix to β-pleat), triggering a volume reduction multiple; and achieves structure control of silk fibroin microspheres by controlling the reduction multiple of the microspheres, adding a porogen to the silk fibroin solution, or changing the aggregation state of the silk fibroin solution (from molecules to nanofibers).

[0027] The embodiments of the present invention have the following advantages:

[0028] 1. Controllable size and structure

[0029] Research has found that water vapor treatment can proportionally reduce the size of microspheres, with the size after water vapor treatment being approximately 1 / 2 to 1 / 6 of the size before treatment, while maintaining their original shape. Furthermore, the lower the silk fibroin concentration, the greater the reduction in size. Therefore, microspheres can be prepared using regenerated silk fibroin solutions of varying concentrations. Furthermore, by varying the syringe pump speed, oscillation frequency, and water vapor treatment conditions, microspheres of varying sizes, smooth and regular shapes, uniform distribution, and non-stickiness can be produced to meet the particle size requirements of different applications. Furthermore, the size and structure of silk fibroin microspheres can be modified by adding pore-forming agents.

[0030] 2. Controllable mechanical properties and water solubility

[0031] The secondary structures of silk fibroin fibers are primarily β-sheets, α-helices, and random coils. The β-sheet structure is formed by relatively extended polypeptide chains interacting through hydrogen bonds. The β-sheet structure formed by these molecular chains contains numerous hydrogen bonds, forming a hydrogen bond network that gives protein polypeptide fibers and materials primarily composed of β-sheets their exceptional strength and rigidity. Water vapor treatment reduces the random coil portion of the microspheres and increases the β-sheet portion, thereby enhancing their mechanical properties. The microspheres retain their shape better, solidify, and decrease their water solubility. Comparison of different water vapor treatment temperatures and times revealed that, at the same water vapor treatment time, higher water vapor treatment temperatures resulted in smaller microsphere size, fewer macropores, and enhanced mechanical properties. At the same water vapor treatment temperature, longer water vapor treatment times led to a decrease in microsphere size followed by a decrease in size, and an increase in β-sheet content followed by a decrease in size. This is likely due to the internal porosity of the microspheres decreasing to a critical value, resulting in a dense state that prevented further shrinkage. After being placed in water for a long time, the water vapor-treated microspheres retained their original shape, demonstrating excellent water insolubility and shape retention. Therefore, the mechanical properties and water solubility of the microspheres can be controlled by adjusting the water vapor treatment conditions.

[0032] 3. Controllable degradation time

[0033] Silk fibroin microspheres are biomaterials with applications in cosmetic cosmetics, joint injections, and other fields. These materials require a non-immune response in vivo, a suitable degradation rate, and safe, non-toxic degradation products. Ideally, the degradation rate should be consistent with the rate of tissue growth in vivo. Silk fibroin microspheres degrade both in vivo and in vitro, maintaining their rounded shape without disintegrating into irregular fragments during degradation. Furthermore, the degradation products are excreted through metabolism and do not irritate or trigger an inflammatory response. Studies have found that a higher β-sheet content in the microspheres makes them less susceptible to protease degradation and slower degradation. Water vapor treatment can alter the β-sheet content of the microspheres, and treatment time and temperature also influence this. Therefore, the degradation time of the microspheres can be controlled by adjusting the water vapor treatment conditions. Furthermore, the silk fibroin concentration and porosity within the microspheres also influence the degradation time: higher silk fibroin concentration and lower porosity result in slower degradation. Therefore, microspheres can be prepared using silk fibroin solutions of different concentrations. The size, β-sheet content, and porosity of the microspheres can also be changed by changing the injection pump propulsion speed, oscillation frequency, water vapor treatment conditions, etc., thereby achieving accurate control of the degradation time of the microspheres.

[0034] 4. The preparation method is simple and efficient

[0035] This method utilizes freeze-drying technology combined with water vapor treatment and is simple to operate. When small microspheres are prepared by machines on the market, the silk fibroin is sticky and easily clogs the fine needle, resulting in low production efficiency. Studies have found that a 30G needle can allow the silk fibroin solution to pass smoothly. The present invention first prepares large-sized freeze-dried microspheres with a large needle, and then reduces them to the required size through water vapor treatment, avoiding the shortcomings of using fine needles that are prone to clogging and low production efficiency, and is more practical. Traditional freeze-dried microspheres are fluffy and easy to rehydrate. The prepared microspheres are soft, easy to deform, and dissolve in water. The water vapor treatment of the present invention can keep the microspheres in water for a longer time. At the same time, the special β-pleated structure of silk fibroin is utilized to increase the β-pleated content of the microspheres and enhance the mechanical properties. No toxic reagents are used in the entire preparation process, which is environmentally friendly and biocompatible.

[0036] 5. Wide range of applications

[0037] Microspheres sized 20-50 μm can be used for superficial fillers (e.g., fine line repair), improving skin texture by stimulating collagen regeneration. Microspheres sized 50-200 μm can be used for deep dermal fillers (e.g., nasolabial folds and nasolabial grooves), achieving long-lasting contouring through mechanical support. Microspheres sized 100-300 μm can serve as scaffolding units for 3D printing, supporting cell growth (e.g., bone marrow mesenchymal stem cells). Microspheres sized 500-1000 μm can be used for immobilized enzyme catalysis, with apparent reaction rates 5-10 times higher than traditional batch reactions. Microspheres with a pore size of 1-10 μm can enhance cell adhesion and accelerate tissue repair. Microspheres with a pore size greater than 100 μm can promote endothelial cell ingrowth and accelerate tissue regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the embodiments or the description of the prior art. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other implementation drawings based on the provided drawings without inventive effort.

[0039] Figure 1 Optical photos and low-magnification scanning electron microscope images of the freeze-dried microspheres prepared from a 5.56% silk fibroin solution degummed for 30 minutes and treated with water vapor, as provided by the present invention;

[0040] Figure 2 Scanning electron microscope images of freeze-dried microspheres prepared from a 1.5% silk fibroin solution degummed for 30 minutes and treated with water vapor, provided by the present invention;

[0041] Figure 3The freeze-dried microspheres provided by the present invention are made from a 5.56% silk fibroin solution degummed for 30 minutes and high-magnification scanning electron microscope images of the local surface of the microspheres before and after water vapor treatment;

[0042] Figure 4 This is a scanning electron microscope image of the cross section of the freeze-dried microspheres provided by the present invention, which were prepared using a 5.56% silk fibroin solution degummed for 30 minutes and then treated with water vapor;

[0043] Figure 5 FT-IR spectra and secondary structure distribution of the freeze-dried microspheres and the microspheres after water vapor treatment provided by the present invention;

[0044] Figure 6 The water solubility test results of the microspheres after water vapor treatment provided by the present invention;

[0045] Figure 7 The curve of the residual mass-time variation of the microspheres after water vapor treatment after in vitro degradation using a 5.56% silk fibroin solution with a degumming concentration of 30 minutes provided by the present invention as a raw material;

[0046] Figure 8 In the figure, A is an optical photograph of freeze-dried microspheres prepared from a 3% silk fibroin solution degummed for 10 min, 30 min, and 90 min as provided by the present invention and the microspheres after water vapor treatment; B is the effect of different degumming times on the particle size of freeze-dried microspheres and the microspheres after water vapor treatment provided by the present invention;

[0047] Figure 9 Optical photographs of freeze-dried microspheres and microspheres treated with water vapor provided by the present invention, prepared from a 3% silk fibroin solution degummed for 90 minutes;

[0048] Figure 10 Optical photographs of the particle size changes of the microspheres after post-treatment with different solutions provided by the present invention;

[0049] Figure 11 Scanning electron microscope images of freeze-dried microspheres and water vapor-treated microspheres made from 1% silk fibroin nanofiber gel degummed for 90 minutes provided by the present invention. DETAILED DESCRIPTION

[0050] The following describes the implementation of the present invention using specific embodiments. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. Obviously, the embodiments described are only a portion of the present invention, not all of it. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0051] Silk: Raw silk fibers were purchased from Zhejiang Xiehe Silk Co., Ltd., China, with specifications / item numbers 20-22.

[0052] The concentration of the silk fibroin solution was determined using the evaporation gravimetric method. The specific procedure was as follows: Weigh a weighing boat, record it as w (g), add a certain amount of silk fibroin solution to the weighing boat, and weigh it as w1 (g). Dry the silk fibroin solution and weigh it, record it as w2 (g). The concentration of the silk fibroin solution was calculated according to formula (1). Each measurement was repeated three times, and the average value was taken.

[0053] Concentration of silk fibroin solution (%) = (w2 -w) / (w 1- w)×100% (1)

[0054] Example 1

[0055] This embodiment provides a method for preparing silk fibroin microspheres, comprising the following steps:

[0056] (1) Preparation of silk fibroin solution

[0057] Prepare four silk-boiling pots. Add 12 L of pure water to each pot. Heat to a boil, then add 25.44 g of anhydrous sodium carbonate. Once the water boils, add 30 g of silk. Degumming is controlled at 10, 30, 60, and 90 minutes (stirring every 10 minutes). Wash the silk three times in pure water. Dry the degummed silk in a fume hood. Then, immerse the silk in a 45% lithium bromide aqueous solution, seal with aluminum foil, and place in a 60°C oven for 4 hours, shaking it every hour. After completion, the obtained silk solution was cooled and then transferred to a dialysis bag (3500 Da). It was dialyzed in pure water for 48 hours (the first 24 hours: the water was changed every 3 hours, and the next 24 hours: the water was changed every 8 hours). The dialyzed silk fibroin solution was centrifuged at 4°C and 9000 r / min for 20 minutes to remove impurities. The concentration of the silk fibroin solution obtained from the above four silk boiling pots was determined by evaporation weighing method. The silk fibroin solution of the required concentration was obtained by dilution with deionized water and stored in a 4°C refrigerator for later use.

[0058] (2) Molding and freezing treatment

[0059] The silk fibroin solution prepared in step (1) was loaded into a 10 ml syringe equipped with a 30G needle. The syringe was then fixed to a syringe pump, and the injection speed of the syringe pump was controlled within the range of 0.16 ml / min to 0.25 ml / min. A container containing liquid nitrogen was placed directly below the syringe needle to collect the droplets squeezed out of the syringe and to rapidly freeze them in the liquid nitrogen. At the same time, an oscillation frequency of 0 to 15 Hz was applied to the needle tip to promote the droplets to drip into the liquid nitrogen faster. The droplets formed in the liquid nitrogen were fished out and loaded into a 50 ml test tube. The test tube was then transferred to a freeze dryer and frozen at -80°C for 12 hours to obtain freeze-dried microspheres, which were then stored in the test tube for later use.

[0060] (3) Water vapor treatment

[0061] The freeze-dried microspheres were evenly spread on a water-repellent culture dish, which was then placed in a desiccator (the lid of the desiccator was sealed with vaseline, the bottom of the desiccator contained ultrapure water, and the inside of the desiccator contained a thermometer and a hygrometer for controlling the temperature and humidity to simulate a constant temperature and humidity environment). The humidity, temperature, and time of the water vapor treatment were controlled to produce silk fibroin microspheres (i.e., microspheres after water vapor treatment).

[0062] Test Example 1

[0063] Based on Example 1, water vapor treatment and the effects of different water vapor treatment conditions on the size and structure of microspheres were investigated.

[0064] The silk fibroin solution with a degumming time of 30 min and a concentration of 5.56% obtained in step (1) was subjected to forming and freezing treatment according to the method of step (2) (injection speed of 0.25 ml / min, and no oscillation was applied to the needle tip) to obtain freeze-dried microspheres; the obtained freeze-dried microspheres were subjected to water vapor treatment (using water vapor as the steam source) according to the method of step (3), wherein the humidity was 90%, the temperature was 37°C and 60°C, and the treatment time was 2 h, 4 h, and 7 h, to obtain water vapor treated microspheres.

[0065] Image J software was used to analyze the particle size of the freeze-dried microspheres and the microspheres obtained under different water vapor treatment conditions. The particle size of the freeze-dried microspheres was 2.24 ± 0.05 mm. The test results of the particle size of the microspheres obtained under different water vapor treatment conditions are shown in Table 1 below.

[0066] Table 1

[0067]

[0068] The results showed that compared with silk fibroin microspheres that were not treated with water vapor (i.e., freeze-dried microspheres), the size of silk fibroin microspheres treated with water vapor was significantly reduced (p < 0.01), and the particle size was reduced to about half of the freeze-dried microspheres.

[0069] Analysis revealed that the reduction in the size of the silk fibroin microspheres was primarily due to changes in their crystal structure, resulting in denser packing of the silk fibroin. Increasing the water vapor treatment temperature and prolonging the treatment time resulted in a decrease in the size of the silk fibroin microspheres, but this change was not significant. This suggests that water vapor treatment has an impact on microsphere size, with water vapor treatment significantly reducing the size. Higher temperatures and longer treatment times further reduced the size, but not significantly.

[0070] Test Example 2

[0071] Based on Example 1, the effects of different concentrations of silk fibroin solutions on the size and structure of microspheres were investigated.

[0072] The silk fibroin solutions obtained in step (1) with a degumming time of 30 min and concentrations of 5.56% and 1.5%, respectively, were subjected to forming and freezing treatment according to the method of step (2) (injection speed of 0.16 ml / min, oscillation frequency of 6 Hz) to obtain freeze-dried microspheres; the obtained freeze-dried microspheres were subjected to water vapor treatment according to the method of step (3), wherein the humidity was 90%, the temperature was 60°C, and the treatment time was 2 h, to obtain water vapor treated microspheres.

[0073] Due to the application of vibration, the microspheres can fall into liquid nitrogen faster, so compared with the microspheres prepared without vibration, their initial size is smaller. The freeze-dried microspheres before and after water vapor treatment were subjected to scanning electron microscopy (SEM) testing, infrared testing, water solubility testing, and injectability testing. The results are as follows:

[0074] 1. SEM test results show that the freeze-dried microspheres made from 5.56% silk fibroin solution have a particle size of about 550 μm (optical photos and scanning electron microscope images are shown in Figure 2). Figure 1 a and 1c), after water vapor treatment, the particle size of the microspheres was reduced to about 250 μm, which was reduced to about 1 / 2 of the original size (optical photos and scanning electron microscope images are shown in Figure 1a and 1c, respectively). Figure 1 b and 1d). The freeze-dried microspheres prepared with 1.5% silk fibroin solution have a particle size of approximately 480 μm ( Figure 2 a), after water vapor treatment, the particle size of the microspheres was reduced to about 100 μm, which was reduced to 1 / 5 of the original size ( Figure 2 b). In addition, the porosity of microspheres without water vapor treatment is higher ( Figure 3 A), after water vapor treatment, the microspheres shrink and the macropores decrease ( Figure 3 B), which may be due to the irregular shrinkage of the microspheres after water vapor treatment, which in turn leads to the reduction of pores. Scanning electron microscopy of the cross section of the microspheres revealed that ( Figure 4), there is a dense layer inside the microsphere, but there are still some small pores in the center, which is not completely dense. This may be because the water vapor molecules are too large to enter the interior of the microsphere.

[0075] 2. According to literature reports, within the range of amide I (1600~1700cm), the maximum absorption peak of β-folding appears at 1616~1637cm -1 The maximum absorption peak of random coil appears at 1638~1655 cm -1 The maximum absorption peak of a-helix appears at 1656~1662cm -1 The maximum absorption peak of the β-turn angle appears at 1663-1696 cm -1 The infrared test results show that Figure 5 a, The absorption characteristic peak of freeze-dried microspheres (SF-MS) appears at 1644 cm -1 After water vapor treatment, the maximum absorption characteristic peak of the obtained microspheres (SF-MS-evaporation) is 1622 cm -1 This indicates that the secondary structure of silk fibroin microspheres can be regulated by water vapor treatment.

[0076] To quantitatively characterize the secondary structure components in silk fibroin microspheres, we used Peak fit software to perform Fourier self-deconvolution (FSD) fitting of silk fibroin amide I. The secondary structure distribution of freeze-dried microspheres made from 5.56% silk fibroin solution before and after water vapor treatment is shown in Figure 2. Figure 5 b. Before water vapor treatment, the β-sheet content of the freeze-dried microspheres was 11±0.36%. After water vapor treatment, the β-sheet content increased to 34±0.45%. The secondary structure of the microspheres changed from random coil to β-sheet, and the mechanical properties of the microspheres were significantly improved.

[0077] 3. The water solubility test results show that the microspheres made from 5.56% silk fibroin solution can maintain their round shape after being placed in water for 14 days after being treated with water vapor, and no hydrolysis occurs ( Figure 6 ). Freeze-dried microspheres that have not been treated with water vapor are highly soluble in water and easily deformed. After water vapor treatment, the stability and shape retention of the microspheres in water are greatly improved.

[0078] 4. Injectability Test: Microspheres prepared from a 5.56% silk fibroin solution were treated with water vapor and then suspended in a 1.5% sodium carboxymethylcellulose (CMC) aqueous solution. The suspension was then loaded into a syringe and injected. The results showed that, under the same conditions, microspheres prepared from a 5.56% silk fibroin solution could be easily injected through a 22G needle, and microspheres prepared from a 1.5% silk fibroin solution could also be easily injected through a 25G needle.

[0079] In summary, the higher the silk fibroin solution concentration, the smaller the shrinkage of the microspheres after water vapor treatment. Furthermore, water vapor treatment not only significantly reduces the microsphere size and changes its structure, but also improves its mechanical properties, water insolubility, and morphological stability.

[0080] Test Example 3

[0081] Based on Example 1, the degradation behavior of silk fibroin microspheres after water vapor treatment was investigated.

[0082] The silk fibroin solution with a degumming time of 30 minutes and a concentration of 5.56% obtained in step (1) was subjected to a forming and freezing treatment according to the method of step (2) (injection speed of 0.16 ml / min, oscillation frequency of 6 Hz) to obtain freeze-dried microspheres; the obtained freeze-dried microspheres were subjected to a water vapor treatment according to the method of step (3), wherein the humidity was 90%, the temperature was 60°C, and the treatment time was 2 hours to obtain water vapor treated microspheres.

[0083] Weigh 50 mg of the microspheres after water vapor treatment, accurately measure their actual mass as M0, and add 10 ml of PBS solution containing 2 U / ml proteinase K as the enzymatic hydrolysis group.

[0084] Weigh 50 mg of the prepared water vapor-treated microspheres and add 10 ml of PBS buffer to serve as the control group.

[0085] The enzymatic hydrolysis group and the control group were placed on a rotary mixer and reacted at 37°C with the microspheres kept in a moving and stirring state at a speed of 20 rpm.

[0086] At 5h, 10h, 20h, 54h, and 120h of reaction, three samples were taken from each of the enzymatic hydrolysis group and the control group to determine the mass of the undegraded components. The determination method is as follows:

[0087] Use a water-based microporous filter membrane with a known dry weight and a pore size of 0.45 μm to filter the enzymatic hydrolysis group or the control group respectively, then transfer the filter membrane to a weighing bottle and keep constant weight at 105°C to obtain the mass M1 of the undegraded component of the microspheres.

[0088] According to the measured mass M0 of the initial added microspheres and the mass of the undegraded components M1, the mass change rate (%) of the silk fibroin microspheres was calculated according to formula (2):

[0089] Mass change rate (%) = M1 / M0 (2)

[0090] The calculated mass change rate and the corresponding sampling time are plotted (e.g. Figure 7The mass change curve of the silk fibroin microspheres showed an initial rapid decline, followed by a slower rate of decline, and finally, a prolonged period of complete degradation of the remaining components. The sample degraded rapidly between 0 and 10 hours, likely due to the rapid degradation of the lightly cross-linked silk fibroin and a small amount of free silk fibroin by proteases into free peptides or amino acids, which were then released into the liquid phase. The degradation rate slowed between 10 and 54 hours, as the remaining silk components were highly β-pleated fragments, which were extremely difficult to degrade. This resulted in a slow degradation trend for the sample during this period.

[0091] Test Example 4

[0092] Based on Example 1, the effect of degumming time on the size and structure of microspheres was investigated.

[0093] The silk fibroin solutions obtained in step (1) with degumming times of 10 min, 30 min, and 90 min and a concentration of 3% were freeze-dried according to the method of step (2) (injection speed of 0.16 ml / min, no oscillation applied to the needle tip) to obtain freeze-dried microspheres; the obtained freeze-dried microspheres were subjected to water vapor treatment according to the method of step (3), wherein the humidity was 90%, the temperature was 60°C, and the treatment time was 2 h, to obtain water vapor treated microspheres.

[0094] like Figure 8 As shown in the optical photograph ( Figure 8 A) Comparison of microsphere size changes before and after water vapor treatment. Particle size analysis was performed using ImageJ software. The results are shown in Table 2. The experimental results show that under the same water vapor treatment conditions and the same concentration (3%), microspheres prepared from a silk fibroin solution degummed for 10 minutes had the smallest shrinkage factor, 2.1-fold. Microspheres prepared from a silk fibroin solution degummed for 30 minutes had a shrinkage factor of 2.9-fold. Microspheres prepared from a silk fibroin solution degummed for 90 minutes had the largest shrinkage factor, 3-fold. Where shrinkage factor = freeze-dried microsphere size / microsphere size after water vapor treatment.

[0095] Differences in silk boiling (degumming) time can change the molecular weight of silk fibroin and the amount of residual sericin. The shorter the degumming time, the higher the molecular weight of silk fibroin, the higher the residual sericin, and the stronger the mechanical properties of the corresponding microspheres. By comparing the shrinkage factor of microspheres prepared from silk fibroin with different degumming times, it was found that at the same concentration, the microspheres prepared from silk fibroin solution degummed for 90 minutes had a greater shrinkage factor than those prepared from silk fibroin solution degummed for 10 minutes and 30 minutes. This may be because the freeze-dried microspheres prepared from silk fibroin with a long degumming time have a looser structure, which is more likely to shrink when treated with water vapor, resulting in a greater shrinkage factor.

[0096] Table 2

[0097]

[0098] Test Example 5

[0099] Based on Examples 1 and 4, the effect of the pore-forming agent on the size and structure of silk fibroin microspheres was investigated.

[0100] The 2% silk fibroin solution obtained in step (1) with a degumming time of 60 min and 90 min was added with different amounts of ammonium bicarbonate, with the mass ratio of ammonium bicarbonate to silk fibroin being 0%, 50%, 100%, and 200%. The solution was then freeze-dried according to the method of step (2) (injection speed of 0.16 ml / min, without oscillation of the needle tip) to obtain freeze-dried microspheres. The obtained freeze-dried microspheres were subjected to water vapor treatment according to the method of step (3), wherein the humidity was 90%, the temperature was 60°C, and the treatment time was 2 h, to obtain water vapor treated microspheres.

[0101] The particle sizes of freeze-dried microspheres obtained under different ammonium bicarbonate addition conditions and the corresponding microspheres after water vapor treatment were tested, and the results are shown in Tables 3 and 4 below. The shrinkage factor of the microspheres after water vapor treatment was calculated, and the results are shown in Table 5 below.

[0102] Table 3

[0103]

[0104] Table 4

[0105]

[0106] Table 5

[0107]

[0108] The results showed that when the ammonium bicarbonate to silk fibroin mass ratio was 200%, the corresponding microsphere size reduction factor was the largest, shrinking to 3.17 and 3.34 times the original size, respectively. This reduction factor was greater than that of silk fibroin microspheres without ammonium bicarbonate added, indicating that adding ammonium bicarbonate as a pore-forming agent can increase the porosity of the microspheres and further increase the reduction factor of the microspheres.

[0109] In addition, during the experiment, it was found that the presence of ammonium bicarbonate can make the microspheres more dispersed and reduce the adhesion between the microspheres. Therefore, it is possible to consider further reducing the concentration of silk fibroin to increase the shrinkage factor of the microspheres.

[0110] The 1% silk fibroin solution obtained in step (1) with a degumming time of 90 minutes was added with different amounts of ammonium bicarbonate, with the mass ratio of ammonium bicarbonate to silk fibroin being 0%, 50%, 100%, and 200%. The solution was then freeze-dried according to the method of step (2) (injection speed of 0.16 ml / min, without oscillation of the needle tip) to obtain freeze-dried microspheres. The obtained freeze-dried microspheres were subjected to water vapor treatment according to the method of step (3), wherein the humidity was 90%, the temperature was 60°C, and the treatment time was 2 hours to obtain water vapor treated microspheres.

[0111] The morphology of freeze-dried microspheres obtained under different ammonium bicarbonate addition conditions and the corresponding microspheres after water vapor treatment were observed ( Figure 9 In the figure, ad are freeze-dried microspheres with ammonium bicarbonate addition of 0%, 50%, 100%, and 200%, respectively; eh are microspheres after water vapor treatment with ammonium bicarbonate addition of 0%, 50%, 100%, and 200%, respectively). It was found that when no ammonium bicarbonate was added, the surfaces of the freeze-dried microspheres prepared from low-concentration silk fibroin solution adhered to each other, and after water vapor treatment, the microspheres showed filamentous connections and had poor morphology. After adding ammonium bicarbonate, the dispersion of the microspheres improved with the increase of ammonium bicarbonate content, and the microspheres after water vapor treatment could still maintain a round shape. The results showed that the presence of ammonium bicarbonate balanced the charges between the microspheres, which was beneficial to the dispersion of the microspheres and more convenient for water vapor treatment.

[0112] The particle size of the microspheres before and after water vapor treatment was tested, and the particle size results are shown in Table 6 below. The shrinkage factor of the microspheres after water vapor treatment was calculated, and the results are shown in Table 7 below.

[0113] Table 6

[0114]

[0115] Table 7

[0116]

[0117] The results showed that when the mass ratio of added ammonium bicarbonate to silk fibroin was 200%, the corresponding microsphere particle size was reduced the most, shrinking to 3.78 times the original particle size. This is because the addition of ammonium bicarbonate can not only make the microsphere pores larger, but also make the freeze-dried microspheres prepared from low-concentration silk fibroin solution more dispersed, solving the problem of freeze-dried microspheres prepared from low-concentration silk fibroin solution sticking together, resulting in poor morphology after water vapor treatment, and further broadening the scope of application of this experimental method.

[0118] Test Example 6

[0119] Based on Example 1 and Test Example 4, the effects of the inducer on the size and structure of the silk fibroin microspheres were investigated.

[0120] The silk fibroin solution with a degumming time of 90 min and a concentration of 3% obtained in step (1) was subjected to a forming and freezing treatment according to the method of step (2) (injection speed of 0.16 ml / min, and no oscillation of the needle tip) to obtain freeze-dried microspheres; the obtained freeze-dried microspheres were evenly spread on a water-repellent culture dish, and then the culture dish was placed in a desiccator, and water, a mixed solution of water and ethanol (the mass ratio of water to ethanol was 10:1), and anhydrous ethanol were added to the bottom of the desiccator respectively, and the desiccator was placed in an oven to simulate the water vapor treatment conditions in step (3) to perform water vapor treatment on the microspheres, and the humidity, temperature and time of the post-treatment were controlled to be 90%, 60°C and 2h.

[0121] like Figure 10 , a is the freeze-dried microspheres without post-treatment, b is the microspheres after water vapor treatment when the inducer is deionized water, c is the microspheres after water vapor treatment when the inducer is a mixture of water and ethanol, and d is the microspheres after water vapor treatment when the inducer is anhydrous ethanol. By comparing the changes in the morphology and particle size of the microspheres before and after post-treatment with water and water and ethanol mixtures, it was found that ( Figure 10 b, 10c), the effects of water vapor treatment and water plus ethanol mixture treatment are similar, showing that the particle size of the microspheres is reduced by 2.75 times after water vapor treatment, and by 2.72 times after water plus ethanol mixture treatment. In addition, water vapor treatment is more environmentally friendly than water plus ethanol treatment; by comparing the changes in the morphology and particle size of the microspheres before and after water and ethanol treatment, it was found that ( Figure 10 b, 10d). The particle size of the microspheres after ethanol treatment was not significantly reduced as that after water vapor treatment. The results are shown in Table 8 below.

[0122] Table 8

[0123]

[0124] Test Example 7

[0125] Based on Example 1 and Test Example 4, the effect of the silk fibroin nanofiber solution on the size and structure of the microspheres was investigated.

[0126] Preparation of porous lamellar silk fibroin microspheres

[0127] The purified silk fibroin solution of 3% degummed for 90 minutes in Example 1 was freeze-dried for three days to ensure that all the water was dried. The freeze-dried silk fibroin was taken out, and a silk fibroin solution with a mass volume concentration of 150 mg / mL was weighed and prepared with water as the solvent. The volume of the silk fibroin solution was 150 mL. The silk fibroin solution of this concentration was subjected to ultrasonic induction treatment with a power of 56w and an ultrasonic time of 24 minutes. Each ultrasonication was 6s and a rest of 4s was one cycle. After the ultrasonication, the silk fibroin solution was diluted with deionized water to obtain a final concentration of 10 mg / mL (1wt%) of the silk fibroin solution. It was placed in a 60°C oven and incubated overnight to form a gel.

[0128] A 1% nanofiber gel was sonicated at 80 Hz for 15 minutes. After the gel was converted into a solution, it was loaded into a 10 ml syringe with a 30 G needle. The syringe was mounted on a syringe pump with an injection rate of 0.16 ml / min. The needle tip was oscillated at 6 Hz to accelerate the droplet's immersion in liquid nitrogen. A container of liquid nitrogen was placed under the syringe needle to collect the droplets, which were then rapidly frozen. The frozen droplets were removed from the liquid nitrogen and placed in a 50 ml test tube. The 50 ml test tube was then transferred to a -80°C freezer and frozen overnight. The resulting freeze-dried microspheres were then freeze-dried using a freeze dryer to produce freeze-dried microspheres, which were then stored in the test tube for future use. The freeze-dried microspheres were spread evenly on a water-repellent culture dish, and then the culture dish was placed in a desiccator (the lid of the desiccator was sealed with vaseline, the bottom of the desiccator was filled with ultrapure water, and the inside of the desiccator contained a thermometer and a hygrometer to control the temperature and humidity to simulate a constant temperature and humidity environment). The temperature was controlled at 60°C and the humidity was 90%. The treatment time was 2 h to obtain silk fibroin microspheres (i.e., microspheres after water vapor treatment).

[0129] The fumigated microspheres were taken out and stored in sealed bags. Figure 11 : It was found that the microspheres prepared by nanofiber gel showed a porous lamellar structure, and because the nanofiber gel itself had been β-folded, no further β-folding would occur. Therefore, the silk protein microspheres prepared using nanofiber gel would not shrink further.

[0130] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made thereto. Therefore, such modifications and improvements, without departing from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A method for preparing silk fibroin microspheres with controllable size and structure, characterized in that: The method comprises: The silk fibroin solution is loaded into a syringe equipped with a needle, and the propulsion speed of the syringe is controlled by a syringe pump, so that the silk fibroin solution is dripped into liquid nitrogen in the form of droplets for quick freezing. After forming, the solution is fished out and freeze-dried. The resulting freeze-dried microspheres are treated with water vapor to reduce the particle size, thereby obtaining silk fibroin microspheres with controllable size and structure.

2. The method for preparing silk fibroin microspheres with controllable size and structure according to claim 1, characterized in that: The concentration of the silk fibroin solution is 0.1% to 20%.

3. The method for preparing silk fibroin microspheres with controllable size and structure according to claim 1, characterized in that: The preparation method of the silk fibroin solution is as follows: (1) The silk was soaked in a sodium carbonate solution with a concentration of 0.02M~0.04M, treated at 90~100℃ for 10min~1.5h, washed and dried to obtain degummed silk; (2) soaking the degummed silk in a lithium bromide solution having a concentration of 40% to 45% and treating the solution at 58 to 62° C. for 3 to 4 hours to obtain a silk solution; (3) The silk solution was transferred to a dialysis bag with a molecular weight cutoff of 3500 Da, dialyzed in pure water for 48 h, and centrifuged to obtain the silk fibroin solution.

4. The method for preparing silk fibroin microspheres with controllable size and structure according to claim 1, characterized in that: The size of the needle is 25~32G; The advancement speed is 0.16 ml / min to 0.25 ml / min; The freeze-drying conditions are: -80°C for at least 2 hours, or quick freezing using liquid nitrogen.

5. The method for preparing silk fibroin microspheres with controllable size and structure according to claim 1, characterized in that: The conditions of the water vapor treatment are: temperature 15-100° C., humidity 70-100%, and time 1-10 hours.

6. The method for preparing silk fibroin microspheres with controllable size and structure according to claim 1, characterized in that: During the process of dripping the silk fibroin solution into liquid nitrogen in the form of droplets, the needle is oscillated at a frequency of 0.1 Hz to 15 Hz.

7. The method for preparing silk fibroin microspheres with controllable size and structure according to claim 1, characterized in that: Before the step of loading the silk fibroin solution into a syringe equipped with a needle, the method further comprises: adding ammonium bicarbonate to the silk fibroin solution, wherein the amount of ammonium bicarbonate added is 0.1% to 250% of the mass of the silk fibroin in the silk fibroin solution.

8. The method for preparing silk fibroin microspheres with controllable size and structure according to claim 1, characterized in that: The water vapor treatment is performed using a mixed solution containing water and ethanol, wherein the mass ratio of water to ethanol in the mixed solution is 8-12:

1.

9. A silk fibroin microsphere with controllable size and structure, characterized in that: It is made by the method according to any one of claims 1 to 8.

10. The size and structure-controllable silk fibroin microspheres according to claim 9, characterized in that: The size of the silk fibroin microspheres ranges from 20 μm to 2000 μm, the structure of the microspheres includes solid or porous, the shape of the porous includes regular circles or irregular layers, the size of the pores ranges from 200 nm to 500 μm, the microsphere size reduction factor is 1 to 20, and the transformation from porous structure to dense structure can be achieved through the reduction factor of the microspheres themselves.

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