A sericin microsphere based on colored cocoons, its preparation method and applications

Abstract

This invention provides a sericin microsphere based on colored cocoons, its preparation method, and its applications, relating to the fields of biomaterials and microfluidic processing technology. The preparation method includes the following steps: (1) dissolving sericin in formic acid or dimethyl sulfoxide, adding an organic solvent, and adding or not adding a polymer to obtain an organic phase containing sericin; (2) dissolving an oil-water interface stabilizer in deionized water to obtain an aqueous phase; (3) using the organic phase as the dispersed phase and the aqueous phase as the continuous phase, preparing oil-in-water microdroplets using a capillary glass tube microfluidic device; (4) solidifying the microdroplets to obtain sericin microspheres. The process for preparing sericin microspheres using this invention is simple, mild, and bio-friendly. The sericin microspheres have uniform particle size, good monodispersity, and abundant autofluorescence properties. The sericin microspheres of this invention can be used in fields such as bioimaging, tissue engineering, cell culture, adsorption separation, drug delivery, and biosensing.

Description

Technical Field

[0001] This invention relates to the fields of biomaterials and microfluidic processing technology, specifically to a sericin microsphere based on colored cocoons, its preparation method, and its applications. Background Technology

[0002] China has a long history of silkworm breeding and utilization, and in recent years has cultivated many excellent colored cocoon varieties, such as the golden cocoon. These colored cocoons are rich in sericin. In the traditional silk industry's degumming process, sericin is often discharged as wastewater, resulting in the ineffective utilization of colored cocoons, causing both resource waste and environmental pressure. In recent years, considering the excellent water solubility of sericin, adding sericin as an auxiliary material to other hydrogel material systems to improve the biocompatibility of the materials has become the main form of sericin utilization.

[0003] However, research on sericin remains limited, resulting in low utilization, low added value, and resource waste. Expanding the forms and applications of sericin materials and their composites is crucial for increasing their added value and promoting innovation and upgrading of the silk industry. Further research is needed on how to expand the forms and applications of sericin materials and their composites. Currently, there are no reports on the preparation of microparticle (microspheres or microcapsules) functional materials using sericin as a raw material in a non-water-soluble form and employing microfluidic technology. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a sericin microsphere based on colored cocoons, its preparation method, and its applications. Using a capillary glass tube microfluidic device, sericin and a high-molecular-weight polymer are used as raw materials. Water-in-oil microdroplets are formed through aqueous phase shearing of the organic phase, followed by solidification via organic solvent evaporation to obtain fluorescent sericin microspheres.

[0005] This invention provides a method for preparing sericin microspheres, comprising the following steps: (1) Preparation of organic phase: Dissolve sericin in formic acid or dimethyl sulfoxide, add organic solvent, and add or not add polymer to obtain organic phase containing sericin; (2) Preparation of aqueous phase: Dissolve the oil-water interface stabilizer in deionized water to obtain the aqueous phase; (3) Microfluidic preparation of microdroplets: Using a capillary glass tube microfluidic device, an organic phase is injected as the dispersed phase into the inner injection capillary, and an aqueous phase is injected as the continuous phase into the outer capillary to prepare oil-in-water microdroplets. (4) Solidify the water-in-oil microdroplets to obtain sericin microspheres.

[0006] Further, in step (1), the sericin is natural sericin extracted from golden silkworm cocoons, and the preparation method includes the following steps: (a) Wash the silkworm cocoons, cut them into small pieces, add them to Na2CO3 solution, heat at 80~120℃ to completely dissolve the sericin on the silkworm cocoons, and filter to obtain a coarse sericin solution. (b) The crude sericin solution was dialyzed and centrifuged. The supernatant and precipitate were freeze-dried separately to obtain sericin protein.

[0007] Further, in step (a), the mass-to-volume ratio of the silkworm cocoon silk to the Na2CO3 solution is 1 g: (100-200) mL; And / or, in step (a), the concentration of the Na2CO3 solution is 0.1-1 wt%; And / or, in step (b), the molecular weight cutoff of the dialysis bag during dialysis is 3~5 kDa.

[0008] Preferably, in step (b), the dialysis time is 5-7 days; the dialysis temperature is 4-25℃; And / or, in step (b), the centrifugation is performed at 7000 rpm for 10 min.

[0009] Further, in step (1), the concentration of the sericin dissolved in formic acid or dimethyl sulfoxide is 0.1-10 wt%; And / or, in step (1), the concentration of formic acid or dimethyl sulfoxide in the organic phase is 10-50 vol% And / or, in step (1), the concentration of the polymer in the organic phase is 0-10 wt%; And / or, in step (2), the solubility of the oil-water interface stabilizer is 0.1-10 wt%.

[0010] Further, in step (1), the concentration of the sericin dissolved in formic acid or dimethyl sulfoxide is 0.5-5 wt%; And / or, in step (1), the concentration of formic acid or dimethyl sulfoxide in the organic phase is 10-30 vol% And / or, in step (1), the concentration of the polymer in the organic phase is 0-2 wt%; And / or, in step (2), the solubility of the oil-water interface stabilizer is 2 wt%.

[0011] Further, in step (1), the polymer is selected from any one or more of polylactic acid-glycolic acid copolymer, polystyrene, polymethyl methacrylate, polyvinyl acetate or polycaprolactone; and the polymer is not limited to the above materials, as long as it is soluble in chloroform.

[0012] And / or, in step (1), the organic solvent is selected from chloroform; And / or, in step (2), the oil-water interface stabilizer is selected from polyvinyl alcohol.

[0013] Further, in step (3), the capillary glass tube microfluidic device is a coaxial capillary glass tube device, with an inner diameter of 20-300 μm for the inner injection capillary and an inner diameter of 500-1500 μm for the outer capillary. And / or, in step (3), the organic phase has a flow rate of 50-1500 μL / h and the aqueous phase has a flow rate of 500-10000 μL / h; And / or, in step (4), the curing temperature is 20-60℃ and the curing time is 0.5-24 hours; And / or, in step (4), the microspheres obtained after curing are washed with deionized water.

[0014] The present invention also provides a sericin microsphere, which is prepared by the aforementioned preparation method.

[0015] Furthermore, the average particle size of the microspheres is 10-400 μm, and the monodispersity coefficient CV < 5%; the microspheres all exhibit autofluorescence properties at excitation wavelengths of 405 nm, 488 nm, 561 nm, and 640 nm.

[0016] The present invention also provides the use of the aforementioned sericin microspheres in the fields of bioimaging, tissue engineering, cell culture, adsorption separation, drug delivery and biosensing.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Uniform particle size: Microfluidic devices can be used to obtain O / W microdroplets with controllable structural height and narrow diameter distribution, thereby obtaining silk protein microspheres with uniform particle size and monodispersity coefficient CV < 5%; 2. By dissolving sericin in the organic phase and introducing polymer components, the space for sericin composite systems and functional design has been expanded. 3. Mild and controllable process: The solvent diffusion and evaporation curing process is mild and does not require high temperature and high pressure conditions; 4. The confined aggregation of sericin in a polymer matrix can realize "self-luminescent" microspheres without the need for additional fluorescent dyes; 5. The process is simple and continuous, making it easy to promote and apply to the high-value utilization of silk fibroin in colored cocoons and the development of functional microsphere products. It can be used in fields such as bioimaging, tissue engineering, cell culture, adsorption separation, drug delivery and biosensing. 6. This invention has enabled the utilization of sericin waste and the transformation of waste into treasure through research, and has enhanced a deeper understanding of the basic physicochemical properties of sericin.

[0018] In summary, this invention provides a microfluidic method for preparing autofluorescent sericin microspheres. The process for preparing these microspheres is simple, mild, and biofriendly. Furthermore, the microspheres exhibit uniform particle size, good monodispersity, and abundant autofluorescence. The sericin microspheres prepared by this invention can be used as fluorescent imaging materials, eliminating the need for exogenous dye loading and reducing toxicity. They can be applied in fields such as bioimaging, tissue engineering, cell culture, adsorption separation, drug delivery, and biosensing.

[0019] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0020] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the preparation of sericin microdroplets and microspheres for a glass capillary microfluidic device.

[0022] Figure 2 Optical photographs of sericin A and sericin B extracted from golden cocoons.

[0023] Figure 3 Optical micrographs of the sericin microdroplets and microspheres prepared in Example 1: a is a microscopic image of the sericin microdroplets; b is a microscopic image of the sericin microspheres (in the receiving liquid).

[0024] Figure 4 Optical micrographs of the sericin microdroplets and microspheres prepared in Example 2: a is a microscopic image of the sericin microdroplets; b is a microscopic image of the sericin microspheres (in the receiving liquid).

[0025] Figure 5 Laser confocal microscopy images of the sericin microspheres prepared in Example 2 under different excitation wavelengths.

[0026] Figure 6 Optical micrographs of the sericin microdroplets and microspheres prepared in Example 3: a is a microscopic image of the sericin microdroplets; b is a microscopic image of the sericin microspheres (in the receiving liquid).

[0027] Figure 7 Laser confocal microscopy images of the sericin microspheres prepared in Example 3 under different excitation wavelengths.

[0028] Figure 8 Optical micrographs of the sericin microdroplets and microspheres prepared in Example 4: a is a microscopic image of the sericin microdroplets; b is a microscopic image of the sericin microspheres (in the receiving liquid).

[0029] Figure 9 Optical micrographs of the sericin microdroplets and microspheres prepared in Example 5: a is a microscopic image of the sericin microdroplets; b is a microscopic image of the sericin microspheres (in the receiving liquid).

[0030] Figure 10 Optical micrographs of the sericin microdroplets and microspheres prepared in Example 6: a is a microscopic image of the sericin microdroplets; b is a microscopic image of the sericin microspheres (in the receiving liquid).

[0031] Figure 11 Laser confocal microscopy images of the sericin microspheres prepared in Example 6 under different excitation wavelengths.

[0032] Figure 12 Optical micrographs of the sericin microdroplets and microspheres prepared in Example 7: a is a microscopic image of the sericin microdroplets; b is a microscopic image of the sericin microspheres (in the receiving liquid).

[0033] Figure 13 Laser confocal microscopy images of the sericin microspheres prepared in Example 7 under different excitation wavelengths.

[0034] Figure 14 Optical micrographs of the sericin microdroplets and microspheres prepared in Example 8: a is a microscopic image of the sericin microdroplets; b is a microscopic image of the sericin microspheres (in the receiving liquid).

[0035] Figure 15 Optical micrographs of the sericin microdroplets and microspheres prepared in Example 9: a is a microscopic image of the sericin microdroplets; b is a microscopic image of the sericin microspheres (in the receiving liquid). Detailed Implementation

[0036] The raw materials and equipment used in the specific embodiments of the present invention are all known products, obtained by purchasing commercially available products.

[0037] The process for preparing sericin microdroplets using a glass capillary microfluidic device in this invention is described in [link to documentation]. Figure 1 The aqueous phase shears the organic phase at the shear port of the microfluidic device, thereby forming microdroplets of sericin.

[0038] I. Device Structure Used in This Invention The capillary glass tube microfluidic device includes: 1. Internal injection capillary: used to introduce an organic phase containing sericin and high molecular weight polymers; 2. Outer square or round capillary glass tube: coaxial or confluenced with the inner injection capillary tube, used to introduce an aqueous phase as a continuous phase; 3. Liquid supply unit: includes at least two precision injection pumps or constant flow pumps, which are connected to the organic phase liquid and the aqueous phase liquid respectively, for precise control of the flow rate of each phase; 4. Collection Unit: Located at the outlet of the microfluidic device, it is used to collect oil-in-water emulsions containing organic phase droplets and complete the solvent evaporation and solidification process.

[0039] The inner diameter of the internal injection capillary is preferably 20-300 μm, and the inner diameter of the outer square capillary glass tube is preferably 500-1500 μm. A shearing and droplet-forming region is formed between the inner and outer capillaries, so that the aqueous phase continuously shears the organic phase dispersion phase to generate a uniform oil-in-water (O / W) emulsion structure (the organic phase is dropped into the aqueous phase).

[0040] II. Preparation Method A method for preparing sericin microspheres based on microfluidic technology includes the following steps: 1. Preparation of organic phase (1) Add sericin to formic acid to form a sericin-formic acid pre-solution; (2) Add chloroform, dichloromethane or a mixture thereof to the presol; (3) Add or remove high molecular weight polymers (PLGA, PS, PMMA, PVAC, PCL, etc.) to the obtained formic acid / chloroform mixed solvent and dissolve to obtain an organic phase containing sericin. PLGA: polylactic acid-glycolic acid copolymer; PS: polystyrene; PMMA: polymethyl methacrylate; PVAC: polyvinyl acetate; PCL: polycaprolactone.

[0041] The concentration of sericin in formic acid is 0.1-10 wt% (preferably 0.5-5 wt%). Concentration of polymer in mixed solvent: 0-10 wt% (preferably 0.5-2 wt%); Formic acid volume fraction in organic phase: 10-50 vol% (preferably 10-30 vol%).

[0042] 2. Prepare the aqueous phase An oil-water interface stabilizer (such as polyvinyl alcohol or surfactants F68, SDS, etc.) is dissolved in deionized water to obtain a 0.1-10 wt% aqueous solution as the aqueous phase. The aqueous phase is used as a continuous phase to stabilize the organic phase droplets.

[0043] 3. Prepare the collection solution Same as water.

[0044] 4. Microfluidic devices and microdroplet preparation A capillary glass tube microfluidic device is employed, preferably with a coaxial fluid focusing structure. A precision injection pump controls the injection of the organic phase at a flow rate of 50–1500 μL / h into the inner injection capillary, while the aqueous phase is injected at a flow rate of 500–10000 μL / h into the outer capillary, shearing the organic phase to form water-in-oil microdroplets of controllable size (i.e., organic phase oil droplets dispersed in the aqueous phase). By adjusting the flow ratio of the inner and outer phases and the capillary size, monodisperse droplets (O / W microdroplets) with a particle size of 10–400 μm and a coefficient of variation (CV) < 5% can be obtained.

[0045] 5. Solvent evaporation and solidification into spheres O / W microdroplets are collected in an aqueous receiving liquid, and the temperature is controlled at 20-60℃. Formic acid in the droplets diffuses into the aqueous phase and chloroform evaporates, thereby solidifying the entire microdroplet. After 0.5-24 hours, sericin microspheres or composite microspheres with fluorescent luminescence properties can be obtained.

[0046] 6. Separation and Cleaning After the solvent evaporates and the microspheres solidify, they are washed with deionized water to remove residual organic solvents and aqueous stabilizers, ultimately yielding sericin microspheres or composite microspheres.

[0047] In this invention, the sericin is a natural high-molecular-weight fibrous protein extracted from golden silkworm cocoons. The preparation method of the sericin used in the following examples is as follows: 1 g of silkworm cocoon silk is washed, cut into small pieces, and added to 100 mL of 0.5 wt% Na2CO3 solution. The solution is heated at 100°C for 0.5 h, and the silk is filtered to obtain a crude sericin solution. After the crude sericin solution cools, it is transferred to a dialysis bag (MWCO 3 kDa) and dialyzed with deionized water for 5 days (temperature 4-25°C). After dialysis, the sericin solution is centrifuged (7000 rpm, 10 min), and the supernatant is freeze-dried to obtain sericin A; the precipitate is freeze-dried to obtain sericin B. Optical photographs of sericin A and sericin B are shown below. Figure 2 As shown in the figure. The obtained sericin was stored in a refrigerator at 4°C for later use.

[0048] Example 1: Preparation of sericin microspheres (using sericin B) The preparation steps of the sericin microspheres in this embodiment are as follows: (1) Solution preparation: Dissolve 0.01 g of sericin B in 100 μL of formic acid, then add 900 μL of chloroform to the mixed solution and stir until homogeneous to obtain the organic phase; prepare a 2.0 wt% polyvinyl alcohol (PVA) aqueous solution as the aqueous phase (continuous phase).

[0049] (2) Preparation of sericin microspheres: First, the aqueous phase was introduced into a capillary glass tube microfluidic device. After stabilization, the organic phase was introduced into the capillary glass tube microfluidic device. The flow rates of the organic phase and the aqueous phase were controlled at 600 μL / h and 2000 μL / h, respectively, to obtain a collection solution containing sericin microdroplets. The collection solution was then placed in a 30℃ constant temperature incubator for 24 h to promote the formation of microdroplets into microspheres, thus obtaining sericin microspheres. Subsequently, the microspheres were repeatedly washed with deionized water and collected for use after freeze-drying, with or without freeze-drying.

[0050] See the process of sericin microsphere formation. Figure 1 . Figure 3 As can be seen, the average particle size of the microdroplets prepared in this embodiment is 140.58 μm, and the monodispersity index (CV) is 2.7%. The average particle size of the shaped sericin microspheres is 21.12 μm, and the CV value is 4.2%. Figure 3 b). It can be seen that the prepared microdroplets and microspheres have uniform particle size and morphology.

[0051] Example 2: Preparation of sericin microspheres (using sericin B) The preparation steps of the sericin microspheres in this embodiment are as follows: (1) Solution preparation: Dissolve 0.01 g of sericin B in 200 μL of formic acid, then add 800 μL of chloroform to the mixed solution and stir until homogeneous to obtain the organic phase; prepare a 2.0 wt% polyvinyl alcohol (PVA) aqueous solution as the aqueous phase (continuous phase).

[0052] (2) Preparation of sericin microspheres: First, the aqueous phase was introduced into a capillary glass tube microfluidic device. After stabilization, the organic phase was introduced into the capillary glass tube microfluidic device. The flow rates of the organic phase and the aqueous phase were controlled at 600 μL / h and 2000 μL / h, respectively, to obtain a collection solution containing sericin microdroplets. The collection solution was then placed in a 30℃ constant temperature incubator for 24 h to promote the formation of microdroplets into microspheres, thus obtaining sericin microspheres. Subsequently, the microspheres were repeatedly washed with deionized water and collected for use after freeze-drying, with or without freeze-drying.

[0053] See the process of sericin microsphere formation. Figure 1 . Figure 4As can be seen, the average particle size of the microdroplets prepared in this embodiment is 159.15 μm, and the monodispersity index (CV) is 1.1%. The average particle size of the shaped sericin microspheres is 35.82 μm, and the CV value is 1.7%. Figure 4 (b) This demonstrates that the size and morphology of the prepared microdroplets and microspheres can be controlled by adjusting the ratio of formic acid and chloroform in the organic phase. Furthermore, the photoluminescence phenomenon of the sericin microspheres was observed using a laser confocal microscope, and the results are as follows... Figure 5 As shown, under laser excitation at 405 nm, 488 nm, 561 nm and 640 nm, the sericin microspheres all exhibited obvious fluorescence, with relative average fluorescence intensities of 1591.86, 98.86, 194.56 and 77.40, respectively.

[0054] Example 3: Preparation of sericin microspheres (using sericin B) The preparation steps of the sericin microspheres in this embodiment are as follows: (1) Solution preparation: Dissolve 0.01 g of sericin B in 200 μL of formic acid, then add 800 μL of chloroform to the mixed solution and stir until homogeneous. Then add 1.0 wt% polylactic acid-glycolic acid copolymer (PLGA) and stir until homogeneous to obtain the organic phase. Prepare a 2.0 wt% polyvinyl alcohol (PVA) aqueous solution as the aqueous phase (continuous phase).

[0055] (2) Preparation of sericin microspheres: First, the aqueous phase was introduced into a capillary glass tube microfluidic device. After stabilization, the organic phase was introduced into the capillary glass tube microfluidic device. The flow rates of the organic phase and the aqueous phase were controlled at 600 μL / h and 2000 μL / h, respectively, to obtain a collection solution containing sericin microdroplets. The collection solution was then placed in a 30℃ constant temperature incubator for 24 h to promote the formation of microdroplets into microspheres, thus obtaining sericin microspheres. Subsequently, the microspheres were repeatedly washed with deionized water and collected for use after freeze-drying, with or without freeze-drying.

[0056] See the process of sericin microsphere formation. Figure 1 . Figure 6 As can be seen, the average particle size of the microdroplets prepared in this embodiment is 156.62 μm, and the monodispersity index (CV) is 1.7%. The average particle size of the shaped sericin microspheres is 45.24 μm, and the CV value is 3.9%. Figure 6 (b) This demonstrates that the addition of the polymer PLGA alters the final morphology of the sericin microspheres, resulting in a distinct hollow structure. Furthermore, the photoluminescence phenomenon of the sericin microspheres was observed using a laser confocal microscope, and the results are as follows... Figure 7As shown, under laser excitation at 405 nm, 488 nm, 561 nm and 640 nm, the sericin microspheres all exhibited obvious fluorescence, with relative average fluorescence intensities of 235.42, 7.88, 99.08 and 28.27, respectively.

[0057] Example 4: Preparation of sericin microspheres (using sericin B) The preparation steps of the sericin microspheres in this embodiment are as follows: (1) Solution preparation: Dissolve 0.01 g of sericin B in 100 μL of formic acid, then add 900 μL of chloroform to the mixed solution and stir until homogeneous. Then add 1.0 wt% polymethyl methacrylate (PMMA) and stir until homogeneous to obtain the organic phase. Prepare a 2.0 wt% polyvinyl alcohol (PVA) aqueous solution as the aqueous phase (continuous phase).

[0058] (2) Preparation of sericin microspheres: First, the aqueous phase was introduced into a capillary glass tube microfluidic device. After stabilization, the organic phase was introduced into the capillary glass tube microfluidic device. The flow rates of the organic phase and the aqueous phase were controlled at 600 μL / h and 2000 μL / h, respectively, to obtain a collection solution containing sericin microdroplets. The collection solution was then placed in a 30℃ constant temperature incubator for 24 h to promote the formation of microdroplets into microspheres, thus obtaining sericin microspheres. Subsequently, the microspheres were repeatedly washed with deionized water and collected for use after freeze-drying, with or without freeze-drying.

[0059] See the process of sericin microsphere formation. Figure 1 . Figure 8 As can be seen, the average particle size of the microdroplets prepared in this embodiment is 146.42 μm, and the monodispersity index (CV) is 2.3%. The average particle size of the shaped sericin microspheres is 44.74 μm, and the CV value is 0.9%. Figure 8 b). It can be seen that by changing the composition of the organic phase, including the solvent ratio and the type of polymer, the final morphology of sericin and sericin composite microspheres can be altered.

[0060] Example 5: Preparation of sericin microspheres (using sericin A) The preparation steps of the sericin microspheres in this embodiment are as follows: (1) Solution preparation: Dissolve 0.01 g of sericin A in 200 μL of formic acid, then add 800 μL of chloroform to the mixed solution and stir until homogeneous to obtain the organic phase; prepare a 2.0 wt% polyvinyl alcohol (PVA) aqueous solution as the aqueous phase (continuous phase).

[0061] (2) Preparation of sericin microspheres: First, the aqueous phase was introduced into a capillary glass tube microfluidic device. After stabilization, the organic phase was introduced into the capillary glass tube microfluidic device. The flow rates of the organic phase and the aqueous phase were controlled at 600 μL / h and 2000 μL / h, respectively, to obtain a collection solution containing sericin microdroplets. The collection solution was then placed in a 30℃ constant temperature incubator for 24 h to promote the formation of microdroplets into microspheres, thus obtaining sericin microspheres. Subsequently, the microspheres were repeatedly washed with deionized water and collected for use after freeze-drying, with or without freeze-drying.

[0062] See the process of sericin microsphere formation. Figure 1 . Figure 9 As can be seen, the average particle size of the microdroplets prepared in this embodiment is 160.78 μm, and the monodispersity index (CV) is 1.7%. The average particle size of the shaped sericin microspheres is 11.43 μm, and the CV value is 4.7%. Figure 9 (b) This shows that changing the type of sericin will alter the final morphology of the resulting sericin microspheres.

[0063] Example 6: Preparation of sericin microspheres (using sericin A) The preparation steps of the sericin microspheres in this embodiment are as follows: (1) Solution preparation: Dissolve 0.01 g of sericin A in 200 μL of formic acid, then add 800 μL of chloroform to the mixed solution and stir until homogeneous. Then add 1.0 wt% PLGA and stir until homogeneous to obtain the organic phase. Prepare a 2.0 wt% polyvinyl alcohol (PVA) aqueous solution as the aqueous phase (continuous phase).

[0064] (2) Preparation of sericin microspheres: First, the aqueous phase was introduced into a capillary glass tube microfluidic device. After stabilization, the organic phase was introduced into the capillary glass tube microfluidic device. The flow rates of the organic phase and the aqueous phase were controlled at 600 μL / h and 2000 μL / h, respectively, to obtain a collection solution containing sericin microdroplets. The collection solution was then placed in a 30℃ constant temperature incubator for 24 h to promote the formation of microdroplets into microspheres, thus obtaining sericin microspheres. Subsequently, the microspheres were repeatedly washed with deionized water and collected for use after freeze-drying, with or without freeze-drying.

[0065] See the process of sericin microsphere formation. Figure 1 . Figure 10 As can be seen, the average particle size of the microdroplets prepared in this embodiment is 158.46 μm, and the monodispersity index (CV) is 0.7%. The average particle size of the shaped sericin microspheres is 36.28 μm, and the CV value is 1.2%. Figure 10(b) Therefore, due to the difference in the types of sericin, the sericin microspheres obtained in this example have a solid structure compared to the hollow sericin microspheres prepared in Example 3. Furthermore, the photoluminescence phenomenon of the sericin microspheres was observed using a laser confocal microscope, and the results are as follows... Figure 11 As shown, under laser excitation at 405 nm, 488 nm, 561 nm and 640 nm, the sericin microspheres all exhibited obvious fluorescence, with relative average fluorescence intensities of 146.83, 15.13, 222.75 and 29.50, respectively.

[0066] Example 7: Preparation of sericin microspheres (using sericin A) The preparation steps of the sericin microspheres in this embodiment are as follows: (1) Solution preparation: Dissolve 0.01 g of sericin A in 200 μL of formic acid, then add 800 μL of chloroform to the mixed solution and stir until homogeneous. Then add 1.0 wt% polystyrene (PS) and stir until homogeneous to obtain the organic phase. Prepare a 2.0 wt% polyvinyl alcohol (PVA) aqueous solution as the aqueous phase (continuous phase).

[0067] (2) Preparation of sericin microspheres: First, the aqueous phase was introduced into a capillary glass tube microfluidic device. After stabilization, the organic phase was introduced into the capillary glass tube microfluidic device. The flow rates of the organic phase and the aqueous phase were controlled at 600 μL / h and 2000 μL / h, respectively, to obtain a collection solution containing sericin microdroplets. The collection solution was then placed in a 30℃ constant temperature incubator for 24 h to promote the formation of microdroplets into microspheres, thus obtaining sericin microspheres. Subsequently, the microspheres were repeatedly washed with deionized water and collected for use after freeze-drying, with or without freeze-drying.

[0068] See the process of sericin microsphere formation. Figure 1 . Figure 12 As can be seen, the average particle size of the microdroplets prepared in this embodiment is 159.04 μm, and the monodispersity index (CV) is 0.6%. The average particle size of the shaped sericin microspheres is 36.40 μm, and the CV value is 1.5%. Figure 12 b). The sericin microspheres obtained in this embodiment have a solid structure and uniform morphology. Furthermore, the photoluminescence phenomenon of the sericin microspheres was observed using a laser confocal microscope, and the results are as follows. Figure 13 As shown, under laser excitation at 405 nm, 488 nm, 561 nm, and 640 nm, the sericin microspheres all exhibited significant fluorescence, with relative average fluorescence intensities of 95.29, 11.09, 195.29, and 26.90, respectively. This indicates that, compared to Example 6, the fluorescence properties of the sericin microspheres obtained in this example were altered by changing the type of polymer added.

[0069] Example 8: Preparation of sericin microspheres (using sericin A) The preparation steps of the sericin microspheres in this embodiment are as follows: (1) Solution preparation: Dissolve 0.01 g of sericin A in 100 μL of formic acid, then add 900 μL of chloroform to the mixed solution and stir until homogeneous to obtain the organic phase; prepare a 2.0 wt% polyvinyl alcohol (PVA) aqueous solution as the aqueous phase (continuous phase).

[0070] (2) Preparation of sericin microspheres: First, the aqueous phase was introduced into a capillary glass tube microfluidic device. After stabilization, the organic phase was introduced into the capillary glass tube microfluidic device. The flow rates of the organic phase and the aqueous phase were controlled at 600 μL / h and 2000 μL / h, respectively, to obtain a collection solution containing sericin microdroplets. The collection solution was then placed in a 30℃ constant temperature incubator for 24 h to promote the formation of microdroplets into microspheres, thus obtaining sericin microspheres. Subsequently, the microspheres were repeatedly washed with deionized water and collected for use after freeze-drying, with or without freeze-drying.

[0071] See the process of sericin microsphere formation. Figure 1 . Figure 14 As can be seen, the average particle size of the microdroplets prepared in this embodiment is 161.02 μm, and the monodispersity index (CV) is 1.3%. The average particle size of the shaped sericin microspheres is 25.60 μm, and the CV value is 2.5%. Figure 14 b). It can be seen that the prepared microdroplets and microspheres have uniform particle size and morphology.

[0072] Example 9: Preparation of sericin microspheres (using sericin A) The preparation steps of the sericin microspheres in this embodiment are as follows: (1) Solution preparation: Dissolve 0.01 g of sericin A in 100 μL of formic acid, then add 900 μL of chloroform to the mixed solution and stir until homogeneous. Then add 1.0 wt% PMMA and stir until homogeneous to obtain the organic phase. Prepare a 2.0 wt% polyvinyl alcohol (PVA) aqueous solution as the aqueous phase (continuous phase).

[0073] (2) Preparation of sericin microspheres: First, the aqueous phase was introduced into a capillary glass tube microfluidic device. After stabilization, the organic phase was introduced into the capillary glass tube microfluidic device. The flow rates of the organic phase and the aqueous phase were controlled at 600 μL / h and 2000 μL / h, respectively, to obtain a collection solution containing sericin microdroplets. The collection solution was then placed in a 30℃ constant temperature incubator for 24 h to promote the formation of microdroplets into microspheres, thus obtaining sericin microspheres. Subsequently, the microspheres were repeatedly washed with deionized water and collected for use after freeze-drying, with or without freeze-drying.

[0074] See the process of sericin microsphere formation. Figure 1 . Figure 15 As can be seen, the average particle size of the microdroplets prepared in this embodiment is 159.92 μm, and the monodispersity index (CV) is 1.1%. The average particle size of the shaped sericin microspheres is 40.42 μm, and the CV value is 3.3%. Figure 15 b).

[0075] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing sericin microspheres, characterized in that: Includes the following steps: (1) Preparation of organic phase: Dissolve sericin in formic acid or dimethyl sulfoxide, add organic solvent, and add or not add polymer to obtain organic phase containing sericin; (2) Preparation of aqueous phase: Dissolve the oil-water interface stabilizer in deionized water to obtain the aqueous phase; (3) Microfluidic preparation of microdroplets: Using a capillary glass tube microfluidic device, an organic phase is injected as the dispersed phase into the inner injection capillary, and an aqueous phase is injected as the continuous phase into the outer capillary to prepare oil-in-water microdroplets. (4) Solidify the water-in-oil microdroplets to obtain sericin microspheres.

2. The preparation method according to claim 1, characterized in that: In step (1), the sericin is a natural sericin extracted from golden silkworm cocoons, and the preparation method includes the following steps: (a) Wash the silkworm cocoons, cut them into small pieces, add them to Na2CO3 solution, heat at 80~120℃ to completely dissolve the sericin on the silkworm cocoons, and filter to obtain a coarse sericin solution. (b) The crude sericin solution was dialyzed and centrifuged. The supernatant and precipitate were freeze-dried separately to obtain sericin protein.

3. The preparation method according to claim 2, characterized in that: In step (a), the mass-to-volume ratio of the silkworm cocoon silk to the Na2CO3 solution is 1 g: (100-200) mL; And / or, in step (a), the concentration of the Na2CO3 solution is 0.1-1 wt%; And / or, in step (b), the molecular weight cutoff of the dialysis bag during dialysis is 3~5 kDa.

4. The preparation method according to claim 1, characterized in that: In step (1), the concentration of the sericin dissolved in formic acid or dimethyl sulfoxide is 0.1-10 wt%; And / or, in step (1), the concentration of formic acid or dimethyl sulfoxide in the organic phase is 10-50 vol% And / or, in step (1), the concentration of the polymer in the organic phase is 0-10 wt%; And / or, in step (2), the solubility of the oil-water interface stabilizer is 0.1-10 wt%.

5. The preparation method according to claim 4, characterized in that: In step (1), the concentration of the sericin dissolved in formic acid or dimethyl sulfoxide is 0.5-5 wt%; And / or, in step (1), the concentration of formic acid or dimethyl sulfoxide in the organic phase is 10-30 vol% And / or, in step (1), the concentration of the polymer in the organic phase is 0-2 wt%; And / or, in step (2), the solubility of the oil-water interface stabilizer is 2 wt%.

6. The preparation method according to claim 1, characterized in that: In step (1), the polymer is selected from any one or more of polylactic acid-glycolic acid copolymer, polystyrene, polymethyl methacrylate, polyvinyl acetate or polycaprolactone; And / or, in step (1), the organic solvent is selected from chloroform; And / or, in step (2), the oil-water interface stabilizer is selected from polyvinyl alcohol.

7. The preparation method according to claim 1, characterized in that: In step (3), the capillary glass tube microfluidic device is a coaxial capillary glass tube device with an inner diameter of 20-300 μm for the inner injection capillary and an inner diameter of 500-1500 μm for the outer capillary. And / or, in step (3), the organic phase has a flow rate of 50-1500 μL / h and the aqueous phase has a flow rate of 500-10000 μL / h; And / or, in step (4), the curing temperature is 20-60℃ and the curing time is 0.5-24 hours; And / or, in step (4), the microspheres obtained after curing are washed with deionized water.

8. A type of sericin microsphere, characterized in that: It is prepared by the preparation method described in any one of claims 1 to 7.

9. The sericin microspheres according to claim 8, characterized in that: The microspheres have an average particle size of 10-400 μm and a monodispersity coefficient (CV) of <5%; the microspheres exhibit autofluorescence at excitation wavelengths of 405 nm, 488 nm, 561 nm, and 640 nm.

10. Use of the sericin microspheres of claim 8 or 9 in the fields of bioimaging, tissue engineering, cell culture, adsorption separation, drug delivery and biosensing.

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