Silk fibroin microspheres as well as preparation method and application thereof

Abstract

The invention provides silk fibroin microspheres as well as a preparation method and application thereof, and belongs to the technical field of biomedical material preparation and micro-nano processing. The preparation method of the silk fibroin microspheres comprises the following steps: S1, obtaining a silk fibroin solution; s2, adopting a confocal micro-fluidic chip, taking oleic acid as a dispersion phase, and taking the silk fibroin solution as a continuous phase to obtain oil-in-water drops; s3, dropwise adding the oil-in-water liquid into a K2HPO4-KH2PO4 phosphate mixed solution, and carrying out salting-out to obtain a solid-liquid mixture; and S4, hatching and separating the solid-liquid mixture to obtain a precipitate which is the silk fibroin microspheres. Through a microfluidic / salting-out synergistic strategy, high monodispersity, mild curing and a green process can be considered, and the high-performance silk fibroin microspheres are provided for the biomedical field.

Description

Technical Field

[0001] This invention belongs to the field of biomedical material preparation and micro / nano fabrication technology, and relates to a highly uniform silk fibroin microsphere with the synergistic effect of microfluidics and salting-out technology, its preparation method and application. Background Technology

[0002] Silkworm silk, a natural protein fiber, consists of a core fibroin (approximately 75%) and an outer sericin (approximately 25%), and has been used in the traditional textile industry for thousands of years. Sericin has good solubility and is usually removed, retaining only the biocompatible and biodegradable fibroin as the raw material. Silkworm cocoons undergo degumming, salt dissolution, dialysis, and purification processes to obtain a regenerated fibroin aqueous solution, which can be further processed into various forms such as gels, fibers, films, porous scaffolds, microneedles, microspheres, and nanoparticles.

[0003] In recent years, silk fibroin microspheres, as a multifunctional biomaterial, have shown broad application prospects in drug delivery, tissue engineering, wound repair, and cosmetics. In drug delivery systems, silk fibroin microspheres can serve as carriers, enabling sustained and targeted drug delivery. They can also load various therapeutic agents, including small molecule drugs, proteins, and nucleic acids. By controlling the particle size, porosity, and surface properties of the microspheres, the release rate and behavior of drugs can be controlled, thereby improving efficacy and reducing side effects. As a tissue engineering scaffold material, silk fibroin microspheres can provide a three-dimensional microenvironment for cell growth, promoting tissue regeneration and repair. Silk fibroin microspheres possess good biocompatibility and biodegradability, supporting cell adhesion, proliferation, and differentiation. Furthermore, silk fibroin microspheres also have application potential in cosmetics and medical aesthetics, offering moisturizing, antioxidant, and UV-protective effects that can improve skin elasticity and radiance. Encapsulating active ingredients within silk fibroin microspheres can enhance their stability and bioavailability, thereby improving the efficacy of cosmetics. In addition to the above fields, silk fibroin microspheres can also be used to construct biosensors to detect biomolecules and pathogens. Their advantages, such as safety, edibility, and good biocompatibility, make them suitable for use in food texture improvement, nutrient encapsulation, and the development of functional foods.

[0004] Traditional preparation methods include emulsification solvent method, electrospray method, and salting-out method. The emulsification solvent method disperses an aqueous solution of silk fibroin (aqueous phase) in an oil phase (such as mineral oil, silicone oil, or organic solvents such as dichloromethane or chloroform), and emulsifies it through mechanical stirring or ultrasonication to form an oil-in-water (O / W) or water-in-oil (W / O) emulsion. The solvent (water or organic solvent) is then removed by heating, depressurization, or solvent extraction, causing the silk fibroin to solidify into microspheres. This method is technically mature, widely used, and simple to operate, but particle size control is difficult, and there is a risk of toxic organic residues (solvent / emulsifier). The electrospray method uses a high-voltage electrostatic spraying device to form protein microdroplets, which are then induced by an organic solvent to form water-insoluble β-sheet structures, thus producing silk fibroin microspheres. This method is rapid, continuous, and has high yield, but the equipment is expensive, the high-temperature process can destroy biological activity, and it is not suitable for heat-sensitive drugs. Salting out involves mixing a silk fibroin solution with inorganic salts. The addition of salt neutralizes the surface charge of the protein and disrupts its hydration membrane, thus destabilizing the protein in aqueous solution and ultimately precipitating silk fibroin microspheres. This method is a mild aqueous process, which is beneficial for preserving bioactivity. However, it suffers from a wide particle size distribution, slow solidification, irregular morphology, and poor reproducibility. The high biosafety of salting out has made it a fundamental method for preparing silk fibroin microspheres, but it is limited by the problem of uneven particle size, resulting in a wide microsphere size distribution. Salting out primarily involves high-concentration salt ions disrupting the hydration layer on the surface of silk fibroin molecules and creating charge shielding, causing liquid-liquid phase separation. This results in a concentrated phase rich in silk fibroin and a low-concentration phase. These concentrated phase droplets form the prototype of microspheres. Under stirring or emulsification conditions, these droplets tend to form spheres with minimized surface energy, ultimately forming solid silk fibroin microspheres. Salting out involves the nucleation and growth of silk fibroin in solution, which has a high thermodynamic energy barrier. Furthermore, the nucleation and growth are affected by multiple factors, such as the type and concentration of salt, the initial concentration of silk fibroin, pH value, temperature, stirring speed / method, and addition method. Ultimately, this makes it difficult to stably control the size and uniformity of the microspheres.

[0005] However, existing methods for preparing silk fibroin microspheres have several shortcomings that limit their clinical translation and practical application, mainly in the following aspects: (1) Poor uniformity of microsphere size and irregular shape: Traditional emulsification solvent method, electro-spraying method and salting out method are difficult to accurately control the synchronicity of microsphere nucleation and growth process, resulting in a wide microsphere size distribution and poor reproducibility; (2) Risk of organic solvent residue: Emulsified solvent methods and microfluidic methods usually rely on oil phase and surfactants, and organic reagent residues can affect biocompatibility; (3) High-concentration silk fibroin is difficult to process: Microfluidic technology is the first choice when the uniformity and precise control of microsphere particle size are required. However, high-concentration silk fibroin solutions usually have high viscosity, making it difficult to flow in the narrow channels of microfluidics, which can easily block the channels and make it difficult to generate stable droplets; while low-concentration silk fibroin solutions are easy to operate in microfluidics, but the resulting microsphere structure is not dense enough and is prone to shrinkage and deformation.

[0006] As can be seen from the above analysis, the current methods for preparing silk fibroin microspheres each have their own advantages and disadvantages. They cannot simultaneously achieve multiple advantages such as effective control of microsphere size and shape, avoidance of the use of toxic and harmful chemical reagents, and mild preparation conditions suitable for biomaterial systems. There is an urgent need to develop new silk fibroin microsphere preparation technologies to overcome the shortcomings of existing technologies. Summary of the Invention

[0007] To address the aforementioned technical challenges, this invention employs a microfluidic / salting-out synergistic strategy that balances high monodispersity, mild curing, and green processes, providing high-performance silk fibroin microspheres for the biomedical field.

[0008] On one hand, the present invention provides a method for preparing silk fibroin microspheres, comprising the steps of: S1 yields a silk fibroin solution; S2 uses a confocal microfluidic chip, with oleic acid as the dispersed phase and silk fibroin solution as the continuous phase, to obtain oil-in-water droplets; S3 oil-in-water solution was added dropwise to a K2HPO4-KH2PO4 phosphate mixed solution, and after salting out, a solid-liquid mixture was obtained. The solid-liquid mixture described in S4 is incubated, and the precipitate obtained by separation is the silk fibroin microspheres.

[0009] As one specific implementation, the confocal microfluidic chip includes inserting a dispersed phase channel (also called a capillary) into the interior of a continuous phase channel (also called a capillary), so that the dispersed phase channel is coaxially surrounded by the continuous phase channel to form a coaxial channel, allowing the dispersed phase and the continuous phase to flow through the coaxial channel respectively.

[0010] In a preferred embodiment, the dispersed phase flow rate is 100~1000 μL / h.

[0011] In a preferred embodiment, the continuous phase flow rate is 20–150 mL / h.

[0012] In a preferred embodiment, the concentration of the silk fibroin solution is 1 wt% to 10 wt%.

[0013] In a preferred embodiment, the pH of the K2HPO4-KH2PO4 phosphate mixed solution is 6.0–9.0. Potassium phosphate salts with high salting-out efficiency in the Hofmeister sequence are selected to improve crystallization efficiency; preferably, the ionic strength is greater than 0.6 M and the pH is greater than 6.

[0014] As a preferred embodiment, the incubation conditions include: 25℃~60℃, 10 min~6 hours.

[0015] On the other hand, the present invention provides silk fibroin microspheres obtained by any of the above preparation methods.

[0016] In a preferred embodiment, the coefficient of variation (CV) of the silk fibroin microspheres is ≤5%.

[0017] The present invention also provides the application of any of the above-mentioned silk fibroin microspheres in the field of medical materials.

[0018] As a preferred embodiment, it is used for drug carriers, tissue engineering scaffold materials, or biosensors.

[0019] The present invention also provides the application of any of the above-mentioned silk fibroin microspheres in the preparation of cosmetic skin care products or medical aesthetic materials.

[0020] In the microfluidic / salting-out synergistic method for preparing silk fibroin microspheres provided by this invention, the microfluidic technology and the salting-out method are not simply superimposed, but rather achieve synergistic effects through precise spatial control and molecular-level phase transition induction. This avoids the use of excessive organic solvents (to stabilize the microsphere interface film) in traditional microfluidics, and the synergistic method results in microspheres with higher biocompatibility.

[0021] (1) Synergistic effect The preparation path of the synergistic process is as follows: microfluidic generation of droplets → collection in a salt solution bath for solidification → centrifugal washing. This process shortens the solidification time, allows for continuous production, and produces microspheres with high monodispersity.

[0022] Microfluidics ensures uniform droplet size and monodispersity; salting out solidifies simultaneously with microdroplets (in situ induced protein solidification), avoiding the aggregation problem of traditional salting out methods; microscale droplets (particle size 100-600 micrometers) shorten the salt ion diffusion path, accelerate phase transition, and increase the solidification rate, forming droplets in 10-30 minutes, while the single salting out method requires several hours.

[0023] (2) Solve the problem of difficulty in accurately controlling the size, morphology and monodispersity of microspheres. Traditional emulsification solvent methods and salting-out methods often have difficulty in precisely controlling the process when inducing silk fibroin precipitation to form microspheres. The microspheres have a wide size distribution (high polydispersity), irregular shape, and poor batch-to-batch reproducibility, which is a fatal weakness for applications such as drug delivery or cell culture that require precise size control.

[0024] The advantage of the preparation method combining microfluidics and salting-out solidification in this invention is that by precisely manipulating the fluid in micron-level channels, highly uniform droplets can be generated, thereby ensuring that the final microspheres have extremely high monodispersity and uniform size and shape. This is one of the core advantages of this method.

[0025] (3) Solve the problem of traditional microsphere preparation methods relying on toxic and harmful organic reagents. Traditional emulsification or microfluidic methods require the use of organic solvents and surfactants to form and stabilize emulsions. The subsequent removal of these chemical residues is cumbersome, and the residues may affect the biocompatibility of the microspheres.

[0026] This invention utilizes salting-out curing to reduce the amount of organic solvents used. Salting-out itself, as a physical cross-linking curing mechanism, avoids the use of organic solvents. The cured microspheres are collected by centrifugation and filtration, primarily removing salts. This method is simpler and gentler than removing large amounts of organic solvents and surfactants. It not only improves biosafety (no toxic solvent residue) but also maintains the natural structure of silk fibroin, making it suitable for applications with high biocompatibility requirements, such as drug delivery and tissue engineering. Attached Figure Description

[0027] Figure 1a This is a schematic diagram of the overall microfluidic preparation platform used in the experiments of Embodiments 1 and 2 of the present invention; Figure 1b This is a partially enlarged schematic diagram of the microfluidic control platform used in the experiments of Embodiments 1 and 2 of the present invention; Figure 2 Optical microscope image of silk fibroin microspheres prepared in Example 1; Figure 3 Scanning electron microscope image of silk fibroin microspheres prepared by the method in Example 1; Figure 4 Scanning electron microscope image of silk fibroin microspheres prepared in Comparative Example 1; Figure 5 Comparison of particle size of silk fibroin microspheres prepared in Example 1 and Comparative Example 1; Figure 6 Comparison of coefficient of variation (CV) of silk fibroin microspheres prepared in Example 1 and Comparative Example 1; Figure 7 Optical microscope image of silk fibroin microspheres prepared in Example 2; Figure 8 Bar chart of particle size and coefficient of variation (CV) of silk fibroin microspheres prepared in Example 2. Detailed Implementation

[0028] The present invention will be further described below with reference to specific embodiments, but this does not constitute any limitation on the present invention.

[0029] The method for obtaining silk fibroin solution typically involves the following steps: Using raw silk as raw material, firstly, boil the silk in a 0.2-1% sodium carbonate solution at 100°C for 30-100 minutes. Then, wash the silk fibroin solution 3-4 times with deionized water to remove the sericin from the outer layer, obtaining degummed silk fibroin fibers. Next, immerse the degummed silk fibroin fibers in a 9-10 M lithium bromide solution and place them in an oven at 80-140°C for 2-6 hours to dissolve the fibers. Then, dialyze the solution for 3 days to remove salt ions. Finally, centrifuge to remove insoluble impurities to obtain an aqueous silk fibroin solution. Adjust the concentration of the aqueous silk fibroin solution to 1 wt%-10 wt%.

[0030] The preparation of silk fibroin microspheres using a microfluidic / salting-out synergistic method includes the following steps: (1) Connect two microinjection pumps, syringes, dispensing needles, Teflon tubes, and confocal focusing microfluidic chips in sequence to build a microfluidic preparation platform. Prepare oil-in-water droplets using the confocal microfluidic chip, selecting oleic acid as the dispersed phase, and using the silk fibroin solution with a concentration of 1wt% to 10wt% prepared in step (1) as the continuous phase. Let these two immiscible liquid phases flow through coaxial channels, as shown in Figure 1. Control the flow rate of the dispersed phase to be 100 to 1000 μL / h and the flow rate of the continuous phase to be 20 to 150 mL / h.

[0031] (2) A K2HPO4-KH2PO4 phosphate mixed solution was prepared using dipotassium hydrogen phosphate (K2HPO4) and potassium dihydrogen phosphate (KH2PO4) as raw materials. The pH value of the phosphate solution was adjusted to 6.0-9.0 by adjusting the volume ratio of the two. The microfluidic outlet pipe in step (2) was introduced into the phosphate solution, and the salting-out cross-linking solidification mechanism was used to promote the precipitation and stabilization of silk fibroin. Then, it was placed in an oven at 25℃-60℃ for 10 min-6 hours for incubation. After washing with deionized water, stable silk fibroin microspheres were finally obtained. The instruments and materials used in the embodiments and comparative examples of this invention are described below.

[0032] Preparation Example The preparation of silk fibroin solution includes the following steps: (1) Using raw silk as raw material, first boil it in 0.5% sodium carbonate solution at 100°C for 90 minutes, then wash it three times with deionized water to remove the sericin on the outside of the silk and obtain degummed silk fibroin fiber.

[0033] (2) Immerse the degummed silk fibroin fiber in a 9.3 M lithium bromide aqueous solution, place it in an oven at 80°C for 4 hours to dissolve the fiber, then dialyze it for 3 days to remove salt ions, and then centrifuge it to remove insoluble impurities to obtain a silk fibroin aqueous solution.

[0034] Example 1 The silk fibroin solution obtained in Example 1 was diluted with water to obtain a silk fibroin solution with a concentration of 5%.

[0035] The preparation of silk fibroin microspheres using a microfluidic / salting-out synergistic method includes the following steps: (1) For example Figure 1a As shown, two microinjection pumps (Lange laboratory injection pump LSP), syringes, dispensing needles, Teflon tubes, and co-current focusing microfluidic chips are connected in sequence to build a microfluidic backup platform.

[0036] A schematic diagram of a co-current focusing microfluidic chip, as shown below. Figure 1b As shown, a dispersed phase capillary is inserted into a continuous phase capillary (inserted approximately 1 cm, with the total length of the continuous phase capillary being about 3-6 cm) so that the dispersed phase capillary is coaxially surrounded by the continuous phase capillary, forming a coaxial channel. The inner diameter of the continuous phase capillary is 800 µm, and the tip diameter of the dispersed phase capillary is 200 µm.

[0037] On the microfluidic preparation platform, the injection pump was turned on, using oleic acid as the dispersed phase and a 5% silk fibroin solution as the continuous phase. Through a confocal microfluidic chip, the two immiscible liquid phases were allowed to flow through coaxial channels. The flow rate of the dispersed phase was controlled at 1 mL / h, and the flow rate of the continuous phase was controlled at 100 mL / h, i.e., the flow rate ratio was 1:100, resulting in oil-in-water droplets. The reaction time was only related to the number of microspheres.

[0038] (2) The water-in-oil droplets enter a K2HPO4-KH2PO4 phosphate solution with a pH of 8 through a microfluidic outlet pipe. The salting-out cross-linking solidification mechanism promotes the precipitation and stabilization of silk fibroin, resulting in a solid-liquid mixture. After incubating the solid-liquid mixture in a 40℃ oven for 20 min, the precipitate is washed with deionized water to finally obtain silk fibroin microspheres.

[0039] from Figure 2 and Figure 3 As can be seen, the silk fibroin microspheres prepared by the microfluidic / salting-out method of this invention have regular shapes, are all spherical, and have uniform size.

[0040] from Figure 5 The particle size histogram shows that the average particle size of the silk fibroin microspheres prepared by the microfluidic / salting-out method is 298.74±4.04 µm.

[0041] Figure 6The microfluidic / salting-out method was used to prepare silk fibroin microspheres, which showed a coefficient of variation (CV) of 1.2%, less than 5%. This indicates that the microspheres have highly uniform particle size, a very narrow particle size distribution, excellent uniformity, and are highly monodisperse. These highly uniform microspheres are likely to exhibit better drug or biomolecule release kinetics in practical applications.

[0042] The key to preparing microspheres lies in controlling the solidification process within the spherical shape. This invention uses microfluidic technology to first form silk fibroin solution microdroplets, and then allows each droplet to undergo salting-out solidification individually. Subsequently, the silk fibroin droplets come into contact with salt ions, initiating the salting-out process.

[0043] Spatial control: The core function of microfluidics is the precise generation of droplet templates to form uniform emulsion droplets and ensure monodispersity; it provides uniformly sized reaction microreactors for subsequent salting out, with each droplet serving as a microsphere template to be prepared, i.e., spatial control.

[0044] Molecular-level phase transition induction: The molecules in silk fibroin droplets mainly exist in random coils and a small amount of α-helical structures, forming a water-soluble, thermodynamically unstable metastable state. Salt ions strongly bind to water molecules, greatly consuming free water in the system and producing a dehydration effect. Simultaneously, salt ions neutralize the surface charge of silk fibroin molecules, weakening the electrostatic repulsion between molecules and creating a charge shielding effect. These two effects work together to significantly reduce the solubility of silk fibroin, providing a thermodynamic driving force for the proximity and alignment of molecular chains. Through hydrogen bonding, the silk fibroin molecular chains spontaneously and irreversibly rearrange into a lower-energy, more stable β-sheet crystalline structure. Numerous β-sheet structures cross-link, forming a three-dimensional, water-insoluble solid network; this process, known as molecular-level phase transition induction, achieves the solidification of microspheres.

[0045] Comparative Example 1 The silk fibroin solution obtained in Example 1 was diluted with water to obtain a silk fibroin solution with a concentration of 5%.

[0046] The preparation of silk fibroin microspheres using a single salting-out method includes the following steps: A 5% silk fibroin solution was mixed with a potassium phosphate solution with an ionic strength of 1.2 M (phosphate ion concentration) and pH of 8.0 and stirred for 5 min. The mixture was then allowed to stand at room temperature for 12 hours. After that, the precipitate was washed with deionized water to obtain silk fibroin microspheres.

[0047] Scanning electron microscope images are shown Figure 4 As can be seen, most of the silk fibroin microspheres obtained by the single salting-out method are spherical, but there are also heterogeneous particles, and the particle size distribution is uneven.

[0048] from Figure 5The particle size histogram shows that the average particle size of the silk fibroin microspheres obtained by the single salting-out method is 2.22 ± 1.17 µm.

[0049] Figure 6 The figure shows the coefficient of variation (CV) of silk fibroin microspheres. This parameter is a key statistical indicator that measures the uniformity or dispersion of the particle size distribution of a microsphere population. It represents the percentage of the standard deviation of the particle size relative to the average particle size. As can be seen from the figure, the CV of silk fibroin microspheres prepared by the salting-out method is 58%, which is much greater than 20%, indicating significant polydispersity. This suggests that the microspheres have a wide particle size distribution and poor particle size uniformity.

[0050] Example 2 The silk fibroin solution obtained in Example 1 was diluted with water to obtain a silk fibroin solution with a concentration of 3%.

[0051] The preparation of silk fibroin microspheres using a microfluidic / salting-out synergistic method includes the following steps: (1) On the same microfluidic preparation platform as in Example 1, turn on the injection pump, use oleic acid as the dispersed phase and a 3% silk fibroin solution as the continuous phase, and use a confocal microfluidic chip to make the two immiscible liquid phases flow through coaxial channels respectively. Control the flow rate of the dispersed phase to 0.2 mL / h and the flow rate of the continuous phase to 60 mL / h, that is, the flow rate ratio is 1:300, and water-in-oil droplets are obtained. The reaction time is only related to the number of microspheres.

[0052] (2) The water-in-oil droplets enter a K2HPO4-KH2PO4 phosphate solution with a pH of 8 through a microfluidic outlet pipe. The salting-out cross-linking solidification mechanism promotes the precipitation and stabilization of silk fibroin, resulting in a solid-liquid mixture. After incubating the solid-liquid mixture in a 30°C oven for 30 min, the precipitate is washed with deionized water to finally obtain silk fibroin microspheres.

[0053] like Figure 7 As shown, the prepared silk fibroin microspheres have regular shapes, are complete spheres, and have uniform overall size.

[0054] from Figure 8 The bar chart shows that the average particle size of the prepared silk fibroin microspheres is 205.08±5.50 µm, and the coefficient of variation (CV) is 2.68% (less than 5%), indicating that the microspheres have a concentrated particle size distribution, highly consistent particle size, and good monodispersity.

[0055] It should be noted that the embodiments described above are only for explaining the present invention and do not constitute any limitation on the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory terms, not limiting terms. Modifications can be made to the present invention within the scope of the claims, and revisions can be made to the present invention without departing from the scope and spirit of the present invention. Although the present invention described herein relates to specific methods, materials, and embodiments, it does not mean that the present invention is limited to the specific examples disclosed herein; on the contrary, the present invention can be extended to all other methods and applications with the same function.

Claims

1. A method for preparing silk fibroin microspheres, characterized by, The method comprises the steps of: S1 obtaining a silk fibroin solution; S2 using a confocal microfluidic chip to obtain oil-in-water droplets by taking oleic acid as a dispersed phase and the silk fibroin solution as a continuous phase; S3 adding the oil-in-water droplets into a K2HPO4-KH2PO4 phosphate mixed solution to obtain a solid-liquid mixture through salting-out; S4 incubating the solid-liquid mixture to separate the precipitate as the silk fibroin microspheres.

2. The method according to claim 1, wherein the confocal microfluidic chip comprises a dispersed phase channel and a continuous phase channel, and the dispersed phase channel is inserted into the inside of the continuous phase channel to form a coaxial channel.

3. The method according to claim 1, wherein the flow rate of the dispersed phase is 100-1000 μL / h; and / or the flow rate of the continuous phase is 20-150 mL / h.

4. The method according to claim 1, wherein the concentration of the silk fibroin solution is 1wt%-10wt%; and / or the pH value of the K2HPO4-KH2PO4 phosphate mixed solution is 6.0-9.

0.

5. The method according to claim 1, wherein the incubation conditions comprise 25-60℃ and 10 min-6 hours.

6. The silk fibroin microspheres obtained by the method of any one of claims 1-5.

7. The silk fibroin microspheres according to claim 6, wherein the coefficient of variation CV of the particle size of the silk fibroin microspheres is ≤5%.

8. The silk fibroin microspheres of claim 6 or 7 for use in the field of medical materials. for use as a drug carrier, a tissue engineering scaffold material or a biosensor.

10. The silk fibroin microspheres of claim 6 or 7 for use in the field of preparing cosmetic skin care products or medical aesthetic materials. ​ ​ ​ ​ ​ 9. Use according to claim 8, characterized in that, ​ ​

Images