A bioactive protein microsphere preparation and a method for preparing the same

Abstract

This invention belongs to the field of biomedical engineering technology, specifically relating to a bioactive protein microsphere formulation and its preparation method. The bioactive protein microsphere formulation comprises the following components: basic fibroblast growth factor, sulfated hyaluronic acid, gelatin, sodium alginate, sodium hyaluronate, trehalose, mannitol, and a permeability protectant. This invention constructs a non-covalent complex aggregate system of bioactive protein and sulfated hyaluronic acid under mild conditions, and combines this with low-shear confined dispersion and calcium salt ion cross-linking to form microspheres. This allows the bioactive protein to disperse within the microspheres in the form of complex aggregated microdomains, thereby achieving the goals of protecting protein activity, regulating release behavior, and prolonging the local action time on the wound surface.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering technology, specifically relating to a bioactive protein microsphere formulation and its preparation method. Background Technology

[0002] Bioactive proteins such as basic fibroblast growth factor and vascular endothelial growth factor play important roles in wound repair, such as promoting cell proliferation, promoting angiogenesis and accelerating tissue regeneration. Therefore, they are widely used in wound dressings and related medical preparations.

[0003] However, existing bioactive protein formulations still have significant shortcomings in practical applications. First, bioactive proteins themselves have poor structural stability and are easily affected by changes in temperature, pH, and ionic strength in solution, leading to conformational changes and a decrease in biological activity. Second, the wound environment contains a large number of proteases and a complex ionic environment, which can further accelerate protein degradation and significantly shorten its effective duration.

[0004] To address these issues, existing technologies typically employ carriers such as microspheres, liposomes, or hydrogels to encapsulate or load bioactive proteins, aiming to achieve a sustained-release effect. However, traditional microsphere preparation methods often involve direct encapsulation or one-time mixing to form microspheres, resulting in a relatively random distribution of bioactive proteins within the system. Some proteins tend to accumulate on the surface of the microspheres, leading to a burst release phenomenon upon contact with wound exudate. This results in an initially rapid release followed by insufficient release later, making it difficult to achieve a stable and sustained therapeutic effect.

[0005] Furthermore, bioactive proteins in existing microsphere systems are prone to aggregation or inactivation during microsphere formation and freeze-drying, especially under high-shear mixing or inappropriate cross-linking conditions, which can easily disrupt the protein's spatial conformation and further reduce its bioactivity. Meanwhile, traditional carrier systems have limited ability to regulate protein distribution within the microstructure, making it difficult to achieve precise control over protein release pathways and kinetics. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a bioactive protein microsphere formulation and its preparation method. This addresses the problems of poor stability, easy inactivation, significant burst release, and short duration of action of bioactive proteins during wound application in existing technologies. The present invention constructs a non-covalent composite aggregation system of bioactive protein and sulfated hyaluronic acid under mild conditions, and combines this with low-shear confined dispersion and calcium salt ion cross-linking to form microspheres. This allows the bioactive protein to disperse within the microspheres in the form of composite aggregated microdomains, thereby achieving the goals of protecting protein activity, regulating release behavior, and prolonging the local action time on the wound surface.

[0007] The technical objective of this invention is achieved through the following technical solution: The first aspect of the present invention is to provide a bioactive protein microsphere formulation, the raw material composition of which includes the following components: basic fibroblast growth factor, sulfated hyaluronic acid, gelatin, sodium alginate, sodium hyaluronate, trehalose, mannitol and a permeation protectant.

[0008] Preferably, the basic fibroblast growth factor is recombinant basic fibroblast growth factor.

[0009] Preferably, the sulfated hyaluronic acid has a weight-average molecular weight of 20–100 kDa and a degree of sulfate substitution of 0.4–1.0.

[0010] Preferably, the gel strength of the gelatin is 80 to 220 Bloom.

[0011] Preferably, the viscosity of a 1 wt% aqueous solution of sodium alginate at 25°C is 50–500 mPa·s.

[0012] Preferably, the sodium hyaluronate has a weight-average molecular weight of 50–300 kDa.

[0013] Preferably, the permeation protectant is selected from one of L-arginine, glycine, proline and betaine; more preferably, it is L-arginine.

[0014] A second aspect of the present invention provides a method for preparing a bioactive protein microsphere formulation, comprising the following steps: S1: Add basic fibroblast growth factor to buffer solution, mix until dissolved, and add trehalose and osmotic protectant to obtain basic fibroblast growth factor protection solution; S2: Add sulfated hyaluronic acid to the buffer solution, dissolve and filter to obtain sulfated hyaluronic acid solution; S3: Add the sulfated hyaluronic acid solution obtained in S2 to the basic fibroblast growth factor protective solution obtained in S1, so that the basic fibroblast growth factor and sulfated hyaluronic acid undergo non-covalent complexation to form a composite coagulation solution containing a composite coagulation microdomain of basic fibroblast growth factor-sulfated hyaluronic acid. S4: Add gelatin, sodium alginate, sodium hyaluronate, trehalose, mannitol and permeation protectant to an aqueous medium, stir and dissolve evenly to obtain a hydrophilic microsphere matrix solution; S5: Add the basic fibroblast growth factor-sulfated hyaluronic acid composite coagulation solution obtained in S3 to the hydrophilic microsphere matrix solution obtained in S4, and mix under low shear conditions to obtain a sphere-forming precursor solution containing composite coagulation microdomains. S6: The sphere-forming precursor solution obtained in S5 is added to the calcium salt crosslinking solution in the form of droplets by electrostatic spraying, so that sodium alginate undergoes ionic crosslinking to obtain primary microspheres containing basic fibroblast growth factor-sulfated hyaluronic acid composite coagulation microdomains. S7: The primary microspheres obtained in S6 are allowed to stand and stabilize, allowing the gelatin matrix to undergo physical gelation. Then, the microspheres are washed with a protective washing solution to remove the high concentration of calcium salt crosslinking liquid and unencapsulated components remaining on the surface of the microspheres. S8: Immerse the washed wet microspheres in the freeze-drying protective solution and allow them to stand for a period of time. S9: The wet microsphere system containing the lyophilization protective solution obtained in S8 is pre-frozen and then vacuum freeze-dried to obtain a bioactive protein microsphere formulation.

[0015] Preferably, in step S1, the trehalose and the permeation protectant are used to reduce the risk of aggregation of basic fibroblast growth factor during dissolution and compounding; in step S4, the trehalose, mannitol, and the permeation protectant are used to improve the protein stability in the pre-spheroidizing solution and the microsphere matrix environment; in step S8, the lyophilization protectant is used to improve the structural stability and protein activity retention rate of the microsphere formulation during lyophilization and reconstitution.

[0016] Preferably, in step S1, the buffer solution is HEPES buffer, the concentration of HEPES buffer is 5-20 mM, and the pH is 6.5-7.0; the basic fibroblast growth factor solution contains a basic fibroblast growth factor concentration of 0.05-0.2 mg / mL, a trehalose concentration of 3-6 wt%, and a permeation protectant concentration of 0.05-0.2 wt%.

[0017] Preferably, in step S2, the buffer solution is a HEPES buffer solution with a concentration of 5–20 mM and a pH of 6.5–7.0; the mass concentration of the sulfated hyaluronic acid solution is 0.1–0.6 mg / mL.

[0018] Preferably, in step S3, the mass ratio of the basic fibroblast growth factor to the sulfated hyaluronic acid is 1:4 to 1:8; in this step, the pH of the system is controlled at 6.5 to 7.0, and the temperature is controlled at 2 to 8°C; after the addition is completed, the mixture is gently mixed at 2 to 8°C for 15 to 40 minutes to keep the resulting composite coagulated liquid in a uniformly dispersed state without obvious flocculent precipitates or sediments.

[0019] Preferably, in step S3, the composite coagulation microdomain is a protein-rich region dispersed in the composite coagulation liquid, formed by basic fibroblast growth factor and sulfated hyaluronic acid through electrostatic interaction, hydrogen bonding and affinity binding.

[0020] Preferably, in step S4, the aqueous medium is deionized water or a buffer solution; in the hydrophilic microsphere matrix solution, the mass concentration of gelatin is 3-6 wt%, the mass concentration of sodium alginate is 0.8-2.0 wt%, the mass concentration of sodium hyaluronate is 0.1-0.6 wt%, the mass concentration of trehalose is 2-6 wt%, the mass concentration of mannitol is 0.5-3 wt%, and the mass concentration of the osmotic protectant is 0.02-0.2 wt%; the pH of the hydrophilic microsphere matrix solution is 6.6-7.2; the stirring and dissolving temperature is 30-40°C, and the stirring time is 30-120 min.

[0021] Preferably, in step S5, the volume ratio of the composite coagulation liquid to the hydrophilic microsphere matrix liquid is 1:4 to 1:7; the mixing temperature is 25 to 32°C, the stirring speed is 80 to 180 rpm, and the mixing time is 8 to 20 min; the composite coagulated microdomains are dynamic aggregates formed by non-covalent interactions and have a certain stability under low shear conditions. Low shear mixing is used in this step to keep the composite coagulated microdomains formed in S3 in a dispersed state in the pre-sphere formation liquid and to confine them inside the microspheres during the subsequent ionic crosslinking process.

[0022] Preferably, in step S6, the calcium salt crosslinking solution is one of calcium chloride solution, calcium gluconate solution, or calcium lactate solution; more preferably, it is a calcium chloride solution.

[0023] Preferably, in step S6, the inner diameter of the electrostatic spraying needle is 100-250 μm, the liquid inlet rate is 0.2-1.0 mL / h, the voltage is 5-10 kV, and the distance from the nozzle outlet to the surface of the calcium salt crosslinking liquid is 8-15 cm.

[0024] Preferably, in step S6, the calcium salt in the calcium salt crosslinking solution has a mass concentration of 1.0–3.0 wt%, a pH of 6.8–7.2, and a temperature of 4–12 °C; 2–4 wt% trehalose is added to the calcium salt crosslinking solution to reduce osmotic impact during the sphere formation process; the crosslinking time after sphere formation is 8–20 min; and the average particle size of the obtained primary microspheres is 30–100 μm.

[0025] Preferably, in step S7, the protective washing solution is a HEPES buffer solution with a pH of 6.8 to 7.2, containing 0.2 to 0.8 wt% trehalose and 0.02 to 0.1 wt% calcium chloride.

[0026] Preferably, in step S8, the freeze-drying protective solution is an aqueous solution with a pH of 6.8 to 7.2, containing 5 to 8 wt% trehalose, 1 to 3 wt% mannitol, and 0.05 to 0.2 wt% permeation protectant; the mass-to-volume ratio of the wet microspheres to the freeze-drying protective solution is 1 g: 5 to 10 mL; and the settling temperature is 4 to 12 °C, and the settling time is 20 to 60 min.

[0027] Preferably, in step S9, the pre-freezing temperature is -40 to -60°C, and the pre-freezing time is 3 to 6 hours; the first drying temperature is -35 to -20°C, the vacuum degree is 0.05 to 0.2 mbar, and the drying time is 18 to 30 hours; the second drying temperature is 10 to 25°C, the vacuum degree is 0.05 to 0.2 mbar, and the drying time is 6 to 12 hours.

[0028] The resulting bioactive protein microsphere formulation is a resolvable and dispersible lyophilized microsphere formulation. It can be dispersed in sterile water, PBS buffer, sodium hyaluronate gel, carboxymethyl chitosan gel, alginate gel, or other wound dressing gels before use.

[0029] Compared with the prior art, the present invention has at least the following beneficial effects: This invention improves protein stability by constructing a non-covalent complex system of bioactive protein and sulfated hyaluronic acid. First, under mild, low-temperature conditions, basic fibroblast growth factor and sulfated hyaluronic acid form a non-covalent complex aggregate system through electrostatic interactions, hydrogen bonds, and affinity bonding. This process encourages the bioactive protein to form a protein-rich aggregate microdomain state. Because sulfated hyaluronic acid has a certain affinity and protective effect on proteins, it can reduce conformational disturbances and aggregation tendencies of proteins in solution to a certain extent, thereby improving their initial stability.

[0030] This invention facilitates the reconstruction of protein spatial distribution through low-shear dispersion and confined spheroidization. After the formation of the composite condensation system, it is introduced into the hydrophilic microsphere matrix fluid under low-shear conditions, allowing the composite condensed microdomains to remain dispersed within the system rather than completely dissociating into uniformly distributed single molecules. Subsequently, calcium salt ion cross-linking forms spheres, enabling sodium alginate to form a three-dimensional network structure, thereby further confining the composite condensed microdomains within the microspheres. This process helps to regulate the spatial distribution of bioactive proteins within the microspheres from a random distribution to a relatively enriched microdomain distribution, improving upon the structural defect of easy surface enrichment of proteins in traditional direct encapsulation systems.

[0031] This invention utilizes a microdomain confinement-diffusion pathway mechanism to facilitate the regulation of release behavior. Since bioactive proteins tend to disperse within microspheres as complex aggregated microdomains, their release process is no longer solely controlled by free diffusion, but is jointly influenced by the gradual dissociation of the complex microdomains and diffusion within the microsphere matrix. Therefore, compared to traditional microsphere systems, this invention can effectively suppress protein burst release in the initial stage and provide a sustained release process in subsequent stages, thereby improving the temporal distribution of bioactive proteins' effects in the wound environment.

[0032] This invention improves formulation stability through the synergistic effect of a permeation protection system and a lyophilization protection system. Trehalose, mannitol, and a permeation protection agent system are introduced at different stages of microsphere preparation. Specifically, stage S1 primarily protects the conformational stability of bioactive proteins during the complexation and aggregation process; stage S4 maintains the stability of the precursor solution system before microsphere formation; and stage S8 optimizes structural protection and reconstitution performance before lyophilization. This multi-stage protection system reduces the risk of protein inactivation at different physicochemical process nodes, thereby improving the effective protein retention and functional activity of the final formulation.

[0033] This invention improves wound adaptability by stabilizing the microsphere structure. A gelatin-sodium alginate-sodium hyaluronate composite hydrophilic microsphere matrix is ​​used, and a stable microsphere structure is formed through calcium salt ion cross-linking, giving it a certain ability to maintain its structure in the wound exudate environment. Simultaneously, the synergistic constraint between the internal composite cohesive microdomains of the microspheres and the external matrix structure prevents the microspheres from rapidly disintegrating in a moist environment, thereby prolonging their retention time in the local wound area. Attached Figure Description

[0034] Figure 1 This is an in vitro cumulative release curve of the bioactive protein microsphere formulations of the embodiments and comparative samples of the present invention; Figure 2 These are cell scratch migration experiments after treatment with release solutions in embodiments and comparative examples of the present invention, wherein... Figure 2 a represents the scratch images of different treatment groups at 0h and 24h. Figure 2 b is a statistical chart of the 24-hour scratch closure rate. Detailed Implementation

[0035] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the raw materials involved in the present invention are purchased through conventional commercial channels. Experimental methods without specific conditions are conventional methods and conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0036] Example 1: In this example, the basic fibroblast growth factor is recombinant basic fibroblast growth factor; the sulfated hyaluronic acid has a weight-average molecular weight of 60 kDa and a sulfate degree of substitution of 0.7; the gel strength of the gelatin is 150 Bloom; the viscosity of a 1 wt% aqueous solution of sodium alginate at 25°C is 250 mPa·s; the weight-average molecular weight of sodium hyaluronate is 150 kDa; and the permeation protectant is L-arginine.

[0037] S1: Add recombinant basic fibroblast growth factor to 10mM HEPES buffer, adjust the pH to 6.8, and gently mix until completely dissolved to obtain a 0.10mg / mL basic fibroblast growth factor solution; then add trehalose and L-arginine to make the trehalose concentration 4.5wt% and the L-arginine concentration 0.10wt%, and gently mix at 4℃ for 10min to obtain a basic fibroblast growth factor protective solution; S2: Add sulfated hyaluronic acid to 10mM HEPES buffer, adjust the pH to 6.8, stir until completely dissolved, and filter through a 0.22μm filter membrane to obtain a sulfated hyaluronic acid solution with a mass concentration of 0.30mg / mL; S3: Add the sulfated hyaluronic acid solution obtained in S2 to the basic fibroblast growth factor protective solution obtained in S1, so that the mass ratio of basic fibroblast growth factor to sulfated hyaluronic acid is 1:6; in this step, the pH of the system is controlled at 6.8 and the temperature at 4℃, so that basic fibroblast growth factor and sulfated hyaluronic acid undergo non-covalent complexation to form a composite coagulation solution containing basic fibroblast growth factor-sulfated hyaluronic acid composite coagulation microdomains; after the addition is completed, continue to gently mix for 20 minutes. The resulting composite coagulation solution remains in a uniformly dispersed state, without obvious flocculent precipitates or sediments; S4: Gelatin, sodium alginate, sodium hyaluronate, trehalose, mannitol, and L-arginine were added to deionized water and stirred at 35°C for 60 min to dissolve, thus obtaining a hydrophilic microsphere matrix solution. The hydrophilic microsphere matrix solution contained 4.5 wt% gelatin, 1.4 wt% sodium alginate, 0.35 wt% sodium hyaluronate, 4 wt% trehalose, 1.5 wt% mannitol, and 0.10 wt% L-arginine. The pH of the hydrophilic microsphere matrix solution was 6.9. S5: Add the basic fibroblast growth factor-sulfated hyaluronic acid composite coagulation solution obtained in S3 to the hydrophilic microsphere matrix solution obtained in S4, wherein the volume ratio of the composite coagulation solution to the hydrophilic microsphere matrix solution is 1:5.5; mix at low shear for 12 min at 28℃ and 120 rpm to obtain a spheroidization precursor solution containing composite coagulated microdomains. S6: The precursor solution obtained in S5 was added to the calcium chloride crosslinking solution in droplet form via electrostatic spraying. The nozzle inner diameter was 180 μm, the injection rate was 0.6 mL / h, the voltage was 8 kV, and the distance was 12 cm. This caused sodium alginate to undergo ionic crosslinking, resulting in primary microspheres containing basic fibroblast growth factor-sulfated hyaluronic acid composite aggregated microdomains. The calcium chloride crosslinking solution had a calcium chloride mass concentration of 2.0 wt%, a pH of 7.0, and a temperature of 8 °C. 3 wt% trehalose was added to the calcium chloride crosslinking solution, and crosslinking continued for 14 min after spheroidization. The average particle size of the resulting primary microspheres was approximately 60 μm. S7: The primary microspheres obtained in S6 were allowed to stand at 8°C for 40 min to stabilize, allowing the gelatin matrix to undergo physical gelation. Subsequently, the microspheres were washed twice with a protective washing solution for 3 min each time to remove the high concentration of calcium salt crosslinking solution and unencapsulated components remaining on the surface of the microspheres. The protective washing solution was a pH 7.0 HEPES buffer solution containing 0.5 wt% trehalose and 0.05 wt% calcium chloride. S8: Immerse the washed wet microspheres in the freeze-drying protective solution and allow them to stand at 8°C for 40 minutes; the freeze-drying protective solution is an aqueous solution with pH 7.0, containing 6.5 wt% trehalose, 2 wt% mannitol and 0.10 wt% L-arginine; the mass-to-volume ratio of the wet microspheres to the freeze-drying protective solution is 1 g: 8 mL; S9: The wet microsphere system containing the lyophilization protective solution obtained in S8 is pre-frozen and then freeze-dried under vacuum. The pre-freezing temperature is -50℃ and the pre-freezing time is 4h. The first drying temperature is -28℃, the vacuum degree is 0.10mbar, and the drying time is 24h. The second drying temperature is 20℃, the vacuum degree is 0.10mbar, and the drying time is 8h, to obtain the bioactive protein microsphere formulation.

[0038] Example 2: In this example, the basic fibroblast growth factor is recombinant basic fibroblast growth factor; the sulfated hyaluronic acid has a weight-average molecular weight of 20 kDa and a degree of sulfate substitution of 0.4; the gel strength of the gelatin is 80 Bloom; the viscosity of the 1 wt% aqueous solution of sodium alginate at 25°C is 50 mPa·s; the weight-average molecular weight of the sodium hyaluronate is 50 kDa; and the permeation protectant is L-arginine.

[0039] S1: Add recombinant basic fibroblast growth factor to 5mM HEPES buffer, adjust the pH to 6.5, and gently mix until completely dissolved to obtain a 0.05mg / mL basic fibroblast growth factor solution; then add trehalose and L-arginine to make the trehalose concentration 3wt% and the L-arginine concentration 0.05wt%, and gently mix at 2℃ for 5min to obtain a basic fibroblast growth factor protective solution; S2: Add sulfated hyaluronic acid to 5mM HEPES buffer, adjust the pH to 6.5, stir until completely dissolved, and filter through a 0.22μm filter membrane to obtain a sulfated hyaluronic acid solution with a mass concentration of 0.10mg / mL; S3: Add the sulfated hyaluronic acid solution obtained in S2 to the basic fibroblast growth factor protective solution obtained in S1, so that the mass ratio of basic fibroblast growth factor to sulfated hyaluronic acid is 1:4. In this step, the pH of the system is controlled at 6.5 and the temperature is 2℃, so that basic fibroblast growth factor and sulfated hyaluronic acid undergo non-covalent complexation to form a composite coagulation solution containing basic fibroblast growth factor-sulfated hyaluronic acid composite coagulation microdomains. After the addition is completed, continue to gently mix for 15 minutes. The resulting composite coagulation solution remains in a uniformly dispersed state without obvious flocculent precipitate or sediment. S4: Gelatin, sodium alginate, sodium hyaluronate, trehalose, mannitol, and L-arginine were added to deionized water and stirred at 30°C for 30 minutes to obtain a hydrophilic microsphere matrix solution. The mass concentrations of the gelatin, sodium alginate, sodium hyaluronate, trehalose, mannitol, and L-arginine in the hydrophilic microsphere matrix solution were 3 wt%, 0.8 wt%, 0.1 wt%, 2 wt%, 0.5 wt%, and 0.02 wt%, respectively. The pH of the hydrophilic microsphere matrix solution was 6.6. S5: Add the basic fibroblast growth factor-sulfated hyaluronic acid composite coagulation solution obtained in S3 to the hydrophilic microsphere matrix solution obtained in S4, wherein the volume ratio of the composite coagulation solution to the hydrophilic microsphere matrix solution is 1:4; mix at low shear for 8 min at 25℃ and 80 rpm to obtain a spheroidization precursor solution containing composite coagulation microdomains. S6: The precursor solution obtained in S5 was added to the calcium chloride crosslinking solution in droplet form via electrostatic spraying. The nozzle inner diameter was 100 μm, the injection rate was 0.2 mL / h, the voltage was 10 kV, and the distance was 15 cm. This caused sodium alginate to undergo ionic crosslinking, resulting in primary microspheres containing basic fibroblast growth factor-sulfated hyaluronic acid composite coagulation microdomains. The calcium chloride crosslinking solution had a calcium chloride mass concentration of 1.0 wt%, a pH of 6.8, and a temperature of 4 °C. 2 wt% trehalose was added to the calcium chloride crosslinking solution, and crosslinking continued for 8 min after spheroidization. The average particle size of the resulting primary microspheres was approximately 30 μm. S7: The primary microspheres obtained in S6 were allowed to stand at 4°C for 20 min to stabilize, allowing the gelatin matrix to undergo physical gelation. Subsequently, the microspheres were washed once with a protective washing solution for 2 min to remove the high concentration of calcium salt crosslinking solution and unencapsulated components remaining on the surface of the microspheres. The protective washing solution was a pH 6.8 HEPES buffer solution containing 0.2 wt% trehalose and 0.02 wt% calcium chloride. S8: Immerse the washed wet microspheres in the freeze-drying protective solution and allow them to stand at 4°C for 20 minutes; the freeze-drying protective solution is an aqueous solution with pH 6.8, containing 5 wt% trehalose, 1 wt% mannitol and 0.05 wt% L-arginine; the mass-to-volume ratio of the wet microspheres to the freeze-drying protective solution is 1 g: 5 mL. S9: The wet microsphere system containing the lyophilization protective solution obtained in S8 is pre-frozen and then freeze-dried under vacuum. The pre-freezing temperature is -40℃ and the pre-freezing time is 3h. The first drying temperature is -35℃, the vacuum degree is 0.05mbar, and the drying time is 18h. The second drying temperature is 10℃, the vacuum degree is 0.05mbar, and the drying time is 6h, to obtain the bioactive protein microsphere formulation.

[0040] Example 3: This example provides a bioactive protein microsphere formulation and its preparation method, the specific steps of which are as follows: In this embodiment, the basic fibroblast growth factor is recombinant basic fibroblast growth factor; the sulfated hyaluronic acid has a weight-average molecular weight of 100 kDa and a degree of sulfate substitution of 1.0; the gel strength of the gelatin is 220 Bloom; the viscosity of a 1 wt% aqueous solution of sodium alginate at 25°C is 500 mPa·s; the weight-average molecular weight of sodium hyaluronate is 300 kDa; and the permeation protectant is L-arginine.

[0041] S1: Add recombinant basic fibroblast growth factor to 20mM HEPES buffer, adjust the pH to 7.0, and gently mix until completely dissolved to obtain a 0.20mg / mL basic fibroblast growth factor solution; then add trehalose and L-arginine to make the trehalose concentration 6wt% and the L-arginine concentration 0.20wt%, and gently mix at 8℃ for 20min to obtain a basic fibroblast growth factor protective solution; S2: Add sulfated hyaluronic acid to 20mM HEPES buffer, adjust the pH to 7.0, stir until completely dissolved, and filter through a 0.22μm filter membrane to obtain a sulfated hyaluronic acid solution with a mass concentration of 0.60mg / mL; S3: Add the sulfated hyaluronic acid solution obtained in S2 to the basic fibroblast growth factor protective solution obtained in S1, so that the mass ratio of basic fibroblast growth factor to sulfated hyaluronic acid is 1:8. In this step, the pH of the system is controlled at 7.0 and the temperature is 8℃, so that basic fibroblast growth factor and sulfated hyaluronic acid undergo non-covalent complexation to form a composite coagulation solution containing basic fibroblast growth factor-sulfated hyaluronic acid composite coagulation microdomains. After the addition is completed, continue to gently mix for 30 minutes. The resulting composite coagulation solution remains in a uniformly dispersed state without obvious flocculent precipitate or sediment. S4: Gelatin, sodium alginate, sodium hyaluronate, trehalose, mannitol, and L-arginine were added to deionized water and stirred at 40°C for 120 min to obtain a hydrophilic microsphere matrix solution. The mass concentrations of the gelatin, sodium alginate, sodium hyaluronate, trehalose, mannitol, and L-arginine in the hydrophilic microsphere matrix solution were 6 wt%, 2.0 wt%, 0.6 wt%, 6 wt%, 3 wt%, and 0.20 wt%, respectively. The pH of the hydrophilic microsphere matrix solution was 7.2. S5: Add the basic fibroblast growth factor-sulfated hyaluronic acid composite coagulation solution obtained in S3 to the hydrophilic microsphere matrix solution obtained in S4. The volume ratio of the composite coagulation solution to the hydrophilic microsphere matrix solution is 1:7. Mix under low shear conditions at 32℃ and 180rpm for 20min to obtain a spheroidization precursor solution containing composite coagulation microdomains. S6: The precursor solution obtained in S5 was added to the calcium chloride crosslinking solution in droplet form via electrostatic spraying. The nozzle inner diameter was 250 μm, the injection rate was 1.0 mL / h, the voltage was 5 kV, and the distance was 8 cm. This caused sodium alginate to undergo ionic crosslinking, resulting in primary microspheres containing basic fibroblast growth factor-sulfated hyaluronic acid composite coagulation microdomains. The calcium chloride crosslinking solution had a calcium chloride mass concentration of 3.0 wt%, a pH of 7.2, and a temperature of 12 °C. 4 wt% trehalose was added to the calcium chloride crosslinking solution, and crosslinking continued for 20 min after spheroidization. The average particle size of the resulting primary microspheres was approximately 100 μm. S7: The primary microspheres obtained in S6 were allowed to stand at 12°C for 60 min to stabilize, allowing the gelatin matrix to undergo physical gelation. Subsequently, the microspheres were washed three times with a protective washing solution for 5 min each time to remove the high concentration of calcium salt crosslinking solution and unencapsulated components remaining on the surface of the microspheres. The protective washing solution was a pH 7.2 HEPES buffer solution containing 0.8 wt% trehalose and 0.10 wt% calcium chloride. S8: Immerse the washed wet microspheres in the freeze-drying protective solution and allow them to stand at 12°C for 60 min; the freeze-drying protective solution is an aqueous solution with pH 7.2, containing 8 wt% trehalose, 3 wt% mannitol and 0.20 wt% L-arginine; the mass-to-volume ratio of the wet microspheres to the freeze-drying protective solution is 1 g: 10 mL; S9: The wet microsphere system containing the lyophilization protective solution obtained in S8 is pre-frozen and then freeze-dried under vacuum. The pre-freezing temperature is -60℃ and the pre-freezing time is 6h. The first drying temperature is -20℃, the vacuum degree is 0.20mbar, and the drying time is 30h. The second drying temperature is 25℃, the vacuum degree is 0.20mbar, and the drying time is 12h to obtain the bioactive protein microsphere formulation.

[0042] Comparative Example 1: This comparative example does not perform the stepwise composite coagulation treatment of S1 to S3 in Example 1; specifically, recombinant basic fibroblast growth factor, sulfated hyaluronic acid, gelatin, sodium alginate, sodium hyaluronate, trehalose, mannitol and L-arginine are added at once to an aqueous phase system composed of deionized water and HEPES buffer, and the pH of the system is adjusted to 6.8; the other conditions are the same as in Example 1.

[0043] Comparative Example 2: This comparative example does not include sulfated hyaluronic acid. Specifically, in S2 of Example 1, an equal volume of 10mM HEPES buffer was used instead of the sulfated hyaluronic acid solution, and the other conditions were the same as in Example 1.

[0044] Comparative Example 3: In S5 of this comparative example, a high-shear mixing method was used. Specifically, a high-speed dispersion method was used to process the mixture at 8000 rpm for 2 min; the remaining conditions were the same as in Example 1.

[0045] Comparative Example 4: This comparative example does not perform the S8 freeze-drying protective liquid immersion and standing treatment as in Example 1. Specifically, after the surface liquid of the washed wet microspheres is drained, they are directly subjected to S9 pre-freezing and vacuum freeze-drying, and the other conditions are the same as in Example 1.

[0046] Comparative Example 5: In this comparative example, the mass concentration of calcium chloride in the calcium chloride crosslinking solution in step S6 was adjusted to 0.3 wt%. The resulting microspheres had a weaker structure, but post-processing was still performed according to S7 to S9 of Example 1; the remaining conditions were the same as in Example 1.

[0047] Performance testing: To verify the performance of the bioactive protein microsphere formulation of this invention, the samples obtained in Examples 1-3 and Comparative Examples 1-5 were subjected to the following tests. For the characterization test of the pre-spheroidization liquid phase system, the test object for Examples 1-3 was the basic fibroblast growth factor-sulfated hyaluronic acid composite coagulation solution obtained in step S3; the test object for Comparative Example 1 was the pre-spheroidization liquid phase system after a single mixing; the test object for Comparative Example 2 was a protein mixture without sulfated hyaluronic acid; the test object for Comparative Example 3 was the pre-spheroidization liquid phase system after high-shear treatment; and the test objects for Comparative Examples 4 and 5 were the composite coagulation solutions obtained in step S3. Before testing, each sample was diluted with PBS buffer to the same theoretical protein concentration and equilibrated at 25°C for 2 minutes before detection. The average particle size and polydispersity index of each system were determined using a dynamic light scattering instrument, and the surface potential in the PBS buffer system was measured using a zeta potential meter. Each sample was then allowed to stand at 25±2°C for 30 minutes to observe whether flocculent precipitation or sedimentation occurred. During the testing process, all samples were gently inverted and mixed to avoid damaging the system architecture. The relevant test results are shown in Table 1.

[0048] To evaluate the encapsulation performance, lyophilization stability, and reconstitution performance of the microspheres, equal volumes of the lyophilized microsphere formulations obtained in Examples 1-3 and Comparative Examples 1-5 were weighed and added to PBS buffer for reconstitution. After complete dispersion, the reconstituted system was centrifuged at 3000 rpm for 10 min to separate the microspheres from the supernatant, and the supernatant was further filtered through a 0.22 μm filter membrane. The content of basic fibroblast growth factor in the supernatant was determined by ELISA, and the encapsulation efficiency was calculated based on the initial feed amount: Encapsulation efficiency = (Initial feed protein amount - Free protein amount in supernatant) / Initial feed protein amount × 100%. Simultaneously, basic fibroblast growth factor was extracted from the wet microspheres before lyophilization and the microspheres after lyophilization and reconstitution. The effective protein content before and after lyophilization and reconstitution was determined by ELISA, and the effective protein retention rate after lyophilization and reconstitution was calculated: Effective protein retention rate after lyophilization and reconstitution = Effective protein amount in the sample after lyophilization and reconstitution / Effective protein amount in the wet microspheres before lyophilization × 100%. The reconstitution performance was evaluated by recording the time required for the microspheres to be completely dispersed in PBS. The absence of obvious agglomeration was used as the criterion for complete reconstitution. The water content of the lyophilized formulation was determined by the Karl Fischer method. The relevant test results are shown in Table 2.

[0049] To evaluate the cellular effects of the bioactive proteins released from the microspheres, the microsphere formulations obtained in Examples 1-3 and Comparative Examples 1-5 were added to PBS buffer and incubated at 37°C and 100 rpm for 24 h with shaking. The released solution was collected and sterilized by filtration through a 0.22 μm filter. The released solutions from each group were added to a mouse fibroblast NIH / 3T3 culture system at the same volume ratio. A blank culture medium was set up as a negative control, and an equal volume of free basic fibroblast growth factor solution was set up as a positive control. Cell proliferation activity was detected by the CCK-8 assay after 24 h and 48 h of culture. Cell morphology was observed and recorded using an inverted microscope. The relevant test results are shown in Table 3.

[0050] To evaluate the in vitro release behavior of the microspheres, the microsphere formulations obtained in Examples 1-3 and Comparative Examples 1, 2, and 5 were placed in PBS buffer and incubated with shaking at 37°C and 100 rpm. Samples of 1 mL were taken at 2 h, 6 h, 12 h, 24 h, 72 h, 120 h, and 168 h, and an equal volume of fresh PBS was added to maintain a constant system volume. The content of basic fibroblast growth factor in the release solution at each time point was determined by ELISA, and the cumulative release rate was calculated. Release curves were plotted, and the test results are shown below. Figure 1 .

[0051] To evaluate the cell migration ability of the released products, 24-hour release solutions of the microsphere formulations obtained in Examples 1, 1, 2, and 3 were used. Blank culture medium and free basic fibroblast growth factor were used as controls. Human skin fibroblasts were cultured to approximately 90% confluence, and then straight lines were drawn using a sterile pipette tip to create scratches. Exfoliated cells were gently washed with PBS, and then the release solutions from each group were added in the same volume ratio for further culture. Images were taken at 0h and 24h, and the scratch area was analyzed using ImageJ software to calculate the cell migration ability, i.e., the scratch closure rate. The test results are shown below. Figure 2 .

[0052] Table 1. Characterization results of the composite condensation system of the examples and comparative samples

[0053] Table 2. Microsphere encapsulation efficiency, freeze-drying stability, and reconstitution properties of the examples and comparative samples.

[0054] Table 3. Effects of microsphere release solutions from the examples and comparative samples on the proliferation activity of NIH / 3T3 cells.

[0055] As shown in Table 1, the composite condensation systems obtained in Examples 1-3 all maintained good dispersion, indicating that under the conditions of low temperature, near-neutral pH, and suitable mass ratio specified in this invention, basic fibroblast growth factor and sulfated hyaluronic acid can form a relatively stable non-covalent composite system. This composite system is not a simple homogeneous mixture, but rather, through the interaction between the anionic groups on sulfated hyaluronic acid and the positively charged and affinity-binding regions on the surface of basic fibroblast growth factor, protein molecules tend to form protein-rich composite condensation microdomains in the liquid phase. These composite condensation microdomains maintain a certain degree of repulsive stability in terms of potential, thus preventing further aggregation into large-sized precipitates and maintaining a good dispersion foundation during subsequent low-shear mixing and spheroidization processes.

[0056] Compared to the examples, Comparative Example 1 used a one-time mixing and spheroidization method, without undergoing the process of first compounding and agglomeration followed by the introduction of a hydrophilic microsphere matrix fluid. Because proteins, sulfated hyaluronic acid, gelatin, sodium alginate, and protective agents are present simultaneously, charge pairing, chain segment entanglement, and polymer thickening processes occur concurrently within the system. Basic fibroblast growth factor and sulfated hyaluronic acid are unlikely to preferentially form stable, independently dispersed compound agglomeration microdomains; instead, they are prone to forming larger, heterogeneous aggregates due to localized concentration unevenness and competition among multiple components. Therefore, Comparative Example 1 exhibits a wider particle size distribution, decreased dispersion stability, and a tendency to aggregate and settle after standing.

[0057] Comparative Example 2, without the addition of sulfated hyaluronic acid, lacked anionic components capable of forming affinity complexes with basic fibroblast growth factor. The protein primarily existed in a free or weakly adsorbed state within the gelatin / sodium alginate system. Due to the absence of the complex fixation and affinity protection provided by sulfated hyaluronic acid, the protein was more prone to random diffusion, localized surface enrichment, or uneven distribution during subsequent globule formation, making it difficult to form protein-rich microdomains with sustained-release regulation capabilities. This phenomenon is related to... Figure 1 The release curves shown in the comparison curve 2 show a trend of rapid initial release followed by insufficient regulatory capacity in subsequent release.

[0058] Comparative Example 3 employed a high-shear mixing treatment in S5. While it initially formed a complex aggregated system, the high shear rate easily disrupted the dynamic non-covalent equilibrium of the complex aggregated microdomains and increased the risk of protein conformational perturbation and local aggregation. The complex aggregated microdomains are essentially maintained primarily by non-covalent interactions such as electrostatic interactions, hydrogen bonds, and affinity binding. Excessive mechanical shearing weakens their dispersion integrity, causing some proteins to dissociate from or re-aggregate within the complex aggregated microdomains, thus leading to a decrease in the system's dispersibility.

[0059] The characterization results of the composite condensation system in Comparative Examples 4 and 5 in Table 1 are similar to those in Example 1. The front-end composite condensation steps of the two comparative examples were not destroyed, and their main defects occurred in the freeze-drying protection stage and the ion cross-linking sphere formation stage, respectively.

[0060] As shown in Table 2, Examples 1-3 are generally superior to the comparative examples in terms of encapsulation efficiency, effective protein retention after lyophilization and reconstitution, reconstitution dispersibility, and water content, indicating a synergistic effect between the composite aggregation, confined sphere formation, and lyophilization protection system of the present invention. The protein-sulfated hyaluronic acid composite aggregation microdomains formed first can reduce the free diffusion and surface loss of protein during the sphere formation process. Subsequently, the hydrophilic microsphere matrix composed of gelatin, sodium alginate, and sodium hyaluronate forms a three-dimensional network through calcium ion cross-linking, further confining the composite microdomains inside the microspheres, thereby improving the protein encapsulation efficiency. At the same time, trehalose, mannitol, and L-arginine play a protective role in the protein dissolution, sphere formation pre-solution solution formation, and pre-lyophilization impregnation stages, respectively, reducing conformational disturbances and aggregation tendency of proteins during freezing, dehydration, and reconstitution, thereby improving the effective protein retention rate after lyophilization and reconstitution.

[0061] Comparative Example 1, lacking pre-treatment for complexation and aggregation, resulted in protein encapsulation and non-specific adsorption within the microsphere system, leading to a significant decrease in both encapsulation effectiveness and effective protein retention after lyophilization. Comparative Example 2, lacking the affinity protection of sulfated hyaluronic acid, lacked stable complexation sites within the microsphere matrix, making it prone to loss during washing and release. Comparative Example 3, affected by high shear stress, potentially damaged the protein microdomain structure and conformation, resulting in decreased effective protein retention. Comparative Example 4, although the pre-encapsulation process was not disrupted, lacked lyophilization protection solution immersion, making it more susceptible to protein inactivation and microsphere aggregation due to ice crystal formation, dehydration stress, and interfacial stress during reconstitution, thus significantly deteriorating its effective protein retention and reconstitution dispersibility. Comparative Example 5 showed insufficient calcium salt cross-linking and low sodium alginate network strength, making the microspheres more prone to swelling and structural loosening in humid and release environments, leading to decreased encapsulation stability and release regulation capabilities.

[0062] Figure 1The in vitro release curves shown further validated the aforementioned structural differences. Examples 1-3 all exhibited a relatively gradual cumulative release trend, indicating that the release of bioactive proteins in the microspheres is not solely dependent on free diffusion, but is jointly regulated by the dissociation of the complex aggregate microdomains and diffusion within the microsphere matrix. For the microspheres of this invention, wound exudate or the release medium first enters the hydrophilic microsphere matrix, causing the microspheres to swell. Subsequently, the protein needs to gradually dissociate from the sulfated hyaluronic acid complex microdomains and then diffuse through the gelatin / sodium alginate / sodium hyaluronate network. Therefore, its release process exhibits a dual regulatory characteristic of complex microdomain dissociation and microsphere matrix diffusion. In contrast, Comparative Examples 1, 2, and 5 all showed a faster release trend, corresponding to the burst release or structural loosening problems caused by the lack of stable complex aggregate microdomains, insufficient sulfated hyaluronic acid affinity fixation, and inadequate microsphere cross-linking, respectively.

[0063] Table 3 and Figure 2 The effectiveness of the released protein was further verified at the cellular level. Table 3 shows that the release solutions from Examples 1-3 can promote the proliferation of NIH / 3T3 cells. Figure 2 The results show that the release solutions from Example 1, the comparative example, and the free bFGF treatment group have different effects on scratch migration of human skin fibroblasts. The release solution from Example 1 showed a good promoting effect on both cell proliferation and scratch migration, indicating that the basic fibroblast growth factor released by the formulation of this invention can still maintain high functional activity after undergoing compound aggregation, spheroidization, washing, lyophilization, and reconstitution. This result is consistent with the effective protein retention rate in Table 2, indicating that this invention not only improves physical encapsulation or release curves, but also protects the functional activity of proteins. The free bFGF control can promote cell proliferation and migration, but it lacks the sustained release and structural protection of the microsphere carrier, and is easily affected by dilution and degradation in long-term action or complex wound environments; while the microsphere release solution from the examples can maintain a strong promoting effect in cell tests at 24h and 48h, indicating that the compound aggregation confinement system of this invention has a synergistic effect on protein activity protection and sustained effective release.

[0064] Figure 2 In Example a, cell migration was more pronounced in the scratched area after treatment with Example 1. Figure 2 Example b shows a higher scratch closure rate, indicating that the release solution of this invention can effectively promote the migration of wound repair-related cells to the damaged area. Comparative Example 1, due to unstable protein distribution and release caused by single-use mixing, exhibits lower effective activity of the release solution; Comparative Example 2 lacks the complex effect of sulfated hyaluronic acid, resulting in insufficient protein protection and sustained-release capacity; Comparative Example 3, after high-shear treatment, has an increased risk of protein activity impairment, thus its cell migration promotion effect is weaker than that of Example 1. These results support the mechanistic logic of this invention—improving the performance of protein microsphere formulations through composite aggregation microdomain construction, low-shear confined spheroidization, and a multi-stage protection system—at the cellular function level.

[0065] Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims.

Claims

1. A bioactive protein microsphere preparation, characterized by, Its raw material composition includes the following components: basic fibroblast growth factor, sulfated hyaluronic acid, gelatin, sodium alginate, sodium hyaluronate, trehalose, mannitol, and penetration protectant; The basic fibroblast growth factor is a recombinant basic fibroblast growth factor. The sulfated hyaluronic acid has a weight-average molecular weight of 20–100 kDa and a degree of sulfate substitution of 0.4–1.

0. The gel strength of the gelatin is 80–220 Bloom. The viscosity of a 1 wt% aqueous solution of sodium alginate at 25°C is 50–500 mPa·s. The weight-average molecular weight of the sodium hyaluronate is 50–300 kDa; The permeation protectant is selected from one of L-arginine, glycine, proline, and betaine.

2. A method for preparing the bioactive protein microspheres preparation according to claim 1, characterized by, Includes the following steps: S1: Add basic fibroblast growth factor to buffer solution, mix until dissolved, and add trehalose and osmotic protectant to obtain basic fibroblast growth factor protection solution; S2: Add sulfated hyaluronic acid to the buffer solution, dissolve and filter to obtain sulfated hyaluronic acid solution; S3: Add the sulfated hyaluronic acid solution obtained in S2 to the basic fibroblast growth factor protective solution obtained in S1, so that the basic fibroblast growth factor and sulfated hyaluronic acid undergo non-covalent complexation to form a composite coagulation solution containing a composite coagulation microdomain of basic fibroblast growth factor-sulfated hyaluronic acid. S4: Add gelatin, sodium alginate, sodium hyaluronate, trehalose, mannitol and permeation protectant to an aqueous medium, stir and dissolve evenly to obtain a hydrophilic microsphere matrix solution; S5: Add the basic fibroblast growth factor-sulfated hyaluronic acid composite coagulation solution obtained in S3 to the hydrophilic microsphere matrix solution obtained in S4, and mix under low shear conditions to obtain a sphere-forming precursor solution containing composite coagulation microdomains. S6: The sphere-forming precursor solution obtained in S5 is added to the calcium salt crosslinking solution in the form of droplets by electrostatic spraying, so that sodium alginate undergoes ionic crosslinking to obtain primary microspheres containing basic fibroblast growth factor-sulfated hyaluronic acid composite coagulation microdomains. S7: The primary microspheres obtained in S6 are allowed to stand and stabilize, allowing the gelatin matrix to undergo physical gelation. Then, the microspheres are washed with a protective washing solution to remove the high concentration of calcium salt crosslinking liquid and unencapsulated components remaining on the surface of the microspheres. S8: Immerse the washed wet microspheres in the freeze-drying protective solution and allow them to stand for a period of time. S9: The wet microsphere system containing the lyophilization protective solution obtained in S8 is pre-frozen and then vacuum freeze-dried to obtain a bioactive protein microsphere formulation.

3. A method for preparing a bioactive protein microsphere formulation according to claim 2, characterized in that, In step S1, the buffer solution is HEPES buffer, the concentration of HEPES buffer solution is 5-20 mM, and the pH is 6.5-7.0; the mass concentration of basic fibroblast growth factor is 0.05-0.2 mg / mL, the mass concentration of trehalose is 3-6 wt%, and the mass concentration of osmotic protectant is 0.05-0.2 wt%.

4. A method for preparing a bioactive protein microsphere formulation according to claim 2, characterized in that, In step S2, the buffer solution is HEPES buffer, the concentration of HEPES buffer is 5-20 mM, and the pH is 6.5-7.0; the mass concentration of the sulfated hyaluronic acid solution is 0.1-0.6 mg / mL.

5. A method for preparing a bioactive protein microsphere formulation according to claim 2, characterized in that, In step S3, the amount of sulfated hyaluronic acid solution added is adjusted so that the mass ratio of basic fibroblast growth factor to sulfated hyaluronic acid is 1:4 to 1:8; in step S3, the pH of the system is controlled to be 6.5 to 7.0 and the temperature is 2 to 8°C.

6. A method for preparing a bioactive protein microsphere formulation according to claim 2, characterized in that, In step S4, the hydrophilic microsphere matrix solution contains gelatin at a mass concentration of 3–6 wt%, sodium alginate at a mass concentration of 0.8–2.0 wt%, sodium hyaluronate at a mass concentration of 0.1–0.6 wt%, trehalose at a mass concentration of 2–6 wt%, mannitol at a mass concentration of 0.5–3 wt%, and a permeation protectant at a mass concentration of 0.02–0.2 wt%; the pH of the hydrophilic microsphere matrix solution is 6.6–7.

2.

7. A method for preparing a bioactive protein microsphere formulation according to claim 2, characterized in that, In step S5, the volume ratio of the composite coagulation liquid to the hydrophilic microsphere matrix liquid is 1:4 to 1:7, and the mixing temperature is 25 to 32°C.

8. A method for preparing a bioactive protein microsphere formulation according to claim 2, characterized in that, In step S6, the calcium salt crosslinking solution is a calcium chloride solution; the calcium chloride concentration in the calcium salt crosslinking solution is 1.0-3.0 wt%, the pH is 6.8-7.2, and the temperature is 4-12℃; the calcium salt crosslinking solution contains 2-4 wt% trehalose, and after spheroidization, crosslinking continues for 8-20 min, and the average particle size of the obtained primary microspheres is 30-100 μm.

9. A method for preparing a bioactive protein microsphere formulation according to claim 2, characterized in that, In step S8, the freeze-drying protective solution is an aqueous solution with a pH of 6.8 to 7.2, containing 5 to 8 wt% trehalose, 1 to 3 wt% mannitol, and 0.05 to 0.2 wt% permeation protectant; the mass-to-volume ratio of the wet microspheres to the freeze-drying protective solution is 1 g: 5 to 10 mL; the temperature for the immersion and settling treatment is 4 to 12 °C, and the time is 20 to 60 min.

10. A method for preparing a bioactive protein microsphere formulation according to claim 2, characterized in that, In step S9, the pre-freezing temperature is -40 to -60℃, and the pre-freezing time is 3 to 6 hours; the first drying temperature is -35 to -20℃, the vacuum degree is 0.05 to 0.2 mbar, and the drying time is 18 to 30 hours; the second drying temperature is 10 to 25℃, the vacuum degree is 0.05 to 0.2 mbar, and the drying time is 6 to 12 hours.

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