Poly-l-lactic acid microspheres, and preparation method and application thereof

Abstract

The application belongs to the technical field of biomedical materials, and relates to a poly-L-lactic acid microsphere as well as a preparation method and application thereof. The poly-L-lactic acid microsphere has an amorphous structure, the shape of the poly-L-lactic acid microsphere is a regular sphere, and the poly-L-lactic acid microsphere comprises a smooth and dense surface layer and an internal porous structure. The poly-L-lactic acid microsphere provided by the application has an amorphous structure, can realize uniform degradation, can avoid non-uniform degradation behaviors such as'massive disintegration', and can reduce the occurrence of local aseptic inflammatory reactions. The poly-L-lactic acid microsphere has a smooth and dense surface layer and an internal porous structure, can reduce resistance during injection, is more easily injected, and has excellent tissue compatibility.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a poly(L-lactic acid) microsphere, its preparation method, and its application. Background Technology

[0002] Poly-L-lactic acid (PLLA) has shown broad application prospects in fields such as cosmetic fillers, tissue engineering scaffolds, and drug-controlled release carriers due to its good biocompatibility, biodegradability, and suitable mechanical strength. In recent years, with the rapid development of regenerative medical aesthetic materials, injectable microspheres based on PLLA have become an important choice for stimulating collagen regeneration and achieving long-term soft tissue filling and texture improvement. However, PLLA microspheres prepared by existing technologies have the following technical defects.

[0003] The crystalline structure leads to degradation behavior and limitations in biocompatibility. Poly-L-lactic acid (PLA) molecular chains have a strong tendency to crystallize. Existing PLA microspheres tend to form high crystallinity during preparation, resulting in an excessively long degradation period in vivo and a "blocky disintegration" pattern. This non-uniform degradation behavior not only triggers localized aseptic inflammatory reactions (such as redness, swelling, and nodules), but also makes it difficult to match the degradation rate with the body's own collagen regeneration rate, affecting the durability and naturalness of the filling effect. Although rapid freezing and solvent quenching can further reduce the crystallinity of PLA microspheres, certain crystalline regions still exist in the material and are difficult to control effectively.

[0004] Traditional preparation processes lack sufficient precision in structural control. Traditional methods, such as homogenization emulsification-solvent evaporation, lack precise means to control the particle size distribution, morphological regularity, and internal pore structure of microspheres. This results in microsphere products with simple structures, wide size distributions, poor monodispersity, and poor injectability, which severely limits drug loading efficiency, bioactive factor loading capacity, and the three-dimensional space required for cell ingrowth. Consequently, their function is mainly limited to "physical occupancy," while their "active regeneration" effect is limited.

[0005] Existing technologies face challenges in synergistically controlling the structure and performance of poly-L-lactic acid (PLA) microspheres. To improve the properties of PLA microspheres, current techniques attempt to prepare porous PLA microspheres through methods such as reemulsification, phase separation, or gas foaming. However, while these methods generally introduce high porosity and interconnected pore structures, they struggle to effectively control the crystallization behavior of PLA microspheres, resulting in a high degree of crystallinity. Because the balance between crystallinity and porous structure remains unresolved, it is difficult to synergistically optimize the degradation behavior, injection performance, mechanical properties, and drug release kinetics of PLA microspheres.

[0006] Therefore, there is a need to develop poly-L-lactic acid microspheres that can achieve uniform degradation, have good injection performance, mechanical properties, and can be used in fields such as drug release. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides poly-L-lactic acid microspheres, their preparation method, and applications. The poly-L-lactic acid microspheres prepared using the method provided by this invention possess an amorphous structure capable of uniform degradation, a smooth and dense surface, and an internal porous structure with high porosity, exhibiting excellent injection performance and mechanical properties.

[0008] To achieve this objective, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a poly-L-lactic acid microsphere, wherein the poly-L-lactic acid microsphere has an amorphous structure, the poly-L-lactic acid microsphere is in the shape of a regular sphere, and the poly-L-lactic acid microsphere includes a smooth and dense surface layer and an internal porous structure.

[0010] In this invention, the poly-L-lactic acid microspheres have an amorphous structure that enables uniform degradation, avoiding non-uniform degradation behavior such as "block disintegration" and reducing the occurrence of local aseptic inflammatory reactions. The poly-L-lactic acid microspheres have a smooth and dense surface layer, which reduces resistance during injection and makes injection easier compared to poly-L-lactic acid microspheres with a rough porous structure or irregular shape. In addition, the internal porous structure of the poly-L-lactic acid microspheres can carry substances such as proteins, peptides, and small molecule chemicals according to actual needs. Furthermore, after the surface layer of the poly-L-lactic acid microspheres degrades, the internal porous structure can serve as an excellent porous scaffold for cell loading and growth.

[0011] In this invention, the smooth and dense surface refers to the surface of the poly-L-lactic acid microspheres observed under a scanning electron microscope at 3000x magnification, where no pore structure communicating with the interior was observed, nor were there any obvious protrusions. The porous structure refers to a three-dimensional interconnected porous sponge-like network.

[0012] Preferably, the porosity of the poly-L-lactic acid microspheres is 10% to 90%, such as 20%, 30%, 40%, 50%, 60%, 70%, or 80%.

[0013] Preferably, the average particle size of the poly-L-lactic acid microspheres is 10~200 μm, such as 20 μm, 40 μm, 60 μm, 80 μm, 100 μm, 120 μm, 140 μm, 160 μm or 180 μm.

[0014] Preferably, the coefficient of variation of the poly-L-lactic acid microspheres is 1% to 10%, such as 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 9%.

[0015] In a second aspect, the present invention provides a method for preparing poly-L-lactic acid microspheres as described in the first aspect, the method comprising the following steps: (1) mixing poly-L-lactic acid, an amorphization inducer, and an organic solvent to obtain an oil phase solution; (2) mixing a surfactant, an amorphization inducer, and water to obtain an aqueous phase solution; (3) mixing the oil phase solution obtained in step (1) and the aqueous phase solution obtained in step (2) to obtain an oil-in-water emulsion; (4) mixing the oil-in-water emulsion obtained in step (3) with an oil containing... The aqueous solution of the surfactant is mixed and stirred to allow the organic solvent to evaporate and the microspheres to solidify. The mixing and stirring temperatures are each independently set at 5~25℃ (e.g., 6℃, 8℃, 10℃, 12℃, 14℃, 16℃, 18℃, 20℃, 22℃, or 24℃, etc.) to obtain the poly-L-lactic acid microspheres. Steps (1) and (2) are performed separately or simultaneously, without any specific order. In step (1), the mass-to-volume ratio of the amorphization inducer to the organic solvent is 0.5~30. g / 100mL (e.g., 1 g / 100mL, 2 g / 100mL, 4 g / 100mL, 6 g / 100mL, 8 g / 100mL, 10 g / 100mL, 12 g / 100mL, 14 g / 100mL, 16 g / 100mL, 18 g / 100mL, 20 g / 100mL, 22 g / 100mL, 24 g / 100mL, 26 g / 100mL, or 28 g / 100mL, etc.); the mass-to-volume ratio of the amorphization inducing agent to water in step (2) is 0.5~25 g / 100mL (e.g., 1 g / 100mL, 2 g / 100mL, 4 g / 100mL, 6 g / 100mL, 8 g / 100mL, 10 g / 100mL, 12 g / 100mL, 14 g / 100mL, 16 g / 100mL, etc.). (g / 100mL, 18 g / 100mL, 20 g / 100mL, 22 g / 100mL or 24 g / 100mL, etc.).

[0016] In this invention, the addition of an amorphization inducer to the oil-phase solution and the aqueous-phase solution can inhibit the crystallization of poly-L-lactic acid (PLA) and induce PLA to achieve confined amorphization. The amorphization inducer in the aqueous-phase solution also stabilizes the oil-in-water emulsion and regulates the interface. Specifically, during the microsphere solidification stage caused by the evaporation of the organic solvent, the amorphization inducer, driven by both organic solvent diffusion and phase separation, not only acts as a dynamic pore-forming template, physically creating pores, but also undergoes directional diffusion and interfacial enrichment in the oil and water phases of the oil-in-water emulsion, forming phase separation and occupying pore space. After solidification, it is selectively removed, forming a three-dimensional interconnected sponge-like porous network. Furthermore, it also plays a role in confined amorphization. Through the interaction between the amorphization inducer molecules and the PLA molecular chains, it inhibits the ordered arrangement and crystallization behavior of the PLA molecular chains, inducing the formation of stable amorphous PLA within the confined space inside the dispersed phase droplets in the oil-in-water emulsion. Furthermore, the amorphization inducer in the aqueous solution enriches at the oil-water interface to form a stable film, thereby reducing interfacial energy and inhibiting bubble nucleation and phase separation pore formation. On the other hand, it forms hydrogen bonds with the poly-L-lactic acid molecular chains to restrict chain segment movement, resulting in a smooth and dense surface after solidification. This achieves a synergistic, one-step controlled preparation of the two key structural features: "amorphous state" and "dense on the outside and sparse on the inside".

[0017] In this invention, the poly-L-lactic acid microspheres are regularly spherical with a smooth, dense surface and good injectability. The interior has a three-dimensional, interconnected, porous, sponge-like structure, and the overall structure is amorphous. This amorphous structure ensures uniform and gradual degradation, avoiding the localized acidic accumulation and severe inflammatory response caused by the blocky disintegration of crystalline poly-L-lactic acid. The high specific surface area and interconnected channels effectively promote cell adhesion, migration, and growth, and significantly improve the loading efficiency of drugs, growth factors, etc. The smooth, dense surface layer has multiple beneficial functions: during preparation, it supports the internal three-dimensional, interconnected porous, sponge-like network to prevent collapse; during injection, it reduces resistance and ensures needle penetration; in vivo, it delays immune clearance and prolongs cell retention; during degradation, it acts as a barrier to regulate the rate and prevent acidosis; and in conjunction with the internal structure of the three-dimensional, interconnected porous, sponge-like network, it achieves the dual functions of "physical filling" and "tissue regeneration."

[0018] In this invention, the mixing and stirring in step (4) are carried out in a low temperature environment of 5~25℃. This low temperature environment achieves low temperature phase separation by regulating phase separation kinetics, suppressing bubble nucleation and stabilizing the amorphous state. It is a key process parameter for achieving a completely amorphous state and a structure with dense outer and sparse inner structure.

[0019] Preferably, the mass ratio of the amorphization inducing agent to poly-L-lactic acid in step (1) is (0.1~6):1, for example, 0.2:1, 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1 or 5.5:1, etc.

[0020] Preferably, the mass-to-volume ratio of the surfactant to water in step (2) is 0.1~5 g / 100mL, for example 0.5g / 100mL, 1 g / 100mL, 1.5 g / 100mL, 2 g / 100mL, 2.5 g / 100mL, 3 g / 100mL, 3.5 g / 100mL, 4 g / 100mL or 4.5 g / 100mL, etc.

[0021] In this invention, the ratio of oil phase solution to aqueous phase solution used in step (3) of preparing the oil-in-water emulsion is not limited in this invention, as long as the oil-in-water emulsion is prepared. This invention only provides illustrative examples here:

[0022] Preferably, the volume ratio of the oil phase solution and the aqueous phase solution in step (3) is 1:(1~50), for example, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40 or 1:45, etc.

[0023] Preferably, the poly-L-lactic acid has a viscosity-average molecular weight of 5 to 500 kDa, such as 10 kDa, 50 kDa, 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, or 450 kDa.

[0024] Preferably, the amorphization inducing agents in steps (1) and (2) each independently include a polymer amorphization inducing agent and / or a small molecule amorphization inducing agent, and more preferably a polymer amorphization inducing agent.

[0025] Preferably, the polymer amorphization inducing agent includes any one or a combination of at least two of polyvinylpyrrolidone, poloxamer, or polyethylene glycol.

[0026] For example, the poloxamer includes poloxamer L62.

[0027] Preferably, the small molecule amorphization inducer includes any one or a combination of at least two of ethanol, ethylene glycol, n-propanol, isopropanol, propylene glycol, or glycerol.

[0028] Preferably, the organic solvent includes any one or a combination of at least two of dichloromethane, chloroform, dichloroethane, ethyl acetate, acetone, dimethyl sulfoxide, or tetrahydrofuran.

[0029] Preferably, the surfactant in step (2) includes any one or a combination of at least two of polyvinyl alcohol, gelatin, Tween, sodium lauryl sulfate, hyaluronic acid, carboxymethyl cellulose, alginate, polylysine, or polyacetylimide.

[0030] Preferably, the mixing in step (3) includes microfluidic mixing or emulsification using a membrane emulsification device, and more preferably microfluidic mixing.

[0031] In this invention, by combining the confined amorphization strategy induced by an amorphization inducer with microfluidics or membrane emulsification technology, the microfluidics or membrane emulsification technology can achieve precise control of the size of the dispersed phase droplets in the oil-in-water emulsion, improving uniformity. As a result, the prepared poly-L-lactic acid microspheres have high particle size uniformity, low coefficient of variation, and smooth and dense surface, ensuring good injectability and preventing clogging of the syringe needle.

[0032] Preferably, the surfactant in the aqueous solution containing surfactant in step (4) includes any one or a combination of at least two of polyvinyl alcohol, gelatin, Tween, sodium dodecyl sulfate, hyaluronic acid, carboxymethyl cellulose, alginate, polylysine, or polyacetylimide.

[0033] For example, the polyvinyl alcohol includes PVA-1788; the Tween includes Tween 40.

[0034] Preferably, the mass percentage concentration of the surfactant in the aqueous solution containing the surfactant in step (4) is 0.1% to 5%, for example, 0.3%, 0.6%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0% or 4.5%.

[0035] Preferably, the stirring speed in step (4) is 20~500 rpm, such as 50 rpm, 100 rpm, 150 rpm, 200 rpm, 250 rpm, 300 rpm, 350 rpm, 400 rpm or 450 rpm.

[0036] Preferably, the stirring time in step (4) is 1 to 48 hours, such as 5 hours, 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours or 45 hours.

[0037] Preferably, the stirring process further includes separation, washing, and drying steps.

[0038] Preferably, the separation includes separation by centrifugation or filtration.

[0039] Preferably, the washing includes washing with water.

[0040] Preferably, the number of washing cycles is 3 to 6, such as 4 or 5 times.

[0041] Preferably, the separation and washing temperatures are each independently 5~25℃, for example 6℃, 8℃, 10℃, 12℃, 14℃, 16℃, 18℃, 20℃, 22℃ or 24℃, etc.

[0042] Preferably, the drying includes any one or a combination of at least two of freeze drying, vacuum drying, supercritical fluid drying, or oven drying.

[0043] Preferably, the pre-freezing temperature for freeze drying is -80℃ to -20℃.

[0044] Preferably, the vacuum drying temperature is 20℃~50℃.

[0045] Preferably, the temperature for drying the supercritical fluid is 30°C to 50°C.

[0046] Preferably, the drying temperature is 20℃~50℃.

[0047] Thirdly, the present invention provides the application of poly-L-lactic acid microspheres as described in the first aspect or poly-L-lactic acid microspheres prepared by the preparation method described in the second aspect in the preparation of soft tissue filling materials, tissue engineering materials or drug delivery materials.

[0048] In this invention, the preparation method induces confined amorphization of poly-L-lactic acid by adding an amorphization inducer, and preferably employs microfluidic or membrane emulsification technology combined with a low-temperature phase separation process. This achieves precise and synergistic control of particle size, porosity, pore size distribution, and amorphous structure, resulting in high process reproducibility and suitability for large-scale production. The poly-L-lactic acid microspheres prepared by this method exhibit superior comprehensive performance. In the field of medical aesthetics and regeneration, they can serve as an ideal soft tissue filler with long-lasting, safe, and regenerative functions; in tissue engineering, they can serve as excellent porous scaffolds for cell loading and growth; and in drug delivery, they can serve as highly efficient controlled-release carriers suitable for delivering various drugs such as proteins, peptides, and small molecule chemicals.

[0049] Compared with the prior art, the present invention has at least the following beneficial effects:

[0050] The poly-L-lactic acid microspheres of this invention have an amorphous structure that enables uniform degradation, avoiding non-uniform degradation behavior such as "block disintegration" and reducing the occurrence of local aseptic inflammatory reactions. The poly-L-lactic acid microspheres have a smooth, dense surface and an internal porous structure. Compared to poly-L-lactic acid microspheres with a porous surface, this reduces resistance during injection and makes injection easier. The internal porous structure of the poly-L-lactic acid microspheres can, on the one hand, carry substances such as proteins, peptides, and small molecule chemicals according to actual needs; on the other hand, after the surface of the poly-L-lactic acid microspheres degrades, the internal porous structure can serve as an excellent porous scaffold for cell loading and growth, exhibiting excellent biocompatibility. The prepared poly-L-lactic acid microspheres have an injection thrust of ≤5N, are not prone to clogging the syringe needle, have good injection performance, and maintain a highly regular spherical shape and uniform particle size during subcutaneous degradation. They do not exhibit non-uniform degradation behavior of "block disintegration", and have a natural transition with the surrounding tissue, demonstrating excellent tissue compatibility and degradation uniformity. Attached Figure Description

[0051] Figure 1 This is a schematic diagram of the formation principle of poly-L-lactic acid microspheres in Example 1 and an optical microscope image of the formation process;

[0052] Figure 2 and Figure 3 This is a scanning electron microscope image of the poly-L-lactic acid microspheres prepared in Example 1;

[0053] Figure 4 and Figure 5 The image shows a scanning electron microscope image of the poly-L-lactic acid microspheres prepared in Comparative Example 1.

[0054] Figure 6 The image shows a comparison of the X-ray diffraction patterns of the poly-L-lactic acid microspheres prepared in Example 1 and those prepared in Comparative Example 1.

[0055] Figure 7 A comparison of the Fourier transform infrared spectra of the poly-L-lactic acid microspheres prepared in Example 1 and the poly-L-lactic acid microspheres prepared in Comparative Example 1.

[0056] Figure 8 The image shows a comparison of the hematoxylin-eosin (H&E) stained histological features of the amorphous poly-L-lactic acid microspheres with a dense outer layer and a sparse inner layer prepared in Example 1 and the poly-L-lactic acid microspheres prepared in Comparative Example 1 after subcutaneous implantation into the back of SD rats for 1-3 months.

[0057] Figure 9 The image shows a comparison of Masson-stained collagen fibers from the subcutaneous implantation of amorphous poly-L-lactic acid microspheres with a dense outer layer and a sparse inner layer prepared in Example 1 and poly-L-lactic acid microspheres prepared in Comparative Example 1 into the backs of SD rats for 1-3 months. Detailed Implementation

[0058] To facilitate understanding of the present invention, the following embodiments are provided. Those skilled in the art should understand that these embodiments are merely illustrative and should not be construed as limiting the scope of the invention.

[0059] Unless otherwise specified, the materials and equipment involved in the following detailed embodiments are all conventional materials and equipment in the art and will not affect the technical effects of the present invention.

[0060] Example 1

[0061] This embodiment provides a method for preparing poly-L-lactic acid microspheres, the preparation method comprising the following steps:

[0062] (1) Add 0.5 g of poly-L-lactic acid (viscosity average molecular weight of 100 kDa) and 0.2 g of amorphization inducer (polyvinylpyrrolidone, weight average molecular weight of 10000) to 10 mL of organic solvent (dichloromethane), and stir magnetically until completely dissolved to obtain a homogeneous oil phase solution.

[0063] (2) Dissolve 2.0 g surfactant (PVA-1788) and 1.0 g amorphization inducer (polyvinylpyrrolidone, weight average molecular weight of 10000) in 100 mL of deionized water, stir at 500 rpm for 2 h in a 90℃ water bath, and cool to room temperature (25℃) to obtain a homogeneous aqueous solution.

[0064] (3) Using a flow-focusing microfluidic chip, the oil phase solution obtained in step (1) and the aqueous phase solution obtained in step (2) are pumped into the chip at flow rates of 0.5 mL / h and 5 mL / h, respectively. The water-in-oil emulsion droplets are formed in the chip by fluid shearing action, thus obtaining the water-in-oil emulsion.

[0065] (4) At a temperature of 20°C, the oil-in-water emulsion obtained in step (3) is slowly added to an aqueous solution containing surfactant (PVA-1788) for dilution. The surfactant concentration in the aqueous solution is 1% by mass. The mixture is stirred at 100 rpm for 24 hours at 20°C to allow the dichloromethane to fully evaporate and the microspheres to solidify completely. Then, at a temperature of 20°C, the solidified microspheres are separated by centrifugation (5000 rpm, 5 min), the supernatant is removed, and the precipitate is collected. Deionized water is added to the precipitate, and the mixture is slowly stirred and washed for 1 hour. The mixture is then centrifuged again. The above "washing with water - stirring - centrifugation" operation is repeated 3 times to remove residual amorphization inducer and surfactant. The precipitate obtained by centrifugation is pre-frozen at -20°C and then freeze-dried for 48 hours to obtain a white powder product, which is the poly-L-lactic acid microsphere.

[0066] Example 2

[0067] This embodiment provides a method for preparing poly-L-lactic acid microspheres, the preparation method comprising the following steps:

[0068] (1) Add 0.5 g of poly-L-lactic acid (viscosity average molecular weight of 100 kDa) and 0.2 g of amorphization inducer (polyvinylpyrrolidone, weight average molecular weight of 10000) to 10 mL of organic solvent (dichloromethane), and stir magnetically until completely dissolved to obtain a homogeneous oil phase solution.

[0069] (2) Dissolve 1.0 g surfactant (sodium dodecyl sulfate) and 1.0 g amorphization inducer (polyvinylpyrrolidone, weight average molecular weight of 10000) in 100 mL of deionized water and stir at 500 rpm for 2 h in a water bath at 25 °C to obtain a homogeneous aqueous solution.

[0070] (3) Using a flow-focusing microfluidic chip, the oil phase solution obtained in step (1) and the aqueous phase solution obtained in step (2) are pumped into the chip at flow rates of 0.5 mL / h and 5 mL / h, respectively. The water-in-oil emulsion droplets are formed in the chip by fluid shearing action, thus obtaining the water-in-oil emulsion.

[0071] (4) At a temperature of 10°C, the oil-in-water emulsion obtained in step (3) is slowly added to an aqueous solution containing a surfactant (sodium dodecyl sulfate) for dilution. The surfactant concentration in the aqueous solution is 4% by mass. The mixture is stirred at 300 rpm for 48 hours at 10°C to allow the organic solvent to evaporate completely and the microspheres to solidify completely. Then, at a temperature of 20°C, the solidified microspheres are separated by centrifugation (5000 rpm, 5 min), the supernatant is removed, and the precipitate is collected. Deionized water is added to the precipitate, and the mixture is slowly stirred and washed for 1 hour. The mixture is then centrifuged again. The above "washing with water - stirring - centrifugation" operation is repeated 3 times to remove residual amorphization inducers and surfactants. The precipitate obtained by centrifugation is pre-frozen at -40°C and then freeze-dried for 48 hours to obtain a white powder product, which is the poly-L-lactic acid microsphere.

[0072] Example 3

[0073] This embodiment provides a method for preparing poly-L-lactic acid microspheres, the preparation method comprising the following steps:

[0074] (1) Add 0.5 g of poly-L-lactic acid (viscosity average molecular weight of 100 kDa) and 0.2 g of amorphization inducer (polyvinylpyrrolidone, weight average molecular weight of 10000) to 10 mL of organic solvent (dichloroethane), and stir magnetically until completely dissolved to obtain a homogeneous oil phase solution.

[0075] (2) Dissolve 5.0 g surfactant (Tween 40) and 1.0 g amorphization inducer (polyvinylpyrrolidone, weight average molecular weight of 10000) in 100 mL of deionized water, stir at 500 rpm for 2 h in a 90℃ water bath, and cool to room temperature (25℃) to obtain a homogeneous aqueous solution.

[0076] (3) Using a flow-focusing microfluidic chip, the oil phase solution obtained in step (1) and the aqueous phase solution obtained in step (2) are pumped into the chip at flow rates of 0.5 mL / h and 5 mL / h, respectively. The water-in-oil emulsion droplets are formed in the chip by fluid shearing action, thus obtaining the water-in-oil emulsion.

[0077] (4) At a temperature of 25°C, the oil-in-water emulsion obtained in step (3) is slowly added to an aqueous solution containing a surfactant (Tween 40) for dilution. The surfactant concentration in the aqueous solution is 1% by mass. The mixture is stirred at 500 rpm for 48 hours at 25°C to allow the organic solvent to evaporate completely and the microspheres to solidify completely. Then, at a temperature of 25°C, the solidified microspheres are separated by centrifugation (5000 rpm, 5 min), the supernatant is removed, and the precipitate is collected. Deionized water is added to the precipitate, and the mixture is slowly stirred and washed for 1 hour. The mixture is then centrifuged again. The above "washing with water - stirring - centrifugation" operation is repeated 3 times to remove residual amorphization inducers and surfactants. The precipitate obtained by centrifugation is pre-frozen at -80°C and then freeze-dried for 48 hours to obtain a white powder product, which is the poly-L-lactic acid microsphere.

[0078] Example 4

[0079] This embodiment provides a method for preparing poly-L-lactic acid microspheres, which differs from Example 1 in that 0.2 g of amorphization inducer (polyvinylpyrrolidone, weight average molecular weight of 10000) in step (1) is replaced with 0.3 g of amorphization inducer (poloxam L62); and the amorphization inducer (polyvinylpyrrolidone, weight average molecular weight of 10000) in step (2) is replaced with the same mass of amorphization inducer (poloxam L62); other conditions are the same as in Example 1.

[0080] Example 5

[0081] This embodiment provides a method for preparing poly(L-lactic acid) microspheres, which differs from Example 1 in that 0.2 g of amorphization inducer (polyvinylpyrrolidone, weight average molecular weight of 10000) in step (1) is replaced with 0.5 g of amorphization inducer (polyethylene glycol, weight average molecular weight of 5000); 1.0 g of amorphization inducer (polyvinylpyrrolidone, weight average molecular weight of 10000) in step (2) is replaced with 0.5 g of amorphization inducer (polyethylene glycol, weight average molecular weight of 5000); other conditions are the same as in Example 1.

[0082] Example 6

[0083] This embodiment provides a method for preparing poly-L-lactic acid microspheres, which differs from Example 1 in that the amorphization inducer (polyvinylpyrrolidone, weight-average molecular weight of 10,000) in step (1) is replaced with the same mass of amorphization inducer (ethylene glycol); the amorphization inducer (polyvinylpyrrolidone, weight-average molecular weight of 10,000) in step (2) is replaced with the same mass of amorphization inducer (ethylene glycol); other conditions are the same as in Example 1.

[0084] Example 7

[0085] This embodiment provides a method for preparing poly-L-lactic acid microspheres, which differs from Example 1 in that the amorphization inducer (polyvinylpyrrolidone, weight-average molecular weight of 10,000) in step (1) is replaced with the same mass of amorphization inducer (glycerol); the amorphization inducer (polyvinylpyrrolidone, weight-average molecular weight of 10,000) in step (2) is replaced with the same mass of amorphization inducer (glycerol); other conditions are the same as in Example 1.

[0086] Example 8

[0087] This embodiment provides a method for preparing poly-L-lactic acid microspheres. The difference between this method and Example 1 is that step (3) is adjusted as follows: the oil phase solution obtained in step (1) and the aqueous phase solution obtained in step (2) are mixed at a volume ratio of 1:10 and emulsified into a crude emulsion by high-speed shearing at 500 rpm; the crude emulsion is transferred to a membrane emulsifier and repeatedly passed through an SPG membrane emulsifier with a pore size of 50 μm under a pressure of 1.0 bar, and the emulsification is repeated 3 times to form monodisperse oil-in-water emulsion droplets, thus obtaining an oil-in-water emulsion. Other conditions are the same as in Example 1.

[0088] Example 9

[0089] This embodiment provides a method for preparing poly-L-lactic acid microspheres. The difference between this method and Example 1 is that the mass of the amorphization inducer (polyvinylpyrrolidone, weight-average molecular weight of 10000) in step (1) is adjusted to 0.1g, while other conditions are the same as in Example 1.

[0090] Example 10

[0091] This embodiment provides a method for preparing poly-L-lactic acid microspheres. The difference between this method and Example 1 is that the mass of the amorphization inducer (polyvinylpyrrolidone, weight-average molecular weight of 10000) in step (1) is adjusted to 1g, while other conditions are the same as in Example 1.

[0092] Example 11

[0093] This embodiment provides a method for preparing poly-L-lactic acid microspheres. The difference between this method and Example 1 is that the mass of the amorphization inducer (polyvinylpyrrolidone, weight-average molecular weight of 10000) in step (1) is adjusted to 3g, while other conditions are the same as in Example 1.

[0094] Example 12

[0095] This embodiment provides a method for preparing poly-L-lactic acid microspheres. The difference between this method and Example 1 is that the mass of the amorphization inducer (polyvinylpyrrolidone, weight-average molecular weight of 10000) in step (2) is adjusted to 5g, while other conditions are the same as in Example 1.

[0096] Example 13

[0097] This embodiment provides a method for preparing poly-L-lactic acid microspheres. The difference between this method and Example 1 is that the mass of the amorphization inducer (polyvinylpyrrolidone, weight-average molecular weight of 10000) in step (2) is adjusted to 18g, while other conditions are the same as in Example 1.

[0098] Example 14

[0099] This embodiment provides a method for preparing poly-L-lactic acid microspheres. The difference between this method and Example 1 is that the mass of the amorphization inducer (polyvinylpyrrolidone, weight-average molecular weight of 10000) in step (2) is adjusted to 25g, while other conditions are the same as in Example 1.

[0100] Comparative Example 1

[0101] This comparative example provides a method for preparing poly-L-lactic acid microspheres. The difference between this method and Example 1 is that no amorphization inducing agent (polyvinylpyrrolidone, weight-average molecular weight of 10,000) is added in steps (1) and (2). Other conditions are the same as in Example 1.

[0102] Comparative Example 2

[0103] This comparative example provides a method for preparing poly-L-lactic acid microspheres. The difference between this method and Example 1 is that the amorphization inducer (polyvinylpyrrolidone, weight-average molecular weight of 10,000) is not added in step (1), while other conditions are the same as in Example 1.

[0104] Comparative Example 3

[0105] This comparative example provides a method for preparing poly-L-lactic acid microspheres. The difference between this method and Example 1 is that the amorphization inducer (polyvinylpyrrolidone with a weight-average molecular weight of 10,000) is not added in step (2), while other conditions are the same as in Example 1.

[0106] Comparative Example 4

[0107] This comparative example provides a method for preparing poly-L-lactic acid microspheres. The difference between this method and Example 1 is that the mass of the amorphization inducer (polyvinylpyrrolidone, weight-average molecular weight of 10000) in step (1) is adjusted to 0.02 g, while other conditions are the same as in Example 1.

[0108] Comparative Example 5

[0109] This comparative example provides a method for preparing poly-L-lactic acid microspheres. The difference between this method and Example 1 is that the mass of the amorphization inducer (polyvinylpyrrolidone, weight-average molecular weight of 10000) in step (1) is adjusted to 4 g, while other conditions are the same as in Example 1.

[0110] Comparative Example 6

[0111] This comparative example provides a method for preparing poly-L-lactic acid microspheres. The difference between this method and Example 1 is that the mass of the amorphization inducer (polyvinylpyrrolidone, weight-average molecular weight of 10000) in step (2) is adjusted to 0.2 g, while other conditions are the same as in Example 1.

[0112] Comparative Example 7

[0113] This comparative example provides a method for preparing poly-L-lactic acid microspheres. The difference between this method and Example 1 is that the mass of the amorphization inducer (polyvinylpyrrolidone, weight-average molecular weight of 10,000) in step (2) is adjusted to 30 g, while other conditions are the same as in Example 1.

[0114] Comparative Example 8

[0115] This comparative example provides a method for preparing poly-L-lactic acid microspheres. The difference between this method and Example 1 is that the temperature of dilution and stirring in step (4) is adjusted to 30°C, while other conditions are the same as in Example 1.

[0116] The following performance tests were performed on the poly-L-lactic acid microspheres provided in Examples 1 to 14 and the poly-L-lactic acid microspheres provided in Comparative Examples 1 to 8.

[0117] (1) Morphology: The surface and internal cross-sectional morphology were observed using a scanning electron microscope.

[0118] (2) Average particle size and coefficient of variation: The microsphere images of the sample to be tested (poly-L-lactic acid microspheres) were obtained by scanning electron microscopy. No less than 100 microspheres were randomly selected from the microsphere images, and the diameter of each microsphere was measured by image analysis software (ImageJ). The average value was calculated, which is the average particle size.

[0119] Based on the above average particle size measurement results, the coefficient of variation (CV) of the particle size distribution is calculated using the following formula: CV (%) = (σ / D) avg ) × 100%, where σ is the standard deviation, D avg The average particle size is denoted as .

[0120] (3) Porosity: Scanning electron microscope was used to take cross-sectional images of the sample (poly-L-lactic acid microspheres), and the images were binarized using image analysis software (ImageJ). The percentage of pore area to the total area of ​​the image was calculated, which is the approximate value of porosity. No less than 5 cross-sectional images were randomly selected for each sample, and the average value was taken.

[0121] (4) Crystallinity: X-ray diffraction is used to test whether there is a crystallization peak. If there is a crystallization peak, it is crystalline; if there is no crystallization peak, it is amorphous.

[0122] (5) Injection thrust: Referring to the typical formulation of soft tissue filling microspheres, 150 mg of poly-L-lactic acid microspheres, 90 mg of carboxymethyl cellulose (CMC) and 127.5 mg of mannitol were reconstituted in 5 mL of sterile water. After mixing, the mixture was drawn into a 1 mL syringe (27G needle) and injected at a rate of 10 mm / min using a universal testing machine. The maximum thrust (N) was recorded. Each sample was measured in parallel 3 times and the average value was taken.

[0123] The test results are shown in Tables 1 and 2 below.

[0124] Table 1

[0125]

[0126] Table 2

[0127]

[0128] According to the test results in Tables 1 and 2, the poly-L-lactic acid microspheres prepared by the preparation methods provided in Examples 1 to 14 have an amorphous structure, are regularly spherical, have a smooth and dense surface, and have a three-dimensional interconnected porous sponge-like network inside. The injection thrust after reconstitution is 2.8N to 4.7N.

[0129] Figure 1This is a schematic diagram of the formation principle of poly-L-lactic acid microspheres in Example 1 of the present invention and an optical microscope image of the formation process. As shown in the figure, the amorphization inducer, driven by the synergistic effect of two-phase diffusion and phase separation, performs the dual functions of confined amorphization and dynamic pore formation. Two-phase diffusion: Driven by a concentration gradient, organic solvents (such as dichloromethane) diffuse unidirectionally from the interior of the oil-phase droplet to the aqueous phase, causing the concentration of poly-L-lactic acid (PLA) in the droplet to gradually increase. At the same time, driven by the concentration difference between the oil and water phases, the amorphization inducing agent undergoes bidirectional dynamic distribution at the oil-water interface, continuously accumulating at the interface and forming a stable interfacial adsorption layer. The amorphization inducing agent enriched at the interface inhibits the ordered arrangement and crystallization behavior of PLA molecular chains within the confined space inside the oil-phase droplet through steric hindrance and intermolecular hydrogen bonding, locking its amorphous conformation to achieve confined amorphization. Phase separation: As the organic solvent (such as dichloromethane) diffuses, liquid-liquid phase separation occurs inside the oil-phase droplet, dividing into a polymer-enriched phase (PLA-enriched) and a polymer-deficient phase (PLA-deficient). The polymer-enriched phase is subsequently solidified into a polymer framework of microspheres, while the polymer-deficient phase is selectively removed during separation, washing, and drying to form three-dimensional interconnected channels, thereby achieving dynamic pore formation.

[0130] The surface and internal structures of the poly-L-lactic acid microspheres prepared in Example 1 are as follows: Figure 2 and 3 As shown, it is a regular sphere with a smooth and dense surface, and an interior consisting of a three-dimensional, interconnected, porous, sponge-like network. Figure 6 As shown in the X-ray diffraction (XRD) patterns, the poly-L-lactic acid microspheres prepared in Example 1 showed no crystallization diffraction peaks, indicating an amorphous state; while the poly-L-lactic acid microspheres prepared in Comparative Example 1 exhibited obvious crystallization peaks, indicating a crystalline state. The Fourier transform infrared (FTIR) spectra of both also showed... Figure 7 The characteristic peak positions correspond perfectly with those of the PLLA raw material (poly-L-lactic acid, with a viscosity-average molecular weight of 100 kDa), confirming that their chemical compositions are the same.

[0131] like Figure 8 and Figure 9As shown, within 3 months of subcutaneous implantation into the back skin of SD rats, the poly-L-lactic acid microspheres provided in Example 1 exhibited significant performance advantages: the poly-L-lactic acid microspheres provided in Example 1 maintained a highly regular spherical shape and uniform particle size, transitioning naturally with the surrounding tissue, indicating excellent tissue compatibility and degradation uniformity. In contrast, the poly-L-lactic acid microspheres provided in Comparative Example 1 showed irregular morphology over time, with many flattened, elongated, and broken particles arranged randomly, indicating uneven degradation in vivo. Furthermore, the irregular morphology may have triggered mechanical friction and local micro-inflammatory reactions, leading to disordered cell arrangement. Masson staining showed that by 3 months, the gaps and periphery of the poly-L-lactic acid microspheres provided in Example 1 were completely filled with a dense blue collagen fiber network, confirming that its "dense on the outside and sparse on the inside" structure could effectively induce collagen ingrowth and form a stable support scaffold. Conversely, the poly-L-lactic acid microspheres provided in Comparative Example 1 had a sparse periphery and lower collagen fiber density, demonstrating that its collagen regeneration induction ability was significantly weaker than that of Example 1.

[0132] Compared to Example 1, if polyvinylpyrrolidone is replaced with the same mass of ethylene glycol (Example 6), the resulting poly-L-lactic acid microspheres are regular spherical, amorphous, with a smooth and dense surface and a three-dimensional interconnected sponge-like structure inside, but the porosity is reduced and the pore size is smaller. If polyvinylpyrrolidone is replaced with the same mass of glycerol (Example 7), the resulting poly-L-lactic acid microspheres are regular spherical, amorphous, with a smooth and dense surface and a three-dimensional interconnected sponge-like structure inside, but the porosity is reduced and the pore size is smaller. It can be seen that different amorphization inducing agents can achieve the dual functions of confined amorphization and dynamic pore formation, but they have different focuses in terms of pore formation efficiency, pore size control and interface stability. The best comprehensive performance can be obtained by compounding or selecting the best types. The poly-L-lactic acid microspheres prepared by selecting the best polymer amorphization inducing agent have higher porosity.

[0133] Compared with Example 1, if no amorphization inducing agent is added in steps (1) and (2) (Comparative Example 1), the prepared poly-L-lactic acid microspheres are as follows: Figure 4 and 5 Although the surface shown is dense and smooth, it is solid and without pores inside, as indicated by the X-ray diffraction pattern. Figure 6 As shown, there are crystalline peaks, indicating a crystalline state. The Fourier transform infrared spectrum peak positions correspond to those of the PLLA standard raw material as follows: Figure 7 As shown.

[0134] Compared with Example 1, if no amorphization inducer is added in step (1) (Comparative Example 2), the prepared poly-L-lactic acid microspheres have a dense and smooth surface, but are solid and crystalline inside. This is because the oil phase solution lacks an amorphization inducer, which on the one hand cannot play a crystallization inhibition role. The poly-L-lactic acid molecular chains can move freely and arrange themselves in an orderly manner during the evaporation of organic solvent, thus forming a crystalline state. On the other hand, because the system lacks an amorphization inducer as a dynamic pore-forming template, liquid-liquid phase separation cannot occur to form a polymer-deficient phase, and therefore cannot construct pores, eventually solidifying into a solid structure.

[0135] Compared to Example 1, if no amorphization inducer is added in step (2) (Comparative Example 3), the prepared poly-L-lactic acid microspheres have a dense and smooth surface, but only a few pores inside and are in a crystalline state, resulting in low porosity. This may be because the lack of an amorphization inducer in the aqueous solution leads to a decrease in interfacial stability, causing the amorphization inducer in the oil solution to diffuse rapidly into the aqueous phase and be lost, thus leading to insufficient phase separation. This not only makes the system only able to form a few local pores and unable to build a through network, but also weakens the confinement effect on the poly-L-lactic acid molecular chains at the interface, resulting in incomplete crystallization inhibition, and the obtained poly-L-lactic acid microspheres are ultimately in a crystalline state.

[0136] Compared to Example 1, if the amount of amorphization inducer added to the oil phase solution is too small (Comparative Example 4), the prepared poly-L-lactic acid microspheres are regular spherical, crystalline, with a smooth and dense surface, a few internal pores, and low porosity. If the amount of amorphization inducer added to the oil phase solution is too large (Comparative Example 5), the resulting oil-in-water emulsion is unstable and prone to instability, breakage, or aggregation. When the droplets become unstable, the distribution of poly-L-lactic acid and amorphization inducer within them is disrupted, making it impossible to maintain a regular spherical morphology. Simultaneously, the phase separation process becomes uncontrolled, resulting in only... The poly-L-lactic acid microspheres are irregularly aggregated with isolated pores, unable to form a three-dimensional interconnected sponge-like network. The resulting microspheres are irregularly clustered with a rough surface and mostly non-interconnected independent pores, resulting in a significant reduction in porosity. Therefore, by controlling the amount of amorphization inducer added to the oil phase solution within a suitable range (the mass-volume ratio of amorphization inducer to organic solvent in the oil phase solution is 0.5~30g / 100mL), the prepared poly-L-lactic acid microspheres exhibit better overall performance in terms of morphological regularity, pore interconnectivity, structural strength, and amorphous state retention.

[0137] Compared to Example 1, if the amount of amorphization inducer added to the aqueous solution is too small (Comparative Example 6), the prepared poly-L-lactic acid microspheres are regular spherical, crystalline, with a smooth and dense surface and a three-dimensional interconnected porous sponge-like network structure, resulting in reduced porosity. If the amount of amorphization inducer added to the aqueous solution is too large (Comparative Example 7), an excessively thick interfacial film is formed at the oil-water interface, significantly hindering the diffusion and volatilization of organic solvents (such as dichloromethane), leading to insufficient phase separation and pore formation in the emulsion droplets. Due to the limited length, the prepared poly-L-lactic acid microspheres, although regular spherical and amorphous, have a rough and porous surface and reduced internal porosity. Therefore, by controlling the amount of amorphization inducing agent added to the aqueous solution within a suitable range (the mass-volume ratio of amorphization inducing agent to water in the aqueous solution is 0.5~25g / 100mL), the prepared poly-L-lactic acid microspheres can achieve higher porosity and better overall performance while maintaining the amorphous state, smooth and dense surface, and three-dimensional interconnected internal structure.

[0138] Compared to Example 1, the mixing and dilution of the oil-in-water emulsion and the aqueous solution containing the surfactant in step (4), and the excessively high temperature during stirring to allow the organic solvent to evaporate (Comparative Example 8), resulted in poly(L-lactic acid) microspheres that, while exhibiting a regular spherical shape and a three-dimensional interconnected porous sponge-like network structure with minimal porosity variation, had a rough, porous, and crystalline surface. Therefore, controlling the mixing, dilution, and stirring temperatures in step (4) within a suitable range (each mixing and stirring temperature is independently 5~25℃) is crucial for maintaining the amorphous structure. Poly(L-lactic acid) microspheres prepared within this temperature range exhibit better overall performance in terms of morphological regularity, pore connectivity, structural strength, and amorphous state retention.

[0139] In summary, the poly-L-lactic acid microspheres prepared by the method provided by this invention possess an amorphous structure and a "dense on the outside and sparse on the inside" morphology, with good monodispersity and controllable particle size and porosity. The amorphization inducer is indispensable in both the oil and aqueous phases. By inhibiting poly-L-lactic acid crystallization, stabilizing the oil-in-water emulsion interface, and regulating the phase separation process, it achieves precise control over the amorphous state, smooth and dense surface, and porous sponge-like internal structure of the poly-L-lactic acid microspheres.

[0140] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A poly-L-lactic acid microsphere, characterized in that, The poly-L-lactic acid microspheres have an amorphous structure, are regularly spherical in shape, and include a smooth and dense surface layer and an internal porous structure. The poly-L-lactic acid microspheres were prepared using the following method: (1) Poly-L-lactic acid, an amorphization inducer and an organic solvent are mixed to obtain an oil phase solution; (2) Mix the surfactant, amorphization inducer and water to obtain an aqueous solution; (3) Mix the oil phase solution obtained in step (1) and the aqueous phase solution obtained in step (2) to obtain an oil-in-water emulsion; (4) The oil-in-water emulsion obtained in step (3) is mixed with an aqueous solution containing a surfactant, and stirred to allow the organic solvent to evaporate and the microspheres to solidify. The mixing and stirring temperatures are each 5~25℃, to obtain the poly-L-lactic acid microspheres. Steps (1) and (2) can be performed in any order, or simultaneously. The mass-to-volume ratio of the amorphization inducing agent to the organic solvent in step (1) is 0.5~30 g / 100mL; the mass-to-volume ratio of the amorphization inducing agent to water in step (2) is 0.5~25 g / 100mL; The amorphization inducing agents mentioned in steps (1) and (2) are each independently a polymer amorphization inducing agent and / or a small molecule amorphization inducing agent; The polymer amorphization inducer is any one or a combination of at least two of polyvinylpyrrolidone, poloxamer, or polyethylene glycol. The small molecule amorphization inducer is any one or a combination of at least two of ethanol, ethylene glycol, n-propanol, isopropanol, propylene glycol, or glycerol; The organic solvent is any one or a combination of at least two of dichloromethane, chloroform, dichloroethane, ethyl acetate, acetone, dimethyl sulfoxide, or tetrahydrofuran.

2. The poly-L-lactic acid microspheres according to claim 1, characterized in that, The porosity of the poly-L-lactic acid microspheres is 10%~90%; The average particle size of the poly-L-lactic acid microspheres is 10~200 μm; The coefficient of variation of the poly-L-lactic acid microspheres is 1% to 10%.

3. A method for preparing poly-L-lactic acid microspheres as described in claim 1 or 2, characterized in that, The preparation method includes the following steps: (1) Poly-L-lactic acid, an amorphization inducer and an organic solvent are mixed to obtain an oil phase solution; (2) Mix the surfactant, amorphization inducer and water to obtain an aqueous solution; (3) Mix the oil phase solution obtained in step (1) and the aqueous phase solution obtained in step (2) to obtain an oil-in-water emulsion; (4) The oil-in-water emulsion obtained in step (3) is mixed with an aqueous solution containing a surfactant, and stirred to allow the organic solvent to evaporate and the microspheres to solidify. The mixing and stirring temperatures are each 5~25℃, to obtain the poly-L-lactic acid microspheres. Steps (1) and (2) can be performed in any order, or simultaneously. The mass-to-volume ratio of the amorphization inducer to the organic solvent in step (1) is 0.5~30 g / 100mL; the mass-to-volume ratio of the amorphization inducer to water in step (2) is 0.5~25 g / 100mL.

4. The preparation method according to claim 3, characterized in that, The mass ratio of the amorphization inducing agent to poly-L-lactic acid in step (1) is (0.1~6):1; The mass-to-volume ratio of the surfactant to water in step (2) is 0.1~5 g / 100mL.

5. The preparation method according to claim 3, characterized in that, The viscosity-average molecular weight of the poly-L-lactic acid is 5~500 kDa; The surfactant in step (2) is any one or a combination of at least two of the following: polyvinyl alcohol, gelatin, Tween, sodium lauryl sulfate, hyaluronic acid, carboxymethyl cellulose, alginate, polylysine, or polyacetylimide.

6. The preparation method according to claim 3, characterized in that, The mixing in step (3) includes microfluidic mixing or emulsification using a membrane emulsification device.

7. The preparation method according to claim 3, characterized in that, In step (4), the surfactant in the aqueous solution containing surfactant is any one or a combination of at least two of the following: polyvinyl alcohol, gelatin, Tween, sodium dodecyl sulfate, hyaluronic acid, carboxymethyl cellulose, alginate, polylysine, or polyacetylimide. In step (4), the mass percentage concentration of the surfactant in the aqueous solution containing the surfactant is 0.1% to 5%.

8. The preparation method according to claim 3, characterized in that, The stirring speed in step (4) is 20~500 rpm; The stirring time in step (4) is 1 to 48 hours.

9. The preparation method according to claim 3, characterized in that, The process after stirring also includes separation, washing, and drying steps; The separation includes separation by centrifugation or filtration; The washing includes washing with water; The separation and washing temperatures are each independently 5~25℃; The drying process includes any one or a combination of at least two of the following: freeze drying, vacuum drying, supercritical fluid drying, or oven drying.

10. The application of poly-L-lactic acid microspheres as described in claim 1 or 2, or poly-L-lactic acid microspheres prepared by the preparation method as described in any one of claims 3 to 9, in the preparation of soft tissue filling materials.

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