Injectable polyhydroxy fatty acid ester microspheres and method for producing same

Abstract

The present invention relates to the field of biomedicine technology, and specifically discloses injectable polyhydroxy fatty acid ester (PHA) microspheres and a method for preparing the same. The method for preparing the polyhydroxy fatty acid ester injectable microspheres includes: a) dissolving a PHA material in an organic solvent to obtain an oil phase; b) dissolving hyaluronic acid in water to obtain an aqueous phase; and c) dropping the oil phase into the aqueous phase under stirring conditions, and after the dropping is completed, continuing the stirring to volatilize the organic solvent, and then performing solid-liquid separation and drying to obtain PHA microspheres, and the order of steps a) and b) is not limited. The above method of the present invention can increase the dispersibility of PHA microspheres, and the obtained microspheres can be quickly dispersed in water, and the microspheres have a perfect shape, are independent of each other, and do not have agglomeration phenomenon, so that clogging of the needle tip can be avoided, and it is easy to inject them as a filler later. In addition, PHA microspheres have excellent biocompatibility and can achieve a long-term filling effect without causing rejection.

Description

[Technical Field]

[0001] The present invention relates to the field of biopharmaceutical technology, and in particular to injectable polyhydroxy fatty acid ester microspheres and methods for producing the same. [Background technology]

[0002] (CROSS-REFERENCE TO RELATED APPLICATIONS) This invention claims priority from a Chinese patent application, application number 202111187296.9, entitled "Injectable polyhydroxy fatty acid ester microspheres and their manufacturing method," filed with the State Intellectual Property Office of China on October 12, 2021, the entire contents of which are incorporated herein by reference.

[0003] As humans age or are affected by certain diseases, the muscle and collagen tissues in the human body undergo a certain degree of functional deterioration, leading to problems such as skin depressions and gastric reflux. Various fillers have been developed to fill in the depressed skin or stimulate muscle and collagen regrowth through foreign bodies. However, these substances only have a short duration of effect, necessitating frequent injections to maintain the effect. To achieve long-term effects, attempts have been made to use non-biodegradable materials such as polyvinyl alcohol (PVA) and polymethyl methacrylate (PMMA) as fillers. While these fillers significantly extend the duration of their effects, if they remain in the body for too long, they release harmful substances, which can cause a series of side effects and pose a risk to human health.

[0004] In recent years, biodegradable polymer materials have come into people's sights. These materials are non-toxic and do not cause rejection reactions in the human body. They can be gradually decomposed and excreted through the human body's metabolism. By adjusting parameters such as the molecular weight of the material, the decomposition time can be extended from one week to several years.

[0005] Polyhydroxy fatty acid esters (PHAs), abbreviated as PHAs, are natural polymeric biomaterials and intracellular polyesters synthesized by microorganisms. PHAs are currently one of the most ideal biomedical materials due to their excellent biocompatibility and biodegradability. PHAs have excellent cytocompatibility with cells in the body, allowing cells to grow well on such stents, and they can decompose into CO₂ and H₂O. To facilitate injection, they are typically made into microspheres and injected through a needle. Due to the presence of phagocytes in the human body, microspheres typically have a diameter of 20 microns or larger. However, microspheres that are too large can clog the needle tip or cause skin rupture. Therefore, microspheres used for injections are typically 60 microns or smaller.

[0006] Conventionally, when PHA materials are made into injectable microspheres, a polyvinyl alcohol (PVA) aqueous solution is typically used as the aqueous phase, and the microspheres are produced using an emulsification method or a microflow control method. However, the microspheres produced by this process tend to aggregate and adhere to each other, forming large clumps. This makes subsequent injections inconvenient and significantly hinders the application of biodegradable microspheres as fillers. Therefore, a new method for producing microspheres is urgently needed to solve the aggregation and adhesion problems of microspheres. Summary of the Invention

[0007] Therefore, an object of the present invention is to provide polyhydroxy fatty acid ester microspheres and a method for producing the same, which can effectively improve the dispersibility of polyhydroxy fatty acid ester microspheres, overcome the problems of aggregation and adhesion of the microspheres, and effectively improve the absorbability and injectability of the polyhydroxy fatty acid ester microspheres.

[0008] The present invention provides a method for producing injectable polyhydroxy fatty acid ester microspheres, comprising the steps of: a) dissolving a PHA material in an organic solvent to obtain an oil phase; b) dissolving hyaluronic acid in water to obtain an aqueous phase; c) adding the oil phase dropwise to the aqueous phase under stirring conditions, and after the completion of the addition, continuing stirring to volatilize the organic solvent, and then performing solid-liquid separation and drying to obtain PHA microspheres; The steps a) and b) can be performed in any order in the present production method.

[0009] Preferably, the molecular weight of the hyaluronic acid is 5 to 500 KDa. Preferably, the mass concentration of hyaluronic acid in the aqueous phase is 0.01% to 1% (w / v). Preferably, in the step c), the volume ratio of the oil phase to the water phase is 1:(10 to 200). Preferably, the mass concentration of the PHA material in the oil phase is 2.5% to 10% (w / v); The organic solvent is one or more selected from the group consisting of dichloromethane, chloroform, and ethyl acetate.

[0010] Preferably, the molecular weight of the PHA material is 10 to 100 KDa. Preferably, the PHA material is a natural or unnatural polyhydroxy fatty acid ester; The natural or unnatural polyhydroxy fatty acid esters include, but are not limited to, one or more of PHB, PHBV, PHBHHx, P34HB, PHBVHHx, PHHx, and PHO.

[0011] Preferably, the stirring speed is 100 to 500 rpm. Preferably, the stirring is performed by a magnetic stirrer; and after the dropwise addition of the oil phase is completed, the stirring is continued for 4 hours or more. The present invention further provides injectable polyhydroxy fatty acid ester microspheres prepared by the method described in the above technical solution.

[0012] In the manufacturing method of the present invention, a PHA material is dissolved in an organic solvent to form an oil phase, and a hyaluronic acid solution is used as the aqueous phase. The oil phase is gradually added to the aqueous phase while stirring the aqueous solution. The stirring is continued to volatilize the organic solvent, followed by solid-liquid separation and drying to produce PHA microspheres. The above method of the present invention can improve the dispersibility of PHA microspheres, and the resulting microspheres can be quickly dispersed in water. The microspheres have a complete, independent morphology and are free of aggregation, which prevents clogging of the needle tip and facilitates their subsequent injection as a filler. At the same time, the polyhydroxy fatty acid ester microspheres obtained by the present invention have excellent absorbability and injectability. Furthermore, PHA microspheres have excellent biocompatibility and can achieve long-term filling effects without causing rejection reactions.

[0013] Experimental results have shown that the manufacturing method provided by the present invention has the following beneficial effects: (1) High microsphere yield: The microsphere yield is 70% or more. (2) Good dispersibility: The PHA microspheres obtained by the present invention can be dispersed in water with appropriate mixing. After standing, the microspheres exhibit good dispersibility in water, do not exhibit phase separation, do not float on the top layer of water, and do not settle on the bottom. They are uniformly dispersed in the water, forming a uniform dispersion. After air-drying the dispersion, observation of the microsphere morphology revealed that the microspheres were perfectly shaped, independent of each other, and free of aggregation. The particle diameter of all or most of the microsphere particles was 60 μm or less, meeting the needs for injection. (3) Good suckability: When the dispersion was sucked into a syringe, the suckability of the microspheres was 85% or more, demonstrating excellent suckability. (4) Good injectability: The injected dispersion was collected by pressing the syringe and the injection rate of the microspheres was calculated. The injection rate of the microspheres reached 88% or more, indicating excellent injectability. [Brief explanation of the drawings]

[0014] In order to more clearly explain the technical means in the embodiments of the present invention or the prior art, the drawings used in the following description of the embodiments or the prior art will be briefly described below. However, the drawings in the following description are merely embodiments of the present invention, and those skilled in the art can obtain other drawings based on the provided drawings without performing any creative work.

[0015] [Figure 1] 1 is an optical microscope photograph of the injectable PHBHHx microspheres obtained in Example 1. [Figure 2] 1 is an optical microscope photograph of the injectable PHBVHHx microspheres obtained in Example 2. [Figure 3] 1 is an optical microscope photograph of the injectable PHB microspheres obtained in Example 3. [Figure 4] 1 is an optical microscope photograph of the injectable PHBV microspheres obtained in Example 4. [Figure 5] 1 is a light microscope photograph of the injectable P34HB microspheres obtained in Example 5. [Figure 6] FIG. 1 is a schematic diagram showing the dispersion stability of the microspheres obtained in Examples 1 to 5 and Comparative Examples 1 to 5. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention provides a method for producing injectable polyhydroxy fatty acid ester microspheres, comprising the steps of: a) dissolving a PHA material in an organic solvent to obtain an oil phase; b) dissolving hyaluronic acid in water to obtain an aqueous phase; c) adding the oil phase dropwise to the aqueous phase under stirring conditions, and after the completion of the addition, continuing stirring to volatilize the organic solvent, and then performing solid-liquid separation and drying to obtain PHA microspheres; The steps a) and b) can be performed in any order in the present production method.

[0017] In the manufacturing method of the present invention, a PHA material is dissolved in an organic solvent to form an oil phase, and a hyaluronic acid solution is used as the aqueous phase. The oil phase is gradually added to the aqueous phase while stirring the aqueous solution. The stirring is continued to volatilize the organic solvent, followed by solid-liquid separation and drying to produce PHA microspheres. The above method of the present invention can improve the dispersibility of PHA microspheres, and the resulting microspheres can be quickly dispersed in water. The microspheres have a complete, independent morphology and are free of aggregation, which prevents clogging of the needle tip and facilitates their subsequent injection as a filler. At the same time, the polyhydroxy fatty acid ester microspheres obtained by the present invention have excellent absorbability and injectability. Furthermore, PHA microspheres have excellent biocompatibility and can achieve long-term filling effects without causing rejection reactions.

[0018] Step a) Dissolving the PHA material in an organic solvent to obtain an oil phase In the present invention, the PHA material, i.e., polyhydroxy fatty acid ester, is not particularly limited and may be any fifth-generation PHA commercial product commonly used in the art, specifically, natural or unnatural polyhydroxy fatty acid ester, more specifically, one or more of PHB (i.e., 3-hydroxybutyrate), PHBV (i.e., copolymer of hydroxybutyrate and hydroxyvalerate), PHBHHx (i.e., P3HB-co-3HHx, copolymer of 3-hydroxybutyrate and 3-hydroxycaproic acid), P34HB (i.e., P3HB-co-4HB, copolymer of 3-hydroxybutyrate and 4-hydroxybutyrate), PHBVHHx (i.e., copolymer of 3-hydroxybutyrate, 3-hydroxycaproic acid, and 3-hydroxyvaleric acid), PHHx (i.e., polyhydroxycaproic acid), and PHO (i.e., polyhydroxycaprylic acid). In the present invention, the molecular weight of the PHA material is preferably 10 to 100 KDa. In some embodiments of the present invention, the molecular weight is 10 to 30 KDa, 20 to 100 KDa, or 10 to 100 KDa. The average molecular weight of the PHA material is preferably 15 to 80 KDa. In some embodiments of the present invention, the average molecular weight is 15 KDa, 40 KDa, 50 KDa, or 80 KDa.

[0019] In some embodiments of the present invention, the PHA material is PHBHHx, has a molecular weight of 20 to 100 KDa, and an average molecular weight of 50 KDa. In some other embodiments of the present invention, the PHA material is PHBVHHx, has a molecular weight of 20 to 100 KDa, and an average molecular weight of 50 KDa. In some other embodiments of the present invention, the PHA material is PHB, has a molecular weight of 20 to 100 KDa, and an average molecular weight of 80 KDa. In some other embodiments of the present invention, the PHA material is PHBV, has a molecular weight of 20 to 100 KDa, and an average molecular weight of 40 KDa. In some other embodiments of the present invention, the PHA material is P34HB, has a molecular weight of 10 to 30 KDa, and an average molecular weight of 15 KDa.

[0020] In the present invention, the organic solvent is preferably one or more of dichloromethane, chloroform, and ethyl acetate. In the present invention, there is no particular limitation on the origin of the organic solvent, and any commercially available product may be used.

[0021] In the present invention, the mass concentration (w / v) of the organic solution obtained by dissolving the PHA material in an organic solvent is preferably 2.5% to 10%. In some embodiments of the present invention, the mass concentration is 2.5%, 5%, or 10%. In the present invention, the organic solution obtained by dissolving the PHA material in an organic solvent is used as the oil phase.

[0022] Step b) Dissolving hyaluronic acid in water to obtain an aqueous phase In the present invention, the molecular weight of the hyaluronic acid, i.e., HA, is preferably 5 to 500 KDa. In some embodiments of the present invention, the molecular weight is 5 to 100 KDa, 10 to 200 KDa, or 100 to 500 KDa. In the present invention, the average molecular weight of the hyaluronic acid is preferably 50 to 300 KDa. In some embodiments of the present invention, the average molecular weight is 50 KDa or 300 KDa.

[0023] In some embodiments of the present invention, the hyaluronic acid has a molecular weight of 5 to 100 KDa and an average molecular weight of 50 KDa. In some other embodiments of the present invention, the hyaluronic acid has a molecular weight of 10 to 200 KDa and an average molecular weight of 50 KDa. In some other embodiments of the present invention, the hyaluronic acid has a molecular weight of 100 to 500 KDa and an average molecular weight of 300 KDa.

[0024] In the present invention, the water is preferably distilled water or deionized water, and more preferably distilled water. In the present invention, the mass concentration (w / v) of the aqueous solution of hyaluronic acid dissolved in water is preferably 0.01% or more and 1% or less. In some embodiments of the present invention, the mass concentration is 0.5%, 5%, or 10%. In the present invention, the aqueous solution of hyaluronic acid dissolved in water is used as the aqueous phase. Conventionally, PHA microspheres have been produced using an aqueous solution of polyvinyl alcohol (PVA) as the aqueous phase, but the resulting PHA microspheres tend to aggregate and adhere in water, making rapid dispersion difficult. In contrast, in the present invention, the use of an aqueous hyaluronic acid solution as the aqueous phase improves the dispersibility of PHA microspheres, allowing them to be rapidly dispersed in water. The microspheres have a complete, independent morphology and are free of aggregation, preventing needle clogging and facilitating their subsequent injection as a filler. At the same time, the polyhydroxy fatty acid ester microspheres obtained in the present invention have excellent absorbability and injectability. Furthermore, PHA microspheres have excellent biocompatibility and can achieve long-term filling effects without causing rejection reactions. In the present invention, there is no restriction on the order of the steps a) and b).

[0025] Step c) Dropping the oil phase into the aqueous phase under stirring conditions, and after the dropwise addition is completed, continuing stirring to volatilize the organic solvent, followed by solid-liquid separation and drying to obtain PHA microspheres. In the present invention, after the oil phase and the aqueous phase are obtained, the aqueous phase is stirred, and under these stirring conditions, the oil phase is slowly added dropwise to the aqueous phase. Here, the stirring is performed using a magnetic stirrer. The stirring speed is 100 to 500 rpm, and typically and non-limitingly, the stirring speed can be 100 rpm, 200 rpm, 300 rpm, 400 rpm, or 500 rpm. The volume ratio of the oil phase to the aqueous phase is preferably 1:(10 to 200). In some embodiments of the present invention, the volume ratio is 1:10, 1:20, 1:50, 1:100, 1:150, or 1:200.

[0026] After the entire oil phase has been added dropwise, the stirring process preferably includes first continuing stirring to form an emulsion, and then continuing stirring to volatilize the organic solvent. During the continuing stirring process, the stirring is performed using a magnetic stirrer at a speed of 100 to 500 rpm. In some embodiments of the present invention, the stirring speed is 400 rpm. During the continuing stirring process, the stirring is performed using a magnetic stirrer at a speed of 100 to 500 rpm. Typical, but non-limiting, stirring speeds can be 100 rpm, 200 rpm, 300 rpm, 400 rpm, or 500 rpm. The stirring is preferably continued for 4 hours or more, more preferably 4 to 6 hours. In some embodiments of the present invention, the stirring is continued (to volatilize the organic solvent) for 4, 5, or 6 hours after forming the emulsion. In the present invention, there is no particular limitation on the temperature at which the oil phase is dropped and stirred, and the temperature may be room temperature, specifically 10 to 37°C, and preferably 25°C.

[0027] Stirring is continued until the solvent is completely evaporated, and then solid-liquid separation is carried out. In the present invention, there are no particular limitations on the method of solid-liquid separation, and a conventional method well known to those skilled in the art, such as filtration, may be used. After solid-liquid separation, the product is dried to remove water. In the present invention, the drying is preferably performed by first freezing and then lyophilization. In the present invention, the drying temperature is -80 to -20°C; the freezing temperature is preferably -80 to -20°C, and the lyophilization temperature is preferably -60 to -40°C. In some embodiments of the present invention, the freezing temperature is preferably -80 to -40°C and the time is preferably 6 to 12 hours; and the lyophilization temperature is preferably -40°C and the time is preferably 24 hours. After the above drying treatment, PHA microspheres are obtained.

[0028] The present invention further provides PHA microspheres prepared by the method described in the above technical solution. In the manufacturing method of the present invention, a PHA material is dissolved in an organic solvent to form an oil phase, and a hyaluronic acid solution is used as the aqueous phase. The oil phase is gradually added to the aqueous phase while stirring the aqueous solution. The stirring is continued to volatilize the organic solvent, followed by solid-liquid separation and drying to produce PHA microspheres. The above method of the present invention can improve the dispersibility of PHA microspheres, and the resulting microspheres can be quickly dispersed in water. The microspheres have a complete, independent morphology and are free of aggregation, which prevents clogging of the needle tip and facilitates their subsequent injection as a filler. At the same time, the polyhydroxy fatty acid ester microspheres obtained by the present invention have excellent absorbability and injectability. Furthermore, PHA microspheres have excellent biocompatibility and can achieve long-term filling effects without causing rejection reactions.

[0029] As a result of the experiment, the following was found: (1) The production method provided by the present invention has a microsphere yield of 70% or more. The PHA microspheres obtained by the present invention can be dispersed in water with appropriate mixing, and after standing, the microspheres exhibit good dispersibility in water, do not exhibit layer separation, do not float on the top layer of water, and do not settle on the bottom, but are uniformly dispersed in the water body to form a uniform dispersion. After air-drying the dispersion, observation of the microsphere morphology revealed that the microspheres were intact, independent of each other, and free of aggregation. The particle diameter of all or most of the microsphere particles was 60 μm or less, meeting the requirements for injection. (2) A disposable 1 mL syringe was fitted with a needle with an inner diameter of 0.5 mm and a length of 19.7 mm (i.e., the needle of a conventional 1 mL syringe) to serve as the aspiration rate detection device, and 1 mL of microsphere dispersion (0.1 g dry weight of microspheres, designated M0) was used as the experimental sample. All of the microsphere dispersion was aspirated at once, and the microspheres that could be aspirated into the syringe tube were removed and freeze-dried. The mass was designated M1, and the aspiration rate was defined as [M1 / M0] × 100%. When the above microsphere dispersion was aspirated using the syringe, the aspiration rate of the microspheres was over 85%, demonstrating excellent aspiration properties. (3) A disposable 1 mL syringe equipped with a needle with an inner diameter of 0.5 mm and a length of 19.7 mm (i.e., the needle of a conventional 1 mL syringe) was used as the aspiration rate detection device, and 1 mL of microsphere dispersion (0.1 g dry weight of microspheres, designated M0) was used as the experimental sample. 1 mL of microsphere dispersion was injected into the syringe tube from the rear of the syringe, and all of the microsphere dispersion was injected at once. The microspheres that could be injected through the needle and out of the syringe tube were collected and freeze-dried. The mass was designated M2, and the injection rate was defined as (M2 / M0) × 100%. The injected dispersion was collected by pressing the syringe, and the microsphere injection rate was calculated. The microsphere injection rate reached over 88%, demonstrating excellent injectability.

[0030] In order to further understand the present invention, preferred embodiments of the present invention will be described below based on examples. However, it should be understood that these descriptions are intended to further illustrate the features and advantages of the present invention and are not intended to limit the scope of the claims of the present invention.

[0031] Example 1 One gram of PHBHHx (molecular weight 20–100 kDa, average molecular weight 50 kDa) was dissolved in 20 mL of dichloromethane to prepare a 5% (w / v) final organic solution, which served as the oil phase. Hyaluronic acid (molecular weight 5–100 kDa, average molecular weight 50 kDa) was dissolved in distilled water to prepare a 0.5% (w / v) final aqueous solution, which served as the aqueous phase. While stirring the aqueous phase at 400 rpm with a magnetic stirrer, the oil phase was slowly added dropwise to the aqueous phase, resulting in a 1:50 volume ratio. Stirring was continued at 400 rpm with a magnetic stirrer until an emulsion formed; the organic solvent was removed by further stirring at this speed for 5 hours. The resulting solution was then first frozen (at -80°C for 6 hours) and then lyophilized (at -40°C for 24 hours) to remove water, yielding injectable PHBHHx microspheres.

[0032] Based on the amount of PHBHHx material added and the amount of PHBHHx microspheres obtained, the yield of injectable PHBHHx microspheres was calculated to be 93%. The freeze-dried PHBHHx microspheres were dissolved in water and mixed appropriately to disperse in water. After air-drying, the microspheres were observed under an optical microscope. The results are shown in Figure 1. Figure 1 is an optical microscope image of the injectable PHBHHx microspheres obtained in Example 1. The microspheres obtained had good dispersibility and were spherical or ellipsoidal. The particle diameter was measured using a particle size analyzer to be 1 to 50 μm.

[0033] Example 2 One gram of PHBVHHx (molecular weight 20-100 kDa, average molecular weight 50 kDa) was dissolved in 20 mL of dichloromethane to prepare a 5% (w / v) final organic solution, which served as the oil phase. Hyaluronic acid (molecular weight 10-200 kDa, average molecular weight 50 kDa) was dissolved in distilled water to prepare a 0.5% (w / v) final aqueous solution, which served as the aqueous phase. While stirring the aqueous phase at 400 rpm with a magnetic stirrer, the oil phase was slowly added dropwise to the aqueous phase, resulting in a 1:50 volume ratio. Stirring was continued at 400 rpm with a magnetic stirrer until an emulsion formed; the organic solvent was removed by further stirring at this speed for 4 hours. The resulting solution was then first frozen (at -40°C for 12 hours) and then lyophilized (at -40°C for 24 hours) to remove water, yielding injectable PHBVHHx microspheres.

[0034] The yield of the obtained injectable PHBVHHx microspheres was 91%. The freeze-dried PHBVHHx microspheres were dissolved in water and mixed appropriately to disperse in water. After air-drying, the microspheres were observed under an optical microscope. The results are shown in Figure 2, which is an optical microscope image of the injectable PHBVHHx microspheres obtained in Example 2. The microspheres obtained had good dispersibility and were spherical or ellipsoidal. The particle diameter was measured using a particle size analyzer to be 4 to 50 μm.

[0035] Example 3 One gram of PHB (molecular weight 20-100 kDa, average molecular weight 80 kDa) was dissolved in 20 mL of dichloromethane to prepare a 5% (w / v) final organic solution, which served as the oil phase. Hyaluronic acid (molecular weight 100-500 kDa, average molecular weight 300 kDa) was dissolved in distilled water to prepare a 0.5% (w / v) final aqueous solution, which served as the aqueous phase. While stirring the aqueous phase at 400 rpm with a magnetic stirrer, the oil phase was slowly added dropwise to the aqueous phase, resulting in a 1:50 volume ratio. Stirring was continued at 400 rpm with a magnetic stirrer until an emulsion formed; stirring was continued at this speed for 6 hours to remove the organic solvent. The resulting solution was then first frozen (at -40°C for 12 hours) and then lyophilized (at -40°C for 24 hours) to remove the water, yielding injectable PHB microspheres.

[0036] The yield of the obtained injectable PHB microspheres was 92%. The freeze-dried PHB microspheres were dissolved in water and mixed appropriately to disperse in water. After air-drying, the microspheres were observed under an optical microscope. The results are shown in Figure 3, which is an optical microscope image of the injectable PHB microspheres obtained in Example 3. The microspheres obtained had good dispersibility and were spherical or ellipsoidal. The particle diameter was measured using a particle size analyzer and was 2 to 55 μm.

[0037] Example 4 One gram of PHBV (molecular weight 20-100 kDa, average molecular weight 40 kDa) was dissolved in 20 mL of dichloromethane to prepare a 5% (w / v) final organic solution, which served as the oil phase. Hyaluronic acid (molecular weight 5-100 kDa, average molecular weight 50 kDa) was dissolved in distilled water to prepare a 0.5% (w / v) final aqueous solution, which served as the aqueous phase. While stirring the aqueous phase at 400 rpm with a magnetic stirrer, the oil phase was slowly added dropwise to the aqueous phase, resulting in a 1:50 volume ratio. Stirring was continued at 400 rpm with a magnetic stirrer until an emulsion formed; the organic solvent was removed by further stirring at this speed for 5 hours. The resulting solution was then first frozen (at -40°C for 12 hours) and then lyophilized (at -40°C for 24 hours) to remove water, yielding injectable PHBV microspheres.

[0038] The yield of the obtained injectable PHBV microspheres was 90%. The freeze-dried PHBV microspheres were dissolved in water and mixed appropriately to disperse in water. After air-drying, the microspheres were observed under an optical microscope. The results are shown in Figure 4. Figure 4 is an optical microscope image of the injectable PHBV microspheres obtained in Example 4. The microspheres obtained had good dispersibility and were spherical or ellipsoidal. The particle diameter was measured using a particle size analyzer and was 1 to 52 μm.

[0039] Example 5 One gram of P34HB (molecular weight 10-30 kDa, average molecular weight 15 kDa) was dissolved in 20 mL of dichloromethane to prepare a 5% (w / v) final organic solution, which served as the oil phase. Hyaluronic acid (molecular weight 5-100 kDa, average molecular weight 50 kDa) was dissolved in distilled water to prepare a 0.5% (w / v) final aqueous solution, which served as the aqueous phase. While stirring the aqueous phase at 400 rpm with a magnetic stirrer, the oil phase was slowly added dropwise to the aqueous phase, resulting in a 1:50 volume ratio. Stirring was continued at 400 rpm with a magnetic stirrer until an emulsion formed; the organic solvent was removed by further stirring at this speed for 5 hours. The resulting solution was then first frozen (at -40°C for 12 hours) and then lyophilized (at -40°C for 24 hours) to remove water, yielding injectable P34HB microspheres.

[0040] The yield of the resulting injectable P34HB microspheres was 90%. The freeze-dried P34HB microspheres were dissolved in water and mixed appropriately to disperse in water. After air-drying, the microspheres were observed under an optical microscope. The results are shown in Figure 5. Figure 5 is an optical microscope image of the injectable P34HB microspheres obtained in Example 5, and it was found that the resulting microspheres had good dispersibility and were spherical or ellipsoidal. Measurement using a particle size analyzer revealed that the particle diameter was 5 to 100 μm, with 68% of the particles having a diameter of 1 to 60 μm, meaning that the majority of the particles were 60 μm or less.

[0041] Example 6: Different oil phase concentrations 0.5 g, 1 g, and 2 g of PHBHHx (molecular weight 20–100 kDa, average molecular weight 50 kDa) were dissolved in 20 mL of dichloromethane to prepare organic solutions with final concentrations of 2.5%, 5%, and 10% (w / v), respectively, which served as the oil phase. Hyaluronic acid (molecular weight 5–100 kDa, average molecular weight 50 kDa) was dissolved in distilled water to prepare an aqueous solution with a final concentration of 0.5% (w / v), which served as the aqueous phase. While stirring the aqueous phase at 400 rpm with a magnetic stirrer, the oil phase was slowly added dropwise to the aqueous phase, resulting in a volume ratio of 1:50. Stirring was continued at 400 rpm with a magnetic stirrer until an emulsion formed; the organic solvent was removed by further stirring at this speed for 5 hours. The resulting solution was then first frozen (freezing at −80°C for 6 hours) and then lyophilized (freeze-dried at −40°C for 24 hours) to remove water, yielding injectable PHBHHx microspheres.

[0042] The yields of the resulting injectable PHBHHx microspheres were 90%, 93%, and 89%, respectively. The freeze-dried PHBHHx microspheres were dispersed in water by dissolving and appropriately mixing. After air-drying, the microspheres were observed under an optical microscope and found to have good dispersibility and spherical or ellipsoidal shapes. The particle sizes, as determined by a particle size analyzer, were 0.4-20 μm, 1-50 μm, and 1-80 μm, respectively (83% of the particles were 1-60 μm in diameter).

[0043] Example 7: Different volume ratios of oil phase and water phase One gram of PHBHHx (molecular weight 20-100 kDa, average molecular weight 50 kDa) was dissolved in 20 mL of dichloromethane to prepare a 5% (w / v) final organic solution, which served as the oil phase. Hyaluronic acid (molecular weight 5-100 kDa, average molecular weight 50 kDa) was dissolved in distilled water to prepare a 0.5% (w / v) final aqueous solution, which served as the aqueous phase. While stirring the aqueous phase at 400 rpm with a magnetic stirrer, the oil phase was slowly added dropwise to the aqueous phase, resulting in volume ratios of 1:10, 1:50, and 1:200, respectively. Stirring was continued at 400 rpm with a magnetic stirrer until an emulsion formed; the organic solvent was removed by further stirring at this speed for 5 hours. The resulting solution was then first frozen (freezing at −80°C for 6 hours) and then lyophilized (freeze-dried at −40°C for 24 hours) to remove water, yielding injectable PHBHHx microspheres.

[0044] The yields of the resulting injectable PHBHHx microspheres were 71%, 93%, and 94%, respectively. The freeze-dried PHBHHx microspheres were dispersed in water by dissolving and appropriately mixing. After air-drying, the microspheres were observed under an optical microscope and found to have good dispersibility and spherical or ellipsoidal shapes. Particle size measurements revealed that the microspheres had particle sizes of 1-110 μm (58% of the particles were in the 1-60 μm range), 1-50 μm, and 1-50 μm, respectively.

[0045] Example 8: Different stirring methods and rotation speeds One gram of PHBHHx (molecular weight 20–100 kDa, average molecular weight 50 kDa) was dissolved in 20 mL of dichloromethane to prepare a 5% (w / v) final organic solution, which served as the oil phase. Hyaluronic acid (molecular weight 5–100 kDa, average molecular weight 50 kDa) was dissolved in distilled water to prepare a 0.5% (w / v) final aqueous solution, which served as the aqueous phase. While stirring the aqueous phase at 400 rpm with a magnetic stirrer, the oil phase was slowly added dropwise to the aqueous phase, resulting in a 1:50 volumetric ratio. Stirring was continued with a magnetic stirrer (rotation speeds of 100 rpm, 400 rpm, and 500 rpm, respectively) and a high-speed homogenizer (rotation speed 12,000 rpm) until an emulsion formed; the organic solvent was removed by further stirring for 5 hours. The resulting solution was then first frozen (freezing at −80°C for 6 hours) and then lyophilized (freeze-dried at −40°C for 24 hours) to remove water, yielding injectable PHBHHx microspheres.

[0046] The yields of the resulting injectable PHBHHx microspheres were 77%, 93%, 89%, and 99%, respectively. The freeze-dried PHBHHx microspheres were dispersed in water by dissolving and appropriately mixing. After air-drying, the microspheres were observed under an optical microscope and found to have good dispersibility and spherical or ellipsoidal shapes. Particle size measurements revealed that the particle sizes were 5-60 μm, 1-50 μm, 1-45 μm, and 0.1-10 μm, respectively (particle sizes were generally below 10 μm, with only 5% of particles ranging from 1 to 60 μm, and the overall particle size distribution was within the 0.1-10 μm range). This demonstrates that, compared with high-speed homogenization, the use of a magnetic stirrer at an appropriate speed (100-500 rpm) in this invention is advantageous for obtaining microspheres with particle sizes more suited to the human body. Example 9: Different types of oil phase organic solvent

[0047] 1 g of PHBHHx (molecular weight 10–20 kDa, average molecular weight 50 kDa) was dissolved in 20 mL of dichloromethane, chloroform, or ethyl acetate to prepare an organic solution with a final concentration of 5% (w / v), which served as the oil phase. Hyaluronic acid (molecular weight 5–100 kDa, average molecular weight 50 kDa) was dissolved in distilled water to prepare an aqueous solution with a final concentration of 0.5% (w / v), which served as the aqueous phase. While stirring the aqueous phase at 400 rpm with a magnetic stirrer, the oil phase was slowly added dropwise to the aqueous phase, resulting in a volume ratio of 1:50. Stirring was continued at 400 rpm with a magnetic stirrer until an emulsion formed; the organic solvent was removed by further stirring at this speed for 5 hours. The resulting solution was then first frozen (freezing at −80°C for 6 hours) and then lyophilized (freeze-dried at −40°C for 24 hours) to remove water, yielding injectable PHBHHx microspheres.

[0048] The yields of the resulting injectable PHBHHx microspheres were 93%, 92%, and 89%, respectively. The freeze-dried PHBHHx microspheres were dissolved in water and dispersed appropriately. After air-drying, the microspheres were observed under an optical microscope and found to have good dispersibility and spherical or ellipsoidal shapes. The particle sizes, as determined by a particle size analyzer, were 1-52 μm, 1-50 μm, and 1-55 μm, respectively.

[0049] Example 10: Different aqueous phase concentrations One gram of PHBHHx (molecular weight 20-100 kDa, average molecular weight 50 kDa) was dissolved in 20 mL of dichloromethane to prepare a 5% (w / v) final organic solution, which served as the oil phase. Hyaluronic acid (molecular weight 10-200 kDa, average molecular weight 50 kDa) was dissolved in distilled water to prepare aqueous solutions with final concentrations of 0.01% (w / v), 0.5% (w / v), and 1% (w / v), respectively, which served as the aqueous phase. While stirring the aqueous phase at 400 rpm with a magnetic stirrer, the oil phase was slowly added dropwise to the aqueous phase, resulting in a 1:50 volume ratio. Stirring was continued at 400 rpm with a magnetic stirrer until an emulsion formed; the organic solvent was removed by further stirring at this speed for 5 hours. The resulting solution was then first frozen (freezing at −80°C for 6 hours) and then lyophilized (freeze-dried at −40°C for 24 hours) to remove water, yielding injectable PHBHHx microspheres.

[0050] The yields of the resulting injectable PHBHHx microspheres were 80%, 93%, and 87%, respectively. The freeze-dried PHBHHx microspheres were dissolved in water and dispersed appropriately by mixing. After air-drying, the microspheres were observed under an optical microscope and found to have good dispersibility and spherical or ellipsoidal shapes. The particle sizes, as determined by a particle size analyzer, were 3-50 μm, 1-50 μm, and 0.7-50 μm, respectively.

[0051] Comparative Example 1 One gram of PHBHHx (molecular weight 20–100 kDa, average molecular weight 50 kDa) was dissolved in 20 mL of dichloromethane to prepare a 5% (w / v) final organic solution, which served as the oil phase. Polyvinyl alcohol (PVA) with an 80% alcoholysis degree was dissolved in distilled water to prepare a 0.5% (w / v) final aqueous solution, which served as the aqueous phase. While stirring the aqueous phase at 400 rpm with a magnetic stirrer, the oil phase was slowly added dropwise to the aqueous phase, resulting in a 1:50 volume ratio. Stirring was continued at 400 rpm with a magnetic stirrer until an emulsion formed; stirring was continued at this speed for 5 hours to remove the organic solvent. The resulting solution was then first frozen (at -80°C for 6 hours) and then lyophilized (at -40°C for 24 hours) to remove the water, yielding PHBHHx microspheres.

[0052] Comparative Example 2 One gram of PHBVHHx (molecular weight 20–100 kDa, average molecular weight 50 kDa) was dissolved in 20 mL of dichloromethane to prepare a 5% (w / v) final organic solution, which served as the oil phase. Polyvinyl alcohol (PVA) with an 80% alcoholysis degree was dissolved in distilled water to prepare a 0.5% (w / v) final aqueous solution, which served as the aqueous phase. While stirring the aqueous phase at 400 rpm with a magnetic stirrer, the oil phase was slowly added dropwise to the aqueous phase, resulting in a 1:50 volumetric ratio. Stirring was continued at 400 rpm with a magnetic stirrer until an emulsion formed; stirring was continued at this speed for 5 hours to remove the organic solvent. The resulting solution was then first frozen (at -80°C for 6 hours) and then lyophilized (at -40°C for 24 hours) to remove the water, yielding PHBVHHx microspheres.

[0053] Comparative Example 3 One gram of PHB (molecular weight 20–100 kDa, average molecular weight 80 kDa) was dissolved in 20 mL of dichloromethane to prepare a 5% (w / v) final organic solution, which served as the oil phase. Polyvinyl alcohol (PVA) with an 80% alcoholysis degree was dissolved in distilled water to prepare a 0.5% (w / v) final aqueous solution, which served as the aqueous phase. While stirring the aqueous phase at 400 rpm with a magnetic stirrer, the oil phase was slowly added dropwise to the aqueous phase, resulting in a 1:50 volume ratio. Stirring was continued at 400 rpm with a magnetic stirrer until an emulsion formed; stirring was continued at this speed for 5 hours to remove the organic solvent. The resulting solution was then first frozen (at −80°C for 6 hours) and then lyophilized (at −40°C for 24 hours) to remove the water, yielding PHB microspheres.

[0054] Comparative Example 4 One gram of PHBV (molecular weight 20–100 kDa, average molecular weight 40 kDa) was dissolved in 20 mL of dichloromethane to prepare a 5% (w / v) final organic solution, which served as the oil phase. Polyvinyl alcohol (PVA) with an 80% alcoholysis degree was dissolved in distilled water to prepare a 0.5% (w / v) final aqueous solution, which served as the aqueous phase. While stirring the aqueous phase at 400 rpm with a magnetic stirrer, the oil phase was slowly added dropwise to the aqueous phase, resulting in a 1:50 volumetric ratio. Stirring was continued at 400 rpm with a magnetic stirrer until an emulsion formed; stirring was continued at this speed for 5 hours to remove the organic solvent. The resulting solution was then first frozen (at -80°C for 6 hours) and then lyophilized (at -40°C for 24 hours) to remove the water, yielding injectable PHBV microspheres.

[0055] Comparative Example 5 One gram of P34HB (molecular weight 10–30 kDa, average molecular weight 15 kDa) was dissolved in 20 mL of dichloromethane to prepare a 5% (w / v) final organic solution, which served as the oil phase. Polyvinyl alcohol (PVA) with an 80% alcoholysis degree was dissolved in distilled water to prepare a 0.5% (w / v) final aqueous solution, which served as the aqueous phase. While stirring the aqueous phase at 400 rpm with a magnetic stirrer, the oil phase was slowly added dropwise to the aqueous phase, resulting in a 1:50 volumetric ratio. Stirring was continued at 400 rpm with a magnetic stirrer until an emulsion formed; stirring was continued at this speed for 5 hours to remove the organic solvent. The resulting solution was then first frozen (at -80°C for 6 hours) and then lyophilized (at -40°C for 24 hours) to remove the water, yielding P34HB microspheres.

[0056] Example 11: Determining the stability of microspheres 0.01 g of each of the microspheres produced in Examples 1 to 5 and Comparative Examples 1 to 5 was collected, dispersed in 1 mL of distilled water, and placed in a 5 mL glass bottle. The mixture was quickly mixed and then allowed to stand.

[0057] After standing for 30 minutes, the dispersion state of each microsphere in water was observed. As a result, as shown in Figure 6, which is a diagram showing the dispersion stability of the microspheres obtained in Examples 1 to 5 and Comparative Examples 1 to 5 (the 10 containers from left to right correspond to the samples of Examples 1 to 5 and Comparative Examples 1 to 5, respectively). It was found that the PHBHHx microspheres, PHBVHHx microspheres, PHB microspheres, PHBV microspheres, and P34HB microspheres in Examples 1 to 5 all had good dispersibility, no phase separation phenomenon was observed, and the microspheres were uniformly dispersed in the water body. The PHBHHx microspheres, PHBVHHx microspheres, PHB microspheres, PHBV microspheres, and P34HB microspheres in Comparative Examples 1 to 5 did not have water dispersibility, and all exhibited serious phase separation phenomenon, with the microspheres floating on the upper layer of the water. This proves that in the present invention, the dispersibility and stability of microspheres are significantly improved when an aqueous solution of hyaluronic acid is used as the aqueous phase substance, rather than when an aqueous solution of polyvinyl alcohol is used.

[0058] Example 12: Measurement of the attractiveness of microspheres A disposable 1 mL syringe equipped with a needle with an inner diameter of 0.5 mm and a length of 19.7 mm (i.e., the needle of a conventional 1 mL syringe) was used as an injection rate detection device, and 1 mL of microsphere dispersion (0.1 g microsphere dry weight, designated MO) was used as the experimental sample. 0.1 g of each of the microspheres prepared in Examples 1 to 5 and Comparative Examples 1 to 5 was collected, dispersed in 1 mL of distilled water, and placed in a glass bottle. After rapid mixing, a microsphere dispersion was formed. 1 mL of the dispersion was aspirated into the syringe, and the microspheres aspirated into the syringe were each freeze-dried. The ratio of the mass of microspheres aspirated into the syringe (M1) to the total mass of microspheres (MO) was defined as the microsphere aspirate rate (M1 / MO) × 100%.

[0059] As a result, the suction rates of PHBHHx microspheres, PHBVHHx microspheres, PHB microspheres, PHBV microspheres, and P34HB microspheres in Examples 1 to 5 were 95%, 93%, 91%, 90%, and 85%, respectively. The suction rates of PHBHHx microspheres, PHBVHHx microspheres, PHB microspheres, PHBV microspheres, and P34HB microspheres in Comparative Examples 1 to 5 were 10%, 13%, 11%, 9%, and 6%, respectively. This demonstrates that the suction rates of the microspheres in Examples 1 to 5 are much higher than those in Comparative Examples 1 to 5. This demonstrates that the suction properties of microspheres are significantly improved when a hyaluronic acid aqueous solution is used as the aqueous phase material compared to when a polyvinyl alcohol aqueous solution is used.

[0060] Example 13: Determining the injectability of microspheres A disposable 1 mL syringe was fitted with a needle with an inner diameter of 0.5 mm and a length of 19.7 mm (i.e., a conventional 1 mL syringe needle) to serve as an injection rate detection device. 1 mL of microsphere dispersion (0.1 g microsphere dry weight, designated MO) was used as the experimental sample. 0.1 g of each of the microspheres prepared in Examples 1 to 5 and Comparative Examples 1 to 5 was dispersed in 1 mL of distilled water and placed in a glass vial. After rapid mixing, a microsphere dispersion was formed. The dispersion was completely injected into the 1 mL syringe, and the syringe was pressed to collect the injected dispersion. The microsphere mass (M2) of the injected dispersion was freeze-dried and the percentage of the total microsphere mass (MO) was calculated as the microsphere injection rate (M2 / MO) × 100%.

[0061] As a result, the injectability of PHBHHx microspheres, PHBVHHx microspheres, PHB microspheres, PHBV microspheres, and P34HB microspheres in Examples 1 to 5 was 91%, 92%, 90%, 90%, and 88%, respectively. The injectability of PHBHHx microspheres, PHBVHHx microspheres, PHB microspheres, PHBV microspheres, and P34HB microspheres in Comparative Examples 1 to 5 was 7%, 11%, 13%, 8%, and 4%, respectively. This demonstrates that the injectability of the microspheres in Examples 1 to 5 is significantly higher than that of Comparative Examples 1 to 5. This demonstrates that the injectability of the microspheres in the present invention is significantly improved when a hyaluronic acid aqueous solution is used as the aqueous phase material compared to when a polyvinyl alcohol aqueous solution is used.

[0062] The above experimental results demonstrate that the manufacturing method provided by the present invention has the following beneficial effects: (1) High microsphere yield: The microsphere yield is 70% or more. (2) Good dispersibility: The PHA microspheres obtained by the present invention can be dispersed in water with appropriate mixing. After standing, the microspheres exhibit good dispersibility in water, without phase separation, without floating on the top layer of the water, or settling on the bottom, and are uniformly dispersed throughout the water, forming a uniform dispersion. After air-drying the dispersion, the microsphere morphology was observed. The microspheres were found to be completely morphological, independent of each other, with no aggregation. All or most of the microsphere particles had a particle size of 60 μm or less, meeting the needs for injection. (3) Good suckability: When the dispersion was sucked into a syringe, the microspheres were sucked up at a rate of 85% or more, demonstrating excellent suckability. (4) Good injectability: The injected dispersion was collected by pressing the syringe and the injection rate of the microspheres was calculated. The injection rate of the microspheres reached 88% or more, indicating excellent injectability.

[0063] The principles and embodiments of the present invention have been described above using specific examples. However, the description of the above examples is merely intended to facilitate understanding of the method and core idea of ​​the present invention, including the best mode, as well as methods for manufacturing and using any device or system and implementing any combination. Those skilled in the art may make minor improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications are also within the scope of protection of the claims of the present invention. The scope of protection of the present invention is defined by the claims and may include other examples that may occur to those skilled in the art. It is clear that various modifications and alterations may be made within the scope of the claims, as long as they are similar to the elements recited in the claims or contain equivalent elements that are not substantially different from the wording of the claims. It is understood that these modifications and alterations are also within the technical scope of the present invention. The present application includes the following aspects. [Section 1] 1. A method for producing polyhydroxy fatty acid ester (PHA) microspheres, comprising: a) dissolving a PHA material in an organic solvent to obtain an oil phase; b) dissolving hyaluronic acid in water to obtain an aqueous phase; c) adding the oil phase dropwise to the aqueous phase under stirring conditions, and after the addition is completed, continuing to stir to volatilize the organic solvent, and then performing solid-liquid separation and drying to obtain PHA microspheres; Including, The steps a) and b) are not limited in order, wherein the molecular weight of the PHA material is 10 to 100 KDa; In the oil phase, the mass concentration of the PHA material is 2.5% to 10%; The molecular weight of the hyaluronic acid is 5 to 500 KDa, In the aqueous phase, the mass concentration of hyaluronic acid is 0.01% to 1%. A manufacturing method characterized by: [Section 2] In the step c), the volume ratio of the oil phase to the aqueous phase is 1:(10 to 200). Item 1. The manufacturing method according to item 1. [Section 3] The organic solvent is one or more selected from the group consisting of dichloromethane, chloroform, and ethyl acetate. Item 1. The manufacturing method according to item 1. [Section 4] The average molecular weight of the PHA material is 15 to 80 KDa. Item 1. The manufacturing method according to item 1. [Section 5] The PHA material is PHBHHx, has a molecular weight of 20 to 100 KDa, and has an average molecular weight of 50 KDa, or The PHA material is PHBVHHx, has a molecular weight of 20 to 100 KDa, and has an average molecular weight of 50 KDa, or The PHA material is PHB, has a molecular weight of 20 to 100 KDa, and has an average molecular weight of 80 KDa; or The PHA material is PHBV, has a molecular weight of 20 to 100 KDa, and has an average molecular weight of 40 KDa; or The PHA material is P34HB, has a molecular weight of 10 to 30 KDa, and an average molecular weight of 15 KDa. Item 1. The manufacturing method according to item 1. [Section 6] The average molecular weight of the hyaluronic acid is 50 to 300 KDa. Item 1. The manufacturing method according to item 1. [Section 7] The hyaluronic acid has a molecular weight of 5 to 100 KDa and an average molecular weight of 50 KDa, or The hyaluronic acid has a molecular weight of 10 to 200 KDa and an average molecular weight of 50 KDa, or The hyaluronic acid has a molecular weight of 100 to 500 KDa and an average molecular weight of 300 KDa. Item 1. The manufacturing method according to item 1. [Section 8] The stirring speed is 100 to 500 rpm. Item 1. The manufacturing method according to item 1. [Section 9] the stirring is performed by a magnetic stirrer; After the dropwise addition of the oil phase is completed, stirring is continued for 4 hours or more. Item 1. The manufacturing method according to item 1. [Section 10] After the dropwise addition of the oil phase is completed, stirring is continued for 4 to 6 hours. Item 10. The manufacturing method according to item 9. [Section 11] The stirring temperature is 10 to 37°C. Item 1. The manufacturing method according to item 1. [Section 12] the drying is first by freezing and then by freeze-drying; The freezing temperature is −80 to −20° C., and the freeze-drying temperature is −60 to −40° C. Item 1. The manufacturing method according to item 1. [Section 13] Item 13. Polyhydroxy fatty acid ester microspheres produced by the method according to any one of Items 1 to 12. [Section 14] the particle size of the polyhydroxy fatty acid ester microspheres is 60 μm or less, and / or The polyhydroxy fatty acid ester microspheres have an absorption rate of 85% or more, and / or The injection rate of the polyhydroxy fatty acid ester microspheres is 88% or more. Item 14. The polyhydroxy fatty acid ester microspheres according to item 13.

Claims

1. 1. A method for producing polyhydroxy fatty acid ester (PHA) microspheres, comprising: a) dissolving a PHA material in an organic solvent to obtain an oil phase; b) dissolving hyaluronic acid in water to obtain an aqueous phase; c) adding the oil phase dropwise to the aqueous phase under stirring conditions, and after the completion of the addition, continuing to stir to volatilize the organic solvent, and then performing solid-liquid separation and drying to obtain PHA microspheres; Including, The steps a) and b) are not limited in order, wherein the mass concentration of the PHA material in the oil phase is 2.5% to 10%; The molecular weight of the hyaluronic acid is 5 to 500 KDa, In the aqueous phase, the mass concentration of hyaluronic acid is 0.01% to 1%; the PHA material is PHBHHx, has a molecular weight of 20-100 KDa, and has an average molecular weight of 50 KDa; or the PHA material is PHBVHHx, has a molecular weight of 20-100 KDa, and has an average molecular weight of 50 KDa; or the PHA material is PHB, has a molecular weight of 20-100 KDa, and has an average molecular weight of 80 KDa; or the PHA material is PHBV, has a molecular weight of 20-100 KDa, and has an average molecular weight of 40 KDa; or The PHA material is P34HB, has a molecular weight of 10 to 30 KDa, and an average molecular weight of 15 KDa; A manufacturing method characterized by:

2. In the step c), the volume ratio of the oil phase to the aqueous phase is 1:(10 to 200). The manufacturing method according to claim 1 .

3. The organic solvent is one or more selected from the group consisting of dichloromethane, chloroform, and ethyl acetate. The manufacturing method according to claim 1 .

4. The average molecular weight of the hyaluronic acid is 50 to 300 KDa. The manufacturing method according to claim 1 .

5. The hyaluronic acid has a molecular weight of 5 to 100 KDa and an average molecular weight of 50 KDa; or The hyaluronic acid has a molecular weight of 10 to 200 KDa and an average molecular weight of 50 KDa, or The hyaluronic acid has a molecular weight of 100 to 500 KDa and an average molecular weight of 300 KDa. The manufacturing method according to claim 1 .

6. The stirring speed is 100 to 500 rpm. The manufacturing method according to claim 1 .

7. the stirring is performed by a magnetic stirrer; After the dropwise addition of the oil phase is completed, the stirring is continued for 4 hours or more. The manufacturing method according to claim 1 .

8. After the dropwise addition of the oil phase is completed, stirring is continued for 4 to 6 hours. The manufacturing method according to claim 7.

9. The stirring temperature is 10 to 37°C. The manufacturing method according to claim 1 .

10. the drying is first by freezing and then by freeze-drying; The freezing temperature is −80 to −20° C., and the freeze-drying temperature is −60 to −40° C. The manufacturing method according to claim 1 .

11. the particle size of the polyhydroxy fatty acid ester microspheres is 60 μm or less, and / or The polyhydroxy fatty acid ester microspheres have an absorption rate of 85% or more, and / or The injectability of the polyhydroxy fatty acid ester microspheres is 88% or more; The manufacturing method according to claim 1 .

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