Silk fibroin elastic porous microsphere with adjustable particle size as well as preparation method and application of silk fibroin elastic porous microsphere

Abstract

According to the adjustable and controllable silk fibroin porous microspheres and the preparation method thereof, the microspheres with the size of 20 microns to 1 mm are prepared by adjusting technological parameters for preparing the microspheres. Pores are of a nano-submicron-micron multi-stage composite pore structure, and the porous microspheres have elasticity. The method provided by the invention shows wide application potential in the aspect of preparing silk fibroin microspheres with diversified sizes, and the microspheres have remarkable advantages in application scenes of medical injectable microspheres and medical embolism microspheres due to unique physical and chemical characteristics; the method has remarkable application value in multiple fields.

Description

Technical Field

[0001] The invention relates to the field of functional materials, and in particular to a silk fibroin elastic porous microsphere with adjustable particle size, and a preparation method and application thereof. Background Art

[0002] Human skin aging is affected by many factors such as genes, age, and environment, but generally speaking, the characteristics of aging include loss of subcutaneous tissue, decreased skin elasticity and moisture, and increased skin folds and wrinkles. These changes are related to thinning of the epidermis, atrophy of the dermis, loss of elastic tissue in the dermis, and loss of dermal collagen. In order to improve these problems, the development of drugs and implantable biomaterials has received a good response. Among them, injectable fillers have become more and more popular due to their advantages such as simple operation, less damage, and quick effect. Degradable microspheres have been proven to be a high-efficiency, low-toxic scaffold material and drug sustained-release control system, and have recently attracted widespread attention from researchers and consumers. Silk fibroin is a natural biomacromolecule derived from silkworms, with a special hydrophilic-hydrophobic block structure. The hydrophilic light chain can form an amorphous random coil or α-helical structure, while the hydrophobic heavy chain can form a β-folded structure. Silk fibroin has abundant sources, excellent mechanical properties, and good biocompatibility, and its research in the biomedical field has received increasing attention. In addition, silk fibroin also has excellent processability and controllability of performance. By changing its secondary structure, the mechanical properties and degradation properties of silk fibroin materials can be regulated to meet different needs for material properties. At present, people have a growing demand for aesthetics, and injectable soft tissue fillers have been widely used to fill wrinkles, depressions, defects and cosmetic purposes on the body surface. Microsphere implants promote collagen synthesis by creating an environment conducive to the growth of fibroblasts. Currently, commonly used medical microspheres can be divided into organic microspheres and inorganic microspheres according to different materials. Organic microspheres include polylactic acid (PLA), poly-L-lactic acid (PLLA), polycaprolactone (PCL), etc., while inorganic microspheres include hydroxyapatite, bioceramics, etc. These materials have good biocompatibility and biodegradability. When organic PLA microspheres are used as injectable fillers, their degradation cycle and the safety of degradation products have been recognized by the medical community. PLA microspheres may cause injection-related side effects such as redness, swelling, and nodules, especially when the injection technique is improper or the product is not diluted enough. PLLA microspheres release lactic acid during biodegradation, which may stimulate macrophage phagocytosis and sometimes lead to chronic inflammation and foreign body reactions, especially in high concentrations or large particles. Although PCL microspheres have good flexibility and processability, PCL microspheres may induce a moderate inflammatory response during the degradation process. Although this helps to stimulate fibroblasts to aggregate on the surface of the microspheres and secrete extracellular matrix, excessive inflammatory response may also lead to adverse reactions. Among inorganic microspheres, although hydroxyapatite microspheres are similar to the inorganic components of human bones and teeth, they degrade slowly and may also cause long-term stiffness or unevenness in the treatment area. Bioceramic microspheres are relatively rarely used in the field of medical beauty, and the problems they have may include complex preparation processes and uncontrollable degradation rates. It can be seen that each type of filling microsphere has its own advantages and disadvantages. Silk fibroin materials have become an ideal choice for preparing injectable microspheres for soft tissue filling due to their good biocompatibility, adjustable degradability, excellent mechanical properties, injectability and broad application prospects. The size of injectable microspheres is generally 25 to 60 μm, which is the optimal size to avoid macrophage phagocytosis and can reduce skin trauma and serious side effects (including granulomas) during injection. At the same time, this size stimulates the production of collagen and elastin, which in turn act via fibroblasts to achieve a skin tightening effect. Embolic microspheres are widely used in transcatheter arterial interventional treatment of tumors. At present, the embolic microsphere products used in clinical practice are still relatively simple, mostly non-degradable polyvinyl alcohol embolic microspheres. In addition, most of the research on the preparation of embolic microspheres focuses on drug loading, and there are few studies on the preparation of large-sized microspheres. In actual clinical practice, the size of embolic microspheres is required to be between 50-1200μm, and embolic microspheres of matching specifications are selected according to the thickness of the blood vessels at the embolization site. There are few reports of natural polymer embolic microspheres larger than 200μm in the current literature. Clinically, there are various types and sizes of tumors, and some tumor blood vessels are large, especially for proximal embolization, which requires large-sized microspheres. However, the current microsphere preparation methods have certain difficulties in preparing large-sized microspheres. Summary of the invention

[0003] Technical problem to be solved: The purpose of the present invention is to provide a kind of elastic porous silk fibroin microspheres with adjustable particle size, solve the problem of unevenness caused by step-by-step cross-linking in the preparation process of existing microsphere fillers, reduce the addition of chemical reagents, and prepare pure natural material, degradable, and size-adjustable silk fibroin microspheres.

[0004] Technical solution: A type of elastic porous microsphere of silk fibroin with adjustable particle size, wherein the diameter of the porous microsphere is 20um-1mm, the pores present a nano-submicron-micron multi-level composite pore structure, and the porous microsphere has elasticity. The method for preparing the above-mentioned elastic porous silk fibroin microspheres with adjustable particle size comprises the following steps: S1. dissolving the degummed silk with a neutral salt to obtain a silk fibroin salt solution, and dialyzing the silk fibroin salt solution to obtain a silk fibroin aqueous solution; S2. After concentrating the silk fibroin aqueous solution prepared in S1, the solution was mixed with an equal volume of ethanol aqueous solution to obtain a silk fibroin ethanol mixed solution; S3. adding the silk fibroin ethanol blend solution prepared in S2 to the oil phase solution or the mixed solution of the oil phase and the surfactant, stirring the reaction, and standing at low temperature until the silk fibroin self-crosslinks to form silk fibroin microspheres; S4. Wash the silk fibroin microspheres prepared in S3, and then freeze-dry them to obtain elastic porous silk fibroin microspheres. Preferably, the neutral salt is any one or more of lithium bromide, calcium chloride, calcium bromide, calcium iodide, zinc chloride, zinc bromide, zinc iodide, magnesium chloride, magnesium bromide, magnesium iodide, calcium nitrate or copper nitrate, the concentration of the neutral salt solution is 5wt% to saturation concentration, the dissolution temperature is 25-100°C, and the dissolution time is 2-8h. Preferably, the concentration of the concentrated silk fibroin aqueous solution is 3-10wt%, and the concentration of the ethanol aqueous solution is 0.2-5wt%. Preferably, the volume ratio of the oil phase solution to the silk fibroin aqueous solution is 1:(1-100), and the volume ratio of the mixed solution of the oil phase and the surfactant to the silk fibroin aqueous solution is 1:(1-100). Preferably, the volume ratio of the surfactant to the oil phase is 1:(5-1000). Preferably, the surfactant includes any one or more of Tween, Span or Sorbitan ester surfactants, and the oil phase is liquid paraffin. Preferably, the low-temperature standing temperature is -196 to 0°C, the rotation speed of the stirring reaction is 100 to 1000 rpm, and the stirring reaction time is 0 to 60 min. Preferably, the freeze-drying temperature in S4 is -80°C to -20°C, and the freeze-drying time is 12h to 48h. The above-mentioned particle size-adjustable silk fibroin elastic porous microspheres are used in medical aesthetic filling and embolic materials. Beneficial effects: The elastic porous microspheres of silk fibroin with adjustable particle size of the present invention have the following advantages: 1. In the process of preparing elastic porous silk fibroin microspheres, the present invention cleverly introduces ethanol as a key additive. This innovative strategy has a significant impact on the microsphere process of silk fibroin. Ethanol plays multiple roles here. It not only acts as a denaturant to induce silk fibroin to undergo specific self-assembly behavior, but also promotes the optimization of microsphere structure and the improvement of performance. The addition of ethanol first induces silk fibroin molecules to self-assemble, which is the basis for forming nano-scale or micron-scale microspheres. Under the action of ethanol, the interaction between silk fibroin molecules is finely regulated, prompting them to arrange and combine in a specific way, thereby forming microspheres with stable structure. Under low temperature conditions, the addition of ethanol further induces the occurrence of phase separation. Phase separation is the process in which different components in a polymer solution are spontaneously separated in space due to differences in solubility. Under the action of ethanol, silk fibroin molecules are gradually enriched in certain specific areas in the solution, and these areas are finally solidified to form a porous structure. This porous structure not only gives the microspheres a higher specific surface area and better adsorption performance, but also provides more possibilities for its application in the biomedical field. 2. The present invention controls the size and particle size distribution of the microspheres by adjusting the surfactant concentration during the preparation process. The surfactant affects the surface tension between the two phases, reduces the polymerization probability, and forms a stable and solid interface film between the two phases, thereby ensuring the stability of the size of the silk microspheres. By precisely controlling the concentration of the surfactant, silk fibroin microspheres with a particle size range of 25 μm to 1 mm are prepared, while showing good elasticity and porous structure, providing a solid foundation for subsequent applications. 3. The method proposed in the present invention shows a wide range of application potential in the preparation of silk fibroin microspheres with diverse sizes. These microspheres have significant application value in multiple fields due to their unique physical and chemical properties. The 25-60 μm elastic porous silk fibroin microspheres prepared in the present invention show great potential as injectable microspheres in the direction of medical beauty. These microspheres can exist stably in the skin for a long time without causing adverse reactions due to their good biocompatibility and degradability. More importantly, their porous structure is conducive to cell infiltration and angiogenesis, thereby stimulating the regeneration of collagen, thereby achieving the effect of improving skin texture, enhancing skin firmness and elasticity. In addition, the controllable degradation characteristics of this microsphere enable its maintenance time in the skin to be extended, providing a more lasting effect for medical beauty. In the application scenario of embolic microspheres, the 100 μm-1 mm silk fibroin microspheres prepared by the present invention can be used as efficient embolic microspheres. These large-sized microspheres have significant advantages in vascular embolization. They can more effectively embolize small arteries, thereby cutting off the nutrient supply to the tumor and inhibiting the growth and spread of the tumor. Compared with traditional embolic materials, silk fibroin microspheres have better biocompatibility and degradability, and can reduce inflammatory reactions and complications after embolization. In addition, by precisely controlling the size and shape of the microspheres, precise embolization of blood vessels of different sizes and locations can be achieved, further improving the therapeutic effect and safety. 4. The silk fibroin microspheres prepared by the present invention fully consider the key needs in practical applications in terms of structural design, especially in the important application scenario of embolization therapy, and the excellent resilience it exhibits has become a highlight of the microsphere material. In the process of embolization therapy, the shape and size of the vascular wall often show significant diversity due to individual differences, different anatomical parts and changes in physiological state. Therefore, for embolic materials, having good elasticity is the key to ensuring that they can adapt to these complex and changeable vascular environments. Because the shape and size of the vascular wall will change in different parts and different physiological states. Microspheres with good elasticity can better adapt to these changes and achieve a tighter embolization effect; at the same time, during the embolization process, the microspheres need to withstand certain pressure and shear force. Microspheres with good elasticity can better adapt to changes in these mechanical environments and reduce the possibility of breakage and failure. Embolic microspheres with good elasticity can pass through the microcatheter more smoothly, and can return to their original round shape after reaching the target blood vessel, thereby ensuring that the embolization effect is permanent and accurate. This characteristic not only improves the safety of embolization therapy, but also improves the durability of the treatment effect. BRIEF DESCRIPTION OF THE DRAWINGS In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present application and should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative work: Figure 1 This is a scanning electron microscope image of the microspheres provided in Example 1 of the present application; Figure 2 This is a scanning electron microscope image of the microspheres provided in Example 2 of the present application; Figure 3 This is a scanning electron microscope image of the microspheres provided in Example 3 of the present application; Figure 4 This is a scanning electron microscope image of the microspheres provided in Example 4 of the present application; Figure 5 This is a scanning electron microscope image of the microspheres provided in Example 5 of the present application; Figure 6 This is a scanning electron microscope image of the microspheres provided in Example 6 of the present application; Figure 7 This is a scanning electron microscope image of the microspheres provided in Example 7 of the present application; Figure 8 This is a scanning electron microscope image of the microspheres provided in Example 8 of the present application; Fig. 9 This is a scanning electron microscope image of the microspheres provided in Example 9 of the present application; Fig.10This is a scanning electron microscope image of the microspheres provided in Example 10 of the present application; Fig.11 This is a scanning electron microscope image of the microspheres provided in Example 11 of the present application; Fig.12 This is a scanning electron microscope image of the microspheres provided in Example 12 of the present application; Fig.13 XRD of the microspheres provided in Example 13 and Comparative Example 1 of the present application; Fig.14 The rebound performance test of microspheres with different particle sizes prepared for this application. DETAILED DESCRIPTION The present invention will be further described below in conjunction with embodiments, which are explanations of the present invention and are not limited to the following embodiments: A method for preparing elastic porous silk fibroin microspheres with adjustable particle size, the preparation method comprising the following steps: Example 1 S1. dissolving the degummed silk with 9.8M LiBr solution to obtain a silk fibroin salt solution, and dialyzing the silk fibroin salt solution to obtain a silk fibroin aqueous solution; S2. After concentrating the silk fibroin aqueous solution prepared in S1, 3 wt % SF and 1 wt % ethanol aqueous solution were mixed in equal volumes to obtain a silk fibroin ethanol mixed solution; S3. Add the silk fibroin ethanol blend solution prepared in S2 to the mixed solution of oil phase and surfactant, the ratio of oil phase to surfactant is 1000:1, add the water phase dropwise to the oil phase, the oil-water ratio is 10:1, stir and stand at -20°C for 15 hours, until the silk fibroin self-crosslinks to form silk fibroin microspheres; S4. The silk fibroin microspheres prepared in S3 were washed with isopropanol and anhydrous ethanol for multiple times, and then freeze-dried to obtain elastic porous silk fibroin microspheres. Example 2 The difference from Example 1 is that in Example 2, the ratio of oil phase to surfactant in the microsphere preparation process is 100:1, and other parameters and operations refer to Example 1. Example 3 The difference from Example 1 is that in Example 3, the ratio of oil phase to surfactant in the microsphere preparation process is 10:1, and other parameters and operations refer to Example 1. Example 4 A method for preparing elastic porous silk fibroin microspheres with adjustable particle size, the preparation method comprising the following steps: S1. dissolving the degummed silk with a LiBr solution to obtain a silk fibroin salt solution, and dialyzing the silk fibroin salt solution to obtain a silk fibroin aqueous solution; S2. After concentrating the silk fibroin aqueous solution prepared in S1, 3 wt % SF and 1 wt % ethanol aqueous solution were mixed in equal volumes to obtain a silk fibroin ethanol mixed solution; S3. Add the silk fibroin ethanol blend solution prepared in S2 to the oil phase or the mixed solution of the oil phase and the surfactant, the ratio of the mixed solution of the oil phase and the surfactant to the silk fibroin ethanol blend solution is 10:1, and the oil-water ratio is 10:1. After stirring, stand at -20°C for 15 hours to allow the silk fibroin to self-crosslink to form silk fibroin microspheres; S4. Wash the silk fibroin microspheres prepared in S3, and then freeze-dry them to obtain elastic porous silk fibroin microspheres. Example 5 The difference from Example 4 is that in Example 5, the ratio of the mixed solution of oil phase and surfactant to the silk fibroin ethanol mixed solution in the microsphere preparation process is 30:1, and other parameters and operations refer to Example 4. Example 6 The difference from Example 4 is that in Example 6, the ratio of the mixed solution of oil phase and surfactant to the silk fibroin ethanol blended solution in the microsphere preparation process is 50:1, and other parameters and operations refer to Example 4. Example 7 A method for preparing elastic porous silk fibroin microspheres with adjustable particle size, the preparation method comprising the following steps: S1. dissolving the degummed silk with a LiBr solution to obtain a silk fibroin salt solution, and dialyzing the silk fibroin salt solution to obtain a silk fibroin aqueous solution; S2. After concentrating the silk fibroin aqueous solution prepared in S1, 3 wt % SF and 1 wt % ethanol aqueous solution were mixed in equal volumes to obtain a silk fibroin ethanol mixed solution; S3. Add the silk fibroin ethanol blend solution prepared in S2 to the oil phase solution, the ratio of the mixed solution of the oil phase and the surfactant to the silk fibroin ethanol blend solution is 10:1, and the oil-water ratio is 10:1. After stirring for 5 minutes, stand at -20°C for 15 hours to allow the silk fibroin to self-crosslink to form silk fibroin microspheres. S4. Wash the silk fibroin microspheres prepared in S3, and then freeze-dry them to obtain elastic porous silk fibroin microspheres. Example 8 The difference from Example 7 is that in Example 8, the reaction is stirred for 15 minutes during the microsphere preparation process. Other parameters and operations refer to Example 7. Example 9 The difference from Example 7 is that in Example 9, the reaction is stirred for 30 minutes during the microsphere preparation process, and other parameters and operations refer to Example 7. Example 10 A method for preparing elastic porous silk fibroin microspheres with adjustable particle size, the preparation method comprising the following steps: S1. dissolving the degummed silk with a neutral salt to obtain a silk fibroin salt solution, and dialyzing the silk fibroin salt solution to obtain a silk fibroin aqueous solution; S2. After concentrating the silk fibroin aqueous solution prepared in S1, 3 wt % SF and 1 wt % ethanol aqueous solution were mixed in equal volumes to obtain a silk fibroin ethanol mixed solution; S3. Add the silk fibroin ethanol blend solution prepared in S2 to the oil phase solution, with the ratio of the oil phase to the silk fibroin ethanol blend solution being 10:1. After stirring for 30 minutes, let stand at -20°C for 15 hours to allow the silk fibroin to self-crosslink and form silk fibroin microspheres. S4. Wash the silk fibroin microspheres prepared in S3, and then freeze-dry them to obtain elastic porous silk fibroin microspheres. Embodiment 11 The difference from Example 10 is that in the process of preparing microspheres in Example 11, the ratio of the oil phase to the silk fibroin ethanol blend solution is 30:1, and other parameters and operations refer to Example 10. Example 12 The difference from Example 10 is that in the process of preparing microspheres in Example 12, the ratio of oil phase to silk fibroin ethanol blend solution is 50:1, and other parameters and operations refer to Example 10. Embodiment 13 A method for preparing elastic porous silk fibroin microspheres with adjustable particle size, the preparation method comprising the following steps: S1. dissolving the degummed silk with a neutral salt to obtain a silk fibroin salt solution, and dialyzing the silk fibroin salt solution to obtain a silk fibroin aqueous solution; S2. After concentrating the silk fibroin aqueous solution prepared in S1, 3 wt % SF and 1 wt % ethanol aqueous solution were mixed in equal volumes to obtain a silk fibroin ethanol mixed solution; S3. Add the silk fibroin ethanol blend solution prepared in S2 to the oil phase solution, with the ratio of the oil phase to the silk fibroin ethanol blend solution being 10:1. After stirring for 30 minutes, let stand at -20°C for 15 hours to allow the silk fibroin to self-crosslink and form silk fibroin microspheres. S4. Wash the silk fibroin microspheres prepared in S3, and then freeze-dry them to obtain elastic porous silk fibroin microspheres. Comparative Example 1 A method for preparing silk fibroin microspheres, the method comprising the following steps: S1. dissolving the degummed silk with a neutral salt to obtain a silk fibroin salt solution, and dialyzing the silk fibroin salt solution to obtain a silk fibroin aqueous solution; S2. After concentrating the silk fibroin aqueous solution prepared in S1, a 3 wt % SF solution was obtained; S3. Add the silk fibroin solution prepared in S2 to the oil phase solution, the ratio of the oil phase to the silk fibroin solution is 10:1, stir and react for 30 minutes, and then stand at -20°C for 15 hours to allow the silk fibroin to self-crosslink to form silk fibroin microspheres; S4. Wash the silk fibroin microspheres prepared in S3, and then freeze-dry them to obtain silk fibroin microspheres. Experiment 1 Observation of the morphology of microspheres The morphological changes of the silk fibroin microspheres after adjusting the parameters were detected by using a field emission scanning electron microscope (Hitachi, Japan, model: S8100) to observe the morphology of the silk fibroin porous microspheres prepared in Examples 1 to 12 of the present invention. Figure 1-12 shown. (1) Effect of surfactant addition on the preparation of microspheres The experimental results are as follows Figures 1 to 3 As shown, the scanning electron microscope images of the microspheres in the freeze-dried state of Example 1, Example 2, and Example 3 of the present invention are shown. The average particle size of Examples 1 and 2 is large, the particle size distribution is uneven, the surface pore structure is small, and the holes are not obvious. It can be seen that in terms of the particle size and surface morphology of the microspheres, the effect of the microspheres prepared in Example 3 is significantly better than that of the microspheres prepared in Example 1 and Example 2. This shows that adjusting the addition of surfactants plays an important role in reducing the particle size of the microspheres, controlling the morphology, and avoiding the agglomeration of the microspheres. (2) Effect of the presence of surfactants and the change of the oil-water ratio on the preparation of microspheres like Figures 4 to 6 The experimental results shown in the figure show the scanning electron microscope (SEM) images of the microspheres in the freeze-dried state in Example 4, Example 5 and Example 6 of the present invention. Specifically, Example 4 and Example 5 successfully prepared microspheres with an average particle size of about 200 microns; while the microspheres prepared in Example 6 had an average pore size of 133.43±30.05 microns, and the surfaces of all microspheres showed obvious porous structural characteristics. This observation shows that the particle size of the microspheres can be effectively controlled by adjusting the mixing ratio of the water phase and the oil phase. (3) Effect of stirring time ratio on the preparation of microspheres like Figures 7 to 9The experimental results shown show scanning electron microscope (SEM) images of the microspheres in the freeze-dried state in Example 7, Example 8 and Example 9 of the present invention, respectively. Specifically, the appropriate stirring time can ensure that the monomer and the emulsifier are evenly dispersed in the water. Too short or too long stirring time may lead to uneven particle size distribution; the microspheres prepared in Examples 7 to 9, as the stirring time increases, the average particle size of the microspheres also increases from 47.12±8.83 to 94.19±29.09, and the surfaces of all microspheres show obvious porous structural characteristics. This observation shows that the particle size of the microspheres can be effectively regulated by adjusting the mixing ratio of the stirring time. (4) Effect of changing the oil-water ratio on the preparation of microspheres in the absence of surfactant like Figures 10-12 The experimental results shown show scanning electron microscope (SEM) images of the microspheres in the freeze-dried state in Example 10, Example 11 and Example 12 of the present invention, respectively. Specifically, without adding a surfactant, only changing the ratio of the oil phase to the water phase, the oil-water ratio will affect the interfacial tension in the emulsion. When the oil-water ratio changes, the interfacial tension will also be adjusted accordingly, thereby affecting the shape, size and distribution of the microspheres. The microspheres prepared in Examples 10 to 12 have a larger average particle size and the surfaces of all microspheres show obvious porous structural characteristics, but as the oil-water ratio increases, the particle size of the microspheres also decreases significantly. This observation shows that without adding a surfactant, adjusting the oil-water ratio can obtain microspheres with larger particle sizes. Experiment 2: Microsphere particle size analysis experiment The experimental process includes: using Image J data analysis software, taking an appropriate amount of microspheres prepared in Examples 1 to 12 to analyze the particle size. Table 1 shows the particle size of the microspheres prepared in Examples 1 to 12. Table 1 Particle size of microspheres in Examples 1 to 12 Experiment 3 XRD analysis The silk fibroin sample was ground into powder with a mortar, a small amount of sample powder was taken out and added to lithium bromide powder, and then ground again with a mortar, and then pressed into a sheet form with a tablet press, and placed on a Nicolet 5700 Fourier infrared spectrometer scanner to measure the infrared spectrum of the sample. The wave number range during the test was 400 to 4000 cm-1, the number of scans was 32, and the data processing software peakfit was used to process the experimental results. like Fig.13 As shown, the FTIR of Example 13 and Comparative Example 1 are shown. As shown in the figure, FTIR is very sensitive to the molecular conformation of silk fibroin. The characteristics of silk fibroin are the random curl absorption peak (amide V) at around 660 cm-1 and the α-helix absorption peak at 1655 cm-1.-1 Around (amide I), 1546cm -1 (amide II), 1270 cm -1 (Amide III), 625 cm -1 (Amide V), β-sheet absorption at about 1630 cm -1 (Amide I), 1520 cm -1 (amide II), 1240 cm -1 (amide III) and 695 cm -1 (Amide V). When no ethanol is added, the absorption peak of the silk fibroin scaffold at amide I is around 1640 cm-1. This proves that the corresponding characteristic peak of the regenerated silk fibroin corresponds to the Silk II structure of the silk fibroin. After adding low concentrations of ethanol, the frozen silk microspheres of different concentrations will form a β-folded structure. Experiment 3: Microball rebound test experiment The resilience of the large silk protein microspheres was tested as follows: In order to evaluate the resilience of the microspheres, the specific process is as follows: Several microsphere samples with different particle sizes were selected, and these samples were carefully selected to ensure their diversity and representativeness in size. Subsequently, the microspheres were clamped with tweezers, a step that requires the operator to have a high degree of stability and precision to avoid unnecessary damage or pre-deformation of the microspheres during the experiment. The results are as follows Fig.14 As shown in the figure, the clamped microspheres are repeatedly squeezed and released. This step simulates the external pressure situation that the microspheres may encounter in actual applications, so as to observe their shape recovery ability after being stressed. Through this experiment, we verified that both large-size and small-size microspheres have good rebound performance, which provides valuable data support for the potential application of microsphere materials. Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A silk fibroin elastic porous microsphere with adjustable particle size, characterized in that: The porous microspheres have a diameter of 20um-1mm, pores present a nano-submicron-micron multi-level composite pore structure, and the porous microspheres have elasticity.

2. The method for preparing the size-adjustable silk fibroin elastic porous microspheres according to claim 1, characterized in that: The preparation method comprises the following steps: S1. dissolving the degummed silk with a neutral salt to obtain a silk fibroin salt solution, and dialyzing the silk fibroin salt solution to obtain a silk fibroin aqueous solution; S2. After concentrating the silk fibroin aqueous solution prepared in S1, the solution was mixed with an equal volume of ethanol aqueous solution to obtain a silk fibroin ethanol mixed solution; S3. adding the silk fibroin ethanol blend solution prepared in S2 to the oil phase solution or the mixed solution of the oil phase and the surfactant, stirring the reaction, and standing at low temperature until the silk fibroin self-crosslinks to form silk fibroin microspheres; S4. Wash the silk fibroin microspheres prepared in S3, and then freeze-dry them to obtain elastic porous silk fibroin microspheres.

3. The method for preparing the size-adjustable silk fibroin elastic porous microspheres according to claim 1, characterized in that: The neutral salt is any one or more of lithium bromide, calcium chloride, calcium bromide, calcium iodide, zinc chloride, zinc bromide, zinc iodide, magnesium chloride, magnesium bromide, magnesium iodide, calcium nitrate or copper nitrate. The concentration of the neutral salt solution is from 5wt% to saturation concentration, the dissolution temperature is 25-100°C, and the dissolution time is 2-8h.

4. The method for preparing the size-adjustable silk fibroin elastic porous microspheres according to claim 1, characterized in that: The concentration of the silk fibroin aqueous solution after concentration is 3-10 wt %, and the concentration of the ethanol aqueous solution is 0.2-5 wt %.

5. The method for preparing the size-adjustable silk fibroin elastic porous microspheres according to claim 1, characterized in that: The volume ratio of the oil phase solution to the silk fibroin aqueous solution is 1:(1-10), and the volume ratio of the mixed solution of the oil phase and the surfactant to the silk fibroin aqueous solution is 1:(1-10).

6. The method for preparing the size-adjustable silk fibroin elastic porous microspheres according to claim 5, characterized in that: The volume ratio of the surfactant to the oil phase is 1:(5-20).

7. The method for preparing the size-adjustable elastic porous silk fibroin microspheres according to claim 5, characterized in that: The surfactant includes any one or more of Tween, Span or Sorbitan ester surfactants, and the oil phase is liquid paraffin.

8. The method for preparing the size-adjustable silk fibroin elastic porous microspheres according to claim 1, characterized in that: The low-temperature standing temperature is -196 to 0°C, the rotation speed of the stirring reaction is 100 rpm to 1000 rpm, and the stirring reaction time is 10 to 60 minutes.

9. The method for preparing the size-adjustable silk fibroin elastic porous microspheres according to claim 1, characterized in that: The freeze drying temperature in S4 is -80°C to -20°C, and the freeze drying time is 12h to 48h.

10. Use of the size-adjustable silk fibroin elastic porous microspheres as claimed in claim 1 in medical cosmetic filling and embolic materials.

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