Silk fibroin microspheres as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological materials, and discloses silk fibroin microspheres as well as a preparation method and application thereof. The preparation method of the silk fibroin microspheres comprises the following steps: taking a silk fibroin solution as a water phase, shearing the water phase by using an oil phase by using a droplet micro-fluidic chip to prepare a collection solution containing silk fibroin micro-droplets, and performing induced molding to obtain the silk fibroin microspheres. The silk fibroin solution is prepared by adding silk fibroin, poloxamer and a cross-linking agent into a solvent, stirring and dissolving, the solvent comprises deionized water and a PBS solution, the cross-linking agent comprises a photo-cross-linking agent and a chemical cross-linking agent, the photo-cross-linking agent is riboflavin and sodium persulfate, and the chemical cross-linking agent is genipin. The preparation method is simple, green and environmentally friendly, and the prepared silk fibroin microspheres are uniform in particle size, controllable in morphology, good in monodispersity and good in biocompatibility, have special autofluorescence and adjustable fluorescence, and can be applied to multiple technical fields such as cell co-culture, drug delivery, tracing and biomedical imaging.

Description

Technical Field

[0001] The invention relates to the technical field of biomaterials, and in particular to a silk fibroin microsphere and a preparation method and application thereof. Background Art

[0002] Silk fibroin accounts for 2 / 3 of silk protein and is mainly composed of 18 amino acids such as glycine, alanine, and serine. It has good biocompatibility, biodegradability, flexibility, and tensile strength. It can be prepared into various forms such as membranes, gels, microspheres, and fibers. Silk fibroin was originally used in medical treatment as surgical sutures, and has been widely studied and applied in the biomedical field. Silk fibroin is used as a burn wound covering film, anticoagulant material, bone repair scaffold, etc. It has the characteristics of being non-toxic, non-irritating, and stable, providing favorable carrier conditions for drugs. Nowadays, people's research on silk fibroin in drug delivery systems is the most mature.

[0003] Polymer microspheres refer to tiny spherical entities with a particle size of 1 to 500 μm prepared by physical or chemical methods using natural polymer materials (such as starch, silk fibroin, gelatin, chitosan, etc.) and synthetic polymer materials (such as polylactic acid, polylactic acid-glycolic acid copolymer, etc.). Microspheres have been widely used in chemistry, physics, biology, materials and medicine due to their huge specific surface area and functional groups, such as dispersion technology, clinical diagnostic analysis, cosmetics and drug delivery systems. Silk fibroin microspheres are ideal materials for drug delivery systems because of their good biocompatibility, biodegradability, large specific surface area and good dispersibility. At present, silk fibroin microspheres are mostly used in drug delivery systems, cell culture and other aspects, and are less used in other aspects. The preparation of polymer microspheres with functional groups is the future development direction of silk microsphere preparation.

[0004] There are various methods for preparing silk fibroin microspheres, including spray drying, self-assembly, emulsification precipitation, salting out, etc. In recent years, due to the development of droplet microfluidics, the technology of preparing silk fibroin microspheres by microfluidics has also gradually developed. Droplet microfluidics is a technology that studies the generation, manipulation and application of droplets at the microscale. It can use immiscible phases to generate monodisperse droplets with controllable size. Compared with traditional chemical analysis platforms, it has the advantages of high throughput and good monodispersity. However, the existing technology for preparing silk microspheres by droplet microfluidics is limited by the slow formation speed of silk fibroin hydrogel and the high viscosity of silk fibroin aqueous solution, which requires the use of solvents and inorganic salts. However, the use of solvents such as formic acid, methanol, ethanol and lithium bromide that are toxic to biological cells will inevitably affect the safety of the use of silk microspheres; or the silk fibroin is methacrylated or phenol-modified. Although the modification can accelerate the formation of silk microspheres, it may also produce new cytotoxicity. In addition, the experimental process is more complicated, which limits the regulation of the structure and morphology of the microspheres, thereby limiting the promotion and application of microfluidic preparation of silk microspheres to a certain extent.

[0005] Functionalization of microspheres is one of the important ways to expand the application of microspheres. For example, the recently developed fluorescent microspheres have been used in the fields of labeling, tracing, detection, standards, immobilized enzymes, immunomedicine, high-throughput drug screening, and genetic research. However, most of the existing fluorescent microspheres are coated with fluorescent substances on the surface or embedded inside, which not only has the problem of fluorescence leakage, but also has the phenomenon of photobleaching and certain toxicity, which is also a shortcoming that the fluorescent microspheres need to overcome. Summary of the invention

[0006] In view of this, the present invention aims to provide a silk fibroin microsphere and a preparation method thereof, so as to solve the problems of limited ability to control microsphere structure morphology, certain biological cell toxicity and slow microsphere forming speed in the existing preparation of silk fibroin microspheres using droplet microfluidics technology. The silk fibroin microspheres provided by the present invention have special spontaneous fluorescence and adjustable fluorescence, which can overcome the shortcomings of fluorescence leakage, photobleaching and biological toxicity of existing fluorescent microspheres. At the same time, the present invention broadens the application field of silk fibroin microspheres.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions.

[0008] In a first aspect, the present invention provides a method for preparing silk fibroin microspheres, which comprises the following steps:

[0009] Step 1, preparing a silk fibroin solution: adding silk fibroin, poloxamer and a cross-linking agent into a solvent and stirring and dissolving to prepare a silk fibroin solution, wherein the solvent comprises deionized water and PBS solution, the cross-linking agent comprises a photo cross-linking agent and a chemical cross-linking agent, the photo cross-linking agent comprises riboflavin and sodium persulfate, and the chemical cross-linking agent comprises genipin;

[0010] Step 2, preparing silk fibroin microspheres: using silk fibroin solution as the aqueous phase, using a droplet microfluidic chip to utilize the oil phase to shear the aqueous phase to obtain a collection liquid containing silk fibroin microdroplets, and then inducing molding to obtain silk fibroin microspheres.

[0011] In the above technical scheme, the present invention adds a high molecular nonionic surfactant poloxamer when dissolving silk fibroin, thereby enhancing the solubility of silk fibroin and avoiding the problems caused by the use of lithium bromide and the use of modifiers. The present invention uses riboflavin and sodium persulfate photocuring system to photocrosslink silk fibroin, and uses genipin to react with free amino groups in silk fibroin for chemical crosslinking, thereby accelerating the formation of silk fibroin microspheres.

[0012] The cross-linking of silk fibroin by riboflavin and sodium persulfate photocuring system is achieved through the dityrosine system, involving two types of reactions: excited state oxidation and free radical oxidation. Under ultraviolet conditions, riboflavin (RF-) transitions to the singlet state (RF- * ), and form a triplet state through intersystem crossing ( 3 RF -* ), persulfate can extract an electron from triplet riboflavin to form two riboflavin free radicals (RF · ) and sulfate radical anion (SO4 -· ). Tyrosine residues can be directly 3 RF -* Oxidation or RF · Oxidation forms tyrosine free radicals, and two tyrosine free radicals can form a dityrosine structure, thereby achieving cross-linking. The formation of dityrosine bonds makes the silk fibroin microspheres fluorescent, but the microspheres can only have obvious fluorescence under the excitation of 405 and 488nm through the action of riboflavin and sodium persulfate. Silk fibroin is a material with fluorescent properties in itself. After adding genipin, the β-folded structure of silk fibroin can be changed, thereby changing the fluorescent luminescence characteristics of silk fibroin, and generating fluorescence under the excitation of 561 and 640nm. Therefore, the silk fibroin microspheres prepared by the present invention have adjustable and controllable autofluorescence.

[0013] At the same time, the microspheres prepared only by the action of riboflavin and sodium persulfate have less internal β-folding structure and a fast degradation rate of the microspheres. The present invention freeze-dries the microspheres or introduces an interpenetrating network strategy on the basis of photo-crosslinking, uses ultraviolet light to achieve photo-crosslinking of tyrosine free radicals in silk fibroin mediated by riboflavin and sodium persulfate to form a layer of hydrogel network, uses genipin to chemically crosslink with silk fibroin amino groups to form another layer of hydrogel network, and then obtains silk fibroin hydrogel microspheres with an interpenetrating network, which can reduce the degradation rate of the microspheres and achieve the long-term effect of the silk fibroin microspheres.

[0014] Furthermore, the concentration of silk fibroin in the silk fibroin solution is 2-5wt%, the concentration of poloxamer is 0.5wt%, the concentration of riboflavin is 0.2wt%, the concentration of sodium persulfate is 0.1wt%, and the concentration of genipin is 0.5wt%. By adjusting the concentration of silk fibroin in the silk fibroin liquid, the internal structure and fluorescence characteristics of the silk fibroin microspheres can be regulated.

[0015] Furthermore, the concentration of the PBS solution is 1 mM, and the pH is 7. Salt ions can promote protein salting out or aggregation. The phosphate buffer solution (PBS) introduced when dissolving silk fibroin in the present invention can affect the cross-linking effect of silk fibroin, thereby regulating the structure of the microspheres.

[0016] Furthermore, the flow rate of the oil phase is 2000-6000 μL / h, and the flow rate of the silk fibroin aqueous phase is 100-500 μL / h. By changing the flow rates of the oil phase and the aqueous phase, silk fibroin microspheres with different particle sizes can be prepared.

[0017] Furthermore, the induction molding method includes ultraviolet irradiation and light-proof constant temperature standing.

[0018] Furthermore, the ultraviolet irradiation time is 1-3 minutes. The fluorescence intensity and microsphere morphology can be regulated by adjusting the photo-crosslinking time of silk fibroin and riboflavin.

[0019] Furthermore, the time of the light-proof constant temperature static state is 1-3 hours, and the constant temperature is 35° C. The morphology and structure of the microspheres can be changed by changing the light-proof constant temperature static state time.

[0020] In summary, the present invention can regulate the structure and autofluorescence properties of silk fibroin hydrogel microspheres by changing the silk fibroin concentration, the flow rate of the water-oil phase, the cross-linking type, the cross-linking sequence, the cross-linking time and the addition of phosphate buffer solution.

[0021] In a second aspect, the present invention provides a silk fibroin microsphere prepared by the above-mentioned preparation method, which has good biocompatibility, spontaneous fluorescence and adjustable fluorescence.

[0022] In a third aspect, the present invention provides the application of the above-mentioned silk fibroin microspheres in drug encapsulation and fluorescence color development. The present invention can achieve drug encapsulation by dissolving the drug into a silk fibroin aqueous solution or soaking the silk fibroin microspheres in a drug solution for a certain period of time. The silk fibroin microspheres provided by the present invention have special autofluorescence and adjustable fluorescence, can be used for fluorescence color development, and are expected to be used in multiple fields such as labeling, tracing, detection, standards, biomedical imaging, tissue engineering, regenerative medicine, and skin repair.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The preparation method provided by the present invention avoids the use of solvents such as formic acid, methanol, ethanol and inorganic salts such as lithium bromide, which may cause biological cell toxicity and the use of modifiers, and is a green and environmentally friendly processing method;

[0025] (2) The preparation method provided by the present invention uses droplet microfluidics combined with cross-linking technology to achieve rapid preparation of silk fibroin microspheres;

[0026] (3) The silk microspheres prepared by the present invention have uniform particle size, controllable morphology, good monodispersity and good biocompatibility;

[0027] (4) The silk microspheres prepared by the present invention have controllable and adjustable spontaneous fluorescence and good fluorescence stability. They can emit fluorescence without coupling with fluorescent substances. They are a new type of fluorescent microspheres that can overcome the shortcomings of existing fluorescent microspheres such as fluorescence leakage, photobleaching and biological toxicity.

[0028] (5) The present invention can effectively regulate the enzymatic degradation rate of silk fibroin hydrogel microspheres by introducing an interpenetrating network hydrogel strategy and freeze-drying post-treatment methods;

[0029] (6) The silk microspheres prepared by the present invention can be used in many fields such as cell co-culture, drug delivery, tissue engineering, tracing, biomedical imaging, regenerative medicine, skin repair, etc., and have a wide range of applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of preparing silk fibroin microspheres for droplet microfluidic chip;

[0031] Figure 2 Characterization images of the morphology of RSs microspheres prepared at different UV irradiation times in Example 1;

[0032] Figure 3 The laser confocal microscopic images of RSs microspheres prepared at different UV irradiation times in Example 1;

[0033] Figure 4 Characterization images of the morphology of RSs microspheres prepared at different UV irradiation times in Example 2;

[0034] Figure 5 The laser confocal microscopic images of RSs microspheres prepared at different UV irradiation times in Example 2;

[0035] Figure 6 Characteristic images of the morphology of GSs microspheres prepared in Example 3 by standing at a constant temperature and in the dark for different periods of time;

[0036] Figure 7Laser confocal microscopic images of GSs microspheres prepared in Example 3 by standing at a constant temperature and in the dark for different periods of time;

[0037] Figure 8 Characteristic images of the morphology of GSs microspheres prepared in Example 4 by standing at a constant temperature and in the dark for different periods of time;

[0038] Fig. 9 Laser confocal microscopic images of GSs microspheres prepared in Example 4 by standing at a constant temperature and in the dark for different periods of time;

[0039] Fig.10 Characterization images of the morphology of RGSs microspheres prepared in Example 5 and Example 6;

[0040] Fig.11 Laser confocal microscopy images of RGSs microspheres prepared in Example 5 and Example 6;

[0041] Fig.12 Characterization images of the morphology of RGSs microspheres prepared in Example 7 and Example 8;

[0042] Fig.13 Laser confocal microscopy images of RGSs microspheres prepared in Example 7 and Example 8;

[0043] Fig.14 Laser confocal microscopy images of RGSs microspheres prepared with different silk fibroin concentrations in Example 9;

[0044] Fig.15 Optical microscopic images of the process of preparing RGSs microspheres at different water phase flow rates in Example 10;

[0045] Fig.16 This is a laser confocal microscopic image of the degradation process of the RSs microspheres (not freeze-dried) prepared in Example 2 in Example 11;

[0046] Fig.17 This is a laser confocal microscopic image of the degradation process of the RSs microspheres (freeze-dried) prepared in Example 2 in Example 11;

[0047] Fig.18 This is a laser confocal microscopic image of the degradation process of the RGSs microspheres prepared in Example 7 in Example 11;

[0048] Fig.19 It is a comparison chart of the drug loading of RSs-DOX drug-loaded microspheres prepared in Example 13 and RGSs-DOX drug-loaded microspheres prepared in Example 15;

[0049] Fig. 20This is a bar chart for quantitatively evaluating the cytotoxicity of RSs microspheres, RGSs microspheres, RSs-DOX drug-loaded microspheres, and RGSs-DOX drug-loaded microspheres in Example 17;

[0050] Fig.21 These are stained microscopic images for qualitatively evaluating the cytotoxicity of RSs microspheres, RGSs microspheres, RSs-DOX drug-loaded microspheres, and RGSs-DOX drug-loaded microspheres in Example 17. DETAILED DESCRIPTION

[0051] The present invention provides a method for preparing silk fibroin microspheres, which comprises the following steps:

[0052] Step 1, preparing a silk fibroin solution: adding silk fibroin, poloxamer and a cross-linking agent into a solvent and stirring and dissolving to prepare a silk fibroin solution, wherein the solvent comprises deionized water and PBS solution, the cross-linking agent comprises a photo cross-linking agent and a chemical cross-linking agent, the photo cross-linking agent comprises riboflavin and sodium persulfate, and the chemical cross-linking agent comprises genipin;

[0053] Step 2, preparing silk fibroin microspheres: using silk fibroin solution as the aqueous phase, using a droplet microfluidic chip to utilize the oil phase to shear the aqueous phase to obtain a collection liquid containing silk fibroin microdroplets, and then inducing molding to obtain silk fibroin microspheres.

[0054] In the present invention, the silk fibroin is a natural polymer fiber protein extracted from silk. The preparation method of the silk fibroin used in the following examples is: accurately weigh 1g of silk cocoon with a balance, add it to 100mL of 0.5% Na2CO3 solution, heat it for 0.5h, then wash it with deionized water to remove the sericin on the surface of the silk, and degumming it repeatedly. After degumming, place it in an oven to obtain dry degummed silk. Dissolve 1g of degummed silk in 5ml of a ternary solvent system (CaCl2 / C2H5OH / H2O) at 72°C, stir to fully dissolve the silk for 2h to obtain a crude silk fibroin solution. After the crude silk fibroin solution is cooled, it is transferred to a dialysis bag (molecular weight cutoff of 10kDa), dialyzed with deionized water for three days, and desalted. After the dialysis is completed, the silk fibroin solution is repeatedly centrifuged (7000rpm, 10min) to remove the solid matter in the solution, and the supernatant is freeze-dried to obtain silk fibroin, which is stored in a refrigerator at 4°C.

[0055] See also Figure 1 In the present invention, the process of preparing silk fibroin microdroplets using a droplet microfluidic chip is as follows: the water phase is injected from the inlet 1 of the chip, and the oil phase is injected from the inlet 2 of the chip as the shear phase to shear the water phase at the shear port 3 to form silk fibroin microdroplets; the size of the silk fibroin microdroplets can be controlled by adjusting the injection speed of the oil phase and the water phase. The flow rate of the oil phase is 2000-6000 μL / h, and the flow rate of the water phase is 100-500 μL / h.

[0056] In the present invention, the oil phase includes one or more of soybean oil, light mineral oil, straight-chain alkane, fluorinated oil HFE7500 and silicone oil; the oil phase also includes a surfactant, and the surfactant preferably includes one or more of PGPR, Span-80, Tween-20, PFPE-PEG, KF-6017, EM-90 and Triton X-100. The concentration of the surfactant in the oil phase is preferably 10wt%. The oil phase used in the following examples is soybean oil containing 10wt% PGPR.

[0057] In the present invention, the induction molding method includes ultraviolet irradiation and light-proof constant temperature standing. When the crosslinking agent added to the silk fibroin liquid is a photocrosslinking agent, the induction molding method is ultraviolet irradiation, and the ultraviolet irradiation time is 1-3 minutes; when the crosslinking agent added to the silk fibroin liquid is a chemical crosslinking agent, the induction molding method is light-proof constant temperature standing, and the light-proof constant temperature standing time is 1-3 hours, and the constant temperature is 35°C.

[0058] In order to make those skilled in the art better understand the technical solution of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments, but it should not be considered as limiting the scope of protection of the present invention. Unless otherwise specified, the materials and equipment used in the following embodiments are all commercially available products in the field.

[0059] Example 1. Preparation of riboflavin-silk fibroin microspheres (RSs microspheres)

[0060] The preparation steps are as follows:

[0061] (1) Preparation of silk fibroin solution: Riboflavin, sodium persulfate, poloxamer (F127) and silk fibroin were added into deionized water and stirred to dissolve at room temperature (stirring speed 800 rpm, stirring time 30 min) to prepare a silk fibroin solution, wherein the silk fibroin concentration was 3 wt%, the riboflavin concentration was 0.2 wt%, the sodium persulfate concentration was 0.1 wt%, and the F127 concentration was 0.5 wt%.

[0062] (2) Preparation of RSs microspheres: The silk fibroin solution prepared above was used as the aqueous phase, and soybean oil containing 10 wt% PGPR was used as the oil phase. The aqueous phase speed was controlled to be 200 μL / h, and the oil phase speed was controlled to be 2000 μL / h. After the collection liquid containing silk fibroin microdroplets was prepared by using the oil phase to shear the aqueous phase using a droplet microfluidic chip, the collection liquid was placed under ultraviolet irradiation, and the microdroplets were formed into microspheres. After the oil phase solution around the microspheres was replaced with n-hexane, they were washed with deionized water, and the microspheres were collected and freeze-dried to obtain RSs microspheres. The particle size of the silk fibroin microdroplets prepared in this example was 101.3±0.6 μm, and the monodispersity coefficient (CV value) was 0.6%.

[0063] The time of UV irradiation was controlled to be 1min, 2min and 3min to prepare RSs microspheres respectively. The morphology of the prepared RSs microspheres was observed by optical microscope and scanning electron microscope. The results are shown in Figure 2 As shown, the luminescence properties of the microspheres were observed using a laser confocal microscope, see Figure 3 It can be found that the microspheres prepared in this example show obvious fluorescence effect under 405nm and 488nm laser excitation, and have good stability. Compared with the fluorescence excited by 405nm, the fluorescence phenomenon of 488nm excitation is stronger. In addition, with the increase of ultraviolet irradiation cross-linking time, the fluorescence intensity of the microspheres gradually increases, and the morphology of the microspheres also varies, indicating that adjusting the reaction time of the silk fibroin hydrogel network can achieve the regulation of fluorescence intensity and microsphere morphology.

[0064] Example 2: Preparation of RSs microspheres

[0065] The preparation steps are as follows:

[0066] (1) Preparation of silk fibroin solution: Riboflavin, sodium persulfate, poloxamer (F127) and silk fibroin were added to a PBS solution (1 mM, pH = 7), and stirred to dissolve at room temperature (stirring speed 800 rpm, stirring time 30 min) to prepare a silk fibroin solution, wherein the silk fibroin concentration was 3 wt %, the riboflavin concentration was 0.2 wt %, the sodium persulfate concentration was 0.1 wt %, and the F127 concentration was 0.5 wt %.

[0067] (2) Preparation of RSs microspheres: The silk fibroin solution prepared above was used as the aqueous phase, and soybean oil containing 10 wt% PGPR was used as the oil phase. The aqueous phase speed was controlled to be 200 μL / h, and the oil phase speed was controlled to be 2000 μL / h. After the collection liquid containing silk fibroin microdroplets was prepared by using the oil phase to shear the aqueous phase using a droplet microfluidic chip, the collection liquid was placed under ultraviolet irradiation, and the microdroplets were formed into microspheres. After the oil phase solution around the microspheres was replaced with n-hexane, they were washed with PBS solution, and the microspheres were collected and freeze-dried to obtain RSs microspheres. The particle size of the silk fibroin microdroplets prepared in this embodiment was 102.4±0.8 μm, and the monodispersity coefficient (CV value) was 0.7%.

[0068] The time of UV irradiation was controlled to be 1min, 2min and 3min to prepare RSs microspheres respectively. The morphology of the prepared RSs microspheres was observed by optical microscope and scanning electron microscope. The results are shown in Figure 4 As shown, the luminescence properties of the microspheres were observed using a laser confocal microscope, see Figure 5 It can be found that the microspheres prepared in this example show obvious fluorescence effect under 405nm and 488nm laser excitation, and have good stability. Compared with the fluorescence excited by 405nm, the fluorescence phenomenon of 488nm excitation is stronger. In addition, with the increase of ultraviolet irradiation cross-linking time, the fluorescence intensity of the microspheres gradually increases, and the morphology of the microspheres also varies, indicating that adjusting the reaction time of the silk fibroin hydrogel network can achieve the regulation of fluorescence intensity and microsphere morphology.

[0069] Example 3: Preparation of genipin-silk fibroin microspheres (GSs microspheres)

[0070] The preparation steps are as follows:

[0071] (1) Preparation of silk fibroin solution: Genipin, poloxamer (F127) and silk fibroin were added into deionized water and stirred to dissolve at room temperature (stirring speed 800 rpm, stirring time 30 min) to prepare a silk fibroin solution, wherein the silk fibroin concentration was 3 wt%, the genipin concentration was 0.5 wt%, and the F127 concentration was 0.5 wt%.

[0072] (2) Preparation of GSs microspheres: The silk fibroin solution prepared above was used as the aqueous phase, and soybean oil containing 10 wt% PGPR was used as the oil phase. The aqueous phase speed was controlled to be 200 μL / h, and the oil phase speed was controlled to be 2000 μL / h. After the collection liquid containing silk fibroin microdroplets was prepared by using the oil phase to shear the aqueous phase using a droplet microfluidic chip, the collection liquid was kept at a constant temperature (35° C.) and kept away from light for a certain period of time, the microdroplets were formed into microspheres, the oil phase solution around the microspheres was replaced with n-hexane, and then washed with deionized water, and the microspheres were collected and freeze-dried to obtain GSs microspheres. The particle size of the silk fibroin microdroplets prepared in this example was 102.2±0.4 μm, and the monodispersity coefficient (CV value) was 0.4%.

[0073] The GSs microspheres were prepared by keeping the samples in a constant temperature and away from light for 1 h, 2 h and 3 h, respectively. The morphology of the prepared GSs microspheres was observed by optical microscopy and scanning electron microscopy. Figure 6 As shown, the luminescence properties of the microspheres were observed using a laser confocal microscope, see Figure 7 It can be found that the microspheres prepared in this example show obvious fluorescence effects under 405nm, 488nm, 561nm, and 640nm laser excitation, and the fluorescence intensity excited by 561nm is the strongest. With the increase of the constant temperature and light-proof standing time, the fluorescence intensity excited by 561nm and 640nm gradually increases, and the internal structure of the microspheres becomes denser, indicating that adjusting the reaction time of the silk fibroin hydrogel network can achieve the regulation of the fluorescence intensity and the morphology of the microspheres.

[0074] Example 4: Preparation of GSs microspheres

[0075] The preparation steps are as follows:

[0076] (1) Preparation of silk fibroin solution: Genipin, poloxamer (F127) and silk fibroin were added to a PBS solution (1 mM, pH = 7), and stirred to dissolve at room temperature (stirring speed 800 rpm, stirring time 30 min) to prepare a silk fibroin solution, wherein the silk fibroin concentration was 3 wt%, the genipin concentration was 0.5 wt%, and the F127 concentration was 0.5 wt%.

[0077] (2) Preparation of GSs microspheres: The silk fibroin solution prepared above was used as the aqueous phase, and soybean oil containing 10 wt% PGPR was used as the oil phase. The aqueous phase speed was controlled to be 200 μL / h, and the oil phase speed was controlled to be 2000 μL / h. After the collection liquid containing silk fibroin microdroplets was prepared by using the oil phase to shear the aqueous phase using a droplet microfluidic chip, the collection liquid was kept at a constant temperature (35° C.) and kept away from light for a certain period of time, the microdroplets were formed into microspheres, and the oil phase solution around the microspheres was replaced with n-hexane, and then washed with PBS solution, and the microspheres were collected and freeze-dried to obtain RSs microspheres. The particle size of the silk fibroin microdroplets prepared in this example was 109.8±0.7 μm, and the monodispersity coefficient (CV value) was 0.6%.

[0078] The GSs microspheres were prepared by keeping the samples in a constant temperature and away from light for 1 h, 2 h and 3 h, respectively. The morphology of the prepared GSs microspheres was observed by optical microscopy and scanning electron microscopy. Figure 8 As shown, the luminescence properties of the microspheres were observed using a laser confocal microscope, see Fig. 9 It can be found that the microspheres prepared in this example show obvious fluorescence effects under 405nm, 488nm, 561nm, and 640nm laser excitation, and the fluorescence intensity under 561nm excitation is the strongest. With the increase of constant temperature standing time, the morphology and structure of the microspheres change, indicating that adjusting the reaction time of the silk fibroin hydrogel network can achieve the regulation of fluorescence intensity and microsphere morphology.

[0079] Example 5: Preparation of riboflavin-genipin-silk fibroin microspheres (RGSs microspheres)

[0080] The preparation steps are as follows:

[0081] (1) Preparation of silk fibroin solution: Riboflavin, sodium persulfate, genipin, poloxamer (F127) and silk fibroin were added into deionized water and stirred to dissolve at room temperature (stirring speed 800 rpm, stirring time 30 min) to prepare a silk fibroin solution, wherein the silk fibroin concentration was 3 wt%, the riboflavin concentration was 0.2 wt%, the sodium persulfate concentration was 0.1 wt%, the genipin concentration was 0.5 wt%, and the F127 concentration was 0.5 wt%.

[0082] (2) Preparation of RGSs microspheres: The silk fibroin solution prepared above was used as the aqueous phase, and soybean oil containing 10 wt% PGPR was used as the oil phase. The aqueous phase speed was controlled to be 200 μL / h, and the oil phase speed was controlled to be 2000 μL / h. After the collection liquid containing silk fibroin microdroplets was prepared by using the droplet microfluidic chip to shear the aqueous phase with the oil phase, the collection liquid was first placed in an ultraviolet irradiation environment for 2 minutes, and then kept at a constant temperature (35°C) and away from light for 3 hours. The microdroplets were formed into microspheres. After replacing the oil phase solution around the microspheres with n-hexane, they were washed with deionized water, and the microspheres were collected and freeze-dried to obtain RGSs microspheres. The particle size of the silk fibroin microdroplets prepared in this example was 98.2±1.1 μm, and the monodispersity coefficient (CV value) was 1.2%; the particle size of the RGSs microspheres prepared was 77.8±2.2 μm, and the monodispersity coefficient (CV value) was 2.8%.

[0083] Example 6: Preparation of RGSs microspheres

[0084] The preparation steps are as follows:

[0085] (1) Preparation of silk fibroin solution: Riboflavin, sodium persulfate, genipin, poloxamer (F127) and silk fibroin were added into deionized water and stirred to dissolve at room temperature (stirring speed 800 rpm, stirring time 30 min) to prepare a silk fibroin solution, wherein the silk fibroin concentration was 3 wt%, the riboflavin concentration was 0.2 wt%, the sodium persulfate concentration was 0.1 wt%, the genipin concentration was 0.5 wt%, and the F127 concentration was 0.5 wt%.

[0086] (2) Preparation of RGSs microspheres: The silk fibroin solution prepared above was used as the aqueous phase, and soybean oil containing 10 wt% PGPR was used as the oil phase. The aqueous phase speed was controlled to be 200 μL / h, and the oil phase speed was controlled to be 2000 μL / h. After the collection liquid containing silk fibroin microdroplets was prepared by using the droplet microfluidic chip to shear the aqueous phase with the oil phase, the collection liquid was kept at a constant temperature (35°C) and kept away from light for 3 hours, and then placed in an ultraviolet irradiation environment for 2 minutes. The microdroplets were formed into microspheres. After replacing the oil phase solution around the microspheres with n-hexane, they were washed with deionized water, and the microspheres were collected and freeze-dried to obtain RGSs microspheres. The particle size of the silk fibroin microdroplets prepared in this example was 101.6±1.4 μm, and the monodispersity coefficient (CV value) was 1.4%.

[0087] The morphology of the RGSs microspheres prepared in Example 5 and Example 6 was observed using an optical microscope and a scanning electron microscope. Fig.10 As shown, the luminescence properties of the RGSs microspheres prepared in Example 5 and Example 6 were observed using a laser confocal microscope. Fig.11It can be found that the microspheres prepared in Examples 5 and 6 all exhibited obvious fluorescence effects under excitation at 405 nm, 488 nm, 561 nm, and 640 nm. Compared with the microspheres prepared in Example 5, the fluorescence of the microspheres prepared in Example 6 under excitation at 405 nm, 488 nm, 561 nm, and 640 nm was significantly weakened, and the internal pores were larger, indicating that the fluorescence and morphology of the microspheres can be controlled by regulating the order of chemical crosslinking and ultraviolet crosslinking.

[0088] Compared with the RSs microspheres prepared in Example 1 (UV irradiation for 2 minutes), the fluorescence intensity of the RGSs microspheres prepared in Examples 5 and 6 under 405nm and 488nm laser excitation is enhanced and has obvious fluorescence at 561nm and 640nm, among which the fluorescence under 488nm excitation is the strongest and 640nm is the weakest. Scanning electron microscopy can observe that the internal structure of the microspheres is denser after the addition of genipin. These phenomena indicate that the fluorescence luminescence characteristics and morphological structure of the microspheres can be controlled by adjusting the components of the aqueous phase.

[0089] Example 7: Preparation of RGSs microspheres

[0090] The preparation steps are as follows:

[0091] (1) Preparation of silk fibroin solution: Riboflavin, sodium persulfate, genipin, poloxamer (F127) and silk fibroin were added to a PBS (1 mM, pH = 7) solution, and stirred and dissolved at room temperature (stirring speed 800 rpm, stirring time 30 min) to prepare a silk fibroin solution, wherein the silk fibroin concentration was 3 wt%, the riboflavin concentration was 0.2 wt%, the sodium persulfate concentration was 0.1 wt%, the genipin concentration was 0.5 wt%, and the F127 concentration was 0.5 wt%.

[0092] (2) Preparation of RGSs microspheres: The silk fibroin solution prepared above was used as the aqueous phase, and soybean oil containing 10 wt% PGPR was used as the oil phase. The aqueous phase speed was controlled to be 200 μL / h, and the oil phase speed was controlled to be 2000 μL / h. After the collection liquid containing silk fibroin microdroplets was prepared by using the droplet microfluidic chip to shear the aqueous phase with the oil phase, the collection liquid was first placed in an ultraviolet irradiation environment for 2 minutes, and then kept at a constant temperature (35°C) and away from light for 3 hours. The microdroplets were formed into microspheres. After replacing the oil phase solution around the microspheres with n-hexane, they were washed with PBS solution, and the microspheres were collected and freeze-dried to obtain RGSs microspheres. The particle size of the microdroplets prepared in this embodiment was 97.2±1.3 μm, and the monodispersity coefficient (CV value) was 1.3%; the particle size of the microspheres prepared was 91.2±2.3 μm, and the monodispersity coefficient (CV value) was 2.5%.

[0093] Example 8: Preparation of RGSs microspheres

[0094] The preparation steps are as follows:

[0095] (1) Preparation of silk fibroin solution: Riboflavin, sodium persulfate, genipin, poloxamer (F127) and silk fibroin were added to a PBS (1 mM, pH = 7) solution, and stirred and dissolved at room temperature (stirring speed 800 rpm, stirring time 30 min) to prepare a silk fibroin solution, wherein the silk fibroin concentration was 3 wt%, the riboflavin concentration was 0.2 wt%, the sodium persulfate concentration was 0.1 wt%, the genipin concentration was 0.5 wt%, and the F127 concentration was 0.5 wt%.

[0096] (2) Preparation of RGSs microspheres: The silk fibroin solution prepared above was used as the aqueous phase, and soybean oil containing 10 wt% PGPR was used as the oil phase. The aqueous phase speed was controlled to be 200 μL / h, and the oil phase speed was controlled to be 2000 μL / h. After the collection liquid containing silk fibroin microdroplets was prepared by using the oil phase to shear the aqueous phase using a droplet microfluidic chip, the collection liquid was kept at a constant temperature (35°C) and kept away from light for 3 hours, and then placed in an ultraviolet irradiation environment for 2 minutes. The microdroplets were formed into microspheres. After replacing the oil phase solution around the microspheres with n-hexane, they were washed with PBS solution, and the microspheres were collected and freeze-dried to obtain RGSs microspheres. The particle size of the silk fibroin microdroplets prepared in this example was 97.2±1.0 μm, and the monodispersity coefficient (CV value) was 1.0%.

[0097] The morphology of the RGSs microspheres prepared in Example 7 and Example 8 was observed using an optical microscope and a scanning electron microscope. Fig.12 The luminescence properties of the microspheres were observed by laser confocal microscopy, see Fig.13 It was found that the microspheres prepared in Examples 7 and 8 showed obvious fluorescence effects under excitation at 405nm, 488nm, and 561nm. Compared with the microspheres prepared in Example 7, the fluorescence of the microspheres prepared in Example 8 under excitation at 405nm, 488nm, and 561nm was significantly weakened, and the internal pores were larger, indicating that the fluorescence and morphology of the microspheres can be controlled by regulating the order of chemical crosslinking and ultraviolet crosslinking.

[0098] Compared with the RSs microspheres prepared in Example 2 (UV irradiation for 2 min), the fluorescence intensity of the RGSs microspheres prepared in Examples 7 and 8 under 405nm and 488nm laser excitation was enhanced and had obvious fluorescence at 561nm, among which the fluorescence under 488nm excitation was the strongest and 561nm was the weakest. Scanning electron microscopy showed that the internal structure of the microspheres was denser after the addition of genipin, which indicated that the fluorescence luminescence characteristics and morphological structure of the microspheres could be controlled by adjusting the components of the aqueous phase.

[0099] Compared with the microspheres prepared in Example 5, the fluorescence intensity of the microspheres prepared in Example 7 is reduced, and the internal structure is not as dense as the microspheres prepared in Example 5; compared with the microspheres prepared in Example 6, the fluorescence intensity of the microspheres prepared in Example 8 is reduced, and the internal structure is not as dense as the microspheres prepared in Example 6; this shows that the introduction of PBS solution can achieve the regulation of the fluorescence and morphology of the microspheres.

[0100] Example 9: Preparation of RGSs microspheres

[0101] The preparation steps were the same as those in Example 7, except that the concentration of silk fibroin in the silk fibroin solution was changed to 2wt%, 3wt%, 4wt%, and 5wt% to prepare RGSs microspheres respectively. The luminescence properties of the prepared microspheres were observed by laser confocal microscopy. The results are shown in Fig.14 It can be found that the fluorescence intensity of the microspheres prepared with different silk fibroin concentrations is different, indicating that the fluorescence luminescence properties and morphology of the microspheres can be controlled by adjusting the concentration of silk fibroin in the aqueous solution.

[0102] Example 10: Preparation of RGSs microspheres

[0103] The preparation steps are the same as those in Example 7, except that the injection speed of the aqueous phase is changed to observe the change in the droplet size, see Fig.15 It can be found that with the increase of water phase flow rate, the particle size of microdroplets formed by shearing gradually increases, and then the particle size of microspheres gradually increases, indicating that the particle size of microspheres can be controlled by adjusting the water phase flow rate.

[0104] Example 11. Detection of the degradation performance of silk fibroin microspheres

[0105] The detection method is as follows:

[0106] (1) Preparation of protease XIV solution: Add protease XIV powder into PBS solution and stir evenly to obtain 1.75 U / mL protease XIV solution.

[0107] (2) The RSs microspheres prepared in Example 2 (UV irradiation for 2 min, before and after freeze-drying) and the RGSs microspheres prepared in Example 7 were added to a protease XIV solution, respectively. The amount of protease XIV used was 2.5 U / 5 mg silk fibroin. The degradation degree of the microspheres was observed using a laser confocal microscope at room temperature.

[0108] See also Figures 16 to 18It can be found that with the increase of time, the fluorescence of the RSs microspheres (not freeze-dried) prepared in Example 2 gradually decreases, the morphology of the microspheres changes, and after 20 minutes, the microspheres are completely degraded. After the microspheres are freeze-dried, the structure of the microspheres changes and the degradation rate is significantly reduced. In addition, RGSs microspheres prepared by adding genipin to the aqueous phase can also delay the degradation of the microspheres. It shows that the enzymatic degradation rate of silk fibroin hydrogel microspheres can be effectively regulated by post-freeze drying treatment and the introduction of interpenetrating network hydrogel strategy.

[0109] Example 12: Preparation of riboflavin-silk fibroin-doxorubicin drug-loaded microspheres (RSs-DOX drug-loaded microspheres)

[0110] The preparation steps are as follows:

[0111] (1) Preparation of drug-loaded silk fibroin solution: Add silk fibroin, F127, riboflavin, and sodium persulfate to PBS (1 mM, pH = 7) solution, and stir and dissolve at room temperature (stirring speed 800 rpm, stirring time 30 min) to obtain a silk fibroin solution, wherein the concentration of silk fibroin is 6 wt%, the concentration of F127 is 0.5 wt%, the concentration of riboflavin is 0.4 wt%, and the concentration of sodium persulfate is 0.2 wt%. Add doxorubicin and F127 to PBS (1 mM, pH = 7) solution, and stir and dissolve to obtain a doxorubicin solution, wherein the concentration of doxorubicin is 0.2 wt%, and the concentration of F127 is 0.5 wt%. Stir and mix the silk fibroin solution and the doxorubicin solution in equal volumes to obtain a drug-loaded silk fibroin solution.

[0112] (2) Preparation of RSs-DOX drug-loaded microspheres: The drug-loaded silk fibroin solution prepared above was used as the aqueous phase, and soybean oil containing 10 wt% PGPR was used as the oil phase. The aqueous phase velocity was controlled to be 300 μL / h and the oil phase velocity was controlled to be 3000 μL / h. A droplet microfluidic chip was used to shear the aqueous phase with the oil phase to obtain a collection liquid containing silk fibroin microdroplets. The collection liquid was then placed in an ultraviolet irradiation environment for 2 min to form microdroplets. The oil phase solution around the microspheres was replaced with n-hexane and then washed with PBS solution. The microspheres were collected and freeze-dried to obtain RSs-DOX drug-loaded microspheres.

[0113] Example 13: Preparation of RSs-DOX drug-loaded microspheres

[0114] The unfreeze-dried RSs microspheres prepared in Example 2 (irradiated with ultraviolet light for 2 min) were immersed in a 10 mg / mL doxorubicin solution, incubated with shaking for 24 h, taken out, washed with deionized water until the solution was transparent, and freeze-dried and stored for later use.

[0115] Example 14: Preparation of riboflavin-genipin-silk fibroin-doxorubicin drug-loaded microspheres (RGSs-DOX drug-loaded microspheres)

[0116] The preparation steps are as follows:

[0117] (1) Preparation of drug-loaded silk fibroin solution: Add silk fibroin, F127, riboflavin, sodium persulfate, and genipin to a PBS (1 mM, pH = 7) solution, and stir to dissolve to obtain a silk fibroin solution, wherein the concentration of silk fibroin is 6 wt%, the concentration of F127 is 0.5 wt%, the concentration of riboflavin is 0.4 wt%, the concentration of sodium persulfate is 0.2 wt%, and the concentration of genipin is 1 wt%. Add doxorubicin and F127 to a PBS (1 mM, pH = 7) solution, and stir to dissolve to obtain a doxorubicin solution, wherein the concentration of doxorubicin is 0.2 wt%, and the concentration of F127 is 0.5 wt%. Stir and mix the silk fibroin solution and the doxorubicin solution in equal volumes to obtain a drug-loaded silk fibroin solution.

[0118] (2) Preparation of RGSs-DOX drug-loaded microspheres: The drug-loaded silk fibroin solution prepared above was used as the aqueous phase, and soybean oil containing 10 wt% PGPR was used as the oil phase. The aqueous phase velocity was controlled to be 300 μL / h, and the oil phase velocity was controlled to be 3000 μL / h. A droplet microfluidic chip was used to shear the aqueous phase with the oil phase to obtain a collection liquid containing silk fibroin microdroplets. The collection liquid was then placed in an ultraviolet irradiation environment for 2 min, and then kept at a constant temperature (35°C) and away from light for 3 h. The microdroplets were formed into microspheres. The oil phase solution around the microspheres was replaced with n-hexane, and then washed with PBS solution. The microspheres were collected and freeze-dried to obtain RGSs-DOX drug-loaded microspheres.

[0119] Example 15: Preparation of RGSs-DOX drug-loaded microspheres

[0120] The unfreeze-dried RGSs microspheres prepared in Example 7 were immersed in a 10 mg / mL doxorubicin solution, incubated with shaking for 24 h, taken out, washed with deionized water until the solution was transparent, and freeze-dried and stored for later use.

[0121] Example 16. Determination of drug loading of drug-loaded microspheres

[0122] Drawing of the standard curve of doxorubicin. Accurately weigh 1 mg of doxorubicin, fully dissolve in 1 mL of deionized water in a dark place, and then dilute to solutions with concentrations of 100, 90, 80, 70, 60, 50, 40, 30, 20, and 10 μg / mL. Measure the OD values ​​of doxorubicin solutions of different concentrations at the characteristic peak of doxorubicin at 480 nm using a multifunctional microplate reader. Draw a standard curve based on the OD values ​​and the corresponding different concentrations of doxorubicin solutions. Repeat the measurement three times for each sample.

[0123] Take the drug-loaded microspheres prepared in Examples 13 and 15 respectively, add dimethyl sulfoxide (DMSO) to prepare a 1 mg / mL solution, use an ultrasonic cell crusher to sonicate for 5 min to completely dissolve doxorubicin in DMSO, then centrifuge it (operating parameters: centrifugal force 8000g, centrifugation time 10 min), and draw 100 μL of supernatant into a 96-well plate. According to the characteristic peak of doxorubicin, use an ELISA reader to measure the OD value at 480nm, and according to the measured doxorubicin standard curve, the concentration of doxorubicin in the DMSO solution (i.e., the drug concentration contained in the drug-loaded microspheres) is obtained. Repeat the experiment three times, and each sample is measured three times.

[0124] The drug loading was calculated according to the following formula:

[0125] Drug loading = (drug concentration in drug-loaded microspheres / drug concentration in drug-loaded microspheres) × 100%

[0126] The drug loading results of the drug-loaded microspheres prepared in Examples 13 and 15 are shown in Fig.19 shown.

[0127] Example 17. Evaluation of cytotoxicity of RSs microspheres (UV irradiation for 2 min in Example 2), RGSs microspheres (Example 7), RSs-DOX drug-loaded microspheres (Example 12), and RGSs-DOX drug-loaded microspheres (Example 14)

[0128] The evaluation method is as follows:

[0129] (1) Cell culture: Human liver tumor cells HepG2 were added to a medium containing 10% fetal bovine serum (FBS), 1 U / mL penicillin, and 1×10 -3 The cells were cultured in high glucose medium (DMEM) containing 100 mg / mL streptomycin.

[0130] (2) Quantitative evaluation of the cytotoxicity of RSs microspheres, RGSs microspheres, RSs-DOX microspheres, and RGSs-DOX microspheres: Human liver tumor HepG2 cells in the logarithmic growth phase were cultured at 1×10 4 The cells were inoculated in a 48-well plate at a ratio of 100 cells / well and placed in an incubator for 24 h. After the cells adhered to the wall and grew, they were added with 10% (FBS), 1 U / mL penicillin, 1×10 -3 The control group was added with an equal amount of DMEM medium containing 10% (FBS), 1U / mL penicillin, 1×10 -3mg / mL streptomycin in DMEM medium, with 3 parallel groups set up for each group. After culturing for 1 and 3 days respectively, the cells were washed with PBS, and then CCK-8 reagent was added for 1 hour. The absorbance value of each well was detected by an ELISA reader at the maximum absorption wavelength of 450nm. The absorbance value reflects the number of living cells. The larger the absorbance value, the more living cells there are. See the results for details. Fig. 20 As shown, it can be seen that the cell activity of the RSs microspheres and RGSs microspheres provided by the present invention after 1 day of culture is not different from that of the control group, and the cell activity after 3 days of culture is better than that of the control group.

[0131] (3) Qualitative evaluation of the cytotoxicity of RSs microspheres, RGSs microspheres, RSs-DOX microspheres, and RGSs-DOX microspheres: Human liver tumor HepG2 cells in the logarithmic growth phase were cultured at 1×10 5 The cells were inoculated in a 12-well plate at a ratio of 100 cells / well and placed in an incubator for 24 h. After the cells adhered to the wall and grew, they were added with 10% (FBS), 1 U / mL penicillin, 1×10 -3 The control group was added with an equal amount of DMEM medium containing 10% (FBS), 1U / mL penicillin, 1×10 -3 mg / mL streptomycin in DMEM medium, with 3 parallel groups set up for each group. After 3 days of continuous culture, the cells were washed with PBS, and then calcein AM and propidium iodide (PI) solution were added for staining, and then the staining was observed under an inverted fluorescence microscope, see Fig.21 shown.

[0132] from Fig. 20 , Fig.21 From the test results, it can be seen that the RSs microspheres and RGSs microspheres provided by the present invention have low cytotoxicity and can be used in many fields such as cell co-culture, drug delivery, tissue engineering, tracing, biomedical imaging, regenerative medicine, and skin repair. The cytotoxicity of the RSs-DOX drug-loaded microspheres and RGSs-DOX drug-loaded microspheres provided by the present invention meets the toxicity requirements for drug-encapsulated microspheres.

[0133] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing silk fibroin microspheres, characterized in that: The following steps are involved: Step 1, preparing a silk fibroin solution: adding silk fibroin, poloxamer and a cross-linking agent into a solvent and stirring and dissolving to prepare a silk fibroin solution, wherein the solvent comprises deionized water and PBS solution, the cross-linking agent comprises a photo cross-linking agent and a chemical cross-linking agent, the photo cross-linking agent comprises riboflavin and sodium persulfate, and the chemical cross-linking agent comprises genipin; Step 2, preparing silk fibroin microspheres: using silk fibroin solution as the aqueous phase, using a droplet microfluidic chip to utilize the oil phase to shear the aqueous phase to obtain a collection liquid containing silk fibroin microdroplets, and then inducing molding to obtain silk fibroin microspheres.

2. The preparation method according to claim 1, characterized in that: The silk fibroin solution has a silk fibroin concentration of 2-5 wt %, a poloxamer concentration of 0.5 wt %, a riboflavin concentration of 0.2 wt %, a sodium persulfate concentration of 0.1 wt %, and a genipin concentration of 0.5 wt %.

3. The preparation method according to claim 1, characterized in that: The concentration of the PBS solution is 1 mM, and the pH is 7.

4. The preparation method according to claim 1, characterized in that: The flow rate of the oil phase is 2000-6000 μL / h, and the flow rate of the silk fibroin water phase is 100-500 μL / h.

5. The preparation method according to claim 1, characterized in that: The induction molding method comprises ultraviolet irradiation and light-proof constant temperature standing.

6. The preparation method according to claim 5, characterized in that: The ultraviolet irradiation time is 1-3 minutes.

7. The preparation method according to claim 5, characterized in that: The light-proof constant temperature standing time is 1-3 hours, and the constant temperature is 35°C.

8. A silk fibroin microsphere, characterized in that: The preparation method is described in any one of claims 1 to 7.

9. Use of the silk fibroin microspheres according to claim 8 in drug encapsulation and fluorescence color development.

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