Monodisperse porous silk fibroin microspheres with controllable pore structure and preparation method thereof

Abstract

This invention belongs to the field of biomaterial preparation and provides monodisperse porous silk fibroin microspheres with controllable pore structure and their preparation method. The preparation method includes the following steps: (1) adding the inner oil phase to an aqueous solution of silk fibroin and emulsifying it, using the resulting oil-in-water emulsion as the inner phase fluid; dissolving an oil-soluble surfactant and an inducer in an oily solvent to obtain an outer phase fluid and a collection liquid; (2) using the outer phase fluid to shear the inner phase fluid in a microfluidic device to obtain a monodisperse oil-in-water emulsion, collecting the oil-in-water emulsion with the collection liquid, allowing it to stand for a sufficient period of time. During the standing process, the oil-in-water emulsion droplets gel under the induction of the inducer, while the inner oil phase acts as a pore template. After complete gelation, the residual inner oil phase in the obtained microspheres is removed by washing with an organic solvent, thus obtaining the microspheres. This invention can simplify the process while achieving controllable regulation of the pore structure and improving the monodispersity of porous silk fibroin microspheres.

Description

Technical Field

[0001] This invention belongs to the field of biomaterial preparation and relates to monodisperse porous silk fibroin microspheres with controllable pore structure and their preparation method. Background Technology

[0002] Silk fibroin (SF) is a natural protein derived from silkworms. In addition to the good biocompatibility typical of natural materials, silk fibroin also possesses excellent mechanical properties, ease of processing, unique enzyme-dependent degradation, and low immunogenicity. It has been prepared into microsphere carriers and is widely used in biomedical fields such as drug delivery and tissue engineering. Preparing silk fibroin into porous microspheres endows them with a larger specific surface area, superior loading efficiency, and better release characteristics, thus demonstrating greater application potential in loading bioactive molecules such as drugs, proteins, growth factors, and cells.

[0003] Although various methods exist for preparing silk fibroin microspheres, such as emulsification, phase separation, spray drying, ethanol and freeze-induced self-assembly, and microfluidic methods, porous silk fibroin microspheres still face challenges such as a lack of preparation strategies and difficulty in precisely controlling pore structure. Currently, high-voltage electrostatic spraying combined with liquid nitrogen freezing is commonly used to prepare porous silk fibroin microspheres. For example, QU et al. (QU J, WANG L, NIU LX, et al. Porous Silk Fibroin Microspheres Sustainably Releasing Bioactive Basic Fibroblast Growth Factor[J]. Materials, 2018, 11(8): 1280.) used this method to develop porous silk fibroin microspheres that sustainably release basic fibroblast growth factor. However, the porous silk fibroin microspheres prepared by this method suffer from low porosity, poor permeability, and poor monodispersity. Although the porous silk fibroin microspheres prepared by this method can be used as drug carriers in the field of drug sustained release, their drug loading capacity and the controllability of drug release behavior are hindered. They are also difficult to use as cell carriers or biomimetic scaffolds to support tissues and provide support for cell migration and ingrowth, thus limiting their application in tissue engineering and regenerative medicine. Researchers improved the above method by introducing the water-soluble porogen glucose. By using glucose to regulate silk fibroin aggregation and ice crystal growth, they successfully constructed porous silk fibroin microspheres with highly interconnected pores. For example, DING et al. (DING J, CHENG ZJ, MA YL, et al. Engineering Injectable and Highly Interconnected Porous Silk Fibroin Microspheres for Tissue Regeneration[J].Advanced Healthcare Materials, 2025, 14(8): e2402932.) used this method to prepare porous silk fibroin microspheres. These microspheres showed excellent cell adhesion and proliferation promotion capabilities in vitro, and could achieve sustained release of the loaded model drugs.However, the porous silk fibroin microspheres prepared by this method have a wide particle size distribution, and glucose, as a small-molecule plasticizer, lowers the glass transition temperature of silk fibroin, causing the microspheres to easily agglomerate and clump together during freeze-drying, making it difficult to obtain monodisperse porous silk fibroin microspheres. Furthermore, the formation of the pore structure in this method is essentially controlled by the growth and sublimation process of thermodynamically unstable ice crystals, resulting in a highly random pore structure that is difficult to precisely and orderly control. In addition, the preparation process is cumbersome, still relying on high-voltage electrostatic spraying equipment, low-temperature storage, and freeze-drying. To date, developing a mild and simple process to prepare monodisperse porous silk fibroin microspheres with highly interconnected pores and controllable pore size remains a significant challenge. Summary of the Invention

[0004] To address the problems of cumbersome preparation processes, difficulty in effectively controlling pore structure, and poor monodispersity of existing porous silk fibroin microspheres, this invention provides monodisperse porous silk fibroin microspheres with controllable pore structure and their preparation method. This simplifies the process while achieving controllable control of pore structure, thereby improving the monodispersity of porous silk fibroin microspheres.

[0005] To achieve the above-mentioned objectives, the technical solution adopted by the present invention is as follows:

[0006] A method for preparing monodisperse porous silk fibroin microspheres with controllable pore structure includes the following steps:

[0007] (1) Preparation of internal phase fluid, external phase fluid, and collection liquid

[0008] Preparation of the inner phase fluid: The inner oil phase is added to the silk fibroin aqueous solution and emulsified to obtain an oil-in-water emulsion, which is used as the inner phase fluid; the concentration of the silk fibroin aqueous solution is 0.02~0.12 g / mL, and the volume ratio of the inner oil phase to the silk fibroin aqueous solution is (0.1~1):1; the inner oil phase is an oil that is inert to silk fibroin, has low biotoxicity, and is non-volatile or low-volatile at room temperature;

[0009] Preparation of external phase fluid: Dissolve oil-soluble surfactant and inducer in an oily solvent to obtain external phase fluid; in external phase fluid, the mass fraction of oil-soluble surfactant is 0.1 wt%~10 wt%, and the volume fraction of inducer is 5%~40%;

[0010] Preparation of the collection solution: Dissolve the oil-soluble surfactant and the inducer in an oily solvent to obtain the collection solution; in the collection solution, the mass fraction of the oil-soluble surfactant is 0.1 wt%~10 wt%, and the volume fraction of the inducer is 5%~40%;

[0011] (2) Preparation of monodisperse porous silk fibroin microspheres

[0012] The inner and outer phase fluids are introduced into a primary microfluidic device. The outer phase fluid shears the inner phase fluid to obtain a monodisperse oil-in-water-in-oil emulsion. The oil-in-water-in-oil emulsion is collected with a collecting liquid and allowed to stand for a while. During the standing process, the oil-in-water-in-oil emulsion droplets gel under the induction of an inducing agent. At the same time, the inner oil phase acts as a pore template to occupy sites. After complete gelation, the residual inner oil phase in the obtained microspheres is removed by washing with an organic solvent to obtain monodisperse porous silk fibroin microspheres.

[0013] In step (2) of the above technical solution, during the standing process, the oil-in-water-in-oil emulsion droplets gel under the induction of the inducing agent. At the same time, the inner oil phase acts as a pore template to occupy the space. As the gelation proceeds, the oil-in-water-in-oil emulsion droplets gradually solidify and the inner oil phase is gradually removed. After complete gelation, only a small amount of inner oil phase remains inside and on the surface of the obtained microspheres. The remaining inner oil phase in the obtained microspheres is removed by washing with an organic solvent to obtain monodisperse porous silk fibroin microspheres.

[0014] In the above technical solutions, the selection of the inner oil phase needs to meet the following principles simultaneously: First, it should be non-volatile or low-volatile at room temperature, which is the basis for ensuring that the inner oil phase serves as a template for the porous structure; second, it should be inert to silk fibroin, that is, it should not react with silk fibroin and should not induce the precipitation of silk fibroin, which is the basis for ensuring the formation of a stable oil-in-water-in-oil emulsion; third, it should have low biotoxicity, which is the basis for ensuring the safety of the prepared porous silk fibroin microspheres.

[0015] In the above technical solution, regarding the limitation of the volatility of the internal oil phase at room temperature, the art classifies the volatility of substances into four levels based on their vapor pressure at 25 °C: highly volatile substances have a vapor pressure >200 Pa at 25 °C; moderately volatile substances have a vapor pressure greater than 20 Pa and less than or equal to 200 Pa at 25 °C; low volatile substances have a vapor pressure of 1~20 Pa at 25 °C; and non-volatile substances have a vapor pressure <1 Pa at 25 °C.

[0016] In the above technical solution, the internal oil phase includes at least one of vegetable oil, mineral oil, synthetic oil, and oleic acid. Typically, feasible vegetable oils include coconut oil or soybean oil, feasible mineral oils include liquid paraffin, and feasible synthetic oils include medium-chain triglycerides (MCTs), which are composed of C6-C6 bonds. 12 Triglycerides are formed by the esterification of medium-chain fatty acids with glycerol.

[0017] In the above technical solution, the inducing agent is methanol, ethanol, n-octanol, or ethyl acetate. Further, the inducing agent is methanol or ethanol.

[0018] In the above technical solution, the oil-soluble surfactant includes at least one of polyglycerol ricinoleate, Span 80, and T154 (polyisobutylene bis(succinimide)).

[0019] In the above technical solution, the oily solvent includes at least one of vegetable oil, mineral oil, synthetic oil, and oleic acid. Typically, feasible vegetable oils include soybean oil, feasible mineral oils include light liquid paraffin, and feasible synthetic oils include medium-chain triglycerides (MCTs), which are composed of C6-C6 bonds. 12 Triglycerides are formed by the esterification of medium-chain fatty acids with glycerol.

[0020] In the above technical solution, the size and size distribution of the oil-in-water emulsion droplets are related to the size and monodispersity of the finally prepared porous silk fibroin microspheres. In practical applications, the diameter and diameter distribution of the oil-in-water emulsion droplets can be selected according to the actual application requirements for the diameter and monodispersity of the porous silk fibroin microspheres. Typically, step (1) controls the diameter of the oil-in-water emulsion droplets to be 100 nm to 10 μm.

[0021] In step (1) of the above technical solution, conventional emulsification methods in the prior art can be used to emulsify the internal phase fluid, such as ultrasonic emulsification, magnetic stirring emulsification, or homogenizing emulsifier emulsification. When it is necessary to prepare water-in-oil emulsion droplets with more uniform size, a homogenizing emulsifier can be used for emulsification.

[0022] In step (2) of the above technical solution, the size of the prepared oil-in-water-in-oil emulsion droplets can be adjusted by regulating the flow rates of the external and internal phase fluids and the channel size of the microfluidic device. Typically, in step (2), when preparing a monodisperse oil-in-water-in-oil emulsion in a primary microfluidic device, the flow rate ratio of the external and internal phase fluids is controlled to be (5~40):1. Further, the flow rate of the external phase fluid can be controlled to be 500~26000 μL / h, and the flow rate of the internal phase fluid can be controlled to be 100~3000 μL / h.

[0023] In step (2) of the above technical solution, the primary microfluidic device includes an injection tube, a connecting tube, and a collection tube, which are used in conjunction with an injection pump. The outlet of the injection tube is tapered, and the outlet end of the injection tube is inserted into the inlet end of the collection tube and connected through the connecting tube. The injection tube, connecting tube, and collection tube are coaxially arranged. Typically, the inner diameter of the tapered opening of the injection tube is 50~300 μm, and the inner diameter of the collection tube is 300~800 μm.

[0024] In step (2) of the above technical solution, the time required for the oil-in-water-in-oil emulsion droplets to gel completely under the induction of the inducing agent is related to factors such as the size of the oil-in-water-in-oil emulsion droplets, the type and concentration of the inducing agent in the external phase fluid and the collecting liquid. The larger the size of the oil-in-water-in-oil emulsion droplets and the lower the concentration of the inducing agent in the external phase fluid and the collecting liquid, the longer the time required. Typically, the settling time in step (2) is controlled to be 0.2~24 h.

[0025] In step (2) of the above technical solution, when washing the microspheres obtained after complete gelation, the microspheres are first washed with an organic solvent and then washed with water to remove the organic solvent. The washing with the organic solvent is mainly to remove the residual internal oil phase, oil-soluble surfactants, and oily solvents. The organic solvent used includes one or more of methanol, ethanol, n-octanol, and ethyl acetate. Furthermore, the organic solvent used is the same as the selected inducing agent.

[0026] In step (2) of the above technical solution, after obtaining monodisperse porous silk fibroin microspheres, they can be dried by air drying, baking or freeze drying to facilitate preservation.

[0027] The present invention also provides a monodisperse porous silk fibroin microsphere, which is prepared by the above method. The porous silk fibroin microsphere has an internal interconnected pore structure and the surface of the porous silk fibroin microsphere has a porous structure.

[0028] In the above-mentioned monodisperse porous silk fibroin microspheres, the coefficient of variation of the particle size of the porous silk fibroin microspheres does not exceed 5%, the porosity of the porous silk fibroin microspheres can be adjusted between 20% and 90%, and the average pore size can be adjusted between 1 and 20 μm.

[0029] In the above-mentioned technical solution of monodisperse porous silk fibroin microspheres, the particle size of the monodisperse porous silk fibroin microspheres is determined at the micrometer scale according to the actual application requirements. Typically, the particle size of the monodisperse porous silk fibroin microspheres is 40~350 μm.

[0030] The monodisperse porous silk fibroin microspheres provided by this invention can be used as drug carriers, cell culture scaffolds, or tissue engineering materials.

[0031] Compared with the prior art, the technical solution of the present invention has the following beneficial technical effects:

[0032] 1. This invention provides a method for preparing porous silk fibroin microspheres with controllable pore structure. The method uses an oil-in-water emulsion formed by emulsifying an aqueous silk fibroin solution with an inner oil phase as the inner phase fluid. An outer phase fluid is used to shear the inner phase fluid in a microfluidic device to form a monodisperse oil-in-water emulsion. The oil-in-water emulsion is then collected using a collecting liquid containing an inducer. Under the action of the inducer, the oil-in-water emulsion droplets gel, with the inner oil phase acting as a pore template. After complete gelation, the resulting microspheres are washed with an organic solvent to obtain monodisperse porous silk fibroin microspheres. The method of this invention uses an internal oil phase that is inert to silk fibroin, has low biotoxicity, and is non-volatile or low-volatile as a template for the pore structure. The size, size distribution, and content of the internal oil phase droplets in the internal fluid have a good correspondence with the pore structure (pore size, porosity) of the porous silk fibroin microspheres. By controlling the content of the internal oil phase in the internal fluid and the emulsification process parameters for preparing the internal fluid, the pore structure of the porous silk fibroin microspheres can be controllably adjusted. This method establishes a correspondence between the physical parameters of the internal fluid emulsion and the pore structure parameters of the porous silk fibroin microspheres, achieving precise control of the pore structure. Because the pore-forming principle differs from the highly random, thermodynamically unstable ice crystal growth and sublimation process in existing technologies, this method effectively solves the problems of uncontrollable or poorly controllable pore structure in the preparation of porous silk fibroin microspheres in existing technologies.

[0033] 2. The method of this invention utilizes the laminar shear mode of microfluidic technology to precisely control the diameter of the oil-in-water-in-oil emulsion droplets, which serve as templates for forming porous silk fibroin microspheres, by means of fluid velocity and the channel size of the microfluidic device. This results in monodisperse oil-in-water-in-oil emulsion droplet templates. After solidification of the emulsion droplet templates, the resulting porous silk fibroin microspheres still exhibit excellent monodispersity. This method avoids the problem of uneven emulsion template size caused by turbulence in traditional emulsification methods, which in turn leads to uneven particle size of porous silk fibroin microspheres. Experiments have demonstrated that the porous silk fibroin microspheres prepared by the method of this invention have uniform morphology, good sphericity, and a particle size variation coefficient that is consistently less than 5%, even reaching below 2%, exhibiting significantly better monodispersity than porous silk fibroin microspheres prepared by traditional methods.

[0034] 3. The method of this invention uses the inner oil phase in an oil-in-water-in-oil emulsion droplet as a template for the pore structure. Microfluidic technology is used to precisely control the size of the emulsion droplet template. Combined with the site-occupancy pore-forming mechanism of the dual emulsion template method, porous silk fibroin microspheres are prepared. The inner oil phase is dispersed in a silk fibroin aqueous solution as micro / nano-scale droplets to form an oil-in-water emulsion. This emulsion is then sheared by an external fluid to form an oil-in-water-in-oil emulsion. As the inducing agent in the external fluid and the collecting liquid diffuses into the oil-in-water-in-oil emulsion droplets, the silk fibroin undergoes a conformational change and gels to form microspheres. This solidification process is a physical crosslinking process, mild and requiring no crosslinking agent. The inner oil phase droplets simultaneously occupy space within the microspheres, thus forming a network of narrow-aperture, well-connected channels. By simply adjusting parameters such as the content of the inner oil phase in the inner phase fluid, the degree of emulsification of the inner oil phase in the silk fibroin aqueous solution, the flow rate of each phase fluid, and the concentration of the inducer, the pore structure of the final porous silk fibroin microspheres can be controlled and predicted, achieving precise and controllable construction of the pore structure. For advanced intelligent drug delivery systems, the pore structure controllably constructed using the method of this invention will facilitate the establishment of stable and reproducible structure-activity relationships; for drug carriers, the controllably constructed pore structure using the method of this invention will help stabilize drug loading and encapsulation efficiency, and improve drug release behavior; for cell scaffolds, the highly interconnected pore structure constructed using the method of this invention will facilitate cell migration, proliferation, nutrient delivery, and vascularization. Therefore, the method described in this invention can promote the application of porous silk fibroin microspheres in drug delivery systems, cell culture scaffolds, and tissue engineering materials.

[0035] 4. The method described in this invention can achieve continuous and stable production of porous silk fibroin microspheres, which is beneficial for the efficient and large-scale preparation of porous silk fibroin microspheres. Attached Figure Description

[0036] Figure 1 This is a schematic diagram of a single-stage capillary microfluidic device. The labels in the attached diagram are as follows: 1 is the injection tube, 2 is the connecting tube, and 3 is the collection tube.

[0037] Figure 2 Figure a is an optical image of the O / W / O emulsion droplets prepared in Example 1. Figure 2 Figure b is an optical image of the porous SF microspheres prepared in Example 1. Figure 2 Figure c is a scanning electron microscope image of the porous SF microspheres prepared in Example 1. Figure 2 Image d is a scanning electron microscope image of the cross-section of the porous SF microspheres prepared in Example 1. Figure 2 Image e is an optical image of a cryosection of porous SF microspheres prepared in Example 1.

[0038] Figure 3This is a diameter distribution diagram of the O / W / O emulsion droplets and porous SF microspheres prepared in Example 1.

[0039] Figure 4 Figure a shows the curves of the diameter of the O / W / O emulsion droplets and porous SF microspheres prepared in Example 2 as a function of the ratio of external phase fluid flow rate to internal phase fluid flow rate. Figure 4 Figure b shows the coefficient of variation (CV) of the diameter of the O / W / O emulsion droplets and porous SF microspheres prepared in Example 2.

[0040] Figure 5 Figure a is a scanning electron microscope image of the SO4 cross section. Figure 5 Image b is a scanning electron microscope image of the SO5 cross section. Figure 5 Image c is a scanning electron microscope image of the SO6 cross section. Figure 5 The d-image is a scanning electron microscope (SEM) image of the SO7 cross section, and the magnification of each SEM image is 3000x.

[0041] Figure 6 Figure a shows the porosity test results for SO4, SO5, SO6, and SO7. Figure 6 Figure b shows the average porosity test results for SO4, SO5, SO6, and SO7.

[0042] Figure 7 Image a1 is a scanning electron microscope image of the 4% SO5 surface. Figure 7 Image a1' is a scanning electron microscope image of the 4% SO5 cross section. Figure 7 Image a2 is a scanning electron microscope image of the 6% SO5 surface. Figure 7 Image a2' is a scanning electron microscope image of the 6% SO5 cross section. Figure 7 Image a3 is a scanning electron microscope image of the 8% SO5 surface. Figure 7 Figure a3' is a scanning electron microscope (SEM) image of the 8% SO5 cross section. All SEM images are magnified 2000 times.

[0043] Figure 8 Image a1 is a scanning electron microscope image of the SO3 surface. Figure 8 Image a1' is a scanning electron microscope image of the SO3 cross section. Figure 8 Image a2 is a scanning electron microscope image of the SO5 surface. Figure 8 Image a2' is a scanning electron microscope image of the SO5 cross section. Figure 8 Image a3 is a scanning electron microscope image of the SO7 surface. Figure 8 Image a3' is a scanning electron microscope image of the SO7 cross section. Figure 8 Image a4 is a scanning electron microscope image of the SO₂ surface. Figure 8 Image a4' is a scanning electron microscope image of the SO- cross section. Figure 8The scanning electron microscope images are all magnified to 2000x. Figure 8 The illustrations in the upper right corner of each figure are magnified views at 8000x magnification.

[0044] Figure 9 This is a pore size distribution diagram of SO3, SO5, and SO7.

[0045] Figure 10 The results are the cell viability test results after co-culturing porous SF microspheres prepared in Example 1 with L929 cells for 24 h and 48 h. Detailed Implementation

[0046] The technical solutions of various embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0047] In the following embodiments, the microfluidic device used is a single-stage capillary microfluidic device, and its structural schematic diagram is shown below. Figure 1 As shown, the assembly includes an injection tube 1, a connecting tube 2, and a collection tube 3, used in conjunction with an injection pump. The injection tube 1 is made of a cylindrical glass capillary tube with an outer diameter of 960 μm and an inner diameter of 550 μm. The tail of the cylindrical glass capillary tube is drawn into a conical shape using a needle puller, and then polished on sandpaper until the inner diameter of the conical opening is approximately 50-300 μm. The collection tube 3 is also made of a cylindrical glass capillary tube, with both ends polished smooth. The outer diameter of the collection tube is 960 μm, and the inner diameter is 200-700 μm. The connecting tube 2 is a square glass tube, with both ends polished smooth. It has a square through-hole in its center, with a size of 1.0 × 1.0 mm. After fabrication, the injection tube 1, connecting tube 2, and collection tube 3 are ultrasonically cleaned in anhydrous ethanol and then dried. Insert the outlet end of injection tube 1 into the inlet end of collection tube 3 and connect them via connecting tube 2. Injection tube 1, connecting tube 2, and collection tube 3 are coaxially arranged and fixed to the glass slide with AB glue. Fix a flat-headed needle to the inlet end of injection tube 1 with AB glue, and fix a flat-headed needle to the inlet end of connecting tube 2 with AB glue. Seal the outlet end of connecting tube 2 with AB glue, and connect each flat-headed needle to the injection pump via fittings.

[0048] Example 1

[0049] In this embodiment, a method for preparing monodisperse porous silk fibroin microspheres with controllable pore structure is provided, the steps of which are as follows:

[0050] (1) Preparation of internal phase fluid, external phase fluid and collection liquid

[0051] Preparation of the inner phase fluid: Oleic acid in the inner oil phase was added to an aqueous solution of silk fibroin (SF). The concentration of the SF aqueous solution was 0.05 g / mL, and the volume ratio of oleic acid to SF aqueous solution was 1:4. The resulting mixture was homogenized and emulsified at 10,000 rpm for 30 s using a homogenizer to obtain an oil-in-water emulsion. This oil-in-water emulsion was used as the inner phase fluid.

[0052] Preparation of external phase fluid: Anhydrous ethanol and polyglycerol ricinoleate (PGPR) are added to oleic acid and magnetically stirred until PGPR is completely dissolved to obtain external phase fluid; in external phase fluid, the volume fraction of anhydrous ethanol is 15% and the mass fraction of PGPR is 4%.

[0053] Preparation of the collection solution: The collection solution is the same as the external phase fluid.

[0054] (2) Preparation of monodisperse porous SF microspheres

[0055] The structure adopted is as follows Figure 1 The first-stage capillary microfluidic device shown was prepared.

[0056] The inner phase fluid was injected into the injection tube of the microfluidic device using a syringe pump, and the outer phase fluid was injected into the collection tube of the microfluidic device using a syringe pump. A monodisperse oil-in-water-in-oil (O / W / O) emulsion was formed in the collection tube. The O / W / O emulsion was collected in a container filled with the collection liquid and allowed to stand for 4 hours. During the standing process, the O / W / O emulsion droplets gelled under the induction of ethanol and gradually transformed into microspheres. At the same time, the inner oil phase acted as a pore template to occupy sites. After complete gelation, the microspheres were washed three times with anhydrous ethanol to remove residual oleic acid and PGPR, resulting in monodisperse porous SF microspheres, which were then air-dried and stored.

[0057] In this step, the inner diameter of the conical inlet of the injection tube of the microfluidic device is 80 μm, the inner diameter of the collection tube is 550 μm, the flow rate of the inner phase fluid is controlled at 800 μL / h, and the flow rate of the outer phase fluid is 12 mL / h.

[0058] Figure 2 Figure a is an optical image of the O / W / O emulsion droplets prepared in Example 1. Figure 2 Figure b is an optical image of the porous SF microspheres prepared in Example 1. Figure 2 Figure c is a scanning electron microscope image of the porous SF microspheres prepared in Example 1. Figure 2 Image d is a scanning electron microscope image of the cross-section of the porous SF microspheres prepared in Example 1. Figure 2 Image e is an optical image of a cryosection of the porous SF microspheres prepared in Example 1. Figure 2It can be seen that the porous SF microspheres prepared in this embodiment have uniform size, good monodispersity, submicron-sized pores on the surface of the microspheres, and interconnected honeycomb-shaped micron-sized pores inside the microspheres.

[0059] Figure 3 This is a diameter distribution diagram of the O / W / O emulsion droplets and porous SF microspheres prepared in Example 1. Figure 3 It can be seen that the O / W / O emulsion droplets and porous SF microspheres have narrow diameter distribution and good uniformity. The diameter distribution of porous SF microspheres is between 110 and 130 μm, mainly concentrated around 120 μm.

[0060] Example 2

[0061] In this embodiment, a method for preparing monodisperse porous SF microspheres with controllable pore structure is provided, the steps of which are as follows:

[0062] (1) Preparation of internal phase fluid, external phase fluid and collection liquid

[0063] Preparation of the inner phase fluid: Oleic acid in the inner oil phase was added to an SF aqueous solution with a concentration of 0.05 g / mL and a volume ratio of oleic acid to SF aqueous solution of 1:4. The resulting mixture was homogenized and emulsified at 10,000 rpm for 60 s using a homogenizer to obtain an oil-in-water emulsion, which was used as the inner phase fluid.

[0064] Preparation of external phase fluid: Add anhydrous ethanol and PGPR to oleic acid and stir magnetically until PGPR is completely dissolved to obtain external phase fluid; in external phase fluid, the volume fraction of anhydrous ethanol is 15% and the mass fraction of PGPR is 2%.

[0065] Preparation of the collection solution: The collection solution is the same as the external phase fluid.

[0066] (2) Preparation of monodisperse porous SF microspheres

[0067] The structure adopted is as follows Figure 1 The first-stage capillary microfluidic device shown was prepared.

[0068] The inner phase fluid was injected into the injection tube of the microfluidic device using a syringe pump, and the outer phase fluid was injected into the collection tube of the microfluidic device using a syringe pump. A monodisperse O / W / O emulsion was formed in the collection tube. The O / W / O emulsion was collected in a container filled with the collection liquid and allowed to stand for 4 hours. During the standing process, the O / W / O emulsion droplets underwent gelation under the induction of ethanol and gradually transformed into microspheres. At the same time, the inner oil phase acted as a pore template to occupy sites. After complete gelation, the microspheres were washed three times with anhydrous ethanol to remove residual oleic acid and PGPR, resulting in monodisperse porous SF microspheres, which were then air-dried and stored.

[0069] In this step, the inner diameter of the conical opening of the injection tube of the microfluidic device is 60 μm, and the inner diameter of the collection tube is 400 μm.

[0070] This step involved five sets of experiments, with the internal phase fluid flow rates controlled at 400, 400, 800, 1000 and 1200 μL / h for Experiment 1 to Experiment 5, and the external phase fluid flow rates at 14, 10, 10, 10 and 6 mL / h, respectively.

[0071] Figure 4 Figure a shows the curves of the diameter of the O / W / O emulsion droplets and porous SF microspheres prepared in Example 2 as a function of the ratio of external phase fluid flow rate to internal phase fluid flow rate. Figure 4 Figure b shows the coefficient of variation (CV) of the diameters of the O / W / O emulsion droplets and porous SF microspheres prepared in Example 2. Figure 4 It can be seen that as the ratio of external fluid flow rate to internal fluid flow rate changes, the diameter of porous SF microspheres varies in the range of 107~162 μm. The diameter of O / W / O emulsion droplets and the diameter of porous SF microspheres are inversely proportional to the ratio of external fluid flow rate to internal fluid flow rate. In this embodiment, the CV value of the diameter of O / W / O emulsion droplets does not exceed 1.5%, and the CV value of the diameter of porous SF microspheres does not exceed 3%. They are uniform in size and morphology and have good monodispersity.

[0072] Example 3

[0073] In this embodiment, a method for preparing monodisperse porous SF microspheres with controllable pore structure is provided, the steps of which are as follows:

[0074] (1) Preparation of internal phase fluid, external phase fluid and collection liquid

[0075] Preparation of the inner phase fluid: Oleic acid in the inner oil phase was added to an SF aqueous solution with a concentration of 0.12 g / mL and a volume ratio of oleic acid to SF aqueous solution of 1:1. The resulting mixture was homogenized and emulsified at 10,000 rpm for 60 s using a homogenizer to obtain an oil-in-water emulsion, which was used as the inner phase fluid.

[0076] Preparation of external phase fluid: Add anhydrous ethanol and PGPR to oleic acid and stir magnetically until PGPR is completely dissolved to obtain external phase fluid; in external phase fluid, the volume fraction of anhydrous ethanol is 40% and the mass fraction of PGPR is 10%.

[0077] Preparation of the collection solution: The collection solution is the same as the external phase fluid.

[0078] (2) Preparation of monodisperse porous SF microspheres

[0079] The structure adopted is as follows Figure 1The first-stage capillary microfluidic device shown was prepared.

[0080] The inner phase fluid was injected into the injection tube of the microfluidic device using a syringe pump, and the outer phase fluid was injected into the collection tube of the microfluidic device using a syringe pump. A monodisperse O / W / O emulsion was formed in the collection tube. The O / W / O emulsion was collected in a container filled with the collection liquid and allowed to stand for 12 h. During the standing process, the O / W / O emulsion droplets underwent gelation under the induction of ethanol and gradually transformed into microspheres. At the same time, the inner oil phase acted as a pore template to occupy sites. After complete gelation, the microspheres were washed three times with anhydrous ethanol to remove residual oleic acid and PGPR, resulting in monodisperse porous SF microspheres, which were then air-dried and stored.

[0081] In this step, the inner diameter of the conical inlet of the injection tube of the microfluidic device is 140 μm, and the inner diameter of the collection tube is 550 μm; the flow rate of the inner phase fluid is controlled at 2000 μL / h, and the flow rate of the outer phase fluid is 24 mL / h.

[0082] Example 4

[0083] In this embodiment, a method for preparing monodisperse porous SF microspheres with controllable pore structure is provided, the steps of which are as follows:

[0084] (1) Preparation of internal phase fluid, external phase fluid and collection liquid

[0085] Preparation of the inner phase fluid: Oleic acid in the inner oil phase was added to an SF aqueous solution with a concentration of 0.02 g / mL and a volume ratio of oleic acid to SF aqueous solution of 0.1:1. The resulting mixture was homogenized and emulsified at 10,000 rpm for 30 s using a homogenizer to obtain an oil-in-water emulsion, which was used as the inner phase fluid.

[0086] Preparation of external phase fluid: Add anhydrous ethanol and PGPR to oleic acid and stir magnetically until PGPR is completely dissolved to obtain external phase fluid; in external phase fluid, the volume fraction of anhydrous ethanol is 5% and the mass fraction of PGPR is 0.1%.

[0087] Preparation of the collection solution: The collection solution is the same as the external phase fluid.

[0088] (2) Preparation of monodisperse porous SF microspheres

[0089] The structure adopted is as follows Figure 1 The first-stage capillary microfluidic device shown was prepared.

[0090] The inner phase fluid was injected into the injection tube of the microfluidic device using a syringe pump, and the outer phase fluid was injected into the collection tube of the microfluidic device using a syringe pump. A monodisperse O / W / O emulsion was formed in the collection tube. The O / W / O emulsion was collected in a container filled with the collection liquid and allowed to stand for 6 hours. During the standing process, the O / W / O emulsion droplets underwent gelation under the induction of ethanol and gradually transformed into microspheres. At the same time, the inner oil phase acted as a pore template to occupy sites. After complete gelation, the microspheres were washed three times with anhydrous ethanol to remove residual oleic acid and PGPR, resulting in monodisperse porous SF microspheres, which were then air-dried and stored.

[0091] In this step, the inner diameter of the conical inlet of the injection tube of the microfluidic device is 60 μm, and the inner diameter of the collection tube is 400 μm; the flow rate of the inner phase fluid is controlled at 1200 μL / h, and the flow rate of the outer phase fluid is 18 mL / h.

[0092] Example 5

[0093] In this embodiment, a method for preparing monodisperse porous SF microspheres with controllable pore structure is provided, and the influence of the content of the internal oil phase in the internal phase fluid on the pore structure of the porous SF microspheres is investigated. The steps are as follows:

[0094] (1) Preparation of internal phase fluid, external phase fluid and collection liquid

[0095] Preparation of the inner phase fluid: Oleic acid from the inner oil phase was added to an SF aqueous solution with a concentration of 0.05 g / mL. The resulting mixture was homogenized and emulsified at 10,000 rpm for 30 s using a homogenizer to obtain an oil-in-water emulsion, which was used as the inner phase fluid. During the preparation of the inner phase fluid, the volume ratio of oleic acid to SF aqueous solution was controlled at 1:4, 1:5, 1:6, and 1:7, respectively. The resulting inner phase fluids were designated as inner phase fluid 1, inner phase fluid 2, inner phase fluid 3, and inner phase fluid 4, respectively.

[0096] Preparation of external phase fluid: Add anhydrous ethanol and PGPR to oleic acid and stir magnetically until PGPR is completely dissolved to obtain external phase fluid; in external phase fluid, the volume fraction of anhydrous ethanol is 15% and the mass fraction of PGPR is 2%.

[0097] Preparation of the collection solution: The collection solution is the same as the external phase fluid.

[0098] (2) Preparation of monodisperse porous SF microspheres

[0099] The structure adopted is as follows Figure 1 The first-stage capillary microfluidic device shown was prepared.

[0100] The inner phase fluid was injected into the injection tube of the microfluidic device using a syringe pump, and the outer phase fluid was injected into the collection tube of the microfluidic device using a syringe pump. A monodisperse O / W / O emulsion was formed in the collection tube. The O / W / O emulsion was collected in a container filled with the collection liquid and allowed to stand for 4 hours. During the standing process, the O / W / O emulsion droplets underwent gelation under the induction of ethanol and gradually transformed into microspheres. At the same time, the inner oil phase acted as a pore template to occupy sites. After complete gelation, the microspheres were washed three times with anhydrous ethanol to remove residual oleic acid and PGPR, resulting in monodisperse porous SF microspheres, which were then air-dried and stored.

[0101] In this step, the inner diameter of the conical inlet of the injection tube of the microfluidic device is 80 μm, the inner diameter of the collection tube is 550 μm, the flow rate of the inner phase fluid is controlled at 800 μL / h, and the flow rate of the outer phase fluid is 12 mL / h.

[0102] In this step, four sets of experiments were conducted. The internal phase fluids introduced in Experiment 1 to Experiment 4 were internal phase fluid 1, internal phase fluid 2, internal phase fluid 3, and internal phase fluid 4, respectively. The porous SF microspheres prepared in Experiment 1 to Experiment 4 were labeled as SO4, SO5, SO6, and SO7, respectively.

[0103] Figure 5 Figure a is a scanning electron microscope image of the SO4 cross section. Figure 5 Image b is a scanning electron microscope image of the SO5 cross section. Figure 5 Image c is a scanning electron microscope image of the SO6 cross section. Figure 5 Image d is a scanning electron microscope (SEM) image of the SO7 cross section; all SEM images are magnified 3000x. Figure 5 It can be seen that SO4, SO5, SO6 and SO7 are all rich in interconnected micron-sized pore structures.

[0104] Scanning electron microscope (SEM) images of the internal cross-sections of SO4, SO5, SO6, and SO7 were randomly selected, processed using ImageJ software, and their porosity was analyzed. The results are as follows: Figure 6 As shown in Figure a. Figure 6 Figure b shows the average porosity of SO4, SO5, SO6, and SO7. Figure 6 It can be seen that by adjusting the content of the inner oil phase in the inner phase fluid, the porosity inside the porous SF microspheres can be effectively adjusted. As the volume ratio of the inner oil phase to the SF aqueous solution in the inner phase fluid changes from 1:4 to 1:7, the average porosity of the porous SF microspheres decreases from 76% to 44%.

[0105] Example 6

[0106] In this embodiment, a method for preparing monodisperse porous SF microspheres with controllable pore structure is provided, and the effect of SF aqueous solution concentration on the pore structure of porous SF microspheres is investigated. The steps are as follows:

[0107] (1) Preparation of internal phase fluid, external phase fluid and collection liquid

[0108] Preparation of the inner phase fluid: Oleic acid in the inner oil phase is added to an SF aqueous solution with a volume ratio of 1:5. The resulting mixture is homogenized and emulsified for 30 s using a homogenizer at 10,000 rpm to obtain an oil-in-water emulsion. This oil-in-water emulsion is used as the inner phase fluid.

[0109] Preparation of external phase fluid: Add anhydrous ethanol and PGPR to oleic acid and stir magnetically until PGPR is completely dissolved to obtain external phase fluid; in external phase fluid, the volume fraction of anhydrous ethanol is 15% and the mass fraction of PGPR is 2%.

[0110] Preparation of the collection solution: The collection solution is the same as the external phase fluid.

[0111] This step involved three sets of experiments, where the concentrations of SF aqueous solution in the internal phase fluid were controlled to be 0.04 g / mL, 0.06 g / mL, and 0.08 g / mL, respectively, for Experiments 1 to 3.

[0112] (2) Preparation of monodisperse porous SF microspheres

[0113] The structure adopted is as follows Figure 1 The first-stage capillary microfluidic device shown was prepared.

[0114] The inner phase fluid was injected into the injection tube of the microfluidic device using a syringe pump, and the outer phase fluid was injected into the collection tube of the microfluidic device using a syringe pump. A monodisperse O / W / O emulsion was formed in the collection tube. The O / W / O emulsion was collected in a container filled with the collection liquid and allowed to stand for 4 hours. During the standing process, the O / W / O emulsion droplets underwent gelation under the induction of ethanol and gradually transformed into microspheres. At the same time, the inner oil phase acted as a pore template to occupy sites. After complete gelation, the microspheres were washed three times with anhydrous ethanol to remove residual oleic acid and PGPR, resulting in porous SF microspheres, which were then air-dried and stored.

[0115] In this step, the inner diameter of the conical inlet of the injection tube of the microfluidic device is 60 μm, the inner diameter of the collection tube is 400 μm, the flow rate of the inner phase fluid is controlled at 800 μL / h, and the flow rate of the outer phase fluid is 12 mL / h.

[0116] The porous SF microspheres obtained in Experiments 1-3 were labeled as 4%SO5, 6%SO5, and 8%SO5, respectively.

[0117] Figure 7 Image a1 is a scanning electron microscope image of the 4% SO5 surface. Figure 7 Image a1' is a scanning electron microscope image of the 4% SO5 cross section. Figure 7 Image a2 is a scanning electron microscope image of the 6% SO5 surface. Figure 7 Image a2' is a scanning electron microscope image of the 6% SO5 cross section. Figure 7 Image a3 is a scanning electron microscope image of the 8% SO5 surface. Figure 7 Figure a3' shows a scanning electron microscope (SEM) image of the cross-section of 8% SO5, with each SEM image magnified at 2000x. As shown in Figure 7, with increasing SF aqueous solution concentration, the number of pores on the surface of the porous SF microspheres decreases, and the pore size distribution inside the porous SF microspheres narrows.

[0118] Example 7

[0119] In this embodiment, a method for preparing monodisperse porous SF microspheres with controllable pore structure is provided, and the influence of the content of the internal oil phase in the internal phase fluid on the pore structure of the porous SF microspheres is investigated. The steps are as follows:

[0120] (1) Preparation of internal phase fluid, external phase fluid and collection liquid

[0121] Preparation of the inner phase fluid: Oleic acid from the inner oil phase was added to an SF aqueous solution with a concentration of 0.05 g / mL. The resulting mixture was homogenized and emulsified at 10,000 rpm for 60 s using a homogenizer to obtain an oil-in-water emulsion, which was used as the inner phase fluid. During the preparation of the inner phase fluid, the volume ratio of oleic acid to SF aqueous solution was controlled at 1:3, 1:5, 1:7, and without oleic acid. The resulting inner phase fluids were designated as inner phase fluid 5, inner phase fluid 6, inner phase fluid 7, and inner phase fluid 8, respectively.

[0122] Preparation of external phase fluid: Add anhydrous ethanol and PGPR to oleic acid and stir magnetically until PGPR is completely dissolved to obtain external phase fluid; in external phase fluid, the volume fraction of anhydrous ethanol is 15% and the mass fraction of PGPR is 2%.

[0123] Preparation of the collection solution: The collection solution is the same as the external phase fluid.

[0124] (2) Preparation of highly monodisperse porous SF microspheres

[0125] The structure adopted is as follows Figure 1 The first-stage capillary microfluidic device shown was prepared.

[0126] The inner phase fluid was injected into the injection tube of the microfluidic device using a syringe pump, and the outer phase fluid was injected into the collection tube of the microfluidic device using a syringe pump. A monodisperse O / W / O emulsion was formed in the collection tube. The O / W / O emulsion was collected in a container filled with the collection liquid and allowed to stand for 4 hours. During the standing process, the O / W / O emulsion droplets underwent gelation under the induction of ethanol and gradually transformed into microspheres. At the same time, the inner oil phase acted as a pore template to occupy sites. After complete gelation, the microspheres were washed three times with anhydrous ethanol to remove residual oleic acid and PGPR, resulting in porous SF microspheres, which were then air-dried and stored.

[0127] In this step, the inner diameter of the conical inlet of the injection tube of the microfluidic device is 60 μm, and the inner diameter of the collection tube is 400 μm; the flow rate of the inner phase fluid is controlled at 800 μL / h, and the flow rate of the outer phase fluid is 14 mL / h.

[0128] In this step, four sets of experiments were conducted. The internal phase fluids introduced in Experiment 1 to Experiment 4 were internal phase fluid 5, internal phase fluid 6, internal phase fluid 7, and internal phase fluid 8, respectively. The porous SF microspheres prepared in Experiment 1 to Experiment 4 were denoted as SO3, SO5, SO7, and SO-, respectively.

[0129] Figure 8 Image a1 is a scanning electron microscope image of the SO3 surface. Figure 8 Image a1' is a scanning electron microscope image of the SO3 cross section. Figure 8 Image a2 is a scanning electron microscope image of the SO5 surface. Figure 8 Image a2' is a scanning electron microscope image of the SO5 cross section. Figure 8 Image a3 is a scanning electron microscope image of the SO7 surface. Figure 8 Image a3' is a scanning electron microscope image of the SO7 cross section. Figure 8 Image a4 is a scanning electron microscope image of the SO₂ surface. Figure 8 Image a4' is a scanning electron microscope image of the SO- cross section. Figure 8 The scanning electron microscope images are all magnified to 2000x. Figure 8 The inset in the upper right corner of each figure is a magnified view, magnified 8000 times. Figure 8 It can be seen that as the content of the internal oil phase in the internal phase fluid increases, the porosity on the surface of the porous SF microspheres increases, the porosity inside the porous SF microspheres increases, and the thickness of the lamellae between the pores decreases.

[0130] SEM images of the internal cross-sections of randomly selected SO3, SO5, and SO7 were processed using ImageJ software, and the pore size distribution was analyzed. The results are as follows: Figure 9 As shown. By Figure 9 It can be seen that by adjusting the content of the inner oil phase in the inner phase fluid, the pore size distribution inside the porous SF microspheres can be effectively adjusted. As the content of the inner oil phase in the inner phase fluid decreases, the pore size of the porous SF microspheres gradually decreases and the pore size distribution gradually narrows.

[0131] Example 8

[0132] In this embodiment, the in vitro cytotoxicity of the porous SF microspheres prepared in Example 1 was investigated.

[0133] The porous SF microspheres prepared in Example 1 were co-cultured with mouse fibroblasts (L929 cells), and the cell viability was tested using the CCK-8 standard method, as follows:

[0134] L929 cells were seeded at a density of 9000 cells / well in 96-well plates and cultured at 37 ℃ in a 5% CO2 incubator for 24 h. SF microspheres were sterilized by UV sterilization and washed with L929 cell culture medium by centrifugation. The washed microspheres were then reconstituted with culture medium to prepare microsphere dispersions at concentrations of 1000, 500, 250, 125, and 62.5 μg / mL. The culture medium in each well of the 96-well plate was replaced with 100 μL of the microsphere dispersion at different concentrations as experimental groups, and 100 μL of the culture medium was replaced with culture medium as a control group. After sample loading, the 96-well plates were placed in a cell culture incubator and cultured at 37 ℃ in a 5% CO2 incubator for 24 h and 48 h, respectively. The culture medium in the 96-well plate was replaced with 100 μL of fresh culture medium containing 10 μL of CCK-8. The plate was incubated for 3 h, and the absorbance of each well at 450 nm was measured using a microplate reader. Cell viability was calculated. The results are as follows: Figure 10 As shown.

[0135] Figure 10 The figure shows the cell viability test results after co-culturing porous SF microspheres prepared in Example 1 with L929 cells for 24 h and 48 h. As can be seen from the figure, the cell viability of the control group and all experimental groups is greater than 90%, indicating that the porous SF microspheres are non-cytotoxic.

[0136] Those skilled in the art will recognize that the embodiments described herein are for the purpose of assisting the reader in understanding the invention, and should be understood that the scope of protection of the invention is not limited to such specific statements and embodiments. Those skilled in the art can make various other specific modifications and combinations based on the technical teachings disclosed in this invention without departing from the spirit of the invention, and these modifications and combinations are still within the scope of protection of this invention.

Claims

1. A method for preparing monodisperse porous silk fibroin microspheres with controllable pore structure, characterized in that, Includes the following steps: (1) Preparation of internal phase fluid, external phase fluid, and collection liquid Preparation of the inner phase fluid: The inner oil phase is added to the silk fibroin aqueous solution and emulsified to obtain an oil-in-water emulsion, which is used as the inner phase fluid; the concentration of the silk fibroin aqueous solution is 0.02~0.12 g / mL, and the volume ratio of the inner oil phase to the silk fibroin aqueous solution is (0.1~1):1; the inner oil phase is an oil that is inert to silk fibroin, has low biotoxicity, and is non-volatile or low-volatile at room temperature, and the inner oil phase is vegetable oil, mineral oil, synthetic oil, or oleic acid; Preparation of external phase fluid: Dissolve oil-soluble surfactants and inducers in an oily solvent to obtain an external phase fluid; In the external phase fluid, the mass fraction of oil-soluble surfactant is 0.1 wt% to 10 wt%, and the volume fraction of inducer is 5% to 40%. Preparation of the collection solution: Dissolve the oil-soluble surfactant and the inducer in an oily solvent to obtain the collection solution; in the collection solution, the mass fraction of the oil-soluble surfactant is 0.1 wt%~10 wt%, and the volume fraction of the inducer is 5%~40%; (2) Preparation of monodisperse porous silk fibroin microspheres The inner and outer phase fluids are introduced into a primary microfluidic device. The outer phase fluid shears the inner phase fluid to obtain a monodisperse oil-in-water-in-oil emulsion. The oil-in-water-in-oil emulsion is collected with a collecting liquid and allowed to stand for a while. During the standing process, the oil-in-water-in-oil emulsion droplets gel under the induction of an inducing agent. At the same time, the inner oil phase acts as a pore template to occupy sites. After complete gelation, the residual inner oil phase in the obtained microspheres is removed by washing with an organic solvent to obtain monodisperse porous silk fibroin microspheres.

2. The method for preparing monodisperse porous silk fibroin microspheres with controllable pore structure according to claim 1, characterized in that, The inducing agent is methanol, ethanol, n-octanol, or ethyl acetate.

3. The method for preparing monodisperse porous silk fibroin microspheres with controllable pore structure according to claim 1, characterized in that, The oil-soluble surfactant includes at least one of polyglycerol ricinoleate, Span 80, and T154, and the oily solvent is vegetable oil, mineral oil, synthetic oil, or oleic acid.

4. A method for preparing monodisperse porous silk fibroin microspheres with controllable pore structure according to any one of claims 1 to 3, characterized in that, In step (1), the diameter of the water-in-oil emulsion droplets is controlled to be 100 nm to 10 μm.

5. A method for preparing monodisperse porous silk fibroin microspheres with controllable pore structure according to any one of claims 1 to 3, characterized in that, In step (2), when preparing a monodisperse oil-in-water-in-oil emulsion in a primary microfluidic device, the flow ratio of the external phase fluid to the internal phase fluid is controlled to be (5~40):

1.

6. The method for preparing monodisperse porous silk fibroin microspheres with controllable pore structure according to claim 5, characterized in that, In step (2), when preparing a monodisperse oil-in-water-in-oil emulsion in a primary microfluidic device, the flow rate of the external phase fluid is controlled at 500~26000 μL / h, and the flow rate of the internal phase fluid is controlled at 100~3000 μL / h.

7. A method for preparing monodisperse porous silk fibroin microspheres with controllable pore structure according to any one of claims 1 to 3, characterized in that, In step (2), the settling time is controlled to be 0.2~24 h.

8. Monodisperse porous silk fibroin microspheres, characterized in that, The porous silk fibroin microspheres are prepared by the method described in any one of claims 1 to 7, wherein the interior of the porous silk fibroin microspheres has an interconnected pore structure and the surface of the porous silk fibroin microspheres has a porous structure.

9. The monodisperse porous silk fibroin microspheres according to claim 8, characterized in that, The coefficient of variation of the particle size of the porous silk fibroin microspheres does not exceed 5%, the porosity of the porous silk fibroin microspheres can be adjusted between 20% and 90%, and the average pore size can be adjusted between 1 and 20 μm.

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