A kind of silk fibroin microcapsule and its preparation method and application

Abstract

The present invention relates to the field of biomaterial technology, and discloses a silk microcapsule and its preparation method and application. The preparation method of the silk microcapsule of the present invention is to use a silk protein solution as an intermediate phase, an oily solution as an inner oil phase, use a droplet microfluidic chip to first shear the inner oil phase with an intermediate phase to obtain an intermediate phase containing inner oil phase droplets, and then use an outer oil phase to shear the intermediate phase containing inner oil phase droplets to obtain a collection liquid containing O / W / O type droplets; or use a polymer aqueous solution as the inner water phase, use an outer oil phase to simultaneously shear the inner water phase and the intermediate phase with the help of a droplet microfluidic chip to obtain a collection liquid containing double-phase droplets; and induce molding of the collection liquid to obtain a silk microcapsule. The particle size and capsule wall thickness of the silk microcapsule prepared by the present invention are adjustable, the number of encapsulation liquid cores is controllable, both water-soluble substances and oil-soluble substances can be encapsulated, and it has special spontaneous fluorescence, and can be used in multiple 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 microcapsule and a preparation method and application thereof. Background Art

[0002] Silk fibroin (SF) 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. As a burn wound covering film, anticoagulant material, bone repair scaffold and other materials, silk fibroin 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, mainly focusing on the following types: silk fibroin microspheres, silk fibroin nanoparticles, silk fibroin microcapsules, and silk fibroin microgels.

[0003] Silk microcapsules refer to hollow vesicles constructed with silk fibroin as the capsule wall material. Unlike other silk fibroin microcarriers, silk microcapsules have a higher drug loading rate, and the vesicle-like structure is also conducive to cell uptake. Layer-by-layer self-assembly technology is currently the most commonly used method for preparing silk microcapsules. Select a suitable template, utilize the self-assembly characteristics of silk fibroin, stack layer by layer on the surface of the template, and finally remove the template to obtain a hollow silk microcapsule. However, when loading drugs into silk microcapsules prepared by layer-by-layer self-assembly, small molecule drugs can enter the microcapsule by diffusion, while macromolecular drugs can only be loaded on the surface of the microcapsule, which greatly affects the utilization of the hollow structure of the microcapsule. Although some studies have first loaded the drug on the template surface, and then stacked SF layer by layer, the drug can also be loaded into the capsule after removing the template, but this method still has a low utilization rate for the hollow structure of the microcapsule.

[0004] Droplet microfluidics is a technology that studies the generation, manipulation and application of droplets at a microscale. It can use immiscible phases to generate monodisperse droplets with controllable size. Compared with traditional chemical analysis platforms, it has the advantages of large flux and good monodispersity. Due to the slow formation speed of silk fibroin hydrogel and the high viscosity of silk fibroin aqueous solution, there are few reports on the method of preparing silk fibroin microcapsules using droplet microfluidics. Chinese patent CN113813893B discloses a method for preparing silk fibroin drug delivery capsules based on microfluidic chips. This method can achieve the regulation of drug loading, capsule wall thickness, etc., but the system structure of the capsule prepared by this method is an oil-in-water form, and it is necessary to add organic solvents such as methanol and ethanol to induce silk fibroin crystallization. Moreover, its encapsulated material can only be an oil-soluble substance, and the encapsulated material is single, which limits the application of silk fibroin microcapsules to a certain extent. Summary of the invention

[0005] In view of this, the present invention aims to provide a silk fibroin microcapsule and a preparation method thereof. The silk fibroin microcapsule is prepared by droplet microfluidics to solve the problem of low utilization rate of hollow structure in the existing layer-by-layer self-assembly preparation of silk fibroin microcapsules. The preparation method of droplet microfluidics provided by the present invention can accelerate the molding speed of silk fibroin microcapsules. The particle size and capsule wall thickness of the prepared silk fibroin microcapsules are adjustable, the number of encapsulation liquid cores is controllable, and both water-soluble substances and oil-soluble substances can be encapsulated. In addition, the microcapsule provided by the present invention has spontaneous fluorescence and adjustable fluorescence, which can broaden the application range of silk fibroin microcapsules.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions.

[0007] In a first aspect, the present invention provides a method for preparing a silk fibroin microcapsule, comprising the following steps:

[0008] 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;

[0009] Step 2, preparing an oily solution and an aqueous solution: using an oily solvent as the oily solution and a polymer aqueous solution as the aqueous solution, wherein the polymer aqueous solution and the silk fibroin solution are insoluble in each other;

[0010] Step 3, preparing an outer oil phase: adding a surfactant to an oily solvent and mixing them uniformly to prepare an outer oil phase;

[0011] Step 4, preparing silk microcapsules: using a silk protein solution as an intermediate phase and an oily solution as an inner oil phase, using a droplet microfluidic chip to first shear the inner oil phase with an intermediate phase to obtain an intermediate phase containing inner oil phase droplets, and then using an outer oil phase to shear the intermediate phase containing inner oil phase droplets to obtain a collection liquid containing O / W / O type droplets; or using a silk protein solution as an intermediate phase and an aqueous solution as an inner water phase, using a droplet microfluidic chip to shear the intermediate phase and the inner water phase with an outer oil phase at the same time to obtain a collection liquid containing double-phase water droplets; inducing molding of the collection liquid to obtain a silk microcapsule;

[0012] The oily solvent is selected from one or more of soybean oil, dimethyl silicone oil, and fluorocarbon oil; the surfactant is selected from one or more of PGPR, Span-80, Tween-20, PFPE-PEG, KF-6017, EM-90, and Triton X-100.

[0013] 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, adds a photocrosslinker and / or a chemical crosslinker when preparing a silk fibroin solution, uses a riboflavin and sodium persulfate photocuring system to photocrosslink the silk fibroin, and uses genipin to react with free amino groups in the silk fibroin to perform chemical crosslinking, thereby accelerating the formation of silk fibroin microcapsules. At the same time, riboflavin and sodium persulfate-mediated tyrosine free radicals in the silk fibroin photocrosslink to form a layer of hydrogel network, and genipin and the amino groups of the silk fibroin undergo chemical crosslinking to form another layer of hydrogel network, thereby obtaining a silk fibroin hydrogel microcapsule with an interpenetrating network structure, thereby enhancing the mechanical properties of the silk fibroin microcapsules.

[0014] 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 microcapsules have fluorescence, but the microcapsules 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 microcapsules prepared by the present invention have special autofluorescence.

[0015] The present invention adopts an oil / water / oil emulsion system or a water / water / oil emulsion system to prepare silk fibroin microcapsules, and can encapsulate both water-soluble substances and oil-soluble substances.

[0016] Furthermore, in some embodiments of the present invention, the polymer aqueous solution is a sodium carboxymethyl cellulose aqueous solution.

[0017] Furthermore, in some embodiments of the present invention, the external oil phase is soybean oil containing 10 wt% PGPR.

[0018] Furthermore, in some embodiments of the present invention, the concentration of the PBS solution is 1 mM, and the pH is 7.

[0019] Furthermore, in some embodiments of the present invention, the concentration of silk fibroin in the silk fibroin solution is 3wt%, 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%.

[0020] Furthermore, the induction molding method includes ultraviolet irradiation and light-proof constant temperature standing.

[0021] Furthermore, the ultraviolet irradiation time is 1-3 minutes.

[0022] Furthermore, the light-proof constant temperature standing time is 1-3 hours, and the constant temperature is 35°C.

[0023] In the second aspect, the present invention provides a silk fibroin microcapsule prepared by the above-mentioned preparation method, which has good biocompatibility, adjustable capsule wall thickness, controllable number of liquid cores, can encapsulate substances with different solubility properties, and has special spontaneous fluorescence.

[0024] In a third aspect, the present invention provides the application of the above-mentioned silk microcapsules in drug encapsulation and fluorescence color development. The present invention can achieve drug encapsulation by dissolving the drug into an oily solution, an aqueous solution or a silk protein solution. The drug-loaded microcapsules prepared by dissolving the drug into an oily solution or an aqueous solution make full use of the hollow structure of the microcapsules and improve their utilization rate. The silk microcapsules provided by the present invention have special autofluorescence and can be used for fluorescence color development, and are expected to be used in many fields such as labeling, tracing, detection, standards, biomedical imaging, tissue engineering, regenerative medicine, and skin repair.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) The preparation method provided by the present invention uses droplet microfluidics combined with cross-linking technology to achieve rapid preparation of silk microcapsules, while improving the capsule wall strength of the microcapsules; the preparation of silk microcapsules using droplet microfluidics can fully utilize the hollow structure of the microcapsules, and can conveniently control the particle size, capsule wall thickness and number of encapsulated liquid cores of the microcapsules;

[0027] (2) The preparation method provided by the present invention adopts an oil / water / oil (O / W / O) emulsion system or a water / water / oil (W1 / W2 / O) emulsion system to prepare silk fibroin microcapsules, which can encapsulate both water-soluble substances and oil-soluble substances;

[0028] (3) The silk microcapsules prepared by the present invention have special spontaneous fluorescence and good fluorescence stability, and can emit fluorescence without coupling with fluorescent substances. They are a new type of fluorescent microcapsules that can overcome the shortcomings of fluorescence leakage, photobleaching and biological toxicity existing in coupling with fluorescent substances;

[0029] (4) The silk microcapsules 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 microcapsules using an oil / water / oil emulsion system using a droplet microfluidic chip in Example 1 of the present invention;

[0031] Figure 2 This is a laser confocal microscopic image of the silk microcapsule prepared in Example 1 of the present invention;

[0032] Figure 3 Optical microscopic images of silk microcapsules encapsulating different numbers of inner oil phase droplets prepared by regulating different flow rates of the outer oil phase in Example 2 of the present invention;

[0033] Figure 4 This is an image of preparing O / W / O type droplets by regulating different flow rates of the internal oil phase in Example 3 of the present invention;

[0034] Figure 5 Optical microscopic images of silk fibroin microcapsules with different capsule wall thicknesses prepared by regulating different flow rates of the internal oil phase in Example 3 of the present invention;

[0035] Figure 6 Schematic diagram of preparing silk microcapsules using a water / water / oil emulsion system using a droplet microfluidic chip in Example 4 of the present invention;

[0036] Figure 7 The morphology characterization images of the silk fibroin microcapsules prepared in Example 4 of the present invention (a is an optical microscopy image, b is a scanning electron microscopy image);

[0037] Figure 8 Laser confocal microscopy images of the silk microcapsules prepared in Examples 4 and 5 of the present invention. DETAILED DESCRIPTION

[0038] The present invention provides a method for preparing a silk fibroin microcapsule, which comprises the following steps:

[0039] 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;

[0040] Step 2, preparing an oily solution and an aqueous solution: using an oily solvent as the oily solution and a polymer aqueous solution as the aqueous solution, wherein the polymer aqueous solution and the silk fibroin solution are mutually insoluble, and in the present invention, the polymer aqueous solution is preferably a sodium carboxymethyl cellulose aqueous solution; the oily solvent is selected from one or more of soybean oil, dimethyl silicone oil, and fluorocarbon oil;

[0041] Step 3, preparing an outer oil phase: adding a surfactant to an oily solvent and mixing them uniformly to obtain an outer oil phase; the surfactant is selected from one or more of PGPR, Span-80, Tween-20, PFPE-PEG, KF-6017, EM-90 and Triton X-100;

[0042] Step 4, preparing silk microcapsules: using silk fibroin solution as the intermediate phase and oily solution as the inner oil phase, using a droplet microfluidic chip to first shear the inner oil phase with the intermediate phase to obtain an intermediate phase containing inner oil phase droplets, and then using an outer oil phase to shear the intermediate phase containing inner oil phase droplets to obtain a collected liquid containing O / W / O type droplets; or using silk fibroin solution as the intermediate phase and aqueous solution as the inner water phase, using a droplet microfluidic chip to shear the intermediate phase and the inner water phase with the outer oil phase at the same time to obtain a collected liquid containing double-aqueous phase droplets; inducing molding of the collected liquid to obtain a silk fibroin microcapsule.

[0043] 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 and boil 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.

[0044] 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 solution 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 solution 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.

[0045] 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.

[0046] Example 1: Preparation of silk microcapsules using an O / W / O emulsion system

[0047] The preparation steps are as follows:

[0048] (1) Preparation of silk fibroin solution: riboflavin, sodium persulfate, 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 obtain 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%.

[0049] (2) Preparation of external oil phase: PGPR was added to soybean oil to prepare the external oil phase, wherein the mass concentration of PGPR was 10 wt%.

[0050] (3) Preparation of silk microcapsules: Using silk fibroin solution as the intermediate phase and dimethyl silicone oil as the inner oil phase, see Figure 1 , the inner oil phase is injected from inlet 1 of the droplet microfluidic chip, the middle phase is injected from inlet 2, and the outer oil phase is injected from inlet 3. The middle phase first shears the inner oil phase to obtain the middle phase containing the inner oil phase droplets, and then the outer oil phase shears the middle phase containing the inner oil phase droplets to obtain the collection liquid containing O / W / O type droplets. Among them, the flow rate of the inner oil phase is 350μL / h, the flow rate of the middle phase is 500μL / h, and the flow rate of the outer oil phase is 2000μL / h. After the collection liquid is placed in an ultraviolet irradiation environment for 2 minutes, it is kept at a constant temperature (35℃) and protected from light for 3 hours. The droplets are formed into microcapsules. After replacing the oil phase solution around the microcapsules with n-hexane, it is washed with PBS solution, collected and freeze-dried.

[0051] After testing, the outer diameter of the silk fibroin microcapsule prepared in this embodiment is 234.8±2.3 μm, the monodispersity coefficient (CV value) is 1.0%, and the inner diameter is 160.0±2.3 μm, the monodispersity coefficient (CV value) is 1.4%.

[0052] The luminescence performance of the microcapsules prepared in this example was observed using a laser confocal microscope. Figure 2 It can be found that the microcapsules prepared in this example show obvious fluorescence effect under 405nm, 488nm, and 561nm laser excitation, and weak fluorescence under 640nm laser excitation, and have good stability.

[0053] Example 2: Effect of different flow rates of the external oil phase on the preparation of silk microcapsules

[0054] The preparation method is the same as in Example 1, except that the flow rate of the external oil phase is different, as shown in Table 1.

[0055] Table 1. Mobile phase flow rate for preparing silk microcapsules by droplet microfluidics

[0056] Serial number Internal oil phase flow rate (μL / h) Interphase flow rate (μL / h) External oil phase flow rate (μL / h) Example 2-1 350 400 2500 Example 2-2 350 400 1500 Example 2-3 350 400 900 Embodiment 2-4 350 400 500

[0057] The appearance of the silk microcapsules prepared in this example was observed by optical microscopy. Figure 3 As shown, it can be seen that as the flow rate of the external oil phase continues to decrease, the number of liquid cores gradually increases, indicating that the multi-core structure can be controlled by adjusting the flow rate of the external oil phase.

[0058] Example 3: Effect of different flow rates of the internal oil phase on the preparation of silk microcapsules

[0059] The preparation method is the same as in Example 1, except that the inner oil phase is soybean oil and the flow rate of the inner oil phase is different. For details, see Tables 2 and Figure 4 shown.

[0060] Table 2. Mobile phase flow rate for preparing silk microcapsules by droplet microfluidics

[0061]

[0062]

[0063] The appearance of the silk microcapsules prepared in this example was observed by optical microscopy. Figure 5 As shown, it can be seen that as the flow rate of the inner oil phase continues to increase, the thickness of the silk fibroin microcapsule wall continues to decrease, which indicates that the thickness of the microcapsule wall can be adjusted by adjusting the flow rate of the inner oil phase.

[0064] Example 4: Preparation of silk microcapsules using W1 / W2 / O emulsion system

[0065] The preparation steps are as follows:

[0066] (1) Preparation of silk fibroin solution: riboflavin, sodium persulfate, 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 obtain 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%.

[0067] (2) Preparation of external oil phase: PGPR was added to soybean oil to prepare the external oil phase, wherein the mass concentration of PGPR was 10 wt%.

[0068] (3) Preparation of an aqueous solution: Sodium carboxymethyl cellulose was dissolved in deionized water to prepare a 2 wt % sodium carboxymethyl cellulose solution as an aqueous solution.

[0069] (4) Preparation of silk microcapsules: Using silk fibroin solution (W2 solution) and aqueous solution (W1 solution) as the inner aqueous phase and 10 wt% PGPR soybean oil as the outer oil phase, the microcapsules were prepared using a droplet microfluidic chip. Figure 6 , the external oil phase, W2 solution and W1 solution are introduced into Figure 6 In the glass capillary microfluidic device shown, the flow rate of W1 solution is 50 μL / h, and it enters from inlet 1 to reach the first-stage shearing port; the speed of W2 solution is 300 μL / h, and it enters from inlet 2, and the two-phase solution forms a laminar flow in the channel; the speed of the external oil phase is 2000 μL / h, and it enters from inlet 3, and the two-phase solution is sheared simultaneously to obtain a collection liquid containing two-phase aqueous droplets. After the collection liquid is placed in an ultraviolet irradiation environment for 2 minutes, it is kept at a constant temperature (35°C) and kept away from light for 3 hours, and the droplets are formed into microcapsules. After replacing the oil phase solution around the microcapsules with n-hexane, it is washed with PBS solution, collected and freeze-dried.

[0070] The internal and external appearance of the microcapsules prepared in this example were observed using an optical microscope and a scanning electron microscope. Figure 7 As shown, it can be seen that the capsule wall of the microcapsule prepared in this embodiment has more pores and an obvious hollow structure exists inside.

[0071] According to the test, the particle size of the silk fibroin microcapsule prepared in this embodiment is 174.8±5.1 μm, and the monodispersity coefficient (CV value) is 2.8%.

[0072] Example 5: Preparation of silk microcapsules using W1 / W2 / O emulsion system

[0073] The preparation method is the same as that of Example 4, except that the collected solution is kept at a constant temperature (35°C) and protected from light for 3 hours, and then irradiated with ultraviolet light for 2 minutes. The particle size of the silk microcapsules prepared in this example is 192.4±5.1μm, and the monodispersity coefficient (CV value) is 2.7%.

[0074] The luminescence properties of the microcapsules prepared in Examples 4 and 5 were observed using a laser confocal microscope. Figure 8 It can be found that the microcapsules prepared in Example 4 exhibited obvious fluorescence effects under 405nm, 488nm, and 561nm laser excitation, and weak fluorescence under 640nm laser excitation; the microcapsules prepared in Example 5 exhibited obvious fluorescence effects under 405nm, 488nm, and 561nm laser excitation, and weak fluorescence under 640nm laser excitation. Compared with the microcapsules prepared in Example 4, the fluorescence intensity of the microcapsules prepared in Example 5 was enhanced, indicating that the fluorescence of the microspheres can be controlled by regulating the order of genipin cross-linking and photo-cross-linking.

[0075] 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 a silk fibroin microcapsule, 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 an oily solution and an aqueous solution: using an oily solvent as the oily solution and a polymer aqueous solution as the aqueous solution, wherein the polymer aqueous solution and the silk fibroin solution are insoluble in each other; Step 3, preparing an outer oil phase: adding a surfactant to an oily solvent and mixing them uniformly to prepare an outer oil phase; Step 4, preparing silk microcapsules: using a silk protein solution as an intermediate phase and an oily solution as an inner oil phase, using a droplet microfluidic chip to first shear the inner oil phase with an intermediate phase to obtain an intermediate phase containing inner oil phase droplets, and then using an outer oil phase to shear the intermediate phase containing inner oil phase droplets to obtain a collection liquid containing O / W / O type droplets; or using a silk protein solution as an intermediate phase and an aqueous solution as an inner water phase, using a droplet microfluidic chip to shear the intermediate phase and the inner water phase with an outer oil phase at the same time to obtain a collection liquid containing double-phase water droplets; inducing molding of the collection liquid to obtain a silk microcapsule; The oily solvent is selected from one or more of soybean oil, dimethyl silicone oil, and fluorocarbon oil; the surfactant is selected from one or more of PGPR, Span-80, Tween-20, PFPE-PEG, KF-6017, EM-90, and Triton X-100.

2. The preparation method according to claim 1, characterized in that: The polymer aqueous solution is a sodium carboxymethyl cellulose aqueous solution.

3. The preparation method according to claim 1, characterized in that: The external oil phase is soybean oil containing 10 wt% PGPR.

4. The preparation method according to claim 1, characterized in that: The silk fibroin solution has a silk fibroin concentration of 3 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 %.

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

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

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

8. The preparation method according to claim 6, characterized in that: The light-proof constant temperature standing time is 1-3 hours, and the constant temperature is 35°C.

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

10. Use of the silk microcapsule according to claim 9 in drug encapsulation and fluorescence color development.

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