Method for regulating the beta sheet content and uniformity of silk fibroin microspheres

Abstract

The application relates to the technical field of natural high-molecular functional material preparation, and discloses a method for regulating the beta-folding content and uniformity of silk fibroin microspheres, which is characterized in that the silk fibroin microspheres are prepared by adopting two-stage series connection coaxial confocal microfluidic chips, and the silk fibroin microspheres are subjected to stage freezing through a specific procedure, so that the beta-folding content of the microspheres can be accurately controlled, and meanwhile, the excellent biocompatibility of the material can be maintained; the method overcomes the industry pain points that the traditional silk fibroin microsphere preparation process needs to add a crosslinking agent or modify the silk fibroin base material to prepare microspheres with uniform performance, so that toxic chemical substances are left, the biocompatibility of the material is reduced, and it is difficult to consider all the comprehensive performances; the method is a pure physical modification method, does not need a chemical crosslinking agent, does not cause molecular damage, is green and safe, and retains the biocompatibility of the silk fibroin.

Description

Technical Field

[0001] This invention relates to the field of natural polymer functional materials preparation technology, specifically to a method for regulating the β-sheet content and uniformity of silk fibroin microspheres. Background Technology

[0002] Silk fibroin is a natural structural protein extracted from silkworm cocoons. Due to its excellent biocompatibility, low immunogenicity, biodegradability, and processability, it has become a core substrate in the fields of biomedicine, flexible electronics, and smart polymer materials. The macroscopic properties of silk fibroin depend on the proportion of its secondary structures. β-sheets, the stable crystalline structure, determine the material's mechanical strength, water resistance, thermal stability, and degradation resistance. Random coils and α-helices are metastable amorphous structures, characterized by loose structure, hydrophilic solubility, poor mechanical properties, and rapid degradation.

[0003] Currently available and industry-standard technologies for the preparation and controllable mechanical properties of silk fibroin microspheres have significant shortcomings: existing processes cannot simultaneously achieve: precise and controllable β-sheet formation, absence of chemically toxic residues, uniform particle size and sphericity, high mechanical stability, and controllable degradation. Therefore, one performance indicator must be sacrificed for another, resulting in shortcomings in the overall performance of the product, primarily in the following aspects:

[0004] (1) Traditional microsphere preparation processes result in uneven particle size, porous and rough surface, and poor sphericity: Mechanically emulsified microspheres can have a particle size range of about 5 to 200 μm, but monodisperse microspheres are required in many applications. Microspheres with a large particle size range do not meet the commercial needs after multi-stage sieving, resulting in high raw material loss and increased production costs; electrospraying and freeze-drying microspheres have porous and rough surface and poor sphericity. These shortcomings become apparent in clinical applications. For example, when used as a subcutaneous filler, it can exacerbate macrophage phagocytosis, thus shortening the in vivo support period.

[0005] (2) Traditional methods of improving the mechanical properties of microspheres reduce the biocompatibility of the materials: Although the original silk fibroin microspheres without cross-linking agents have excellent biocompatibility, they have low β-sheet content, are easy to disintegrate after water solubility, have weak mechanical strength, and degrade rapidly in the human body in 1-2 weeks. Therefore, they cannot meet the needs of multiple fields such as long-lasting filling in medical aesthetics and sustained drug delivery. Although the mechanical stability can be improved and the degradation cycle can be extended by adding cross-linking / organic solvent modification (ethanol, methanol, etc.), the residual substances are very likely to cause inflammation, which reduces the biocompatibility.

[0006] (3) Insufficient conformation controllability: Existing silk fibroin microsphere preparation processes are difficult to stably and accurately control the β-sheet content of the product to a high level. Although spray drying, mechanical emulsification and other processes that can be mass-produced have high production efficiency, the high temperature environment and strong shearing during the preparation process will destroy the ordered secondary structure of silk fibroin, resulting in uneven β-sheet content. At the same time, due to the strict control of the amount of various additives, it is difficult to flexibly control the β-sheet ratio. Even if chemical cross-linking is used to adjust the β-sheet content, it can only be increased within a limited and narrow range. This defect greatly restricts the expansion of silk fibroin materials in the medical field. Summary of the Invention

[0007] This invention aims to address the problems of low and uneven β-sheet content in existing silk fibroin microsphere preparation processes. This leads to poor controllability of protein conformation, significant differences in intra-batch and inter-batch mechanical properties of microspheres, and consequently, variations in in vivo degradation rates, posing numerous clinical risks. Therefore, this invention provides a highly efficient, controllable, and scalable preparation process for silk fibroin microspheres that allows for precise control of the β-sheet content while maintaining excellent biocompatibility. It overcomes the industry pain points of traditional silk fibroin microsphere preparation processes, which require the addition of cross-linking agents or modification of the silk fibroin substrate to obtain uniformly performing microspheres, resulting in toxic chemical residues, decreased biocompatibility, and difficulty in achieving a balance of various comprehensive performance characteristics.

[0008] To achieve the above objectives, in a first aspect, the present invention provides a method for regulating the β-sheet content and uniformity of silk fibroin microspheres, comprising the following steps:

[0009] S1. Semi-finished silk fibroin microspheres were prepared using a coaxial confocal microfluidic chip consisting of a primary coaxial confocal unit and a secondary coaxial confocal unit connected in series, wherein:

[0010] Within the primary coaxial confocal unit, an aqueous solution of silk fibroin is used as the dispersed phase as the inner core fluid, and an oil phase solution containing nonionic surfactants is used as the first continuous phase as the outer annular sheath fluid. The two fluids are supplied in the same direction, coaxially, and in the same direction. When the two flow together, the dispersed phase is sheared by the first continuous phase to form a water-in-oil (W / O) emulsion, resulting in a mixed fluid containing silk fibroin droplets.

[0011] Within the secondary coaxial confocal unit, a mixed fluid containing silk fibroin droplets serves as the inner core fluid, while an oil-phase solution containing nonionic surfactants serves as the second continuous phase and the outer annular sheath fluid. The two fluids are supplied coaxially and in the same direction, and after converging, they press the silk fibroin droplets into the end channel, causing the droplets to form elongated or nearly elongated liquid strips within the end channel. After the liquid strips flow out of the end channel, they rebound and return to a spherical shape, and are then solidified to form semi-finished silk fibroin microspheres.

[0012] The relevant dimensions are as follows: the dispersed phase feed capillary is a hollow cylindrical structure with an inner diameter of 30-500 μm at its narrow end; the transition connecting capillary is a hollow cylindrical structure with an inner diameter of 70-700 μm; the end flow channel is a hollow cylindrical structure with an inner diameter of 70-700 μm and a channel length of not less than 50 mm.

[0013] S2. The cured semi-finished silk fibroin microspheres are subjected to the following post-processing in sequence:

[0014] Stage freezing: Cool to -50~-45℃, pre-freeze for 90~120min, raise temperature to -12~-8℃, hold for 90~150min, cool again to -50~-45℃, pre-freeze for 120~180min; then sublimation drying: adjust temperature to -45~-30℃, hold under vacuum for 120~300min, then raise temperature to 0~2℃, hold under vacuum for 60~120min; finally desorption drying: adjust temperature to 15~30℃, hold under vacuum for 180~240min.

[0015] As a further preferred embodiment of the present invention, the coaxial confocal microfluidic chip includes a dispersed phase feed capillary, a primary coaxial focusing cavity, a transition connecting capillary, a secondary coaxial focusing cavity, and an end channel, which are coaxially connected sequentially along the fluid flow direction. The dispersed phase feed capillary, the primary coaxial focusing cavity, and the transition connecting capillary constitute a primary coaxial confocal unit, and the transition connecting capillary, the secondary coaxial focusing cavity, and the end channel constitute a secondary coaxial confocal unit; wherein:

[0016] The dispersed phase feed capillary is coaxially inserted through the inlet end of the primary coaxial focusing cavity and extends into the primary coaxial focusing cavity to transport the dispersed phase fluid as the inner core layer.

[0017] The inlet end of the primary coaxial focusing cavity is provided with a first continuous phase feed port. The first continuous phase fluid input through the first continuous phase feed port merges with the dispersed phase fluid in the primary coaxial focusing cavity to shear the dispersed phase into silk fibroin droplets.

[0018] The transition capillary coaxially connects the outlet of the primary coaxial focusing cavity and the inlet of the secondary coaxial focusing cavity, and is used to transport the mixed fluid containing silk fibroin droplets. The transition capillary coaxially extends into the interior of the secondary coaxial focusing cavity.

[0019] The inlet end of the secondary coaxial focusing cavity is provided with a second continuous phase feed port. The second continuous phase input through the second continuous phase feed port increases the flow velocity of the external annular sheath fluid.

[0020] The end channel is coaxially connected to the outlet of the secondary coaxial focusing cavity, and a gap is reserved between the inlet of the end channel and the capillary outlet. The second continuous phase flows into the mixed fluid containing silk fibroin droplets through this gap to achieve fluid acceleration. The accelerated silk fibroin droplets are squeezed into the end channel and simultaneously formed into long strips or near-long strips by the constraints of the end channel, the compression of the continuous phase fluid, and shear stretching. The silk fibroin molecular chains inside the strips are stretched, the original secondary structure and hydrogen bonds break and molecular chain rearrangement occurs, inducing the generation of silk fibroin with regular enriched β-sheet structure. When the long strips flow out of the end channel, after the channel constraint and shear stress disappear, the long strips rebound and recover into spherical shape. After solidification, silk fibroin microsphere semi-finished products are obtained.

[0021] As a further preferred technical solution of the present invention, the dispersed phase feed capillary extends into the primary coaxial focusing cavity and forms a flow channel with a converging throat. The cross-sectional area of ​​the converging throat flow channel continuously narrows along the fluid flow direction. The inner diameter of the narrow opening at the end of the throat is smaller than the inner diameter of the inlet end of the dispersed phase feed capillary, and the narrow opening at the end of the throat is coaxially connected to the transition connecting capillary.

[0022] As a further preferred embodiment of the present invention, the first continuous phase inlet is vertically opened on the inlet sidewall of the primary coaxial focusing cavity; the second continuous phase inlet is vertically opened on the inlet sidewall of the secondary coaxial focusing cavity.

[0023] As a further preferred technical solution of the present invention, the curing is carried out at a temperature of 30~50℃. At the same time, in order to ensure uniform heating, slow stirring can be used as an aid. After the curing reaction is completed, the silk fibroin microspheres can be collected by filtration and washed multiple times with ethanol, deionized water, etc.

[0024] As a further preferred embodiment of the present invention, the silk fibroin content in the dispersed phase fluid is 0.5-10 wt%; and / or, the concentration of nonionic surfactant in the oil phase solution is 0.5-20 wt%.

[0025] As a further preferred embodiment of the present invention, the nonionic surfactant is selected from at least one of PGPR, SPAN80, SPAN60, SPAN20, PFPE-PEG, Brij-35, Brij-78, Tween 20, Tween 60, or Tween 80; the oil phase in the oil phase solution is selected from at least one of soybean oil, castor oil, liquid paraffin, mineral oil, fluorinated oil, and silicone oil. In the present invention, the first continuous phase and the second continuous phase are independently formulated using the above-mentioned oil phase and nonionic surfactant.

[0026] As a further preferred technical solution of the present invention, the size of the semi-finished silk fibroin microspheres prepared by the two-stage series coaxial confocal microfluidic chip is 50-1200 μm.

[0027] As a further preferred technical solution of the present invention, the inner diameter of the end channel and the continuous phase work together to constrain the silk fibroin droplets in the end channel, forming a long strip or near-long strip of liquid.

[0028] As a further preferred technical solution of the present invention, in the staged freezing step, the cooling time to -50~-45℃ is within 50~120 min, the heating time to -12~-8℃ is within 100~150 min, and the cooling time to -50~-45℃ is within 100~150 min; the vacuum degree of the staged sublimation drying is 50 μbar~500 mbar, and the vacuum degree of the desorption drying is 100~990 mbar; in the staged sublimation drying step, the heating time to -45~-30℃ is within 30~200 min, and the heating time to 0~2℃ is within 100~600 min.

[0029] This invention utilizes the operating principle of a two-stage tandem coaxial confocal microfluidic chip: A dispersed phase of silk fibroin aqueous solution flows into the dispersed phase feed capillary, passes through the throat-constricting section within the first-stage coaxial focusing chamber, and is sheared into water-in-oil (W / O) emulsion silk fibroin droplets by the first continuous phase at the transition capillary junction. As the fluid further propels, it enters the second-stage coaxial focusing chamber, where the addition of a second continuous phase increases the flow rate, accelerating the silk fibroin droplets into the terminal channel. The droplets are compressed by the channel size while being sheared and stretched again by the continuous phase, resulting in an elongated shape. During this process, under the stretching force, the silk fibroin molecular chains undergo orientation extension, breaking the original intramolecular and intermolecular hydrogen bonds, and rearranging and orderly stacking of the molecular chain conformation, thus forming a thermodynamically stable β-sheet conformation and becoming enriched. After the droplets flow out, as the channel constraint stress disappears, they recover from an elongated shape to a spherical shape, and finally, after solidification, a semi-finished silk fibroin microsphere is obtained.

[0030] Compared with the prior art, the present invention can achieve the following beneficial effects:

[0031] Microsphere forming stage: The dispersed phase fluid flows out through the feed capillary, while the first continuous phase flows in the same direction coaxially in the primary coaxial focusing cavity. Due to the axial velocity difference between the two phases, axisymmetric viscous shear stress is generated at the throat of the feed capillary. The shear force continuously stretches the outflowing dispersed phase fluid and competes with the interfacial tension. When the shear stretching exceeds the interfacial tension constraint, the dispersed phase liquid neck undergoes necking and fracture, forming microdroplet emulsion. In this process, the tensile stress induced by the flow field can promote the orientation of silk fibroin molecular chain segments, reduce the proportion of random coil conformation, and induce some chain segments to transform into β-sheet conformation. Microdroplets are propelled into the transitional capillary by the first continuous phase liquid. At the outlet end, the inflow of the second continuous phase increases the overall continuous phase velocity, squeezing the microdroplets into the terminal channel. The microdroplets are constrained by the channel diameter and compressed by the fluid on the inner wall, while simultaneously subjected to the thrust of the high-velocity continuous phase. This causes restricted stretching and deformation of the droplets as they pass through the narrow channel, further extending and oriented the silk fibroin molecular chains within the droplets, thus promoting the formation and stabilization of β-sheet structures. Therefore, through the induction of a two-stage continuous flow field, the conformational transformation process of silk fibroin molecules can be gradually controlled, increasing the β-sheet content within the microspheres. Furthermore, adjusting the inner diameter or length of the terminal channel can regulate the droplet stretching, thereby initially controlling the β-sheet content. As the liquid escapes the constraint of the terminal channel opening, due to gas-liquid interfacial tension and high molecular viscoelasticity, the droplets regain their spherical shape. The entire process achieves microscopic β-sheet enhancement and macroscopic relaxation into spherical formation.

[0032] Post-processing stage: The microspheres undergo a specific freeze-drying and annealing process. Low-temperature freeze-drying ensures the protein molecules' properties are not damaged, maintaining their complete bioactivity. Simultaneously, an annealing process is introduced during the freeze-drying cycle, raising the frozen silk fibroin microspheres to a specific temperature below their eutectic point and holding it for a period. The protein polypeptide chains, previously frozen and locked, gain freedom of movement. After movement, hydrophobic segments move closer together, and numerous inter-chain hydrogen bonds reform within and between molecules. Some random coils and other amorphous conformational regions undergo segment rearrangement, gradually forming a more stable β-sheet crystal structure, further increasing the proportion of β-sheets and enhancing their stability.

[0033] This invention achieves efficient and uniform transformation of random coils and α-helices into stable antiparallel β-sheets through multi-step synergistic regulation of silk fibroin molecules, resulting in the stable preparation of silk fibroin microspheres with high mechanical controllability, breaking away from traditional single-modification models. Drawing inspiration from the flow field-induced orientation and crystallization regulation mechanisms during natural silkworm cocoon spinning, it utilizes the dual shearing effects of the macroscopic channel stretching flow field of a microfluidic chip and the closed vortex field inside the microdroplets to achieve full untangling, straightening, and orderly arrangement of silk fibroin peptide chains. This actively drives intramolecular hydrogen bond breaking and intermolecular hydrogen bond reconstruction, completing in-situ efficient nucleation of β-sheets. Simultaneously, low-temperature annealing solidifies the crystalline structure, eliminates intramolecular stress, reduces conformational reversion tendency, and improves structural stability, achieving simultaneous completion of microscale conformational transformation and structure locking. The entire process employs a purely physical modification method, avoiding the potential toxicity and irreversible changes in molecular structure caused by chemical cross-linking, ensuring green safety and fully preserving the biocompatibility of silk fibroin. Attached Figure Description

[0034] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0035] Figure 1 This is a schematic diagram of a two-stage series coaxial confocal microfluidic chip.

[0036] Figure 2 The image shows a scanning electron microscope (SEM) image of the freeze-dried silk fibroin microspheres prepared in Example 1.

[0037] The objectives, features, and advantages of this invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0038] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0039] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art to which this invention pertains. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0040] Example 1

[0041] This embodiment provides a method for preparing silk fibroin microspheres with regulated β-sheet content and uniformity. The specific steps are as follows:

[0042] S1. Semi-finished silk fibroin microspheres were prepared using a two-stage series coaxial confocal microfluidic chip.

[0043] like Figure 1As shown, the coaxial confocal microfluidic chip in this example includes a dispersed phase feed capillary 1, a primary coaxial focusing chamber 2, a transition connecting capillary 3, a secondary coaxial focusing chamber 4, and an end flow channel 5, which are coaxially connected in sequence along the fluid flow direction; wherein the dispersed phase feed capillary 1, the primary coaxial focusing chamber 2, and the transition connecting capillary 3 constitute the primary coaxial confocal unit, and the transition connecting capillary 3, the secondary coaxial focusing chamber 4, and the end flow channel 5 constitute the secondary coaxial confocal unit.

[0044] A dispersed phase feed capillary coaxially passes through the inlet end of the primary coaxial focusing chamber and extends into the chamber to form a converging, tapering throat channel for conveying the dispersed phase as the internal core fluid. The cross-sectional area of ​​this tapering throat channel continuously narrows along the fluid flow direction, with the inner diameter of its narrow throat end smaller than the inner diameter of the dispersed phase feed capillary. The narrow throat end coaxially connects to a transition capillary. The inlet end of the primary coaxial focusing chamber has an inlet for inputting the first continuous phase, which is used to input the dispersed phase relative to the tapering throat channel as the first continuous phase of the external annular sheath fluid. The transition capillary coaxially connects the outlet of the primary coaxial focusing chamber to the inlet of the secondary coaxial focusing chamber, and extends coaxially into the interior of the secondary coaxial focusing chamber. The inlet end of the secondary coaxial focusing chamber has an inlet for inputting the second continuous phase, which is used to input the mixed fluid containing silk fibroin droplets relative to the transition capillary as the second continuous phase of the external annular sheath fluid. The end flow channel is coaxially connected to the outlet of the secondary coaxial focusing cavity. For example... Figure 1 As shown, all fluids are transported from left to right.

[0045] The relevant dimensions are as follows: the dispersed phase feed capillary is a hollow cylindrical structure, and the inlet inner diameter of its tapered throat channel is 30 μm at the narrow end of the throat; the transition connecting capillary is a hollow cylindrical structure with an inner diameter of 70 μm; the end channel is a hollow cylindrical structure with an inner diameter of 70 μm and a channel length of 50 mm.

[0046] The specific process for preparing silk fibroin microspheres using the above-mentioned two-stage cascaded coaxial confocal microfluidic chip is as follows:

[0047] Within the primary coaxial confocal unit: a 3% (w / w) silk fibroin aqueous solution is used as the dispersed phase, which is fed into the primary coaxial focusing cavity via the dispersed phase feed capillary as the internal core fluid; soybean oil containing 10% (w / w) PGPR is used as the first continuous phase, which is fed into the primary coaxial focusing cavity via the first continuous phase feed inlet as the external annular sheath fluid; the two fluid layers are supplied coaxially and in the same direction, with the dispersed silk fibroin solution flowing at a rate of 50 μL / min and the first continuous phase flowing at a rate of 200 μL / min. When the two flow together, the dispersed phase is sheared by the first continuous phase to form silk fibroin droplets, resulting in a mixed fluid containing silk fibroin droplets that is transported along the transition connecting capillary. The droplet size is 50 μm.

[0048] Within the secondary coaxial confocal unit: a mixed fluid containing silk fibroin droplets serves as the inner core fluid, transported to the secondary coaxial focusing cavity via a transitional capillary; soybean oil containing 10% PGPR by mass serves as the second continuous phase, fed into the secondary coaxial focusing cavity through the inlet of the second continuous phase, serving as the outer annular sheath fluid; these two fluids are supplied coaxially and in the same direction, with the second continuous phase flowing at a rate of 100 μL / min. After converging, the silk fibroin droplets are forced into the end channel, forming elongated liquid strips within the end channel; as the elongated liquid strips flow out of the end channel, they rebound and return to spherical droplets, falling into a receiving liquid equipped with a top stirrer. The receiving liquid consists of soybean oil containing 10% PGPR by mass. After being slowly stirred at 40°C for more than 8 hours, a mixed solution containing solidified silk fibroin microspheres is obtained; finally, the mixture is filtered and washed with ethanol and deionized water to obtain a semi-finished silk fibroin microsphere product.

[0049] S2. Microsphere post-treatment (freeze-drying)

[0050] The semi-finished silk fibroin microspheres were subjected to staged freezing, staged sublimation drying, and desorption drying according to the process in Table 1, finally yielding freeze-dried silk fibroin microspheres (e.g., Figure 2 ).

[0051] Table 1

[0052]

[0053] Three batches of silk fibroin microspheres were prepared in parallel using the method described in Example 1. The secondary conformation of silk fibroin at each stage was tested, and the results are shown in Table 2.

[0054] Table 2

[0055]

[0056] The results above show that after shearing and solidification, the silk fibroin solution forms microspheres with a significant increase in β-sheet content. After freeze-drying, the β-sheet content increases to an average of over 80%, and the β-sheet content of the three batches of silk fibroin microspheres after solidification and freeze-drying shows a high degree of parallelism.

[0057] Example 2

[0058] S1. Semi-finished silk fibroin microspheres were prepared using a two-stage cascaded coaxial confocal microfluidic chip. The difference between this step and Example 1 is as follows: ① Chip dimensions: The dispersed phase feed capillary is a hollow cylinder with an inlet inner diameter of 60 μm at the narrow throat; the transition capillary is a hollow cylinder with an inner diameter of 200 μm; the end channel is a hollow cylinder with an inner diameter of 70 μm and a channel length of 50 mm. ② The flow rate of the dispersed phase silk fibroin solution is 100 μL / min, the first continuous phase flow rate is 200 μL / min, and the second continuous phase flow rate is 100 μL / min. The droplet size after exiting the capillary is 180 μm.

[0059] S2, Microsphere Post-processing

[0060] The semi-finished silk fibroin microspheres were subjected to staged freezing, staged sublimation drying, and desorption drying according to the process in Table 3, and finally freeze-dried silk fibroin microspheres were obtained.

[0061] Table 3

[0062]

[0063] Three batches of silk fibroin microspheres were prepared in parallel using the method described in Example 2. The secondary conformation of silk fibroin at each stage was tested, and the results are shown in Table 4.

[0064] Table 4

[0065]

[0066] The results above show that after shearing and solidification, the silk fibroin solution forms microspheres with a significant increase in β-sheet content. After freeze-drying, the β-sheet content rises to over 85%, and the β-sheet content of the three batches of silk fibroin microspheres after solidification and freeze-drying shows a high degree of parallelism.

[0067] Example 3

[0068] S1. Semi-finished silk fibroin microspheres were prepared using a two-stage cascaded coaxial confocal microfluidic chip. The difference between this step and Example 2 is that ① only the channel length of the end flow channel was adjusted to 100 mm. The droplet size after flowing out through the capillary was 180 μm.

[0069] S2, Microsphere Post-processing

[0070] The semi-finished silk fibroin microspheres were subjected to staged freezing, staged sublimation drying, and desorption drying in sequence according to the process in Table 5, and finally freeze-dried silk fibroin microspheres were obtained.

[0071] Table 5

[0072]

[0073] Three batches of silk fibroin microspheres were prepared in parallel using the method described in Example 3. The secondary conformation of silk fibroin at each stage was tested, and the results are shown in Table 6.

[0074] Table 6

[0075]

[0076] The results above show that after shearing and solidification, the silk fibroin solution forms microspheres with a significant increase in β-sheet content. After freeze-drying, the β-sheet content rises stably and uniformly to over 90%, and the β-sheet content of the three batches of silk fibroin microspheres after solidification and freeze-drying has a high degree of parallelism.

[0077] Example 4

[0078] S1. Semi-finished silk fibroin microspheres were prepared using a two-stage tandem coaxial confocal microfluidic chip. The difference from Example 1 is as follows: ① Chip dimensions: The dispersed phase feed capillary is a hollow cylinder with an inlet inner diameter of 500 μm at the narrow throat; the transition capillary is a hollow cylinder with an inner diameter of 700 μm; the terminal channel is a hollow cylinder with an inner diameter of 400 μm and a channel length of 200 mm. ② The mass percentage concentration of silk fibroin in the dispersed phase was adjusted to 6%, and the first and second continuous phases contained castor oil with a mass percentage concentration of 10% Span 80. ③ The dispersed phase flow rate was 300 μL / min, the first continuous phase flow rate was 1000 μL / min, and the second continuous phase flow rate was 500 μL / min. The droplet size after exiting the capillary was 1200 μm.

[0079] S2, Microsphere Post-processing

[0080] The semi-finished silk fibroin microspheres were subjected to staged freezing, staged sublimation drying, and desorption drying according to the process in Table 7, and finally freeze-dried silk fibroin microspheres were obtained.

[0081] Table 7

[0082]

[0083] Three batches of silk fibroin microspheres were prepared in parallel using the method described in Example 4. The secondary conformation of silk fibroin at each stage was tested, and the results are shown in Table 8.

[0084] Table 8

[0085]

[0086] The results above show that the β-sheet content of the silk fibroin solution formed after shearing and solidification is significantly increased. After freeze-drying, the β-sheet content rises to over 90%, and the β-sheet content of the three batches of silk fibroin microspheres after solidification and freeze-drying has a high degree of parallelism.

[0087] Comparative Example 1

[0088] S1. Semi-finished silk fibroin microspheres were prepared using a coaxial confocal microfluidic chip. The only difference between this chip and the two-stage tandem coaxial confocal microfluidic chip in Example 1 is that it lacks a secondary coaxial focusing cavity and end channel. The dimensions of the dispersed phase feed capillary, the primary coaxial focusing cavity, and the transition connecting capillary are consistent with those in Example 1. The droplet size after flowing out of the intermediate capillary is 50 μm.

[0089] S2, Microsphere Post-processing

[0090] The semi-finished silk fibroin microspheres were subjected to staged freezing, staged sublimation drying, and desorption drying according to the process in Table 1, and finally freeze-dried silk fibroin microspheres were obtained.

[0091] Three batches of silk fibroin microspheres were prepared in parallel using the method of Comparative Example 1, and the secondary conformation of silk fibroin at each stage was tested. The results are shown in Table 9.

[0092] Table 9

[0093]

[0094] Three batches of silk fibroin microspheres were prepared in parallel using the above method and then freeze-dried. The results showed that although the β-sheet content of the silk fibroin microspheres prepared by the microfluidic device without a secondary coaxial focusing cavity and end channel also increased, the batch-to-batch uniformity was poor. After freeze-drying, the β-sheet content increased, but it failed to increase stably and uniformly to 80%.

[0095] Comparative Example 2

[0096] S1. Semi-finished silk fibroin microspheres were prepared using a two-stage cascaded coaxial confocal microfluidic chip, identical to that in Example 1. The droplet size after flowing out through the capillary was 50 μm.

[0097] S2. Microsphere post-processing:

[0098] The semi-finished silk fibroin microspheres were thoroughly dried at 25°C and under a vacuum of 200 mbar to obtain the dried silk fibroin microspheres.

[0099] Three batches of silk fibroin microspheres were prepared in parallel using the method described in Comparative Example 2. The secondary conformation of silk fibroin at each stage was tested, and the results are shown in Table 10.

[0100] Table 10

[0101]

[0102] Three batches of silk fibroin microspheres were prepared in parallel using the above method and then freeze-dried. The results showed that the solidified silk fibroin microspheres, without freeze-drying annealing, only underwent simple drying. Although the β-sheet content of the microspheres increased, the batch-to-batch uniformity was poor, and the β-sheet content could not be stably and uniformly increased to 80%.

[0103] Comparative Example 3

[0104] S1. Semi-finished silk fibroin microspheres were prepared using a two-stage series coaxial confocal microfluidic chip, the same as in Example 1; the droplet size after flowing out through the capillary was 50 μm.

[0105] S2, Microsphere Post-processing

[0106] The semi-finished silk fibroin microspheres were subjected to freezing, staged sublimation drying, and desorption drying in sequence according to the process in Table 11, and finally freeze-dried silk fibroin microspheres were obtained.

[0107] Table 11

[0108]

[0109] Three batches of silk fibroin microspheres were prepared in parallel using the method described in Comparative Example 3. The secondary conformation of silk fibroin at each stage was tested, and the results are shown in Table 12.

[0110] Table 12

[0111]

[0112] The results show that although the β-sheet content of microspheres dried using the freeze-drying process without annealing increases, the batch-to-batch uniformity is poor, the increase in β-sheet content is insufficient, and the average level fails to reach 80%.

[0113] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. The scope of protection of the present invention is defined only by the appended claims.

Claims

1. A method for regulating the β-sheet content and uniformity of silk fibroin microspheres, characterized in that, Includes the following steps: S1. Semi-finished silk fibroin microspheres were prepared using a two-stage cascaded coaxial confocal microfluidic chip, wherein: Within its primary coaxial confocal unit, a silk fibroin aqueous solution is used as the dispersed phase as the inner core fluid, and an oil phase solution containing nonionic surfactants is used as the first continuous phase as the outer annular sheath fluid. The two fluids are supplied in the same direction, coaxially, and in the same direction. When the two flow together, the dispersed phase is sheared by the first continuous phase to form silk fibroin droplets, thus obtaining a mixed fluid containing silk fibroin droplets. Within its secondary coaxial confocal unit, the mixed fluid containing silk fibroin droplets obtained above is used as the inner core fluid, and an oil phase solution containing nonionic surfactants is used as the second continuous phase as the outer annular sheath fluid. The two fluids are supplied coaxially and in the same direction in layers. After the two flow into each other, the silk fibroin droplets are pressed into the end channel, and the silk fibroin droplets form long strips or near-long strips of liquid as they are transported along the end channel. When the liquid strips flow out of the end channel, they rebound and return to a spherical shape. After solidification, a semi-finished product of silk fibroin microspheres is formed. S2. The semi-finished silk fibroin microspheres obtained in S1 are then post-processed according to the following procedure: Stage freezing: First, lower the temperature to -50~-45℃ and hold for 90~120 min, then raise the temperature to -12~-8℃ and hold for 90~150 min, then lower the temperature to -50~-45℃ and hold for 120~180 min; Stage sublimation drying: First adjust the temperature to -45~-30℃, maintain it under vacuum for 120~300min, then raise the temperature to 0~2℃, maintain it under vacuum for 60~120min; Desorption and drying: Adjust the temperature to 15~30℃ and maintain under vacuum for 180~240 min.

2. The method for regulating the β-sheet content and uniformity of silk fibroin microspheres according to claim 1, characterized in that, The coaxial confocal microfluidic chip includes a dispersed phase feed capillary, a primary coaxial focusing cavity, a transition connecting capillary, a secondary coaxial focusing cavity, and an end channel that are coaxially connected in sequence along the fluid flow direction. The dispersed phase feed capillary is coaxially inserted through the inlet end of the primary coaxial focusing cavity and extends into the interior of the primary coaxial focusing cavity to transport the dispersed phase, which serves as the internal core fluid; the primary coaxial focusing cavity is provided with a feed port for inputting the first continuous phase. The transition capillary coaxially connects the outlet of the primary coaxial focusing cavity and the inlet of the secondary coaxial focusing cavity, and is used to transport the mixed fluid containing silk fibroin droplets. The transition capillary coaxially extends into the interior of the secondary coaxial focusing cavity. The secondary coaxial focusing cavity is provided with an inlet for inputting the second continuous phase. The end flow channel coaxially connects to the outlet of the secondary coaxial focusing cavity. The first-stage coaxial confocal unit consists of a dispersed phase feed capillary, a first-stage coaxial focusing cavity, and a transition connecting capillary; the second-stage coaxial confocal unit consists of a transition connecting capillary, a second-stage coaxial focusing cavity, and an end flow channel.

3. The method for regulating the β-sheet content and uniformity of silk fibroin microspheres according to claim 1, characterized in that, The dispersed phase feed capillary extends into the primary coaxial focusing cavity to form a converging throat flow channel. The cross-sectional area of ​​the converging throat flow channel continuously narrows along the fluid flow direction, and the inner diameter of the narrow opening at the end of the throat is smaller than the inner diameter of the dispersed phase feed capillary.

4. The method for regulating the β-sheet content and uniformity of silk fibroin microspheres according to claim 3, characterized in that, The narrow opening at the end of the throat is coaxially connected to the transition connecting capillary.

5. The method for regulating the β-sheet content and uniformity of silk fibroin microspheres according to claim 1, characterized in that, The feed inlet of the first continuous phase is vertically opened at the inlet end of the primary coaxial focusing cavity and located outside the dispersed phase feed capillary; the feed inlet of the second continuous phase is vertically opened at the inlet end of the secondary coaxial focusing cavity and located outside the transition connecting capillary.

6. The method for regulating the β-sheet content and uniformity of silk fibroin microspheres according to claim 1, characterized in that, The silk fibroin content in the dispersed phase fluid is 0.5-10 wt%; and / or, the concentration of nonionic surfactant in the oil phase solution is 0.5-20 wt%.

7. The method for regulating the β-sheet content and uniformity of silk fibroin microspheres according to claim 1, characterized in that, The nonionic surfactant is selected from at least one of PGPR, SPAN80, SPAN60, SPAN20, PFPE-PEG, Brij-35, Brij-78, Tween 20, Tween 60 or Tween 80.

8. The method for regulating the β-sheet content and uniformity of silk fibroin microspheres according to claim 1, characterized in that, The oil phase in the oil phase solution is selected from at least one of soybean oil, castor oil, liquid paraffin, mineral oil, fluorinated oil, and silicone oil.

9. The method for regulating the β-sheet content and uniformity of silk fibroin microspheres according to claim 1, characterized in that, The semi-finished silk fibroin microspheres prepared by the two-stage tandem coaxial confocal microfluidic chip have a size of 50-1200 μm.

10. The method for regulating the β-sheet content and uniformity of silk fibroin microspheres according to claim 1, characterized in that, In the staged freezing step, the cooling time to -50~-45℃ is within 50~120 min, the heating time to -12~-8℃ is within 100~150 min, and the cooling time to -50~-45℃ is within 100~150 min; the vacuum degree of the staged sublimation drying is 50 μbar~500 mbar, and the vacuum degree of the desorption drying is 100~990 mbar; in the staged sublimation drying step, the heating time to -45~-30℃ is within 30~200 min, and the heating time to 0~2℃ is within 100~600 min.

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