Polyhydroxyalkanoate microspheres as well as preparation method and application thereof

Abstract

The invention provides a polyhydroxyalkanoate microsphere and a preparation method thereof. The preparation method comprises the following steps: preparation of a dispersion phase: dissolving polyhydroxyalkanoate in an organic solvent to serve as the dispersion phase; preparation of a continuous phase: dissolving an emulsifier in water to serve as the continuous phase; dispersing the dispersed phase into the continuous phase, stirring to form liquid drops, curing, and removing the solvent to obtain polyhydroxyalkanoate microspheres; the molecular weight of the polyhydroxyalkanoate is 160 to 220 kDa. The prepared microspheres are smoother in surface, free of holes and wrinkles and beneficial to injection filling.

Description

Technical Field

[0001] This application relates to the field of polymer materials science, and particularly relates to a polyhydroxyalkanoate microsphere, a preparation method thereof, and an application thereof. Background Art

[0002] Polymer microspheres have a wide range of applications in fields such as injection filling, cell culture, drug controlled release, vaccine delivery, and immunotherapy due to their small particle size and large specific surface area. Polyhydroxyalkanoate (PHA) is a natural polymer biomaterial with good biocompatibility and biodegradability, and is one of the ideal polymer microsphere materials.

[0003] At present, more than one hundred PHA materials have been discovered, and different polyhydroxyalkanoates have different characteristics. Among them, poly(3-hydroxybutyrate) (P3HB) is a PHA material with relatively high cost performance. However, P3HB is prone to hydrolysis, and the preparation process requires sterilization, which greatly wastes energy; poly(4-hydroxybutyrate) (P4HB) is expensive, and due to the relatively precise requirements for molecular weight and high purity requirements, there are no relevant medical-grade products on the market in China. Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P34HB), a new type of PHA polymer material, has a 4-hydroxybutyrate (4HB) content between 0 and 100%, has good thermal stability, is completely biodegradable, and can be used as a polymer microsphere material. Summary of the Invention

[0004] In the actual production process, the inventors found that even when using P34HB raw materials, due to differences in the preparation process, the properties of the obtained microspheres vary greatly. For example, the microspheres are prone to agglomeration, adhesion, caking, forming large irregular lumps, etc., which are likely to cause rejection of in vivo cell tissues. In order to make the microspheres non-aggregated and of uniform size, it is usually necessary to additionally add a dispersant. Based on the prior art, the inventors of this application creatively discovered that by controlling the viscosity and concentration range of polyvinyl alcohol, PHA microspheres that are non-aggregated and of uniform size can be obtained without additionally adding other dispersants. The inventors further found that P34HB raw materials with different 4HB contents have a great influence on the morphology and smoothness of the microspheres, and the morphology and smoothness of the microspheres directly affect the injection difficulty and injection effect of the microspheres as injection fillers. The microspheres prepared in this application using P34HB with a 4HB content of 14% to 25% have a smoother surface, no holes and wrinkles, which is beneficial for injection filling.

[0005] The technical solution of this application is as follows:

[0006] 1. A polyhydroxyalkanoate microsphere, wherein the polyhydroxyalkanoate is a copolyester of 3-hydroxybutyric acid and 4-hydroxybutyric acid, the size D10 of the polyhydroxyalkanoate microsphere is 20 - 35 μm, D50 is 30 - 50 μm, D90 is 50 - 70 μm, and the microsphere is spherical and has a smooth surface.

[0007] 2. The polyhydroxyalkanoate microsphere according to item 1, wherein the polyhydroxyalkanoate microsphere satisfies the following: D50 - D10 is less than 20 μm, and D90 - D50 is less than 20 μm.

[0008] 3. The polyhydroxyalkanoate microsphere according to item 1 or 2, wherein the content of 4-hydroxybutyric acid in the polyhydroxyalkanoate is 14% - 25%.

[0009] 4. The polyhydroxyalkanoate microsphere according to claim 1 or 2, wherein the molecular weight of the polyhydroxyalkanoate is 160 - 220 kDa.

[0010] 5. A method for a polyhydroxyalkanoate microsphere according to any one of items 1 - 4, which comprises the following steps:

[0011] Preparation of the dispersed phase: Dissolve the polyhydroxyalkanoate in an organic solvent as the dispersed phase;

[0012] Preparation of the continuous phase: Dissolve the emulsifier in water as the continuous phase;

[0013] Disperse the dispersed phase into the continuous phase and stir to form droplets, then solidify and remove the solvent to obtain the polyhydroxyalkanoate microsphere.

[0014] 6. The method according to item 5, wherein the concentration of the polyhydroxyalkanoate in the dispersed phase is 10 - 50 mg / mL.

[0015] 7. The method according to item 5, wherein the organic solvent is selected from one or more of acetone, dichloromethane, and ethyl acetate.

[0016] 8. The method according to item 5, wherein the concentration of the emulsifier in the continuous phase is 15 - 32 mg / mL, and preferably, the emulsifier is selected from one or more of polyvinyl alcohol, gelatin, tween, and span.

[0017] 9. The method according to item 5, wherein the viscosity of the emulsifier is 18 - 45 mPa·s.

[0018] 10. The method according to item 5, wherein the volume ratio of the dispersed phase to the continuous phase is 1:(5 - 10).

[0019] 11. Use of the polyhydroxyalkanoate microspheres according to any one of Items 1-4 or the polyhydroxyalkanoate microspheres prepared by the method according to any one of Items 5-10 in the preparation of biomedical materials, preferably in injection fillers.

[0020] Compared with the prior art, the beneficial effects of this application are as follows:

[0021] This application solves the problems of uncontrolled particle size and morphology and low yield in the preparation of microspheres by the existing stirring method, and at the same time avoids the problems of high requirements for raw materials, equipment and process conditions and high energy consumption in other methods such as membrane emulsification method and microfluidic method. Compared with other methods, the method for preparing polymer microspheres with uniform and controllable particle size proposed in this application has simple equipment and low investment cost. At the same time, the polymer microspheres prepared by this application have a smooth appearance, good sphericity, and a narrow particle size distribution.

[0022] The microspheres prepared in this application can be used in aesthetic medical injection fillers and non-aesthetic drug controlled release carrier materials, and have broad application prospects.

[0023] For the P34HB microspheres in this application, by controlling the viscosity and concentration range of aqueous polyvinyl alcohol, microspheres that do not aggregate and have uniform size are obtained; it is also found in this application that the microspheres prepared from P34HB with a 4HB content of 14% to 25% have a smoother surface, without holes and wrinkles, which is beneficial for injection filling. Description of the Drawings

[0024] Figure 1 It is a flowchart of the polymer microsphere preparation process;

[0025] Figure 2 It is a SEM image of the polymer microspheres;

[0026] Figure 3 It is a SEM image (200 times) of the polymer microspheres prepared in Example 1 of this application;

[0027] Figure 4 It is a SEM image (200 times) of the polymer microspheres prepared in Example 2 of this application;

[0028] Figure 5 It is a SEM image (200 times) of the polymer microspheres prepared in Example 3 of this application;

[0029] Figure 6 It is a SEM image (200 times) of the polymer microspheres prepared in Example 4 of this application;

[0030] Figure 7 It is a SEM image (200 times) of the polymer microspheres prepared in Example 5 of this application;

[0031] Figure 8SEM image (200x) of the polymer microspheres prepared in Example 6 of this application;

[0032] Figure 9 SEM image (200x) of the polymer microspheres prepared in Example 9 of this application;

[0033] Figure 10 SEM image (200x) of the polymer microspheres prepared in Example 10 of this application;

[0034] Figure 11 SEM image (200x) of the polymer microspheres prepared in Comparative Example 1 of this application;

[0035] Figure 12 SEM image (200x) of the polymer microspheres prepared in Comparative Example 2 of this application. Detailed implementation manners

[0036] The following further illustrates the present application in conjunction with examples. It should be understood that the examples are only used to further illustrate and explain the present application, and are not used to limit the present application.

[0037] Unless otherwise defined, the technical and scientific terms in this specification have the same meaning as commonly understood by those skilled in the art. Although methods and materials similar or identical to those described herein can be used in experiments or practical applications, the materials and methods are described below. In case of conflict, the present specification, including its definitions, shall prevail. Additionally, the materials, methods, and examples are for illustrative purposes only and are not restrictive. The following further illustrates the present application with specific examples, but does not limit the scope of the present application.

[0038] The present application provides a polyhydroxyalkanoate microsphere, wherein the polyhydroxyalkanoate is a copolyester of 3-hydroxybutyric acid and 4-hydroxybutyric acid, the size D10 of the polyhydroxyalkanoate microsphere is 20 - 35 μm, D50 is 30 - 50 μm, D90 is 50 - 70 μm, and the microspheres are spherical and have a smooth surface.

[0039] In the present application, D50 represents the average particle size of the microspheres, that is, the particle size corresponding to when the cumulative particle size distribution percentage of the microspheres reaches 50%, 50% of the microspheres have a particle size greater than D50, and 50% of the microspheres have a particle size less than D50. D50 is also called the median particle size or median value particle size. Similarly, D90 represents the particle size corresponding to when the cumulative particle size distribution percentage of the microspheres reaches 90%, and 90% of the microspheres have a particle size less than D90; D10 represents the particle size corresponding to when the cumulative particle size distribution percentage of the microspheres reaches 10%, and 10% of the microspheres have a particle size less than D10.

[0040] D50 - D10 represents the difference in particle size when the cumulative particle size distribution percentage reaches 50% and when it reaches 10%. Similarly, D90 - D50 represents the difference in particle size when the cumulative particle size distribution percentage reaches 90% and when it reaches 50%. The smaller the values of D50 - D10 and D90 - D50, the smaller the particle size distribution range and the more uniform the particle size distribution.

[0041] In this application, the tests for D90, D50 and D10 can be carried out by using the conventional detection methods of those skilled in the art, for example, directly tested by using a Malvern laser particle size analyzer.

[0042] In some embodiments of this application, the D50 of the polyhydroxyalkanoate microspheres is 30 - 50 μm. For example, the D50 of the polyhydroxyalkanoate microspheres can be 30 μm, 31 μm, 32 μm, 33 μm, 34 μm, 35 μm, 36 μm, 37 μm, 38 μm, 39 μm, 40 μm, 41 μm, 42 μm, 43 μm, 44 μm, 45 μm, 46 μm, 47 μm, 48 μm, 49 μm, 50 μm or any range therebetween.

[0043] In some embodiments of this application, the D10 of the polyhydroxyalkanoate microspheres is 20 - 35 μm. For example, the D10 of the polyhydroxyalkanoate microspheres can be 20 μm, 21 μm, 22 μm, 23 μm, 24 μm, 25 μm, 26 μm, 27 μm, 28 μm, 29 μm, 30 μm, 31 μm, 32 μm, 33 μm, 34 μm, 35 μm or any range therebetween.

[0044] In some embodiments of this application, the D90 of the polyhydroxyalkanoate microspheres is 50 - 70 μm. For example, the D90 of the polyhydroxyalkanoate microspheres can be 50 μm, 51 μm, 52 μm, 53 μm, 54 μm, 55 μm, 56 μm, 57 μm, 58 μm, 59 μm, 60 μm, 61 μm, 62 μm, 63 μm, 64 μm, 65 μm, 66 μm, 67 μm, 68 μm, 69 μm, 70 μm or any range therebetween.

[0045] In some embodiments of the present application, the polyhydroxyalkanoate microspheres satisfy the following: D50 - D10 is less than 20 μm, and D90 - D50 is less than 20 μm; for example, D50 - D10 can be 0.001 μm, 0.005 μm, 0.01 μm, 0.05 μm, 0.1 μm, 0.2 μm, 0.3 μm, 0.4 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, 20 μm or any range therebetween; D90 - D50 can be 0.001 μm, 0.005 μm, 0.01 μm, 0.05 μm, 0.1 μm, 0.2 μm, 0.3 μm, 0.4 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, 20 μm or any range therebetween.

[0046] In some embodiments of the present application, the polyhydroxyalkanoate is a copolymer of 3 - hydroxybutyric acid and 4 - hydroxybutyric acid, abbreviated as P34HB.

[0047] In some embodiments of the present application, the content of 4 - hydroxybutyric acid (4HB) in the copolymer of 3 - hydroxybutyric acid and 4 - hydroxybutyric acid is 14% - 25%.

[0048] Wherein, the 4HB content refers to the mass percentage of 4 - hydroxybutyric acid (4HB) in P34HB in the total mass of P34HB.

[0049] For example, the 4HB content can be 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25% or any range therebetween.

[0050] In some embodiments of the present application, the molecular weight of the polyhydroxyalkanoate is 160 - 220 kDa.

[0051] In some embodiments of the present application, the molecular weight of the polyhydroxyalkanoate can be 160 kDa, 170 kDa, 180 kDa, 190 kDa, 200 kDa, 210 kDa, 220 kDa or any range therebetween.

[0052] The inventors of the present application found that when the molecular weight of polyhydroxyalkanoate is 160-220 kDa, the obtained polyhydroxyalkanoate microspheres have a smoother surface without holes and wrinkles, which is beneficial for injection filling. When the molecular weight of polyhydroxyalkanoate is lower than 160 kDa, the obtained polyhydroxyalkanoate microspheres are uneven in size and have a rough surface.

[0053] The present application provides a method for preparing polyhydroxyalkanoate microspheres, which comprises the following steps: Preparation of the dispersed phase: dissolving polyhydroxyalkanoate in an organic solvent as the dispersed phase; Preparation of the continuous phase: dissolving an emulsifier in water as the continuous phase; dispersing the dispersed phase into the continuous phase and stirring to form droplets, followed by curing and removing the solvent to obtain polyhydroxyalkanoate microspheres.

[0054] In some embodiments of the present application, the concentration of polyhydroxyalkanoate in the dispersed phase is 10-50 mg / mL. For example, the concentration of polyhydroxyalkanoate in the dispersed phase can be 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL, 29 mg / mL, 30 mg / mL, 31 mg / mL, 32 mg / mL, 33 mg / mL, 34 mg / mL, 35 mg / mL, 36 mg / mL, 37 mg / mL, 38 mg / mL, 39 mg / mL, 40 mg / mL, 41 mg / mL, 42 mg / mL, 43 mg / mL, 44 mg / mL, 45 mg / mL, 46 mg / mL, 47 mg / mL, 48 mg / mL, 49 mg / mL, 50 mg / mL or any range therebetween.

[0055] In some embodiments of the present application, the organic solvent is selected from one or more of acetone, dichloromethane and ethyl acetate.

[0056] In some embodiments of the present application, the concentration of the emulsifier in the continuous phase is 15-32 mg / mL, preferably 20-28 mg / mL. For example, the concentration of the emulsifier in the continuous phase is 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL, 29 mg / mL, 30 mg / mL, 31 mg / mL, 32 mg / mL or any range therebetween.

[0057] In some embodiments of the present application, the emulsifier is selected from one or more of polyvinyl alcohol, gelatin, Tween, and Span.

[0058] In some embodiments of the present application, the viscosity of the emulsifier is 18 - 45 mPa·s; for example, the viscosity of the emulsifier is 18 mPa·s, 19 mPa·s, 20 mPa·s, 21 mPa·s, 22 mPa·s, 23 mPa·s, 24 mPa·s, 25 mPa·s, 26 mPa·s, 27 mPa·s, 28 mPa·s, 29 mPa·s, 30 mPa·s, 31 mPa·s, 32 mPa·s, 33 mPa·s, 34 mPa·s, 35 mPa·s, 36 mPa·s, 37 mPa·s, 38 mPa·s, 39 mPa·s, 40 mPa·s, 41 mPa·s, 42 mPa·s, 43 mPa·s, 44 mPa·s, 45 mPa·s or any range therebetween.

[0059] In some embodiments of the present application, the volume ratio of the dispersed phase to the continuous phase is 1:(5 - 10). For example, the volume ratio of the dispersed phase to the continuous phase can be 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 or any range therebetween.

[0060] In some embodiments of the present application, the stirring speed is 100 - 1000 rpm / min, preferably 300 - 500 rpm / min. For example, the stirring speed can be 100 rpm / min, 150 rpm / min, 200 rpm / min, 250 rpm / min, 300 rpm / min, 350 rpm / min, 400 rpm / min, 450 rpm / min, 500 rpm / min or any range therebetween.

[0061] In some embodiments of the present application, the inventors found that the curing temperature refers to the boiling point of the organic phase solvent. According to the system of the present application, the curing temperature is 25 - 50 °C, preferably 25 - 40 °C. For example, the curing temperature can be 25 °C, 30 °C, 35 °C, 40 °C, 45 °C, 50 °C or any range therebetween.

[0062] As Figure 1 shown, the technical solution for preparing the polymer microspheres described in the present application is as follows:

[0063] 1) Polyhydroxyalkanoate is dissolved in an organic solvent as the dispersed phase, and the concentration of polyhydroxyalkanoate in the dispersed phase is 10 - 50 mg / mL; the emulsifier is dissolved in water as the continuous phase, and the concentration of the emulsifier in the continuous phase is 15 - 32 mg / mL;

[0064] 2) The dispersed phase is stably injected into the continuous phase at an injection rate of 0.01 - 10 mL / min. The height of the mechanical stirring paddle is adjusted to form a stable flow field in the stirring system. The rotational speed of the stirring paddle is controlled at 100 - 1000 rpm / min, the system temperature is 10 - 60 °C, and the volume ratio of the dispersed phase injection volume to the continuous phase volume is 1:(5 - 10). As the stirring proceeds, the organic phase solution is dispersed into droplets and distributed in the aqueous phase. Under the action of stirring shear, coalescence from small to large and dispersion from large to small occur among the droplets. After a certain period, this process reaches a dynamic equilibrium, and the emulsion droplets finally form tiny droplets with uniform particle sizes.

[0065] 3) The tiny droplets are desolvated and solidified in the continuous phase to form polymer microspheres. The desolvation time is 1 - 24 hours, the solidification temperature is 20 - 50 °C, and the stirring speed is 100 - 500 rpm / min. The microspheres are washed and collected by methods such as centrifugation or filtration, and then dried to obtain polymer microspheres with uniform morphology, particle size, and controllable size. The results are as Figure 2 、 Figure 3 and Figure 4 shown. It can be seen that the obtained polyhydroxyalkanoate microspheres are of uniform size and have a smooth surface.

[0066] This application provides a polyhydroxyalkanoate microsphere, wherein the polyhydroxyalkanoate is a copolyester of 3 - hydroxybutyrate and 4 - hydroxybutyrate, the molecular weight of the polyhydroxyalkanoate is 160 - 220 kDa, and the 4HB content in the copolyester of 3 - hydroxybutyrate and 4 - hydroxybutyrate is 14% - 25%.

[0067] In some embodiments of this application, the D50 of the copolyester microspheres of 3 - hydroxybutyrate and 4 - hydroxybutyrate is 30 - 50 μm, the D10 is 20 - 35 μm, and the D90 is 50 - 70 μm.

[0068] In some embodiments of this application, the copolyester microspheres of 3 - hydroxybutyrate and 4 - hydroxybutyrate satisfy that D50 - D10 is less than 20 μm and D90 - D50 is less than 20 μm.

[0069] In some embodiments of this application, the copolyester microspheres of 3 - hydroxybutyrate and 4 - hydroxybutyrate are prepared by the above - mentioned method.

[0070] This application provides the use of the above - mentioned polyhydroxyalkanoate microspheres in the preparation of biomedical materials.

[0071] In this application, the biomedical material has special properties and is used in medical and health care fields such as surgical repair of human organs, physical therapy and rehabilitation, diagnosis, examination, and treatment of patients, and is a material that does not cause adverse effects on human tissues and blood. The "biomaterial" defined by the International Organization for Standardization (ISO) is a biomedical material or a biomedicine material, which refers to "an inanimate material used to contact tissues to form a function". Biomedical materials are classified according to their application properties into: anticoagulant materials (cardiovascular materials), dental materials, orthopedic materials, ophthalmic materials, adsorption and detoxification materials (for hemoperfusion), prosthesis materials, sustained-release materials, bioadhesive materials, dialysis and ultrafiltration membrane materials, disposable medical materials, and so on. Classified according to the usage requirements of medical materials, they are divided into: non-implantable materials, implantable materials, blood-contact materials, degradable and absorbable materials.

[0072] This application provides the use of the above-mentioned polyhydroxyalkanoate microspheres in the preparation of an injectable filler.

[0073] As the most widely used skin filler at present, the injectable filler can be used in the field of medical aesthetics for nasolabial fold filling, rhinoplasty, tear trough filling, and filling and improving facial wrinkles, etc., and is an ideal material for tissue filling. The injectable filler containing microspheres contains microspheres and a gel carrier. Compared with traditional injectable fillers, the injectable filler containing microspheres has an excellent effect of inducing the human body to secrete and synthesize collagen, and has better effects such as immediate filling of facial depressions and long-term support.

[0074] The polyhydroxyalkanoate microspheres of this application have a smooth surface, and when used as an injectable filler, they can reduce skin trauma and the occurrence of serious side effects during the injection process. For example, they can reduce the occurrence of granulomas.

[0075] The polyhydroxyalkanoate microspheres of this application have a suitable size and the size can be controlled, which can prevent the microspheres from being phagocytosed by macrophages and fibroblasts.

[0076] The polyhydroxyalkanoate microspheres of this application have a more uniform particle size, which can ensure the stability between product batches, have a high screening recovery rate, and greatly reduce production costs. If the microspheres have uneven sizes and a too wide particle size distribution, it is easy to block the needle during injection, the injection pain increases, and even subcutaneous nodules may occur after injection, seriously affecting the product experience. A too wide particle size distribution is also likely to lead to uneven local distribution of the collagen regeneration effect; while the microspheres with uniform sizes of this application are easy to stably stimulate fibroblasts and macrophages at the filling site to secrete collagen, forming an expected regeneration effect.

[0077] Example 1

[0078] 1) Take P34HB (Mw 210 kDa) with a 4HB content of 14.5%, dissolve it in dichloromethane at a concentration of 30 mg / mL as the dispersed phase, take polyvinyl alcohol raw material (degree of alcoholysis 87 - 89%, viscosity 20.5 - 24.5 mPa·s), and dissolve it in ultrapure water at a concentration of 20 mg / mL as the continuous phase for the reaction;

[0079] 2) Steadily inject the dispersed phase into the continuous phase at an injection rate of 10 mL / min. Adjust the height of the mechanical stirring paddle so that it is located at the phase separation interface of the oil - water two - phase. Control the stirring paddle speed at 350 rpm / min, the system temperature at 25 °C, and the volume ratio of the dispersed phase to the continuous phase at 1:7;

[0080] 3) After stirring for 30 min, while maintaining a suitable humidity and air circulation in the environment, heat the stirring system to 40 °C as the curing temperature, adjust the stirring speed to 300 rpm / min, and continue for 4 h. Then, centrifuge to discard the supernatant, wash the collected powder repeatedly at 80 °C, and finally freeze - dry to obtain polyhydroxyalkanoate microspheres.

[0081] The parameters of Examples 2 - 10 and Comparative Examples 1 - 2 are shown in Table 1, and the rest are the same as in Example 1.

[0082] Table 1

[0083]

[0084] Experimental Example 1

[0085] Take the polyhydroxyalkanoate microspheres prepared in Examples 1 - 5 and Comparative Examples 1 - 2, test D90, D50, and D10, and calculate the yield of qualified products.

[0086] D90, D50, and D10 are directly measured by a Malvern laser particle size analyzer (Mastersizer 3000). The specific test method is as follows: Using dry measurement, add a certain amount of polymer microsphere powder to the sample injection chamber of the particle size analyzer, use pure water as the medium, and set the specific test parameters as follows: air pressure 2 bar, injection speed 50%, hopper gap 1 mm, lower limit of obscuration 0.1%, measurement time 10 seconds, and the relevant values can be obtained.

[0087] The yield of qualified products is calculated as follows: Yield of qualified products (%) = (mass of finally collected microspheres - mass of initially input polymer raw material) × 100%

[0088] The results are shown in Table 2.

[0089] Table 2

[0090]

[0091] As can be seen from Table 2, the particle sizes of the microspheres obtained in Comparative Examples 1-2 are uneven, and the yield of qualified products is low.

[0092] As can be seen from Examples 2 and 3, the viscosity and concentration of polyvinyl alcohol also affect the morphology of the microspheres, resulting in uneven microsphere sizes and a low yield of qualified products.

[0093] As can be seen from Example 1 and Examples 4-5, D50 is in the range of 30-50 μm, D10 is in the range of 20-35 μm, and D90 is in the range of 50-70 μm. And D50 - D10 is less than 20 μm, and D90 - D50 is less than 20 μm. This shows that the particle size distribution range is small and the particle size distribution is uniform.

[0094] Experimental Example 2

[0095] Take the polyhydroxyalkanoate microspheres prepared in Examples 1-10 and Comparative Examples 1-2 for scanning electron microscope testing. Among them, spread the dry polymer microsphere powder evenly on the conductive adhesive, and sputter coat the dry microspheres with a thin gold layer under vacuum, with a current of 20 mA and a time of 90 s; then observe the surface morphology, size and microstructure of the microspheres on a scanning electron microscope (COXEM, HS-OP-810, resolution: 100 μm, acceleration voltage: 15 kV) to obtain SEM images.

[0096] The results are shown in Table 3.

[0097] Table 3

[0098]

[0099]

[0100] As can be seen from Table 3, the particle sizes of the microspheres obtained in Comparative Examples 1-2 are uneven and the surface is not smooth.

[0101] As can be seen from Examples 2 and 3, the viscosity and concentration of polyvinyl alcohol also affect the morphology of the microspheres. For example, the microspheres in Example 2 have a phenomenon of aggregation, and the microspheres obtained in Example 3 are uneven in size.

[0102] As can be seen from Example 1 and Examples 4-8, the microspheres prepared in this application are uniform in size and smooth on the surface.

[0103] As can be seen from Examples 9-10, when the 4HB content in P34HB is not within 14% - 25%, the surface of the obtained polyhydroxyalkanoate microspheres has holes, wrinkles on the surface, and is not smooth.

[0104] Although this case has been disclosed as above by way of embodiments, it is not intended to limit this case. Any person with ordinary knowledge in the relevant technical field may make some modifications and refinements without departing from the spirit and scope of this case. Therefore, the protection scope of this case shall be subject to that defined by the appended patent application scope.

Claims

1. A polyhydroxyalkanoate microsphere, wherein, The polyhydroxyalkanoate is a copolyester of 3-hydroxybutyric acid and 4-hydroxybutyric acid. The size D10 of the polyhydroxyalkanoate microspheres is 20 - 35 μm, D50 is 30 - 50 μm, and D90 is 50 - 70 μm. The microspheres are spherical and have a smooth surface.

2. The polyhydroxyalkanoate microspheres according to claim 1, wherein, The polyhydroxyalkanoate microspheres satisfy the following: D50 - D10 is less than 20 μm, and D90 - D50 is less than 20 μm.

3. The polyhydroxyalkanoate microspheres according to claim 1 or 2, wherein the content of 4-hydroxybutyric acid in the polyhydroxyalkanoate is 14% - 25%.

4. The polyhydroxyalkanoate microspheres according to claim 1 or 2, wherein, The molecular weight of the polyhydroxyalkanoate is 160 - 220 kDa.

5. A method for preparing the polyhydroxyalkanoate microspheres according to any one of claims 1 - 4, which comprises the following steps: Preparation of the dispersed phase: Dissolving the polyhydroxyalkanoate in an organic solvent as the dispersed phase; Preparation of the continuous phase: Dissolving an emulsifier in water as the continuous phase; Dispersing the dispersed phase into the continuous phase and stirring to form droplets, followed by curing and removing the solvent to obtain the polyhydroxyalkanoate microspheres.

6. The method according to claim 5, wherein, The concentration of the polyhydroxyalkanoate in the dispersed phase is 10 - 50 mg / mL.

7. The method according to claim 5, wherein, The organic solvent is selected from one or more of acetone, dichloromethane, and ethyl acetate.

8. The method according to claim 5, wherein The concentration of the emulsifier in the continuous phase is 15 - 32 mg / mL. Preferably, the emulsifier is selected from one or more of polyvinyl alcohol, gelatin, Tween, and Span. Preferably, the viscosity of the emulsifier is 18 - 45 mPa·s.

9. The method according to claim 5, wherein, The volume ratio of the dispersed phase to the continuous phase is 1:(5 - 10).

10. Use of the polyhydroxyalkanoate microspheres according to any one of claims 1 - 4 or the polyhydroxyalkanoate microspheres prepared by the method according to any one of claims 5 - 9 in the preparation of biomedical materials, preferably in the use as an injectable filler.

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