Injectable silk fibroin microspheres as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biomedical materials, and relates to injectable silk fibroin microspheres as well as a preparation method and application thereof. The preparation method comprises the following steps: firstly, forming dispersed phase liquid drops with the particle size of 20-75 microns by utilizing a microfluidic technology; enabling the dispersed phase liquid drops to be in contact with the reaction phase, and carrying out sufficient cross-linking reaction to obtain cross-linked microspheres; and finally carrying out centrifugation, purification and vacuum drying on the cross-linked microspheres to obtain injectable silk fibroin microspheres with a particle size of 20-75 [mu] m and a pore size of 2-50 nm. The silk fibroin microspheres are uniform in size, narrow in diameter distribution, smooth and regular in surface, spherical, small in inflammatory response after entering the body, and not easy to be swallowed by macrophages; the surface aperture is small, and the drug sustained release function is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials and relates to an injectable silk fibroin microsphere and a preparation method and application thereof. Background Art

[0002] As the largest tissue in the human body, the skin plays an important role in protecting the human body. It is a natural biological barrier that can isolate factors such as viruses, microorganisms, and temperature changes. Usually, lighter or shallower skin wounds can heal themselves. However, larger and deeper skin wounds are not easy to heal. In addition, damaged skin is difficult to prevent harmful bacteria from invading the tissue, leading to wound infection and severe tissue damage.

[0003] In recent years, injectable hydrogel microspheres have broad application prospects as wound dressings with multifunctional properties such as biodegradability, biocompatibility, responsiveness, and injectability. The porous structure of injectable hydrogel microspheres has multiple functions, such as enhancing the mechanical strength, stability, and ductility of the material, regulating drug release, providing more anchor points for cross-linking with other materials, and solving the shortcomings of hydrogels such as brittleness. Antibacterial injectable hydrogel microspheres play an irreplaceable role in wound healing, especially in infected wounds. Injectable silk fibroin microspheres have a large surface area and can store ultra-high antibacterial drugs, so they have attracted the attention of researchers.

[0004] In the research field of injectable hydrogel microspheres, the preparation materials of microspheres include chitosan, alginate, silk, gelatin, polyacrylic acid, or two different materials such as chitosan and silk protein, chitosan and gelatin for composite preparation, and the preparation techniques include emulsification cross-linking method, membrane emulsification technology, and electrostatic spraying technology. However, the size of microspheres formed by different preparation methods is limited, and it is impossible to prepare microspheres with a narrow size range. For example, the size of microspheres formed by emulsification and cross-linking methods is mostly around 100μm; the size of microspheres formed by electrostatic spraying is difficult to control, and the preparation process often requires high temperature conditions, which may cause the inactivation of the loaded active substances.

[0005] Chinese invention patent application CN114306737A (publication date: April 12, 2022) discloses a silk fibroin-based porous microsphere and its preparation method. The prepared microspheres have a diameter of 25 to 500 μm, and the size range of the microspheres is widely distributed, resulting in uncontrollable effects after the microspheres are injected into the implantation site, and larger microspheres will cause a more severe inflammatory response after being implanted subcutaneously. In addition, smaller microspheres are easily directly engulfed by phagocytes after being implanted subcutaneously. Summary of the invention

[0006] The present invention provides an injectable silk fibroin microsphere and a preparation method and application thereof, aiming to solve the problem that the particle size distribution range of silk fibroin microspheres prepared by the prior art is wide, and the larger microspheres will cause a more severe inflammatory reaction after being implanted subcutaneously. In addition, the smaller microspheres are easily directly phagocytosed by phagocytes after being implanted subcutaneously.

[0007] In order to achieve the above objectives, the present invention provides a method for preparing injectable silk fibroin microspheres in a first aspect, comprising the following steps:

[0008] Providing a microfluidic device and a dispersed phase, a continuous phase and a reaction phase for the microfluidic device;

[0009] The dispersed phase is sheared into dispersed phase droplets with a particle size of 20 to 75 μm by using the continuous phase;

[0010] The dispersed phase droplets are brought into contact with the reactive phase to undergo a sufficient cross-linking reaction to obtain cross-linked microspheres;

[0011] The cross-linked microspheres are centrifuged, purified, and vacuum dried to obtain injectable silk fibroin microspheres;

[0012] in:

[0013] The dispersed phase is a silk fibroin solution;

[0014] The silk fibroin solution contains antibacterial drugs;

[0015] The continuous phase is a mixed solution of emulsifier liquid paraffin;

[0016] The reaction phase is a cross-linking agent liquid paraffin mixed solution.

[0017] In some embodiments of the present invention, the vacuum drying temperature is 20 to 40° C. and the time is 12 to 72 hours.

[0018] In some embodiments of the present invention, the emulsifier is span-80.

[0019] In some embodiments of the present invention, the step of shearing the dispersed phase into dispersed phase droplets with a particle size of 20 to 75 μm using the continuous phase comprises: controlling the flow rate of the dispersed phase to 0.5 to 1.5 mL / h and controlling the flow rate of the continuous phase to 40 to 180 mL / h.

[0020] In some embodiments of the present invention, the mass concentration of silk fibroin in the silk fibroin solution is 0.5 wt % to 5.0 wt %.

[0021] The second aspect of the present invention provides injectable silk fibroin microspheres prepared by the above-mentioned method for preparing injectable silk fibroin microspheres.

[0022] The third aspect of the present invention provides an injectable hydrogel, which includes a first hydrogel and injectable silk fibroin microspheres dispersed in the first hydrogel; the injectable silk fibroin microspheres include a silk fibroin cross-linked gel and an antibacterial drug uniformly distributed in the silk fibroin cross-linked gel, and the particle size of the injectable silk fibroin microspheres is distributed between 20μm and 75μm, and the pore size is distributed between 2nm and 50nm.

[0023] In some embodiments of the present invention, the antibacterial drug is a polyphenol antibacterial drug; the polyphenol antibacterial drug includes one or more of tea polyphenols, rhubarb polyphenols, pomegranate peel polyphenols, date palm pollen polyphenols, burdock polyphenols, qinguan leaf polyphenols, and blueberry leaf polyphenols.

[0024] In some embodiments of the present invention, the antibacterial drug accounts for 1 wt% to 20 wt% of the injectable silk fibroin microspheres; and / or,

[0025] The weight ratio of the injectable silk fibroin microspheres to the first hydrogel is 1:1.5-9.

[0026] In some embodiments of the present invention, the colloid in the first hydrogel is composed of polyethylene glycol and silk fibroin in a weight ratio of 1:2 to 8, and the concentration of the silk fibroin is 1wt% to 6wt%.

[0027] In some embodiments of the present invention, the injectable silk fibroin microspheres further include other therapeutic components uniformly distributed in the silk fibroin cross-linked gel, and the other therapeutic components include one or more of cells, growth factors, and analgesics. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 : The microfluidic device used in the present invention.

[0029] Figure 2 : In vitro release profiles of injectable silk fibroin microspheres. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] For simplicity, the present invention only explicitly discloses some numerical ranges. However, any lower limit can be combined with any upper limit to form an unclearly recorded range; and any lower limit can be combined with other lower limits to form an unclearly recorded range. Similarly, any upper limit can be combined with any other upper limit to form an unclearly recorded range. In addition, although not clearly recorded, each point or single value between the range endpoints is included in the range. Thus, each point or single value can be combined with any other point or single value as its own lower limit or upper limit or with other lower limits or upper limits to form an unclearly recorded range.

[0032] It should be noted that, in the description of the present invention, unless otherwise specified, "above" and "below" are inclusive numbers, and the meaning of "multiple" in "one or more" is two or more. Relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply that there is any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that the process, method, article or equipment including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or equipment. In the absence of more restrictions, the elements defined by the sentence "including one..." do not exclude the existence of other identical elements in the process, method, article or equipment including the elements.

[0033] In the description of the present invention, the description with reference to the terms "any embodiment / method", "one embodiment / method", "some embodiments / methods", "example", "specific example" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment / method or example are included in at least one embodiment / method or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment / method or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments / methods or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments / methods or examples described in this specification and the features of the different embodiments / methods or examples, without contradiction.

[0034] In the description of the present invention, "silk fibroin solution" refers to an aqueous solution in which the solute is mainly silk fibroin, but the solute includes not only silk fibroin, but also a small amount of antibacterial drugs and / or other therapeutic ingredients. "Emulsifier liquid paraffin mixed solution" refers to a solution formed by a mixture of emulsifier and liquid paraffin, without containing other solvents; "cross-linking agent liquid paraffin mixed solution" refers to a solution formed by a mixture of cross-linking agent and liquid paraffin, without containing other solvents. "Polyethylene glycol solution" refers to a solution formed by a mixture of polyethylene glycol and water, without containing other solvents.

[0035] The above summary of the invention of the present invention is not intended to describe each disclosed embodiment or each implementation in the present invention. The following description more specifically illustrates exemplary embodiments. These embodiments can be used in various combinations. In each example, the enumeration is only used as a representative group and should not be interpreted as exhaustive.

[0036] Preparation method:

[0037] As described in the background art, the diameter of silk fibroin-based porous microspheres prepared by the prior art is 25 to 500 μm, and the size range is widely distributed, resulting in uncontrollable effects after the microspheres are injected into the implantation site, and larger microspheres will cause more severe inflammatory reactions after being implanted subcutaneously.

[0038] In addition, the inventors found that the microspheres prepared by freeze-drying, crushing, screening, spray drying, etc. have irregular and angular surfaces and are too large in size. After being injected into the human body, the inflammatory reaction is more serious, while microspheres that are too small in size are easily adsorbed and phagocytosed by phagocytes.

[0039] In view of the above problems, the present invention provides a method for preparing injectable silk fibroin microspheres, comprising the following steps:

[0040] Providing a microfluidic device and a dispersed phase, a continuous phase and a reaction phase for the microfluidic device;

[0041] The dispersed phase is sheared into dispersed phase droplets with a particle size of 20 to 75 μm by using the continuous phase;

[0042] The dispersed phase droplets are brought into contact with the reactive phase to undergo a sufficient cross-linking reaction to obtain cross-linked microspheres;

[0043] The cross-linked microspheres are centrifuged, purified, and vacuum dried to obtain injectable silk fibroin microspheres;

[0044] in:

[0045] The dispersed phase is a silk fibroin solution;

[0046] The silk fibroin solution contains antibacterial drugs;

[0047] The continuous phase is a mixed solution of emulsifier liquid paraffin;

[0048] The reaction phase is a cross-linking agent liquid paraffin mixed solution.

[0049] The present invention uses a microfluidic device to accurately control the size of the obtained injectable silk fibroin microspheres, and can easily carry out drug loading and enzyme fixation, etc. The preparation method is simple, and the obtained injectable silk fibroin microspheres are uniform in size and have a narrow particle size distribution range.

[0050] The preparation method provided by the present invention controls the particle size of dispersed phase droplets to be between 20 and 75 μm in the microfluidic stage, and the particle size of the cross-linked microspheres obtained after the cross-linking reaction does not change and remains between 20 and 75 μm; the present invention then uses a vacuum drying method to treat the purified product. The vacuum drying method can effectively prevent the surface collapse of the microspheres on the one hand, thereby obtaining spherical particles with a particle size between 20 and 75 μm and a smooth and regular surface, namely, injectable silk fibroin microspheres, to avoid being adsorbed and engulfed by phagocytes; on the other hand, vacuum drying forms nanoscale pores on the surface of the injectable silk fibroin microspheres, providing channels for the injectable silk fibroin microspheres to release drugs.

[0051] In some embodiments of the present invention, the vacuum drying time is 12 to 72 hours, and the temperature is 20 to 40° C. The temperature and time of vacuum drying are controlled within the above range. On the one hand, it can ensure that the injectable silk fibroin microspheres can be fully dried, and avoid excessive drying temperature from having a certain impact on the injectable silk fibroin microspheres; on the other hand, a normal temperature vacuum drying device with low equipment requirements can be used, which is conducive to the mass production of injectable silk fibroin microspheres; on the other hand, the vacuum drying temperature is selected at 20 to 40° C., which is close to the transportation and storage temperature of the injectable silk fibroin microspheres, and can reduce the impact of temperature changes on the injectable silk fibroin microspheres.

[0052] In some embodiments of the present invention, the method of using a continuous phase to shear the dispersed phase into dispersed phase droplets with a particle size of 20 to 75 μm includes: the flow rate of the dispersed phase is 0.5 to 1.5 mL / h, preferably 1.0 mL / h; the flow rate of the continuous phase is 40 to 180 mL / h, preferably 100 mL / h. The present invention controls the size of the dispersed phase droplets between 20 and 75 μm by controlling the flow rate of the dispersed phase and the flow rate of the continuous phase, so that the particle size of the injectable silk fibroin microspheres after vacuum drying can be distributed between 20 and 75 μm, and the pore size is distributed between 2 nm and 50 nm. Furthermore, the flow rate ratio of the continuous phase and the dispersed phase is controlled to be 100:1.

[0053] In some embodiments of the present invention, the concentration of silk fibroin in the silk fibroin solution is 0.5wt% to 5.0wt%. The concentration of silk fibroin is controlled within the above range, which is conducive to the dispersion of the dispersed phase in the continuous phase to form dispersed phase droplets. Too high a concentration of the silk fibroin solution will cause the solution viscosity to be too large, which is not conducive to the formation of dispersed phase droplets. Optionally, the concentration of silk fibroin in the silk fibroin solution is 0.5wt%, 1.0wt%, 1.5wt%, 2.0wt%, 2.5wt%, 3.0wt%, 3.5wt%, 4.0wt%, 4.5wt%, 5.0wt%, or a concentration range consisting of any two of the points as endpoints.

[0054] In some embodiments of the present invention, the concentration of the emulsifier in the continuous phase is 1wt%-10wt%, optionally, the concentration of the emulsifier in the continuous phase is 1.0wt%, 1.5wt%, 2.0wt%, 2.5wt%, 3.0wt%, 3.5wt%, 4.0wt%, 4.5wt%, 5.0wt%, 6.0wt%, 7.0wt%, 8.0wt%, 9.0wt%, 10.0wt%, or a concentration range consisting of any two points as endpoints. Preferably, the emulsifier is span-80.

[0055] In some embodiments of the present invention, the crosslinking agent in the reaction phase is glutaraldehyde, polyethylene glycol or EDC. The above crosslinking agents can react with silk fibroin to form a stable silk fibroin crosslinked gel.

[0056] In some embodiments of the present invention, the reaction phase is placed in a stirring device, the stirring speed is 50-200r / min, and the stirring time is between 1 and 5h. The stirring speed and stirring time are within the above ranges. On the one hand, it can ensure that the dispersed phase droplets and the reaction phase undergo sufficient crosslinking reaction, and on the other hand, it can avoid the generation of bubbles due to too long stirring time. If the stirring speed is too slow, the crosslinked microspheres will agglomerate and precipitate; if the stirring speed is too fast, the surface morphology of the crosslinked microspheres will change and become irregular.

[0057] In some embodiments of the present invention, the centrifugal speed is 3000-8000 r / min, the temperature is 4° C., and the time is 10-60 min.

[0058] Injectable Silk Fibroin Microspheres:

[0059] The injectable silk fibroin microspheres prepared by the preparation method of the injectable silk fibroin microspheres of the present invention have a particle size distribution between 20 μm and 75 μm, a pore size distribution between 2 nm and 50 nm, and a uniform particle size of the silk fibroin microspheres, a narrow diameter distribution, and can be injected into the body. The surface is smooth and regular, and is spherical. After entering the body, it will not cause a more severe inflammatory reaction and is not easily swallowed by macrophages. At the same time, the injectable silk fibroin microspheres use a stable silk fibroin cross-linked gel as a skeleton material, uniformly load antibacterial drugs as antibacterial active substances, have a high drug loading rate, and the microspheres have a small pore size, which can achieve long-term sustained release of antibacterial drugs.

[0060] In some embodiments of the present invention, the antibacterial drug is a polyphenol antibacterial drug; the polyphenol antibacterial drug includes one or more of tea polyphenol, rhubarb polyphenol, pomegranate peel polyphenol, date palm pollen polyphenol, burdock polyphenol, qinguan leaf polyphenol, and blueberry leaf polyphenol. Polyphenol antibacterial drugs have excellent antibacterial properties and a wide antibacterial spectrum, and have obvious antibacterial activity against Staphylococcus aureus, Bacillus subtilis, and Bacillus cereus. Therefore, the injectable silk fibroin microspheres of the present invention have a wide range of antibacterial activity.

[0061] Injectable hydrogels:

[0062] The present invention loads the above-mentioned injectable silk fibroin microspheres into an injectable first hydrogel to prepare an injectable hydrogel, which includes the first hydrogel and the injectable silk fibroin microspheres dispersed in the first hydrogel; the injectable silk fibroin microspheres include silk fibroin cross-linked gel and antibacterial drugs uniformly distributed in the silk fibroin cross-linked gel, and the particle size distribution of the injectable silk fibroin microspheres is between 20μm and 75μm, and the pore size distribution is between 2nm and 50nm.

[0063] The above-mentioned injectable hydrogel can be directly injected into the target site when used. Compared with dispersing the injectable silk fibroin microspheres in an aqueous solution for injection, the injectable hydrogel of the present invention can effectively avoid the injectable silk fibroin microspheres flowing with body fluids, resulting in uneven distribution of antibacterial drugs. In addition, the above-mentioned injectable hydrogel can fix the injectable silk fibroin microspheres at the target site, so that the antibacterial drugs are slowly released at the target site. If the antibacterial drugs are loaded into the first hydrogel, the drugs will be released too quickly, and it is impossible to achieve slow release of the drugs in the entire metabolic cycle of the injectable silk fibroin microspheres.

[0064] In some embodiments of the present invention, the antibacterial drug accounts for 1 wt% to 20 wt% of the injectable silk fibroin microspheres. The proportion of the antibacterial drug can be selected according to actual needs, such as the degree of infection.

[0065] In some embodiments of the present invention, the weight ratio of the injectable silk fibroin microspheres to the first hydrogel is 1: 1.5 to 9. When the weight ratio of the injectable silk fibroin microspheres to the first hydrogel is within this range, the injectable silk fibroin microspheres have a long degradation cycle, which can ensure that the injectable hydrogel has a long drug release cycle.

[0066] In some embodiments of the present invention, the colloid in the first hydrogel is composed of polyethylene glycol and silk fibroin in a weight ratio of 1:2 to 8, and the concentration of silk fibroin is 1wt% to 6wt%. The first hydrogel composed of polyethylene glycol and silk fibroin in a weight ratio of 1:2 to 8 can form an in-situ gel after being injected into the target part, which can achieve an immediate filling effect, and the above concentration can ensure that the first hydrogel can be pushed smoothly during use without blocking the needle, and can form a gel immediately after being injected into the target part.

[0067] In some embodiments of the present invention, the injectable hydrogel is used as an injectable filler in the field of medical plastic surgery. The injectable hydrogel can be fixed at the filling site and does not flow arbitrarily.

[0068] In some embodiments of the present invention, the injectable hydrogel is used as a wound dressing. The injectable hydrogel has a drug sustained release function, and thus has a long-term anti-infection effect.

[0069] Example:

[0070] Hereinafter, embodiments of the present invention are described. The embodiments described below are exemplary and are only used to explain the present invention, and cannot be construed as limiting the present invention. If specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in the art or the product instructions are followed. The reagents or instruments used without indicating the manufacturer are all conventional products that can be obtained commercially. The vacuum drying oven used in the following embodiments was purchased from Shanghai Yiheng Scientific Instrument Co., Ltd., with a brand name of D2F-6050; the polyethylene glycol is PEG-400; the ferrous tartrate solution is configured by the following method: take 1g of ferrous sulfate and 5g of potassium sodium tartrate, and dilute to 1L with ultrapure water to obtain a ferrous tartrate solution.

[0071] The microfluidic device used in the present invention is as follows Figure 1 As shown, the channel is Y-shaped, with two continuous phase inlets and one dispersed phase inlet. The dispersed phase inlet is located at the intersection of the Y-shape. When the dispersed phase flows through the intersection of the Y-shape, it is sheared into dispersed phase droplets by the continuous phases on both sides and is evenly dispersed in the continuous phase. A reaction phase container (not shown) is provided at the end of the channel of the microfluidic device. After the dispersed phase droplets enter the reaction phase container, they fully react with the cross-linking agent in the reaction phase to form cross-linked microspheres.

[0072] Example 1

[0073] The method for preparing injectable silk fibroin microspheres provided in this embodiment comprises the following steps:

[0074] S1. Preparation of dispersed phase: According to the weight ratio of silk fibroin to tea polyphenols being 5:1, a silk fibroin solution with a concentration of 5.0 wt% and a tea polyphenol solution with a concentration of 10.0 wt% were mixed to obtain a dispersed phase.

[0075] S2. Preparation of the continuous phase: Span-80 is added to liquid paraffin to prepare an emulsifier liquid paraffin mixed solution with a span-80 concentration of 2.0 wt%.

[0076] S3. Preparation of reaction phase: adding glutaraldehyde into liquid paraffin to prepare a cross-linking agent liquid paraffin mixed solution with a glutaraldehyde concentration of 4.0 wt%.

[0077] S4. Use a syringe to inject the dispersed phase and the continuous phase into the microfluidic device at a certain flow rate, wherein the flow rate of the dispersed phase is 1.0 mL / h, and the flow rate of the continuous phase is 100 mL / h.

[0078] S5. After the continuous phase and the dispersed phase can form stable dispersed phase droplets, adjust the speed of the reaction phase to 160r / min to receive the dispersed phase droplets, and fully react for 2h to obtain cross-linked microspheres. Then, the cross-linked microspheres after stirring the reaction are centrifuged (speed 6000r / min, temperature 4°C, time 15min), remove the upper paraffin mixture, add ethanol to fully clean the impurities on the surface of the cross-linked microspheres, and then add deionized water to repeat the cleaning, collect the cleaning waste liquid, and put the filtrate containing the cross-linked microspheres into a vacuum drying oven for drying (vacuum drying temperature is 30°C, time is 42h, vacuum degree is 0.1MPa) to obtain injectable silk fibroin microspheres.

[0079] The injectable hydrogel provided in this embodiment is composed of a first hydrogel and injectable silk fibroin microspheres dispersed in the first hydrogel. The first hydrogel is composed of silk fibroin and polyethylene glycol in a weight ratio of 2:1. The injectable silk fibroin microspheres are the injectable silk fibroin microspheres obtained in step S5 of this embodiment. The weight ratio of the injectable silk fibroin microspheres to the first hydrogel is 3:7.

[0080] The method for preparing the injectable hydrogel provided in this embodiment comprises the following steps:

[0081] (1) Preparation of the first hydrogel: A silk fibroin solution with a concentration of 8.0 wt% and a polyethylene glycol solution with a concentration of 10.0 wt% were mixed at a weight ratio of 2:1, and the mixture was stirred evenly to form a silk fibroin polyethylene glycol solution.

[0082] (2) Take 0.7 g of silk fibroin polyethylene glycol solution and 0.3 g of injectable silk fibroin microspheres, mix them thoroughly and perform ultrasonic oscillation to obtain an injectable hydrogel.

[0083] The injectable hydrogel was stored in a refrigerator at 4°C when not in use.

[0084] Comparative Example 1

[0085] The method for preparing injectable silk fibroin microspheres provided in this embodiment is the same as that in Example 1 except that step S5 is different as follows:

[0086] S5: The aqueous solution containing the cross-linked microspheres is placed in a -80°C refrigerator and frozen for 4 hours, followed by freeze drying. The freezing temperature is -40°C, the freeze drying time is 48 hours, and the low pressure is 100Pa-400Pa. After freeze drying, injectable silk fibroin microspheres are obtained.

[0087] Comparative Example 2

[0088] The preparation method of injectable silk fibroin microspheres provided in this comparative example is the same as that of Example 1 except that step S5 is different as follows:

[0089] S5: After filtering out the cross-linked microspheres from the aqueous solution containing the cross-linked microspheres, the solution is naturally dried to obtain injectable silk fibroin microspheres.

[0090] Comparative Example 3

[0091] The preparation method of injectable silk fibroin microspheres provided in this comparative example is the same as that of Example 1 except that step S1 is different as follows:

[0092] S1. Preparation of dispersed phase: A 5.0 wt% silk fibroin solution was taken as the dispersed phase.

[0093] Comparative Example 4

[0094] The preparation method of injectable silk fibroin microspheres provided in this comparative example is the same as that of Example 1 except that step S1 is different as follows:

[0095] S1. Preparation of dispersed phase: According to the weight ratio of silk fibroin to tea polyphenols being 8:1, a silk fibroin solution with a concentration of 5.0 wt% and a tea polyphenol solution with a concentration of 10.0 wt% were mixed to obtain a dispersed phase.

[0096] Comparative Example 5

[0097] The preparation method of injectable silk fibroin microspheres provided in this comparative example is the same as that of Example 1 except that step S1 is different as follows:

[0098] S1. Preparation of dispersed phase: According to the weight ratio of silk fibroin to tea polyphenols of 12:1, a silk fibroin solution with a concentration of 5.0 wt% and a tea polyphenol solution with a concentration of 10.0 wt% were mixed to obtain a dispersed phase.

[0099] Results Analysis

[0100] 1. Particle size analysis

[0101] The specific surface area and particle size test results of the injectable silk fibroin microspheres prepared in Example 1 and Comparative Examples 1 to 5 are shown in Table 1. The size of the injectable silk fibroin microspheres of Comparative Example 1 after freeze-drying will be significantly reduced, its appearance is irregular, and its surface is porous. It can be seen from the pore size distribution of Example 1 and Comparative Example 1 that the surface of the injectable silk fibroin microspheres of Comparative Example 1 after freeze-drying is a macroporous structure, while the surface of the injectable silk fibroin microspheres of Example 1 through vacuum drying is mainly a mesoporous structure. By comparing the specific surface area data, it is found that the specific surface area of ​​the injectable silk fibroin microspheres of Comparative Example 1 after freeze-drying is large, so it is easy to accelerate the release of drugs loaded by the injectable silk fibroin microspheres; while the specific surface area of ​​the injectable silk fibroin microspheres of Example 1 through vacuum drying is small, which has the effect of sustained release of drugs.

[0102] Table 1 Specific surface area, pore size distribution and particle size of injected silk fibroin microspheres

[0103]

[0104] 2. Encapsulation efficiency and drug loading rate test

[0105] Take 0.5 mL of the supernatant of the cleaning waste liquid collected in step S5 of Example 1, Comparative Examples 1, 2, 4, and 5, and prepare the sample solution according to the volume ratio of PBS: ferrous tartrate solution: supernatant = 3:1:1. Take 0.5 mL of the supernatant of the cleaning waste liquid collected in step S5 of Comparative Example 3, and prepare the blank solution according to the volume ratio of PBS: ferrous tartrate solution: supernatant = 3:1:1. Use an ultraviolet spectrophotometer to measure the absorbance of the sample solution and the zero sample solution at 540 nm. Substitute the measured absorbance value into the tea polyphenol standard curve equation to calculate its concentration, then calculate the volume of the collected cleaning waste liquid, and finally calculate the content of tea polyphenols in the supernatant. The tea polyphenol content in the injectable silk fibroin microspheres can be obtained by subtracting the content of tea polyphenols in the waste liquid from the amount of tea polyphenols added. And the dried injectable silk fibroin microspheres are immediately weighed and recorded. The drug loading rate and encapsulation rate of injectable silk fibroin microspheres can be calculated according to the following formula:

[0106]

[0107] Where M t is the mass of tea polyphenols added (mg); M wis the mass of tea polyphenols in the supernatant (mg); M m is the mass of injectable silk fibroin microspheres (mg). The test results are shown in Table 2.

[0108] Table 2 Encapsulation efficiency and drug loading rate of injectable silk fibroin microspheres in different embodiments and comparative examples

[0109]

[0110] 3. In vitro release rate of antimicrobial drugs

[0111] Three portions of injectable silk fibroin microspheres were dried to constant weight, placed in beakers, and appropriate amounts of saline were added. The beakers were then placed on a constant temperature shaker with the temperature set to 37°C and the speed set to 100 rpm. 1 mL of supernatant was drawn at 0.5, 1, 3, 5, 10, 24, 36, 48, 72, 96, and 120 h, and an equal amount of saline was added. The concentration of antimicrobial drugs in the supernatant at each time point was determined by a UV-spectrophotometer, and the cumulative release rate of antimicrobial drugs was calculated according to the following formula.

[0112]

[0113] Where n is the number of sampling times, C n is the concentration of antimicrobial drugs in the release medium after n samplings, C i is the antimicrobial drug concentration of the i-th sampling, V0 is the initial volume of the drug release medium, V is the sampling volume, and w is the mass of the antimicrobial drug in the injectable silk fibroin microspheres.

[0114] In vitro release curve of injectable silk fibroin microspheres Figure 2 As shown. The injectable silk fibroin microspheres are a burst release of drugs in the stage of just releasing. In Example 1, the release amount in 3h is 15.3%±0.9%. This is because some antibacterial drugs are on the outermost surface of the injectable silk fibroin microspheres and are released due to the concentration gradient. The 3rd to 72th hour is the slow release stage. At this time, the adsorption and diffusion effects of the injectable silk fibroin microspheres on the antibacterial drugs gradually reach a dynamic equilibrium, and the release amount in 72h is 49.7%±2.1%. The remaining antibacterial drugs can be continuously released during the degradation of the microspheres. According to Comparative Examples 1 and 2, the larger pore size on the surface of the injectable silk fibroin microspheres will cause the drug to be released too quickly, which cannot meet the effect of sustained drug release.

[0115] The foregoing is merely a specific embodiment of the present invention, which enables those skilled in the art to understand or implement the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A method for preparing injectable silk fibroin microspheres, characterized in that: The steps include: Providing a microfluidic device and a dispersed phase, a continuous phase and a reaction phase for the microfluidic device; The dispersed phase is sheared into dispersed phase droplets with a particle size of 20 to 75 μm by using the continuous phase; The dispersed phase droplets are brought into contact with the reactive phase to undergo a sufficient cross-linking reaction to obtain cross-linked microspheres; The cross-linked microspheres are centrifuged, purified, and vacuum dried to obtain injectable silk fibroin microspheres; in: The dispersed phase is a silk fibroin solution; The silk fibroin solution contains antibacterial drugs; The continuous phase is a mixed solution of emulsifier liquid paraffin; The reaction phase is a cross-linking agent liquid paraffin mixed solution.

2. The method for preparing injectable silk fibroin microspheres according to claim 1, characterized in that: The vacuum drying temperature is 20-40°C and the time is 12-72h.

3. The method for preparing injectable silk fibroin microspheres according to claim 1, characterized in that: The method of shearing the dispersed phase into dispersed phase droplets with a particle size of 20 to 75 μm by using the continuous phase includes: controlling the flow rate of the dispersed phase to 0.5 to 1.5 mL / h and controlling the flow rate of the continuous phase to 40 to 180 mL / h.

4. The method for preparing injectable silk fibroin microspheres according to claim 1, characterized in that: The concentration of silk fibroin in the silk fibroin solution is 0.5 wt % to 5.0 wt %.

5. An injectable silk fibroin microsphere, characterized in that: The injectable silk fibroin microspheres are prepared by the preparation method of any one of claims 1 to 4.

6. An injectable hydrogel, characterized in that: It includes a first hydrogel and injectable silk fibroin microspheres dispersed in the first hydrogel; the injectable silk fibroin microspheres include a silk fibroin cross-linked gel and an antibacterial drug uniformly distributed in the silk fibroin cross-linked gel, the particle size of the injectable silk fibroin microspheres is distributed between 20μm and 75μm, and the pore size is distributed between 2nm and 50nm.

7. The injectable hydrogel according to claim 6, characterized in that: The antibacterial drug is a polyphenol antibacterial drug; the polyphenol antibacterial drug includes one or more of tea polyphenols, rhubarb polyphenols, pomegranate peel polyphenols, date palm pollen polyphenols, burdock polyphenols, qinguan leaf polyphenols, and blueberry leaf polyphenols.

8. The injectable hydrogel according to claim 6, characterized in that: The antibacterial drug accounts for 1wt% to 20wt% of the injectable silk fibroin microspheres; and / or the weight ratio of the injectable silk fibroin microspheres to the first hydrogel is 1:1.5-9.

9. The injectable hydrogel according to claim 6, characterized in that: The colloid in the first hydrogel is composed of polyethylene glycol and silk fibroin in a weight ratio of 1:2-8, and the concentration of the silk fibroin is 1wt%-6wt%.

10. The injectable hydrogel according to claim 6, characterized in that: The injectable silk fibroin microspheres further include other therapeutic components uniformly distributed in the silk fibroin cross-linked gel.

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