Degradable microspheres, preparation method and application thereof

Abstract

This invention provides a biodegradable microsphere, its preparation method, and its application. The biodegradable microsphere comprises a porous microsphere with a continuous internal pore structure and a gelatin component loaded on the porous microsphere. The biodegradable polyester microspheres prepared by this invention using a thermally induced phase separation-crystallization method have an excellent interconnected porous structure. The microspheres formed by combining with gelatin biomacromolecules can provide an excellent adhesion and survival environment for cells.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and more specifically, to a biodegradable microsphere, its preparation method, and its application. Background Technology

[0002] Porous microspheres, due to their large specific surface area and high porosity, have attracted widespread attention from researchers in recent years. Porous microspheres prepared from biodegradable materials have broad application prospects in many fields, such as cell culture carriers, controlled drug release, and tissue engineering repair.

[0003] Polylactic acid (PLA) biodegradable polyester materials have good biocompatibility and have been approved for use by the US FDA. However, due to their strong hydrophobicity, these biodegradable polyester materials are not conducive to cell adhesion during cell culture. Therefore, hydrophilic modification is required to improve their surface wettability and facilitate cell adhesion.

[0004] Currently, the average size of many artificially cultured cells is 1–10 micrometers. It is believed that porous spherical cell culture carriers with a particle size of 60–400 μm and a pore size greater than 10 μm, and with a connected pore structure, are more suitable for cell growth and can better meet the needs of cell adhesion, spreading, proliferation, and nutrient exchange. However, the preparation method of microspheres with the above-mentioned suitable pore structure is relatively complex and needs further development. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a biodegradable microsphere and its preparation method. The biodegradable microsphere has an excellent interconnected porous structure, and the microsphere formed by combining with gelatin biomacromolecules can provide cells with an excellent adhesion and survival environment.

[0006] Firstly, one of the objectives of this invention is to provide a biodegradable microsphere.

[0007] Specifically, the biodegradable microspheres comprise porous microspheres with a continuous internal pore structure, and a biomolecular gelatin component loaded onto the porous microspheres. Loading natural macromolecular gelatin onto the porous microspheres does not disrupt the pore structure and enhances the biofunctionality of the microspheres, promoting cell adhesion and growth.

[0008] Furthermore, in the biodegradable microspheres, based on porous microspheres, the mass percentage of the gelatin component loaded on the porous microspheres is 0.01–5%, preferably 0.5–3%.

[0009] Furthermore, the pore structure inside the biodegradable microspheres is interconnected, wherein the pore size is 10–1000 μm, preferably 100–500 μm; the pore size is 1–100 μm, preferably 10–60 μm; and the porosity is 70–95%, preferably 80–95%.

[0010] Secondly, another objective of this invention is to provide a method for preparing biodegradable microspheres, which is one of the objectives of this invention.

[0011] Specifically, the method includes: mixing a biodegradable polyester solution with a preheated mixture of polyol and dispersant to form an emulsion, performing thermal phase separation, drying the separated precipitate to obtain porous microspheres, adding the porous microspheres to a gelatin solution, loading the porous microspheres with gelatin to obtain biodegradable microspheres.

[0012] The biodegradable polyester has a molecular weight of 2000–2000000 g / mol; preferably selected from one or a combination of poly(L-lactic acid), poly(D-lactic acid), glycolide-lactide copolymer, polycaprolactone-lactide copolymer, and polyethylene glycol-lactide copolymer; the polyol is selected from one or a combination of ethylene glycol, glycerol, butanediol, hexanediol, pentanediol, pentaerythritol, diethylene glycol, dipropylene glycol, trimethylolpropane, and isopropanol; the dispersant is selected from one or a combination of polyethylene glycol (e.g., polyethylene glycol 200, polyethylene glycol 400) and polyethyleneimine. This invention uses a polyol, a dispersant, and an organic solution of the biodegradable polyester to form an emulsion. The polyol is the continuous phase, and the organic solution is the dispersed phase. The dispersant promotes better droplet dispersion of the continuous phase, and during freezing, the biodegradable polyester separates from the organic solvent, crystallizes, and forms porous microspheres.

[0013] More specifically, the method includes the following steps:

[0014] Step 1: Dissolve the biodegradable polyester in an organic solvent, heat and stir to mix evenly to obtain a biodegradable polyester solution;

[0015] Step 2: Mix the polyol and dispersant evenly, and preheat to obtain a mixture;

[0016] Step 3: While stirring, add the biodegradable polyester solution from Step 1 to the mixture from Step 2 to form an emulsion;

[0017] Step 4: Place the emulsion from Step 3 in a low-temperature environment and allow it to stand for thermal phase separation.

[0018] Step 5: Remove the frozen emulsion, pour it into distilled water, stir to separate the precipitate, and dry to obtain porous microspheres;

[0019] Step 6: Place the porous microspheres obtained in Step 5 into a gelatin aqueous solution, repeatedly evacuate the vacuum to release the gas, and freeze-dry to obtain biodegradable microspheres.

[0020] Specifically, the method includes the following steps:

[0021] Step 1: Dissolve the biodegradable polyester in an organic solvent, heat and stir to mix it evenly, and obtain a biodegradable polyester solution.

[0022] Step 2: Mix the polyol and dispersant evenly, and preheat to obtain a mixture;

[0023] Step 3: Add the biodegradable polyester solution from Step 1 to the polyol and dispersant mixture from Step 2 at a certain stirring speed, and maintain a certain temperature, stirring speed, and time to emulsify and form an emulsion.

[0024] Step 4: Place the emulsion from Step 3 in a low-temperature environment and allow it to stand for thermal phase separation.

[0025] Step 5: Remove the emulsion that has been left to stand in Step 4 from the low-temperature environment, pour in a large amount of distilled water, stir slowly for a period of time, let it stand for a period of time, filter the mixture, separate the porous microspheres, and dry the porous microspheres at 30-60℃ for later use.

[0026] Step 6: Prepare a gelatin aqueous solution of a certain concentration, add the porous microspheres obtained in Step 5, transfer the system to a colorimetric tube, place a sponge above the liquid surface, ensuring the sponge is in close contact with the tube wall, repeatedly evacuate the colorimetric tube to allow the gelatin aqueous solution to fully enter the microsphere pores, remove the microspheres, and rapidly cool them to solidify the gelatin solution loaded inside and on the surface of the microspheres. After freeze-drying, biodegradable microspheres are obtained.

[0027] Furthermore, in step one, the conditions for preparing the biodegradable polyester solution are as follows:

[0028] The organic solvent is selected from one or a combination of tetrahydrofuran, dioxane, N,N-dimethylformamide, dimethyl sulfoxide, acetonitrile, and dimethyl ether; the heating temperature is 40–90°C, preferably 50–70°C; and the concentration of the biodegradable polyester solution is 0.1–8 wt%, preferably 0.5–5 wt%.

[0029] Furthermore, in step two, the preheating conditions for the mixed solution of polyol and dispersant are as follows:

[0030] The preheating temperature is 30–80°C, preferably 40–60°C; the mass concentration of the dispersant in the polyol is 0.05–10 wt%, preferably 0.1–5 wt%.

[0031] Furthermore, in step three, the conditions for emulsion formation are as follows:

[0032] The mass ratio of the biodegradable polyester solution to the polyol is 1:1 to 1:10, preferably 1:2 to 1:4; the emulsification temperature is 40 to 60°C, preferably 40 to 60°C; the stirring speed is 100 to 1000 rpm, preferably 200 to 600 rpm; and the stirring time is 1 to 8 hours, preferably 3 to 5 hours.

[0033] Furthermore, in step four, the conditions for thermally induced phase separation are:

[0034] The low temperature is -10 to -86℃, preferably -20 to -40℃; the standing separation time is 12 to 96 hours, preferably 24 to 36 hours.

[0035] Furthermore, in step five, the conditions for the formation of porous microspheres are as follows:

[0036] The mass ratio of the frozen emulsion to distilled water is 1:1 to 1:15, preferably 1:2 to 1:10; the stirring speed is 50 to 200 rpm, preferably 100 to 200 rpm; and the stirring time is 10 to 40 min, preferably 10 to 20 min.

[0037] Further, in step six, the concentration of the gelatin aqueous solution is 0.1–5 wt%, preferably 0.5–3 wt%.

[0038] Furthermore, a third objective of this invention is to provide applications of the biodegradable microspheres, which is one of the objectives of this invention.

[0039] The biodegradable microspheres provided by this invention have a connected pore structure, which is suitable for cell adhesion, spreading, proliferation and nutrient exchange. Furthermore, the biodegradable microspheres are loaded with natural gelatin, which increases the biofunctionality of the biodegradable microspheres and further promotes cell adhesion and growth.

[0040] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0041] 1. This invention combines biodegradable polyester, polyol, and dispersant, and obtains biodegradable polyester microspheres with suitable pore structure through thermally induced phase separation-crystallization. The microspheres have a through-hole structure inside and out, which is conducive to multi-dimensional cell adhesion and nutrient exchange.

[0042] 2. The biodegradable polyester molecular chains used in this invention have relatively few reactive groups, making surface modification difficult. For example, using alkaline hydrolysis or aminolysis to introduce reactive groups into the backbone can damage the microsphere structure itself, and the degree of reaction is difficult to control. This invention uses a vacuum impregnation method that does not damage the microsphere structure and can load natural macromolecular collagen onto the surface and interior of the microspheres, increasing biofunctionality and promoting cell adhesion.

[0043] 3. The microsphere preparation and modification method proposed in this invention is simple to operate, has mild conditions, and has promising industrial application prospects. Attached Figure Description

[0044] Figure 1 This is a SEM image of the biodegradable microspheres provided in Example 1 of the present invention;

[0045] Figure 2 SEM image of the biodegradable microspheres provided in Comparative Example 1 of this invention;

[0046] Figure 3 This is an electron microscope image of the biodegradable microspheres provided in Comparative Example 1 of the present invention after 3 days of cell culture.

[0047] Figure 4 This is an electron micrograph of the biodegradable microspheres provided in Example 1 of the present invention after 3 days of cell culture.

[0048] Figure 5 This is a staining image of live cells after 3 days of cell culture using the biodegradable microspheres provided in Comparative Example 1 of the present invention.

[0049] Figure 6 The image shows the staining of live cells after 3 days of cell culture using the biodegradable microspheres provided in Example 1 of this invention. Detailed Implementation

[0050] The present invention will now be described in detail with reference to the accompanying drawings and embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the content of the present invention are still within the scope of protection of the present invention.

[0051] In the following examples and comparative examples, the raw materials were all commercially available products.

[0052] The testing methods for the following embodiments are as follows:

[0053] The average particle size of the microspheres was tested using an Anton Paar PSA900 laser particle size analyzer. A laser particle size analyzer is an instrument that analyzes particle size based on the spatial distribution (scattering spectrum) of diffracted or scattered light. It employs Furanhofer diffraction and Mie scattering theories, and the testing process is unaffected by factors such as temperature changes, medium viscosity, sample density, and surface condition. As long as the sample is uniformly displayed in the laser beam, the test results can be obtained. The microspheres of this invention are tested using a wet method, where the sample is directly added to ethanol for dispersion, and then analyzed for particle size through an optical path system.

[0054] The average pore size and porosity of the microspheres were obtained by mercury porosimetry using an AUTOPORE 9500 mercury porosimeter from Microchip Technology, USA. The mercury porosimetry test was performed in accordance with GB / T 21650.3-2011.

[0055] Example 1

[0056] This embodiment illustrates the preparation of biodegradable microspheres, and the specific steps are as follows:

[0057] Step 1: Dissolve 0.4g of poly-L-lactic acid (PLLA) (molecular weight 400,000) in 10g of tetrahydrofuran and heat to 40℃ to form a homogeneous and transparent solution.

[0058] Step 2: Disperse 0.4g of polyethylene glycol 200 evenly in 35g of butanediol, and preheat in a 45℃ water bath to keep the system temperature constant.

[0059] Step 3: In a 45℃ water bath environment, at a rotation speed of 550 rpm, add the PLLA tetrahydrofuran solution to butanediol containing polyethylene glycol 200 and emulsify and disperse for 3 hours.

[0060] Step 4: Quickly transfer the above system to an environment of -20°C and let it stand for 24 hours.

[0061] Step 5: Remove the above mixture from the low-temperature environment, pour in 500g of distilled water, and stir at 50rpm for 40min. Stop stirring, let it stand, and then filter the mixture to obtain biodegradable polyester porous microspheres. Dry the microspheres at 40℃ for later use.

[0062] Step Six: Prepare a 1.2% gelatin aqueous solution, add the microspheres mentioned above, and transfer the system to a colorimetric tube. Place a sponge above the liquid surface, ensuring the sponge is in close contact with the tube wall. Repeatedly evacuate the colorimetric tube to allow the gelatin aqueous solution to fully penetrate the microsphere pores. Remove the microspheres and place them in a freezer to solidify the gelatin solution loaded inside and on the surface of the microspheres. Then freeze-dry to obtain the biodegradable microspheres.

[0063] Figure 1 The image shows a SEM image of the biodegradable microspheres prepared in this embodiment.

[0064] The detailed evaluation results of the pore structure of the biodegradable microspheres in this embodiment are shown in Table 1.

[0065] Example 2

[0066] This embodiment illustrates the preparation of biodegradable microspheres, and the specific steps are as follows:

[0067] Step 1: Dissolve 0.5g of glycolide-lactide copolymer PLGA (molecular weight 50000) in 20g of acetonitrile and heat to 50℃ to form a homogeneous and transparent solution.

[0068] Step 2: Disperse 0.8g of polyethylene glycol 400 in 40g of glycerol and place it in a 50°C water bath until the temperature is constant.

[0069] Step 3: In a 50℃ water bath environment, add the PLGA acetonitrile solution to the glycerol in which polyethylene glycol 400 is dispersed in Step 2, and emulsify and disperse for 4 hours.

[0070] Step 4: Quickly transfer the above system to an environment of -40℃ and let it stand for 30 hours.

[0071] Step 5: Remove the above mixture from the low-temperature environment, pour in 750g of distilled water, and stir at 100rpm for 20min. Stop stirring, let it stand, and then filter the mixture to obtain biodegradable polyester porous microspheres. Dry the microspheres at 40℃ for later use.

[0072] Step Six: Prepare a 2% gelatin aqueous solution, add the microspheres mentioned above, and transfer the system to a colorimetric tube. Place a sponge above the liquid surface, ensuring the sponge is in close contact with the tube wall. Repeatedly evacuate the colorimetric tube to allow the gelatin aqueous solution to fully penetrate the microsphere pores. Remove the microspheres and rapidly cool them to solidify the gelatin solution loaded inside and on the surface of the microspheres. Then, freeze-dry them to obtain the biodegradable microspheres.

[0073] The detailed evaluation results of the pore structure of the biodegradable microspheres in this embodiment are shown in Table 1.

[0074] Example 3

[0075] This embodiment illustrates the preparation of biodegradable microspheres, and the specific steps are as follows:

[0076] Step 1: Dissolve 0.5g of polyethylene glycol-lactide copolymer PEGLA (molecular weight 120000) in 10g of dimethyl sulfoxide and heat to 55℃ to form a homogeneous and transparent solution.

[0077] Step 2: Disperse 0.2g of polyethyleneimine into 40g of pentaerythritol and place it in a 55℃ water bath until the temperature is constant.

[0078] Step 3: In a 55°C water bath environment, add the PEGLA dimethyl sulfoxide solution to the pentaerythritol in which polyethyleneimine is dispersed in Step 2, and emulsify and disperse for 5 hours.

[0079] Step 4: Quickly transfer the above system to an environment of -30°C and let it stand for 24 hours.

[0080] Step 5: Remove the above mixture from the low-temperature environment, pour in 400g of distilled water, and stir at 150rpm for 30min. Stop stirring, let it stand, and then filter the mixture to obtain biodegradable polyester porous microspheres. Dry the microspheres at 40℃ for later use.

[0081] Step Six: Prepare a 0.8% gelatin aqueous solution, add the microspheres mentioned above, and transfer the system to a colorimetric tube. Place a sponge above the liquid surface, ensuring the sponge is in close contact with the tube wall. Repeatedly evacuate the colorimetric tube to allow the gelatin aqueous solution to fully penetrate the microsphere pores. Remove the microspheres and rapidly cool them to solidify the gelatin solution loaded inside and on the surface of the microspheres. Then freeze-dry to obtain the biodegradable microspheres.

[0082] The detailed evaluation results of the pore structure of the biodegradable microspheres in this embodiment are shown in Table 1.

[0083] Example 4

[0084] This embodiment illustrates the preparation of biodegradable microspheres, and the specific steps are as follows:

[0085] Step 1: Dissolve 0.15g of polycaprolactone-lactide copolymer PCLA (molecular weight 250,000) in 10g of dioxane and heat to 60℃ to form a homogeneous and transparent solution.

[0086] Step 2: Disperse 0.9g of polyethylene glycol 200 into 30g of ethylene glycol and place it in a 40℃ water bath until the temperature is constant.

[0087] Step 3: In a 42℃ water bath environment, add the PCLA dioxane solution to the ethylene glycol in which polyethylene glycol 200 is dispersed in Step 2, and emulsify and disperse for 3.5 hours.

[0088] Step 4: Quickly transfer the above system to an environment of -25°C and let it stand for 36 hours.

[0089] Step 5: Remove the above mixture from the low-temperature environment, pour in 250g of distilled water, and stir at 150rpm for 20min. Stop stirring, let it stand, and then filter the mixture to obtain biodegradable polyester porous microspheres. Dry the microspheres at 40℃ for later use.

[0090] Step Six: Prepare a 2.2% gelatin aqueous solution, add the microspheres mentioned above, and transfer the system to a colorimetric tube. Place a sponge above the liquid surface, ensuring the sponge is in close contact with the tube wall. Repeatedly evacuate the colorimetric tube to allow the gelatin aqueous solution to fully penetrate the microsphere pores. Remove the microspheres and rapidly cool them to solidify the gelatin solution loaded inside and on the surface of the microspheres. Then freeze-dry to obtain the biodegradable microspheres.

[0091] The detailed evaluation results of the pore structure of the biodegradable microspheres in this embodiment are shown in Table 1.

[0092] Example 5

[0093] This embodiment illustrates the preparation of biodegradable microspheres, and the specific steps are as follows:

[0094] Step 1: Dissolve 0.22g of poly(D-lactic acid) PDLA (molecular weight 300,000) in 10g of N,N-dimethylformamide and heat to 70℃ to form a homogeneous and transparent solution.

[0095] Step 2: Disperse 1.8g of polyethylene glycol 400 into 40g of trimethylolpropane and place it in a 60°C water bath until the temperature is constant.

[0096] Step 3: In a 60°C water bath environment, at a rotation speed of 600 rpm, add the N,N-dimethylformamide solution of PDLA to the trimethylolpropane in which polyethylene glycol 400 is dispersed in Step 2, and emulsify and disperse for 3 hours.

[0097] Step 4: Quickly transfer the above system to an environment of -35°C and let it stand for 28 hours.

[0098] Step 5: Remove the above mixture from the low-temperature environment, pour in 300g of distilled water, and stir at 100rpm for 20min. Stop stirring, let it stand, and then filter the mixture to obtain biodegradable polyester porous microspheres. Dry the microspheres at 40℃ for later use.

[0099] Step Six: Prepare a 2.8% gelatin aqueous solution, add the microspheres mentioned above, and transfer the system to a colorimetric tube. Place a sponge above the liquid surface, ensuring the sponge is in close contact with the tube wall. Repeatedly evacuate the colorimetric tube to allow the gelatin aqueous solution to fully penetrate the microsphere pores. Remove the microspheres and rapidly cool them to solidify the gelatin solution loaded inside and on the surface of the microspheres. Then freeze-dry to obtain the biodegradable microspheres.

[0100] The detailed evaluation results of the pore structure of the biodegradable microspheres in this embodiment are shown in Table 1.

[0101] Comparative Example 1

[0102] This comparative example is used to illustrate the preparation of microspheres. The preparation method is basically the same as that in Example 1, except that step six is ​​not included, that is, the porous microspheres in this comparative example are not loaded with gelatin.

[0103] Figure 2 The SEM image of the porous microspheres prepared in this comparative example is shown.

[0104] The detailed evaluation results of the pore structure of the microspheres in this comparative example are shown in Table 1.

[0105] Table 1 shows the test data for the average particle size, average pore size, and porosity of the microspheres provided in Examples 1-5 and Comparative Example 1.

[0106] Table 1:

[0107] sample Example 1 Example 2 Example 3 Example 4 Example 5 Comparative Example 1 Average particle size (μm) 97.2 247.9 182.5 239.6 151.9 101.3 Average pore size (μm) 19.3 31.1 25.6 41.5 34.8 18.9 Porosity (%) 84.12 86.57 87.38 90.25 88.43 82.94

[0108] As shown in Table 1, the biodegradable polyester microspheres prepared in Examples 1-5 and Comparative Example 1 all have large pore sizes and high porosity. The gelatin coating did not affect the pore size or particle size of the microspheres.

[0109] Furthermore, by Figure 1 and Figure 2 A comparison of electron micrographs of Comparative Example 1 and Example 1 shows that the microspheres prepared in Comparative Example 1 have a fibrous porous structure. In Example 1, after coating the microspheres with gelatin, obvious blocky coatings appeared on the microspheres, indicating that the gelatin was loaded onto the microspheres, but the pore structure of the microspheres remained within the range suitable for cell survival.

[0110] The following describes the cell culture experiments using the microspheres provided in Example 1 and Comparative Example 1:

[0111] The experimental methods used are as follows:

[0112] (1) Sterilization of polymer microsphere carriers: Before inoculating cells, the polymer microsphere carriers were sterilized by radiation at 60°C; then soaked in 75% alcohol for 30 minutes, washed 3 times with PBS, washed 3 times with complete culture medium, and soaked in this culture medium for use the next day.

[0113] (2) In vitro cell seeding with polymer microsphere carriers: 2×10 6 One cell and 10 mg of polymer microsphere carrier were placed in a centrifuge tube and centrifuged at 1000 r / min for 4 min. After the cells and microspheres were evenly mixed, the supernatant was discarded and complete culture medium was added. The cells were then cultured in a cell culture incubator at 37℃, 5% CO2, and saturated humidity for 2 hours. After that, the cells were removed, pipetted and mixed evenly, and then transferred to a well plate for further culture.

[0114] (3) CCK-8 assay: Cells were seeded onto microspheres and cultured in complete culture medium under standard cell culture conditions. Cell proliferation was characterized using a CCK-8 assay kit after 1, 3, and 5 days of culture.

[0115] (4) Live and dead cell staining test

[0116] Cells were loaded at 2×10 5Cells were seeded at a density of [number] cells / mg onto different microsphere carriers and cultured in complete culture medium under standard cell culture conditions. After a period of culture, live cells were stained using the LIVE / DEAD cell viability assay kit to characterize cell viability.

[0117] Figures 3-4 SEM images of the microsphere carriers and cells cultured for 3 days provided in Example 1 and Comparative Example 1 are shown.

[0118] Figures 5-6 The images show staining patterns of live cells after 3 days of cell culture using the microsphere carriers provided in Example 1 and Comparative Example 1.

[0119] As shown in the figure, loading gelatin onto microspheres can effectively increase the adhesion rate of cells on and inside the microspheres, which is more conducive to cell adhesion and proliferation.

[0120] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A biodegradable microsphere comprising a porous microsphere with a through-hole internal pore structure, and a gelatin component loaded on the porous microsphere.

2. The biodegradable microspheres according to claim 1, characterized in that, The mass percentage of the gelatin component loaded on the porous microspheres is 0.01–5%, preferably 0.5–3%.

3. The biodegradable microspheres according to claim 1, characterized in that, The porous microspheres have a particle size of 10–1000 μm, preferably 100–500 μm; a pore size of 1–100 μm, preferably 10–60 μm; and a porosity of 70–95%, preferably 80–95%.

4. The method for preparing biodegradable microspheres according to any one of claims 1 to 3, wherein the method comprises: A biodegradable polyester solution and a preheated mixture of polyol and dispersant are mixed to form an emulsion. Thermal phase separation is then performed, and the separated precipitate is dried to obtain porous microspheres. The porous microspheres are then added to a gelatin solution, and gelatin is loaded onto the porous microspheres to obtain the biodegradable microspheres.

5. The method for preparing biodegradable microspheres according to claim 4, characterized in that, The biodegradable polyester has a molecular weight of 2000–2000000 g / mol; preferably selected from one or a combination of poly(L-lactic acid), poly(D-lactic acid), glycolide-lactide copolymer, polycaprolactone-lactide copolymer, and polyethylene glycol-lactide copolymer; and / or, The polyol is selected from one or a combination of ethylene glycol, glycerol, butanediol, hexanediol, pentanediol, pentaerythritol, diethylene glycol, dipropylene glycol, trimethylolpropane, and isopropanol; and / or, The dispersant is selected from one or a combination of polyethylene glycol and polyethyleneimine.

6. The method for preparing biodegradable microspheres according to claim 4, characterized in that, The method includes the following steps: Step 1: Add the biodegradable polyester to an organic solvent, heat and stir to mix evenly to obtain a biodegradable polyester solution; Step 2: Mix the polyol and dispersant evenly, preheat, and obtain a mixture; Step 3: While stirring, add the biodegradable polyester solution from Step 1 to the mixture from Step 2 to form an emulsion; Step 4: Place the emulsion from Step 3 in a low-temperature environment and allow it to stand until thermal phase separation occurs; Step 5: Remove the frozen emulsion and pour it into distilled water. Stir to separate the precipitate and dry to obtain porous microspheres. Step 6: Place the porous microspheres obtained in Step 5 into a gelatin aqueous solution, repeatedly evacuate the vacuum to release the gas, and freeze-dry to obtain biodegradable microspheres.

7. The method for preparing biodegradable microspheres according to claim 6, characterized in that, In step one, the organic solvent is selected from one or a combination of tetrahydrofuran, dioxane, N,N-dimethylformamide, dimethyl sulfoxide, acetonitrile, and dimethyl ether; and / or, the concentration of the biodegradable polyester solution is 0.1 to 8 wt%, preferably 0.5 to 5 wt%.

8. The method for preparing biodegradable microspheres according to claim 6, characterized in that, In step one, the heating temperature is 40–90°C, preferably 50–70°C.

9. The method for preparing biodegradable microspheres according to claim 6, characterized in that, In step two, the preheating temperature is 30–80°C, preferably 40–60°C; and / or, the mass concentration of the dispersant in the polyol is 0.05–10 wt%, preferably 0.1–5 wt%.

10. The method for preparing biodegradable microspheres according to claim 6, characterized in that, In step three, the conditions for emulsion formation are as follows: The mass ratio of the biodegradable polyester solution to the polyol is 1:1 to 1:10, preferably 1:2 to 1:4; and / or, The emulsification temperature is 40–60°C, preferably 40–60°C; and / or, The stirring speed is 100–1000 rpm, preferably 200–600 rpm; and / or, The stirring time is 1 to 8 hours, preferably 3 to 5 hours.

11. The method for preparing biodegradable microspheres according to claim 6, characterized in that, In step four, the conditions for thermally induced phase separation are: a low temperature of -10 to -86°C, preferably -20 to -40°C; and / or a settling time of 12 to 96 hours, preferably 24 to 36 hours.

12. The method for preparing biodegradable microspheres according to claim 6, characterized in that, In step five, the conditions for the formation of the porous microspheres are as follows: The mass ratio of the frozen emulsion to distilled water is 1:1 to 1:15, preferably 1:2 to 1:10; and / or, The stirring speed is 50–200 rpm, preferably 100–200 rpm; and / or, The stirring time is 10 to 40 minutes, preferably 10 to 20 minutes.

13. The method for preparing biodegradable microspheres according to claim 6, characterized in that, In step six, the concentration of the gelatin aqueous solution is 0.1–5 wt%, preferably 0.5–3 wt%.

14. The application of the biodegradable microspheres according to any one of claims 1 to 3 for cell adhesion and growth.

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