WO2023061201A1 - 一种可注射聚羟基脂肪酸酯微球及其制备方法 - Google Patents

一种可注射聚羟基脂肪酸酯微球及其制备方法 Download PDF

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WO2023061201A1
WO2023061201A1 PCT/CN2022/121527 CN2022121527W WO2023061201A1 WO 2023061201 A1 WO2023061201 A1 WO 2023061201A1 CN 2022121527 W CN2022121527 W CN 2022121527W WO 2023061201 A1 WO2023061201 A1 WO 2023061201A1
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microspheres
molecular weight
water
preparation
pha
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PCT/CN2022/121527
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English (en)
French (fr)
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魏岱旭
李腾
张浩千
谈畅
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北京蓝晶微生物科技有限公司
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Priority to KR1020247015668A priority Critical patent/KR20240068802A/ko
Publication of WO2023061201A1 publication Critical patent/WO2023061201A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1641—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poloxamers
    • A61K9/1647—Polyesters, e.g. poly(lactide-co-glycolide)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1682—Processes

Definitions

  • the invention relates to the technical field of biomedicine, in particular to an injectable polyhydroxyalkanoate microsphere and a preparation method thereof.
  • biodegradable polymer materials have entered people's field of vision. This type of material is non-toxic and non-rejecting to the human body, and can be gradually degraded and excreted with the body's metabolism. It can also adjust the molecular weight and other parameters of the material. Degradation times vary from a week to as long as several years.
  • PHA Polyhydroxyalkanoate
  • PHA is a natural polymer biomaterial, a kind of intracellular polyester synthesized by microorganisms. Due to its good biocompatibility and biodegradability, PHA is one of the most ideal biomedical materials at present. PHA has good cytocompatibility with cells in vivo, cells can grow well on this kind of scaffold, and this kind of scaffold can be degraded into CO 2 and H 2 O.
  • it is usually made into microspheres, which can be injected through a needle. Due to the presence of human phagocytes, the diameter of the microspheres is usually more than 20 microns, but too large microspheres will block the needle and even cause skin rupture. The size of the microspheres used for injection is therefore usually below 60 microns.
  • the object of the present invention is to provide an injectable polyhydroxyalkanoate microsphere and a preparation method thereof.
  • the preparation method provided by the invention can effectively improve the dispersibility of polyhydroxyalkanoate microspheres, overcome the problems of microsphere agglomeration and adhesion; meanwhile, can effectively improve the absorbability and injectability of polyhydroxyalkanoate microspheres.
  • the invention provides a preparation method of injectable polyhydroxyalkanoate microspheres, comprising the following steps:
  • steps a) and b) are not limited in sequence.
  • the molecular weight of the hyaluronic acid is 5-500KDa.
  • the mass concentration of hyaluronic acid in the water phase is 0.01%-1% (w/v).
  • the volume ratio of the oil phase to the water phase is 1: (10-200).
  • the mass concentration of the PHA material in the oil phase is 2.5% to 10% (w/v);
  • the organic solvent is selected from one or more of dichloromethane, chloroform and ethyl acetate.
  • the molecular weight of the PHA material is 10-100 KDa.
  • the PHA material is natural or non-natural polyhydroxyalkanoate
  • the natural or unnatural polyhydroxyalkanoates include but are not limited to one or more of PHB, PHBV, PHBHHx, P34HB, PHBVHHx, PHHx and PHO.
  • the stirring rate is 100-500 rpm.
  • the stirring is carried out by using a magnetic stirrer; after the oil phase is added dropwise, the stirring time is ⁇ 4h.
  • the present invention also provides an injectable polyhydroxyalkanoate microsphere prepared by the preparation method described in the above technical scheme.
  • the PHA material is dissolved in an organic solvent to obtain an oil phase
  • the hyaluronic acid aqueous solution is used as the water phase
  • the water phase solution is stirred
  • the oil phase is slowly added to the water phase
  • the organic solvent is continuously stirred. Volatilization, followed by solid-liquid separation and drying, to prepare PHA microspheres.
  • the above method of the present invention can improve the dispersibility of the PHA microspheres, and the obtained microspheres can be quickly dispersed in water, the microspheres are complete in shape, independent of each other, and have no agglomeration phenomenon, thereby avoiding clogging of the needle and facilitating subsequent injection as a filler.
  • the polyhydroxyalkanoate microspheres prepared by the invention have good absorbability and injectability.
  • PHA microspheres have excellent biocompatibility and will not cause rejection, and can achieve long-term filling effects.
  • the preparation method provided by the invention has the following beneficial effects: 1 High yield of microspheres: the yield of microspheres is above 70%. 2 Good dispersibility: the PHA microspheres prepared by the present invention can be dispersed in water after proper mixing. It does not deposit on the bottom of the water, but is evenly dispersed in the water body to form a uniform dispersion. After the above dispersion was air-dried, the morphology of the microspheres was observed. The results showed that the microspheres were complete and independent of each other, without agglomeration. All or most of the microsphere particles had a particle size below 60 ⁇ m, which could meet the injection requirements.
  • Fig. 1 is the optical micrograph of embodiment 1 gained injectable PHBHHx microsphere
  • Fig. 2 is the optical micrograph of embodiment 2 gained injectable PHBVHHx microspheres
  • Fig. 3 is the optical micrograph of embodiment 3 gained injectable PHB microspheres
  • Fig. 4 is the optical micrograph of embodiment 4 gained injectable PHBV microspheres
  • Fig. 5 is the optical micrograph of the obtained injectable P34HB microsphere of embodiment 5;
  • FIG. 6 is a schematic diagram of the dispersion stability of the microspheres obtained in Examples 1-5 and Comparative Examples 1-5.
  • the invention provides a preparation method of injectable polyhydroxyalkanoate microspheres, comprising the following steps:
  • steps a) and b) are not limited in sequence.
  • the PHA material is dissolved in an organic solvent to obtain an oil phase
  • the hyaluronic acid aqueous solution is used as the water phase
  • the water phase solution is stirred
  • the oil phase is slowly added to the water phase
  • the organic solvent is continuously stirred. Volatilization, followed by solid-liquid separation and drying, to prepare PHA microspheres.
  • the above method of the present invention can improve the dispersibility of the PHA microspheres, and the obtained microspheres can be quickly dispersed in water, the microspheres are complete in shape, independent of each other, and have no agglomeration phenomenon, thereby avoiding clogging of the needle and facilitating subsequent injection as a filler.
  • the polyhydroxyalkanoate microspheres prepared by the invention have good absorbability and injectability.
  • PHA microspheres have excellent biocompatibility and will not cause rejection, and can achieve long-term filling effects.
  • the type of the PHA material i.e., polyhydroxyalkanoate
  • the type of the PHA material is not particularly limited, it can be a conventional five-generation PHA commercial product in the field, specifically natural or non-natural polyhydroxyalkanoate; more specifically PHB (i.e. 3-hydroxybutyrate), PHBV (i.e. copolymer of hydroxybutyrate and hydroxyvalerate), PHBHHx (i.e. P3HB-co-3HHx, copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate ), P34HB (i.e. P3HB-co-4HB, 3-hydroxybutyric acid and 4-hydroxybutyrate copolyester), PHBVHHx (i.e.
  • the molecular weight of the PHA material is preferably 10-100KDa; in some embodiments of the present invention, the molecular weight is 10-30KDa, 20-100KDa, 10-100KDa.
  • the average molecular weight of the PHA material is preferably 15-80KDa, and in some embodiments of the present invention, the average molecular weight is 15KDa, 40KDa, 50KDa or 80KDa.
  • the PHA material is PHBHHx with a molecular weight of 20-100 KDa and an average molecular weight of 50 KDa. In other embodiments of the present invention, the PHA material is PHBVHHx, with a molecular weight of 20-100 KDa and an average molecular weight of 50 KDa. In other embodiments of the present invention, the PHA material is PHB with a molecular weight of 20-100 KDa and an average molecular weight of 80 KDa. In other embodiments of the present invention, the PHA material is PHBV with a molecular weight of 20-100 KDa and an average molecular weight of 40 KDa. In other embodiments of the present invention, the PHA material is P34HB with a molecular weight of 10-30 KDa and an average molecular weight of 15 KDa.
  • the organic solvent is preferably one or more of dichloromethane, chloroform and ethyl acetate.
  • the source of the organic solvent there is no special limitation on the source of the organic solvent, as long as it is a commercially available product.
  • the mass concentration (w/v) of the organic solution formed 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%.
  • the organic solution formed by dissolving the PHA material in the organic solvent is used as the oil phase.
  • the molecular weight of the hyaluronic acid is preferably 5-500KDa; in some embodiments of the present invention, the molecular weight is 5-100KDa, 10-200KDa or 100-500KDa.
  • the average molecular weight of the hyaluronic acid is preferably 50-300 KDa. In some embodiments of the present invention, the average molecular weight is 50KDa or 300KDa.
  • the molecular weight of the hyaluronic acid is 5-100KDa, and the average molecular weight is 50KDa. In some other embodiments of the present invention, the molecular weight of the hyaluronic acid is 10-200KDa, and the average molecular weight is 50KDa. In some other embodiments of the present invention, the molecular weight of the hyaluronic acid is 100-500 KDa, and the average molecular weight is 300 KDa.
  • the water is preferably distilled water or deionized water, more preferably distilled water.
  • the mass concentration (w/v) of the aqueous solution formed by dissolving the hyaluronic acid in water is preferably 0.01% to 1%; in some embodiments of the present invention, the mass concentration is 0.5%, 5% or 10%.
  • the aqueous solution formed by dissolving hyaluronic acid in water is used as the water phase.
  • polyvinyl alcohol (PVA) aqueous solution is usually used as the water phase to prepare PHA microspheres, and the resulting PHA microspheres are easy to agglomerate and stick together in water, and it is difficult to disperse quickly.
  • PVA polyvinyl alcohol
  • the present invention uses hyaluronic acid aqueous solution as the water phase.
  • the dispersibility of the PHA microspheres can be improved, and the obtained microspheres can be quickly dispersed in water, the microspheres are complete in shape, independent of each other, and have no agglomeration phenomenon, thereby avoiding clogging of needles and facilitating subsequent injection as a filler.
  • the polyhydroxyalkanoate microspheres prepared by the invention have good absorbability and injectability.
  • PHA microspheres have excellent biocompatibility and will not cause rejection, and can achieve long-term filling effects.
  • the present invention has no order limitation on the above step a) and step b).
  • step c) under stirring conditions, the oil phase is added dropwise to the water phase, after the dropwise addition, the organic solvent is volatilized by continuous stirring, and then solid-liquid separation and drying are performed to obtain PHA microspheres.
  • the water phase is stirred, and under the stirring condition, the oil phase is slowly added dropwise into the water phase.
  • the stirring is carried out by using a magnetic stirrer.
  • the stirring rate is 100-500 rpm, typically but not limited, the stirring rate may be 100 rpm, 200 rpm, 300 rpm, 400 rpm or 500 rpm.
  • the volume ratio of the oil phase to the water phase is preferably 1: (10-200); in some embodiments of the present invention, the volume ratio is 1:10, 1:20, 1:50, 1:100, 1:150 or 1:200.
  • the stirring process preferably includes: firstly continue stirring to form an emulsion, and then continue stirring to volatilize the organic solvent.
  • the stirring is performed using a magnetic stirrer.
  • the stirring rate is 100-500 rpm; in some embodiments of the present invention, the stirring rate is 400 rpm.
  • the stirring is carried out by using a magnetic stirrer; the stirring rate is 100-500rpm, typically non-limiting, the stirring rate can be 100rpm, 200rpm, 300rpm, 400rpm or 500rpm;
  • the time for continuous stirring is preferably ⁇ 4 hours, more preferably 4-6 hours.
  • the time for continuous stirring (to volatilize the organic solvent) after forming the emulsion is 4 hours, 5 hours or 6 hours.
  • the temperature of the above-mentioned dropping of the oil phase and stirring which can be carried out at room temperature, specifically 10-37°C, preferably 25°C.
  • solid-liquid separation was carried out.
  • the method of the solid-liquid separation in the present invention is not particularly limited, and conventional methods known to those skilled in the art can be used, such as filtration and the like.
  • the drying is preferably firstly frozen and then freeze-dried.
  • the drying temperature is preferably -80 ⁇ -20°C; wherein, the freezing temperature is preferably -80 ⁇ -20°C, and the freeze drying temperature is preferably -60 ⁇ -40°C.
  • the freezing temperature is -80°C to -40°C, and the time is 6 to 12 hours; the freeze-drying temperature is preferably -40°C, and the time is 24h.
  • the present invention also provides a PHA microsphere prepared by the preparation method described in the above technical scheme.
  • the PHA material is dissolved in an organic solvent to obtain an oil phase
  • the hyaluronic acid aqueous solution is used as the water phase
  • the water phase solution is stirred
  • the oil phase is slowly added to the water phase
  • the organic solvent is continuously stirred. Volatilization, followed by solid-liquid separation and drying, to prepare PHA microspheres.
  • the above method of the present invention can improve the dispersibility of the PHA microspheres, and the obtained microspheres can be quickly dispersed in water, the microspheres are complete in shape, independent of each other, and have no agglomeration phenomenon, thereby avoiding clogging of the needle and facilitating subsequent injection as a filler.
  • the polyhydroxyalkanoate microspheres prepared by the invention have good absorbability and injectability.
  • PHA microspheres have excellent biocompatibility and will not cause rejection, and can achieve long-term filling effect.
  • the microsphere yield is above 70%.
  • the PHA microspheres prepared by the present invention can be dispersed in water after proper mixing, and after standing, the microspheres show good dispersion in water, no delamination phenomenon occurs, neither float on the upper layer of water nor deposit at the bottom of the water, and It is uniformly dispersed in water to form a uniform dispersion. After the above dispersion was air-dried, the morphology of the microspheres was observed. The results showed that the microspheres were complete and independent of each other, without agglomeration. All or most of the microsphere particles had a particle size below 60 ⁇ m, which could meet the injection requirements.
  • Dissolve 1 g of PHBHHx (molecular weight 20-100 KDa, average molecular weight 50 KDa) in 20 mL of dichloromethane to prepare an organic solution with a final concentration of 5% (w/v), which is the oil phase.
  • Dissolve hyaluronic acid (molecular weight 5-100KDa, average molecular weight 50KDa) in distilled water to form an aqueous solution with a final concentration of 0.5% (w/v), which is the aqueous phase.
  • a magnetic stirrer was used to stir the water phase at a speed of 400 rpm, and at the same time, the oil phase was slowly added dropwise to the water phase under stirring, and the volume ratio of the two solutions was 1:50.
  • the yield of injectable PHBHHx microspheres was calculated to be 93%. Dissolve the freeze-dried PHBHHx microspheres in water, and mix them properly to disperse them in water. After the water is air-dried, observe them with an optical microscope. The results are shown in Figure 1, which is the injectable PHBHHx microspheres obtained in Example 1 From the optical microscope image, it can be seen that the obtained microspheres have good dispersion and are spherical or ellipsoidal. Using a particle size analyzer, the particle size is 1-50 ⁇ m.
  • Dissolve 1 g of PHBVHHx (molecular weight 20-100 KDa, average molecular weight 50 KDa) in 20 mL of dichloromethane to prepare an organic solution with a final concentration of 5% (w/v), which is the oil phase.
  • Dissolve hyaluronic acid (molecular weight 10-200KDa, average molecular weight 50KDa) in distilled water to form an aqueous solution with a final concentration of 0.5% (w/v), which is the aqueous phase.
  • a magnetic stirrer was used to stir the water phase at a speed of 400 rpm, and at the same time, the oil phase was slowly added dropwise to the water phase under stirring, and the volume ratio of the two solutions was 1:50.
  • the yield of the obtained injectable PHBVHHx microspheres was 91%. Dissolve the freeze-dried PHBVHHx microspheres in water, and mix them properly to disperse them in water. After the water is air-dried, observe them with an optical microscope. The results are shown in Figure 2, which is the injectable PHBVHHx microspheres obtained in Example 2 From the optical microscope image, it can be seen that the obtained microspheres have good dispersion and are spherical or ellipsoidal. Using a particle size analyzer, the particle size is 4-50 ⁇ m.
  • Dissolve hyaluronic acid molecular weight 100-500KDa, average molecular weight 300KDa
  • a magnetic stirrer was used to stir the water phase at a speed of 400 rpm, and at the same time, the oil phase was slowly added dropwise to the water phase under stirring, and the volume ratio of the two solutions was 1:50.
  • the yield of the obtained injectable PHB microspheres was 92%. Dissolve the freeze-dried PHB microspheres in water, and mix them properly to disperse them in water. After the water is air-dried, observe them with an optical microscope. The results are shown in Figure 3, which is the injectable PHB microspheres obtained in Example 3. From the optical microscope image, it can be seen that the obtained microspheres have good dispersion and are spherical or ellipsoidal. Detected by a particle size analyzer, the particle size is 2-55 ⁇ m.
  • a magnetic stirrer was used to stir the water phase at a speed of 400 rpm, and at the same time, the oil phase was slowly added dropwise to the water phase under stirring, and the volume ratio of the two solutions was 1:50.
  • the yield of the obtained injectable PHBV microspheres was 90%. Dissolve the freeze-dried PHBV microspheres in water, and mix them properly to disperse them in water. After the water is air-dried, observe them with an optical microscope. The results are shown in Figure 4, which is the injectable PHBV microspheres obtained in Example 4. From the optical microscope image, it can be seen that the obtained microspheres have good dispersion and are spherical or ellipsoidal. Using a particle size analyzer, the particle size is 1-52 ⁇ m.
  • a magnetic stirrer was used to stir the water phase at a speed of 400 rpm, and at the same time, the oil phase was slowly added dropwise to the water phase under stirring, and the volume ratio of the two solutions was 1:50.
  • the yield of the obtained injectable P34HB microspheres was 90%. Dissolve the lyophilized P34HB microspheres in water, and mix them properly to disperse them in water. After the water is air-dried, observe them with an optical microscope. The results are shown in Figure 5, which shows the injectable P34HB microspheres obtained in Example 5. From the optical microscope image, it can be seen that the obtained microspheres have good dispersion and are spherical or ellipsoidal. Using a particle size analyzer, the particle size is 5-100 ⁇ m, and 68% of the particles have a particle size of 1-60 ⁇ m, that is, most of the particles have a particle size below 60 ⁇ m.
  • Embodiment 6 the concentration of oil phase is different
  • Dissolve 0.5g, 1g, and 2g of PHBHHx (molecular weight: 20 to 100KDa, average molecular weight: 50KDa) in 20mL of dichloromethane to prepare 2.5% (w/v), 5% (w/v), and 10% ( w/v)
  • the organic solution of the final concentration is the oil phase.
  • Dissolve hyaluronic acid (molecular weight 5-100KDa, average molecular weight 50KDa) in distilled water to form an aqueous solution with a final concentration of 0.5% (w/v), which is the aqueous phase.
  • a magnetic stirrer was used to stir the water phase at a speed of 400 rpm.
  • the oil phase was slowly added dropwise to the water phase under stirring, and the volume ratio of the two solutions was 1:50.
  • the resulting solution was first frozen (-80°C for 6 h), and then freeze-dried (-40°C for 24 h) to remove water to obtain injectable PHBHHx microspheres.
  • the yields of the obtained injectable PHBHHx microspheres were 90%, 93%, 89%, respectively.
  • the freeze-dried PHBHHx microspheres were dissolved in water and dispersed in water after proper mixing. After the water was air-dried, observed with an optical microscope, the results showed that the obtained microspheres had good dispersibility and were spherical or ellipsoidal. Detected by a particle size analyzer, the particle sizes are 0.4-20 ⁇ m, 1-50 ⁇ m, and 1-80 ⁇ m (83% of the particles have a particle size of 1-60 ⁇ m).
  • Embodiment 7 Oil phase and water phase volume ratio are different
  • Dissolve 1 g of PHBHHx (molecular weight 20-100 KDa, average molecular weight 50 KDa) in 20 mL of dichloromethane to prepare an organic solution with a final concentration of 5% (w/v), which is the oil phase.
  • Dissolve hyaluronic acid (molecular weight 5-100KDa, average molecular weight 50KDa) in distilled water to form an aqueous solution with a final concentration of 0.5% (w/v), which is the aqueous phase.
  • a magnetic stirrer was used to stir the water phase at a speed of 400 rpm. At the same time, the oil phase was slowly added dropwise to the water phase under stirring.
  • the volume ratios of the two solutions were 1:10, 1:50, and 1:200, respectively.
  • the resulting solution was first frozen (-80°C for 6 h), and then freeze-dried (-40°C for 24 h) to remove water to obtain injectable PHBHHx microspheres.
  • the yields of the obtained injectable PHBHHx microspheres were 71%, 93%, 94%, respectively.
  • the freeze-dried PHBHHx microspheres were dissolved in water and dispersed in water after proper mixing. After the water was air-dried, observed with an optical microscope, the results showed that the obtained microspheres had good dispersibility and were spherical or ellipsoidal.
  • the particle sizes are 1-110 ⁇ m (58% of the particles have a particle size of 1-60 ⁇ m), 1-50 ⁇ m, and 1-50 ⁇ m.
  • Embodiment 8 Stirring mode and rotating speed are different
  • Dissolve 1 g of PHBHHx (molecular weight 20-100 KDa, average molecular weight 50 KDa) in 20 mL of dichloromethane to prepare an organic solution with a final concentration of 5% (w/v), which is the oil phase.
  • Dissolve hyaluronic acid (molecular weight 5-100KDa, average molecular weight 50KDa) in distilled water to form an aqueous solution with a final concentration of 0.5% (w/v), which is the aqueous phase.
  • a magnetic stirrer was used to stir the water phase at a speed of 400 rpm, and at the same time, the oil phase was slowly added dropwise to the water phase under stirring, and the volume ratio of the two solutions was 1:50.
  • the yields of the obtained injectable PHBHHx microspheres were 77%, 93%, 89%, 99%, respectively.
  • the freeze-dried PHBHHx microspheres were dissolved in water and dispersed in water after proper mixing. After the water was air-dried, observed with an optical microscope, the results showed that the obtained microspheres had good dispersibility and were spherical or ellipsoidal.
  • the particle sizes are 5-60 ⁇ m, 1-50 ⁇ m, 1-45 ⁇ m, and 0.1-10 ⁇ m (the particle size is basically below 10 ⁇ m, and only 5% of the particles have a particle size of 1-60 ⁇ m.
  • the overall particle size distribution belongs to the level of 0.1 to 10 ⁇ m). This proves that compared with the high-speed homogenization stirring method, the present invention uses a magnetic stirrer to stir at an appropriate speed (100-500 rpm), which is beneficial to obtain a microsphere particle size more suitable for the human body.
  • Embodiment 9 The types of oil phase organic solvents are different
  • Dissolve 1 g of PHBHHx (molecular weight 10-20 KDa, average molecular weight 50 KDa) in 20 mL of dichloromethane, chloroform, and ethyl acetate to form an organic solution with a final concentration of 5% (w/v), which is the oil phase.
  • Dissolve hyaluronic acid (molecular weight 5-100KDa, average molecular weight 50KDa) in distilled water to form an aqueous solution with a final concentration of 0.5% (w/v), which is the aqueous phase.
  • a magnetic stirrer was used to stir the water phase at a speed of 400 rpm, and at the same time, the oil phase was slowly added dropwise to the water phase under stirring, and the volume ratio of the two solutions was 1:50. Continue stirring at a speed of 400 rpm with a magnetic stirrer to form an emulsion; then continue stirring at this speed for 5 h to remove the organic solvent. Afterwards, the resulting solution was first frozen (-80°C for 6 h), and then freeze-dried (-40°C for 24 h) to remove water to obtain injectable PHBHHx microspheres.
  • the yields of the obtained injectable PHBHHx microspheres were 93%, 92%, 89%, respectively.
  • the freeze-dried PHBHHx microspheres were dissolved in water and dispersed in water after proper mixing. After the water was air-dried, observed with an optical microscope, the results showed that the obtained microspheres had good dispersibility and were spherical or ellipsoidal.
  • the particle sizes were 1-52 ⁇ m, 1-50 ⁇ m, and 1-55 ⁇ m, respectively.
  • Embodiment 10 the concentration of aqueous phase is different
  • Dissolve 1 g of PHBHHx (molecular weight 20-100 KDa, average molecular weight 50 KDa) in 20 mL of dichloromethane to prepare an organic solution with a final concentration of 5% (w/v), which is the oil phase.
  • Dissolve hyaluronic acid (molecular weight 10-200KDa, average molecular weight 50KDa) in distilled water to prepare aqueous solutions with final concentrations of 0.01% (w/v), 0.5% (w/v), and 1% (w/v) , is the water phase.
  • a magnetic stirrer was used to stir the water phase at a speed of 400 rpm, and at the same time, the oil phase was slowly added dropwise to the water phase under stirring, and the volume ratio of the two solutions was 1:50.
  • the resulting solution was first frozen (-80°C for 6 h), and then freeze-dried (-40°C for 24 h) to remove water to obtain injectable PHBHHx microspheres.
  • the yields of the obtained injectable PHBHHx microspheres were 80%, 93%, 87%, respectively.
  • the freeze-dried PHBHHx microspheres were dissolved in water, mixed properly and dispersed in water. After the water was air-dried, observed with an optical microscope, the results showed that the obtained microspheres had good dispersibility and were spherical or ellipsoidal.
  • the particle sizes are 3-50 ⁇ m, 1-50 ⁇ m, and 0.7-50 ⁇ m, respectively.
  • PHBHHx molecular weight 20-100 KDa, average molecular weight 50 KDa
  • dichloromethane aqueous solution with a final concentration of 0.5% (w/v)
  • PVA polyvinyl alcohol
  • a magnetic stirrer was used to stir the water phase at a speed of 400 rpm, and at the same time, the oil phase was slowly added dropwise to the water phase under stirring, and the volume ratio of the two solutions was 1:50.
  • PVA polyvinyl alcohol
  • a magnetic stirrer was used to stir the water phase at a speed of 400 rpm, and at the same time, the oil phase was slowly added dropwise to the water phase under stirring, and the volume ratio of the two solutions was 1:50.
  • Embodiment 11 Test the stability of microsphere
  • Figure 6 is a schematic diagram of the dispersion stability of the microspheres obtained in Examples 1 to 5 and Comparative Examples 1 to 5 (10 containers from left to right correspond to Examples 1 to 5 and Comparative Examples 1 to 5 respectively). 5 samples).
  • the PHBHHx microspheres, PHBVHHx microspheres, PHB microspheres, PHBV microspheres and P34HB microspheres in Examples 1 to 5 all have good dispersibility, no stratification occurs, and the microspheres are evenly dispersed in the water body ;
  • PHBHHx microspheres, PHBVHHx microspheres, PHB microspheres, PHBV microspheres and P34HB microspheres in Comparative Examples 1 to 5 do not possess water dispersibility, and serious delamination phenomenon occurs, and the microspheres float in the water upper layer.
  • the present invention uses the hyaluronic acid aqueous solution as the water phase material, which obviously improves the dispersibility and stability of the microspheres.
  • a disposable 1mL syringe needle with an inner diameter of 0.5mm and a length of 19.7mm (that is, the commonly used 1mL syringe tip) is used as the injection rate detection device, and 1mL of microsphere dispersion (the dry weight of the microsphere is 0.1g, recorded M0) is the experimental sample.
  • 1mL of microsphere dispersion the dry weight of the microsphere is 0.1g, recorded M0
  • a disposable 1mL syringe needle with an inner diameter of 0.5mm and a length of 19.7mm (that is, the commonly used 1mL syringe tip) is used as the injection rate detection device, and 1mL of microsphere dispersion (the dry weight of the microsphere is 0.1g, recorded M0) is the experimental sample.
  • 1mL of microsphere dispersion the dry weight of the microsphere is 0.1g, recorded M0
  • the preparation method provided by the present invention has the following beneficial effects: 1 High yield of microspheres: the yield of microspheres is above 70%. 2 Good dispersibility: the PHA microspheres prepared by the present invention can be dispersed in water after proper mixing. It does not deposit on the bottom of the water, but is evenly dispersed in the water body to form a uniform dispersion. After the above dispersion was air-dried, the morphology of the microspheres was observed. The results showed that the microspheres were complete and independent of each other, without agglomeration. All or most of the microsphere particles had a particle size below 60 ⁇ m, which could meet the injection requirements.

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Abstract

一种可注射聚羟基脂肪酸酯（PHA）微球的制备方法，包括a）将PHA材料溶于有机溶剂中，得到油相；b）将透明质酸溶于水中，得到水相；c）在搅拌条件下，将所述油相滴加到所述水相中，滴加完后，持续搅拌使有机溶剂挥发，之后，进行固液分离和干燥，得到PHA微球；所述步骤a）和步骤b）没有顺序限制。所述方法得到的PHA微球在水中能够快速分散，微球形态完整、彼此独立，没有团聚现象，从而避免堵塞针头，便于后续作为填充剂进行注射。

Description

一种可注射聚羟基脂肪酸酯微球及其制备方法
优先权信息
本发明请求于2021年10月12日向中国国家知识产权局提交的、申请号为202111187296.9、发明名称为“一种可注射聚羟基脂肪酸酯微球及其制备方法”的中国专利申请的优先权，并且其全部内容通过引用结合在本发明中。
技术领域
本发明涉及生物医药技术领域，特别涉及一种可注射聚羟基脂肪酸酯微球及其制备方法。
背景技术
随着人类年龄增长或受某些疾病影响，人体中肌肉和胶原蛋白组织会产生不同程度的功能性退化，造成皮肤凹陷、胃液反流等问题，因此人们发明了多种填充剂，以此来填补凹陷皮肤或通过异物刺激肌肉和胶原蛋白的再生长，如玻尿酸、牛胶原蛋白等。但这些物质作为填充剂所起到的填充效果维持时间较短，需要频繁在注射保持其填充效果。为达到长期填充效果，人们又尝试使用生物的不可降解的材料制作成微球作为填充剂，如聚乙烯醇(PVA)，聚甲基丙基酸甲酯(PMMA)等。尽管这些填充剂的填充效果维持时间明显增长，但这些材料在体内留存过久，会释放有害物质，并因此引发一系列的副反应，危害人体健康。
近年来，生物可降解的高分子材料走入人们的视野，该类型材料对人体无毒无排斥反应，并可以随着人体代谢逐渐降解后排出体外，且可以通过调节材料的分子量等参数，使降解时间从一周到长达数年不等。
聚羟基脂肪酸酯，简称PHA，是一种天然的高分子生物材料，由微生物合成的一种细胞内聚酯。由于PHA具有良好的生物相容性能、生物可降解性，是当下最为理想的生物医学材料之一。PHA在体内与细胞具有良好的细胞相容性，细胞可以在此种支架上良好生长，且该种支架可以降解为CO 2和H 2O。为了便于注射，人们通常将其制为微球，可以通过针头进行注射。因为人体吞噬细胞的存在，微球直径通常在20微米以上，但微球过大又会堵塞针头，甚 至引起皮肤破裂。因此用于注射的微球大小通常为60微米以下。
现有技术中，在将PHA材料制备可注射微球时，通常使用聚乙烯醇(PVA)水溶液作为水相，采用乳化法或微流控法进行微球的制作。但是，以此种工艺生产出来的微球，十分容易团聚、粘连，形成较大的块状物质，给后续注射带来不便，极大的阻碍了生物可降解材料微球作为填充剂的应用。因此，急需一种新型的微球制备方案，解决微球易团聚、粘连的问题。
发明内容
有鉴于此，本发明的目的在于提供一种可注射聚羟基脂肪酸酯微球及其制备方法。本发明提供的制备方法，能够有效提高聚羟基脂肪酸酯微球的分散性，克服微球团聚、粘连的问题；同时能够有效提高聚羟基脂肪酸酯微球的可吸收性和可注射性。
本发明提供了一种可注射聚羟基脂肪酸酯微球的制备方法，包括以下步骤：
a)将PHA材料溶于有机溶剂中，得到油相；
b)将透明质酸溶于水中，得到水相；
c)在搅拌条件下，将所述油相滴加到所述水相中，滴加完后，持续搅拌，使有机溶剂挥发，之后，进行固液分离和干燥，得到PHA微球；
所述步骤a)和步骤b)没有顺序限制。
优选的，所述透明质酸的分子量为5～500KDa。
优选的，所述水相中透明质酸的质量浓度为0.01％～1％(w/v)。
优选的，所述步骤c)中，所述油相与水相的体积比为1∶(10～200)。
优选的，所述油相中PHA材料的质量浓度为2.5％～10％(w/v)；
所述有机溶剂选自二氯甲烷、氯仿和乙酸乙酯中的一种或几种。
优选的，所述PHA材料的分子量为10～100KDa。
优选的，所述PHA材料为天然或非天然聚羟基脂肪酸酯；
所述天然或非天然聚羟基脂肪酸酯包括但不限于PHB、PHBV、PHBHHx、P34HB、PHBVHHx、PHHx和PHO中的一种或几种。
优选的，所述搅拌的速率为100～500rpm。
优选的，所述搅拌为利用磁力搅拌器进行搅拌；滴加完油相后，持续搅拌的时间为≥4h。
本发明还提供了一种上述技术方案中所述的制备方法制得的可注射聚羟基脂肪酸酯微球。
本发明提供的制备方法中，将PHA材料溶于有机溶剂制得油相，将透明质酸水溶液作为水相，搅拌水相溶液，并将油相缓慢加入水相中，再持续搅拌使有机溶剂挥发，随后进行固液分离和干燥，从而制得PHA微球。本发明的上述方法，能够提高PHA微球的分散性，所得微球在水中能够快速分散，微球形态完整、彼此独立，没有团聚现象，从而避免堵塞针头，便于后续作为填充剂进行注射。同时，本发明制得的聚羟基脂肪酸酯微球具有较好的可吸收性和可注射性。而且PHA微球具有极佳的生物相容性不会引起排异反应，可以达到长效填充效果。
实验结果表明，本发明提供的制备方法具有以下有益效果：①微球收率高：微球收率在70％以上。②分散性好：本发明制得的PHA微球适当混合即可分散于水中，静置后，微球在水中表现出良好的分散性，未出现分层现象，既不浮在水上层、也不沉积水底，而是均匀分散在水体中，形成均匀分散液。将上述分散液风干后，观察微球形态，结果显示，微球形态完整、彼此独立，没有团聚现象，微球颗粒中全部或大部分颗粒粒径在60μm以下，能够满足注射需求。③可吸取性好：利用注射器抽吸上述分散液，微球的吸取率达到85％以上，表现出优异的可吸取性。④可注射性好：挤推注射器并收集注射出的分散液，计算微球的注射率，结果表明，微球注射率达到88％以上，表现出优异的可注射性。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据提供的附图获得其他的附图。
图1为实施例1所得可注射PHBHHx微球的光学显微镜图；
图2为实施例2所得可注射PHBVHHx微球的光学显微镜图；
图3为实施例3所得可注射PHB微球的光学显微镜图；
图4为实施例4所得可注射PHBV微球的光学显微镜图；
图5为实施例5所得可注射P34HB微球的光学显微镜图；
图6为实施例1～5及对比例1～5所得微球的分散稳定性示意图。
具体实施方式
本发明提供了一种可注射聚羟基脂肪酸酯微球的制备方法，包括以下步骤：
a)将PHA材料溶于有机溶剂中，得到油相；
b)将透明质酸溶于水中，得到水相；
c)在搅拌条件下，将所述油相滴加到所述水相中，滴加完后，持续搅拌使有机溶剂挥发，之后，进行固液分离和干燥，得到PHA微球；
所述步骤a)和步骤b)没有顺序限制。
本发明提供的制备方法中，将PHA材料溶于有机溶剂制得油相，将透明质酸水溶液作为水相，搅拌水相溶液，并将油相缓慢加入水相中，再持续搅拌使有机溶剂挥发，随后进行固液分离和干燥，从而制得PHA微球。本发明的上述方法，能够提高PHA微球的分散性，所得微球在水中能够快速分散，微球形态完整、彼此独立，没有团聚现象，从而避免堵塞针头，便于后续作为填充剂进行注射。同时，本发明制得的聚羟基脂肪酸酯微球具有较好的可吸收性和可注射性。而且PHA微球具有极佳的生物相容性不会引起排异反应，可以达到长效填充效果。
关于步骤a)：将PHA材料溶于有机溶剂中，得到油相。
本发明中，所述PHA材料(即聚羟基脂肪酸酯)的种类没有特殊限制，为本领域常规的五代PHA商业品即可，具体为天然或非天然聚羟基脂肪酸酯；更具体为PHB(即3-羟基丁酸酯)、PHBV(即羟基丁酸酯和羟基戊酸酯的共聚物)、PHBHHx(即P3HB-co-3HHx，3-羟基丁酸和3-羟基己酸共聚酯)、P34HB(即P3HB-co-4HB，3-羟基丁酸和4-羟基丁酸酯共聚酯)、PHBVHHx(即3-羟基丁酸、3-羟基己酸和3-羟基戊酸的共聚物)、PHHx(即聚羟基己酸酯)和PHO(即聚羟基辛酸酯)中的一种或几种。本发明中，所述PHA材料的分子量优选为10～100KDa；在本发明的一些实施例中，所述分子量为10～30KDa、20～100KDa、10～100KDa。所述PHA材料的分子量均值优选为15～80KDa，在本发明的一些实施例中，所述分子量均值为15KDa、40KDa、50KDa或80KDa。
在本发明的一些实施例中，所述PHA材料为PHBHHx，分子量20～100KDa，分子量均值为50KDa。在本发明的另一些实施例中，所述PHA材料为PHBVHHx，分子量20～100KDa，分子量均值为50KDa。在本发明的另一些实施例中，所述PHA材料为PHB，分子量20～100KDa，分子量均值为80KDa。在本发明的另一些实施例中，所述PHA材料为PHBV，分子量20～100KDa，分子量均值为40KDa。在本发明的另一些实施例中，所述PHA材料为P34HB， 分子量10～30KDa，分子量均值为15KDa。
本发明中，所述有机溶剂优选为二氯甲烷、氯仿和乙酸乙酯中的一种或几种。本发明对所述有机溶剂的来源没有特殊限制，为市售商业品即可。
本发明中，所述PHA材料溶于有机溶剂中形成的有机溶液的质量浓度(w/v)优选为2.5％～10％；在本发明的一些实施例中，所述质量浓度为2.5％、5％或10％。本发明将PHA材料溶于有机溶剂中形成的有机溶液，作为油相。
关于步骤b)：将透明质酸溶于水中，得到水相。
本发明中，所述透明质酸(即HA)的分子量优选为5～500KDa；在本发明的一些实施例中，所述分子量为5～100KDa、10～200KDa或100～500KDa。本发明中，所述透明质酸的分子量均值优选为50～300KDa。在本发明的一些实施例中，所述分子量均值为50KDa或300KDa。
在本发明的一些实施例中，所述透明质酸的分子量5～100KDa，分子量均值为50KDa。在本发明的另一些实施例中，所述透明质酸的分子量10～200KDa，分子量均值为50KDa。在本发明的另一些实施例中，所述透明质酸的分子量100～500KDa，分子量均值为300KDa。
本发明中，所述水优选为蒸馏水或去离子水，更优选为蒸馏水。
本发明中，所述透明质酸溶于水中形成的水溶液的质量浓度(w/v)优选为0.01％～1％；在本发明的一些实施例中，所述质量浓度为0.5％、5％或10％。本发明将透明质酸溶于水中形成的水溶液，作为水相。现有技术中，通常使用聚乙烯醇(PVA)水溶液作为水相来制备PHA微球，所得PHA微球在水中容易团聚、粘连，难以快速分散，而本发明采用透明质酸水溶液作为水相，能够提高PHA微球的分散性，所得微球在水中能够快速分散，微球形态完整、彼此独立，没有团聚现象，从而避免堵塞针头，便于后续作为填充剂进行注射。同时，本发明制得的聚羟基脂肪酸酯微球具有较好的可吸收性和可注射性。而且PHA微球具有极佳的生物相容性不会引起排异反应，可以达到长效填充效果。
本发明对上述步骤a)和步骤b)没有顺序限制。
关于步骤c)：在搅拌条件下，将所述油相滴加到所述水相中，滴加完后，持续搅拌使有机溶剂挥发，之后，进行固液分离和干燥，得到PHA微球。
本发明在得到油相和水相后，对水相进行搅拌，在该搅拌条件下，将油相缓慢滴加到水相中。其中，所述搅拌为利用磁力搅拌器进行搅拌。所述搅拌的速率为100～500rpm，典型非限制性的，搅拌速率可为100rpm、200rpm、300rpm、 400rpm或500rpm。所述油相与水相的体积比优选为1∶(10～200)；在本发明的一些实施例中，所述体积比为1∶10、1∶20、1∶50、1∶100、1∶150或1∶200。
将油相全部滴加完毕后，搅拌过程优选包括：先继续搅拌形成乳化液，然后再持续搅拌使有机溶剂挥发。在所述继续搅拌的过程中：所述搅拌为利用磁力搅拌器进行搅拌。所述搅拌的速率为100～500rpm；在本发明的一些实施例中，所述搅拌的速率为400rpm。在所述持续搅拌的过程中：所述搅拌为利用磁力搅拌器进行搅拌；所述搅拌的速率为100～500rpm，典型非限制性的，搅拌速率可为100rpm、200rpm、300rpm、400rpm或500rpm；所述持续搅拌的时间优选为≥4h，更优选为4～6h，在本发明的一些实施例中，形成乳化液后持续搅拌(使有机溶剂挥发)的时间为4h、5h或6h。本发明对上述滴加油相和搅拌的温度没有特殊限制，在室温下进行即可，具体可为10～37℃，优选为25℃。
经持续搅拌使溶剂完全挥发后，进行固液分离。本发明对所述固液分离的方式没有特殊限制，按照本领域技术人员熟知的常规方式即可，如过滤等。
经固液分离后，进行干燥除去水分。本发明中，所述干燥优选为先冷冻，再冷冻干燥。本发明中，所述干燥的温度优选为-80～-20℃；其中，所述冷冻的温度优选为-80～-20℃，所述冷冻干燥的温度优选为-60～-40℃。在本发明的一些实施例中，所述冷冻的温度为-80～-40℃，时间为6～12h；所述冷冻干燥的温度优选为-40℃，时间为24h。经上述干燥处理后，得到PHA微球。
本发明还提供了一种上述技术方案中所述的制备方法制得的PHA微球。
本发明提供的制备方法中，将PHA材料溶于有机溶剂制得油相，将透明质酸水溶液作为水相，搅拌水相溶液，并将油相缓慢加入水相中，再持续搅拌使有机溶剂挥发，随后进行固液分离和干燥，从而制得PHA微球。本发明的上述方法，能够提高PHA微球的分散性，所得微球在水中能够快速分散，微球形态完整、彼此独立，没有团聚现象，从而避免堵塞针头，便于后续作为填充剂进行注射。同时，本发明制得的聚羟基脂肪酸酯微球具有较好的可吸收性和可注射性。而且PHA微球具有极佳的生物相容性不会引起排异反应，可以达到长效填充效果。
实验结果表明：
(1)本发明提供的制备方法，微球收率在70％以上。本发明制得的PHA微球适当混合即可分散于水中，静置后，微球在水中表现出良好的分散性，未出现分层现象，既不浮在水上层、也不沉积水底，而是均匀分散在水体中，形 成均匀分散液。将上述分散液风干后，观察微球形态，结果显示，微球形态完整、彼此独立，没有团聚现象，微球颗粒中全部或大部分颗粒粒径在60μm以下，能够满足注射需求。
(2)以一次性1mL注射器套用针头内径为0.5mm、长度为19.7mm的针头(即常用的1mL注射器枪头)为吸取率检测设备，以1mL的微球分散液(微球干重为0.1g，记为M0)为实验样品。一次性吸取所有微球分散液，能被吸取到注射器管内的微球被取出并冷冻干燥，质量为M1，定义吸取率为(M1/M0)×100％。利用上述注射器抽吸上述微球分散液，微球的吸取率达到85％以上，表现出优异的可吸取性。
(3)以一次性1mL注射器套用针头内径为0.5mm、长度为19.7mm的针头(即常用的1mL注射器枪头)为注射率检测设备，以1mL的微球分散液(微球干重为0.1g，记为M0)为实验样品。将1mL的微球分散液从注射器后方注入注射器管内，一次性注射出所有微球分散液，能通过针头被注射到注射器管外的微球被收集并冷冻干燥，质量为M2，定义注射率为(M2/M0)×100％。挤推注射器并收集注射出的分散液，计算微球的注射率，结果表明，微球注射率达到88％以上，表现出优异的可注射性。
为了进一步理解本发明，下面结合实施例对本发明优选实施方案进行描述，但是应当理解，这些描述只是为进一步说明本发明的特征和优点，而不是对本发明权利要求的限制。
实施例1
将1g的PHBHHx(分子量20～100KDa，分子量均值为50KDa)溶于20mL的二氯甲烷，配成5％(w/v)终浓度的有机溶液，为油相。将透明质酸(分子量5～100KDa，分子量均值为50KDa)溶于蒸馏水，配成终浓度为0.5％(w/v)的水溶液，为水相。利用磁力搅拌器以400rpm的速度搅拌水相，同时，将油相在搅拌下缓慢滴加到水相中，两种溶液的体积比为1∶50。利用磁力搅拌器以400rpm的速度继续搅拌，形成乳化液；再以该速度持续搅拌5h除去有机溶剂。之后，将所得溶液先冷冻(-80℃冷冻6h)，再冷冻干燥(-40℃冷冻干燥24h)除去水分，得到可注射PHBHHx微球。
根据投入的PHBHHx材料量及所得PHBHHx微球量，计算得到可注射PHBHHx微球的收率为93％。将冻干处理后的PHBHHx微球溶于水，适当混合即可分散于水中，待水风干后，利用光学显微镜观察，结果如图1所示，图1为实施例1所得可注射PHBHHx微球的光学显微镜图，可以看出，所得微 球具有良好的分散性，为球型或椭球形。利用粒径仪检测，其粒径为1～50μm。
实施例2
将1g的PHBVHHx(分子量20～100KDa，分子量均值为50KDa)溶于20mL的二氯甲烷，配成5％(w/v)终浓度的有机溶液，为油相。将透明质酸(分子量10～200KDa，分子量均值为50KDa)溶于蒸馏水，配成终浓度为0.5％(w/v)的水溶液，为水相。利用磁力搅拌器以400rpm的速度搅拌水相，同时，将油相在搅拌下缓慢滴加到水相中，两种溶液的体积比为1∶50。利用磁力搅拌器以400rpm的速度继续搅拌，形成乳化液；再以该速度持续搅拌4h除去有机溶剂。之后，将所得溶液先冷冻(-40℃冷冻12h)，再冷冻干燥(-40℃冷冻干燥24h)除去水分，得到可注射PHBVHHx微球。
所得可注射PHBVHHx微球的收率为91％。将冻干处理后的PHBVHHx微球溶于水，适当混合即可分散于水中，待水风干后，利用光学显微镜观察，结果如图2所示，图2为实施例2所得可注射PHBVHHx微球的光学显微镜图，可以看出，所得微球具有良好的分散性，为球型或椭球形。利用粒径仪检测，其粒径为4～50μm。
实施例3
将1g的PHB(分子量20～100KDa，分子量均值为80KDa)溶于20mL的二氯甲烷，配成5％(w/v)终浓度的有机溶液，为油相。将透明质酸(分子量100～500KDa，分子量均值为300KDa)溶于蒸馏水，配成终浓度为0.5％(w/v)的水溶液，为水相。利用磁力搅拌器以400rpm的速度搅拌水相，同时，将油相在搅拌下缓慢滴加到水相中，两种溶液的体积比为1∶50。利用磁力搅拌器以400rpm的速度继续搅拌，形成乳化液；再以该速度持续搅拌6h除去有机溶剂。之后，将所得溶液先冷冻(-40℃冷冻12h)，再冷冻干燥(-40℃冷冻干燥24h)除去水分，得到可注射PHB微球。
所得可注射PHB微球的收率为92％。将冻干处理后的PHB微球溶于水，适当混合即可分散于水中，待水风干后，利用光学显微镜观察，结果如图3所示，图3为实施例3所得可注射PHB微球的光学显微镜图，可以看出，所得微球具有良好的分散性，为球型或椭球形。利用粒径仪检测，其粒径为2～55μm。
实施例4
将1g的PHBV(分子量20～100KDa，分子量均值为40KDa)溶于20mL的二氯甲烷，配成5％(w/v)终浓度的有机溶液，为油相。将透明质酸(分子量 5～100KDa，分子量均值为50KDa)溶于蒸馏水，配成终浓度为0.5％(w/v)的水溶液，为水相。利用磁力搅拌器以400rpm的速度搅拌水相，同时，将油相在搅拌下缓慢滴加到水相中，两种溶液的体积比为1∶50。利用磁力搅拌器以400rpm的速度继续搅拌，形成乳化液；再以该速度持续搅拌5h除去有机溶剂。之后，将所得溶液先冷冻(-40℃冷冻12h)，再冷冻干燥(-40℃冷冻干燥24h)除去水分，得到可注射PHBV微球。
所得可注射PHBV微球的收率为90％。将冻干处理后的PHBV微球溶于水，适当混合即可分散于水中，待水风干后，利用光学显微镜观察，结果如图4所示，图4为实施例4所得可注射PHBV微球的光学显微镜图，可以看出，所得微球具有良好的分散性，为球型或椭球形。利用粒径仪检测，其粒径为1～52μm。
实施例5
将1g的P34HB(分子量10～30KDa，分子量均值为15KDa)溶于20mL的二氯甲烷，配成5％(w/v)终浓度的有机溶液，为油相。将透明质酸(分子量5～100KDa，分子量均值为50KDa)溶于蒸馏水，配成终浓度为0.5％(w/v)的水溶液，为水相。利用磁力搅拌器以400rpm的速度搅拌水相，同时，将油相在搅拌下缓慢滴加到水相中，两种溶液的体积比为1∶50。利用磁力搅拌器以400rpm的速度继续搅拌，形成乳化液；再以该速度持续搅拌5h除去有机溶剂。之后，将所得溶液先冷冻(-40℃冷冻12h)，再冷冻干燥(-40℃冷冻干燥24h)除去水分，得到可注射P34HB微球。
所得可注射P34HB微球的收率为90％。将冻干处理后的P34HB微球溶于水，适当混合即可分散于水中，待水风干后，利用光学显微镜观察，结果如图5所示，图5为实施例5所得可注射P34HB微球的光学显微镜图，可以看出，所得微球具有良好的分散性，为球型或椭球形。利用粒径仪检测，其粒径为5～100μm，其中，68％的颗粒粒径在1～60μm，即大部分颗粒粒径在60μm以下。
实施例6：油相浓度不同
分别将0.5g、1g、2g的PHBHHx(分子量20～100KDa，分子量均值为50KDa)溶于20mL的二氯甲烷，配成2.5％(w/v)、5％(w/v)、10％(w/v)终浓度的有机溶液，为油相。将透明质酸(分子量5～100KDa，分子量均值为50KDa)溶于蒸馏水，配成终浓度为0.5％(w/v)的水溶液，为水相。利用磁力搅拌器以400rpm的速度搅拌水相，同时，将油相在搅拌下缓慢滴加到水相中，两种溶 液的体积比为1∶50。利用磁力搅拌器以400rpm的速度继续搅拌，形成乳化液；再以该速度持续搅拌5h除去有机溶剂。之后，将所得溶液先冷冻(-80℃冷冻6h)，再冷冻干燥(-40℃冷冻干燥24h)除去水分，得到可注射PHBHHx微球。
所得可注射PHBHHx微球的收率分别为90％、93％、89％。将冻干处理后的PHBHHx微球溶于水，适当混合即可分散于水中，待水风干后，利用光学显微镜观察，结果显示所得微球具有良好的分散性，为球型或椭球形。利用粒径仪检测，其粒径分别为0.4～20μm、1～50μm、1～80μm(83％的颗粒粒径在1～60μm)。
实施例7：油相与水相体积比不同
将1g的PHBHHx(分子量20～100KDa，分子量均值为50KDa)溶于20mL的二氯甲烷，配成5％(w/v)终浓度的有机溶液，为油相。将透明质酸(分子量5～100KDa，分子量均值为50KDa)溶于蒸馏水，配成终浓度为0.5％(w/v)的水溶液，为水相。利用磁力搅拌器以400rpm的速度搅拌水相，同时，将油相在搅拌下缓慢滴加到水相中，两种溶液的体积比分别为1∶10、1∶50、1∶200。利用磁力搅拌器以400rpm的速度继续搅拌，形成乳化液；再以该速度持续搅拌5h除去有机溶剂。之后，将所得溶液先冷冻(-80℃冷冻6h)，再冷冻干燥(-40℃冷冻干燥24h)除去水分，得到可注射PHBHHx微球。
所得可注射PHBHHx微球的收率分别为71％、93％、94％。将冻干处理后的PHBHHx微球溶于水，适当混合即可分散于水中，待水风干后，利用光学显微镜观察，结果显示所得微球具有良好的分散性，为球型或椭球形。利用粒径仪检测，其粒径分别为1～110μm(58％的颗粒粒径在1～60μm)、1～50μm、1～50μm。
实施例8：搅拌方式及转速不同
将1g的PHBHHx(分子量20～100KDa，分子量均值为50KDa)溶于20mL的二氯甲烷，配成5％(w/v)终浓度的有机溶液，为油相。将透明质酸(分子量5～100KDa，分子量均值为50KDa)溶于蒸馏水，配成终浓度为0.5％(w/v)的水溶液，为水相。利用磁力搅拌器以400rpm的速度搅拌水相，同时，将油相在搅拌下缓慢滴加到水相中，两种溶液的体积比为1∶50。分别利用磁力搅拌器(转速分别为100rpm、400rpm、500rpm)和高速匀浆机(转速12000rpm)继续搅拌，形成乳化液；再持续搅拌5h除去有机溶剂。之后，将所得溶液先冷冻(-80℃冷冻6h)，再冷冻干燥(-40℃冷冻干燥24h)除去水分，得到可 注射PHBHHx微球。
所得可注射PHBHHx微球的收率分别为77％、93％、89％、99％。将冻干处理后的PHBHHx微球溶于水，适当混合即可分散于水中，待水风干后，利用光学显微镜观察，结果显示所得微球具有良好的分散性，为球型或椭球形。利用粒径仪检测，其粒径分别为5～60μm、1～50μm、1～45μm、0.1～10μm(颗粒粒度基本都在10μm以下，仅5％的颗粒粒径在1～60μm，整体粒度分布属于0.1～10μm的水平)。由此证明，相比高速匀浆搅拌方式，本发明采用磁力搅拌器以适当速率(100～500rpm)搅拌，有利于获得更适宜人体的微球粒度。
实施例9：油相有机溶剂种类不同
分别将1g的PHBHHx(分子量10～20KDa，分子量均值为50KDa)溶于20mL的二氯甲烷、氯仿、乙酸乙酯中，配成5％(w/v)终浓度的有机溶液，为油相。将透明质酸(分子量5～100KDa，分子量均值为50KDa)溶于蒸馏水，配成终浓度为0.5％(w/v)的水溶液，为水相。利用磁力搅拌器以400rpm的速度搅拌水相，同时，将油相在搅拌下缓慢滴加到水相中，两种溶液的体积比为1∶50。利用磁力搅拌器以400rpm的速度继续搅拌，形成乳化液；再再以该速度持续搅拌5h除去有机溶剂。之后，将所得溶液先冷冻(-80℃冷冻6h)，再冷冻干燥(-40℃冷冻干燥24h)除去水分，得到可注射PHBHHx微球。
所得可注射PHBHHx微球的收率分别为93％、92％、89％。将冻干处理后的PHBHHx微球溶于水，适当混合即可分散于水中，待水风干后，利用光学显微镜观察，结果显示所得微球具有良好的分散性，为球型或椭球形。利用粒径仪检测，其粒径分别为1～52μm、1～50μm、1～55μm。
实施例10：水相浓度不同
将1g的PHBHHx(分子量20～100KDa，分子量均值为50KDa)溶于20mL的二氯甲烷，配成5％(w/v)终浓度的有机溶液，为油相。将透明质酸(分子量10～200KDa，分子量均值为50KDa)溶于蒸馏水，配成终浓度分别为0.01％(w/v)、0.5％(w/v)、1％(w/v)的水溶液，为水相。利用磁力搅拌器以400rpm的速度搅拌水相，同时，将油相在搅拌下缓慢滴加到水相中，两种溶液的体积比为1∶50。利用磁力搅拌器以400rpm的速度继续搅拌，形成乳化液；再以该速度持续搅拌5h除去有机溶剂。之后，将所得溶液先冷冻(-80℃冷冻6h)，再冷冻干燥(-40℃冷冻干燥24h)除去水分，得到可注射PHBHHx微球。
所得可注射PHBHHx微球的收率分别为80％、93％、87％。将冻干处理后的PHBHHx微球溶于水，适当混合即可分散于水中，待水风干后，利用光 学显微镜观察，结果显示所得微球具有良好的分散性，为球型或椭球形。利用粒径仪检测，其粒径分别为3～50μm、1～50μm、0.7～50μm。
对比例1
将1g的PHBHHx(分子量20～100KDa，分子量均值为50KDa)溶于20mL的二氯甲烷，配成5％(w/v)终浓度的有机溶液，为油相。将醇解度80％的聚乙烯醇(PVA)溶于蒸馏水，配成终浓度为0.5％(w/v)的水溶液，为水相。利用磁力搅拌器以400rpm的速度搅拌水相，同时，将油相在搅拌下缓慢滴加到水相中，两种溶液的体积比为1∶50。利用磁力搅拌器以400rpm的速度继续搅拌，形成乳化液；再以该速度持续搅拌5h除去有机溶剂。之后，将所得溶液先冷冻(-80℃冷冻6h)，再冷冻干燥(-40℃冷冻干燥24h)除去水分，得到PHBHHx微球。
对比例2
将1g的PHBVHHx(分子量20～100KDa，分子量均值为50KDa)溶于20mL的二氯甲烷，配成5％(w/v)终浓度的有机溶液，为油相。将醇解度80％的聚乙烯醇(PVA)溶于蒸馏水，配成终浓度为0.5％(w/v)的水溶液，为水相。利用磁力搅拌器以400rpm的速度搅拌水相，同时，将油相在搅拌下缓慢滴加到水相中，两种溶液的体积比为1∶50。利用磁力搅拌器以400rpm的速度继续搅拌，形成乳化液；再以该速度持续搅拌5h除去有机溶剂。之后，将所得溶液先冷冻(-80℃冷冻6h)，再冷冻干燥(-40℃冷冻干燥24h)除去水分，得到PHBVHHx微球。
对比例3
将1g的PHB(分子量20～100KDa，分子量均值为80KDa)溶于20mL的二氯甲烷，配成5％(w/v)终浓度的有机溶液，为油相。将醇解度80％的聚乙烯醇(PVA)溶于蒸馏水，配成终浓度为0.5％(w/v)的水溶液，为水相。利用磁力搅拌器以400rpm的速度搅拌水相，同时，将油相在搅拌下缓慢滴加到水相中，两种溶液的体积比为1∶50。利用磁力搅拌器以400rpm的速度继续搅拌，形成乳化液；再以该速度持续搅拌5h除去有机溶剂。之后，将所得溶液先冷冻(-80℃冷冻6h)，再冷冻干燥(-40℃冷冻干燥24h)除去水分，得到PHB微球。
对比例4
将1g的PHBV(分子量20～100KDa，分子量均值为40KDa)溶于20mL的二氯甲烷，配成5％(w/v)终浓度的有机溶液，为油相。将醇解度80％的聚乙 烯醇(PVA)溶于蒸馏水，配成终浓度为0.5％(w/v)的水溶液，为水相。利用磁力搅拌器以400rpm的速度搅拌水相，同时，将油相在搅拌下缓慢滴加到水相中，两种溶液的体积比为1∶50。利用磁力搅拌器以400rpm的速度继续搅拌，形成乳化液；再以该速度持续搅拌5h除去有机溶剂。之后，将所得溶液先冷冻(-80℃冷冻6h)，再冷冻干燥(-40℃冷冻干燥24h)除去水分，得到可注射PHBV微球。
对比例5
将1g的P34HB(分子量10～30KDa，分子量均值为15KDa)溶于20mL的二氯甲烷，配成5％(w/v)终浓度的有机溶液，为油相。将醇解度80％的聚乙烯醇(PVA)溶于蒸馏水，配成终浓度为0.5％(w/v)的水溶液，为水相。利用磁力搅拌器以400rpm的速度搅拌水相，同时，将油相在搅拌下缓慢滴加到水相中，两种溶液的体积比为1∶50。利用磁力搅拌器以400rpm的速度继续搅拌，形成乳化液；再以该速度持续搅拌5h除去有机溶剂。之后，将所得溶液先冷冻(-80℃冷冻6h)，再冷冻干燥(-40℃冷冻干燥24h)除去水分，得到P34HB微球。
实施例11：测试微球的稳定性
分别称取0.01g实施例1～5及对比例1～5中所制备的微球，分别分散于1mL蒸馏水中，放置于5mL玻璃瓶中。快速混匀后，静置。
静置30分钟后，分别观察微球在水中的分散情况。结果如图6所示，图6为实施例1～5及对比例1～5所得微球的分散稳定性示意图(从左到右的10个容器分别对应实施例1～5及对比例1～5的样品)。可以看出，实施例1～5中的PHBHHx微球、PHBVHHx微球、PHB微球、PHBV微球和P34HB微球均具有良好的分散性，未出现分层现象，微球均匀分散在水体中；对比例1～5中的PHBHHx微球、PHBVHHx微球、PHB微球、PHBV微球和P34HB微球不具备水分散性，均出现严重的分层现象，微球漂浮在出现在水上层。由此证明，相比于采用聚乙烯醇水溶液作为水相物质，本发明采用透明质酸水溶液作为水相物质，明显提高了微球的分散性和稳定性。
实施例12：测试微球的可吸取性
以一次性1mL注射器套用针头内径为0.5mm、长度为19.7mm的针头(即常用的1mL注射器枪头)为注射率检测设备，以1mL的微球分散液(微球干重为0.1g，记为M0)为实验样品。分别称取0.1g实施例1～5及对比例1～5中所制备的微球，分别分散于1mL蒸馏水中，放置于玻璃瓶中。快速混匀后，形成微球分 散液。利用1mL注射器抽吸1mL分散液，并分别冷冻干燥吸取在注射器中的微球；以吸取在注射器中的微球质量(M1)占总微球质量(M0)的百分比，为微球的可吸取率(M1/M0)×100％。
结果表明，实施例1～5中的PHBHHx微球、PHBVHHx微球、PHB微球、PHBV微球和P34HB微球的可吸取率分别为95％、93％、91％、90％和85％。对比例1～5中的PHBHHx微球、PHBVHHx微球、PHB微球、PHBV微球和P34HB微球的可吸取率分别为10％、13％、11％、9％和6％。可见，实施例1～5的微球的可吸取率远远高于对比例1～5。由此证明，相比于采用聚乙烯醇水溶液作为水相物质，本发明采用透明质酸水溶液作为水相物质，显著提升了微球的可吸取性。
实施例13：测试微球的可注射性
以一次性1mL注射器套用针头内径为0.5mm、长度为19.7mm的针头(即常用的1mL注射器枪头)为注射率检测设备，以1mL的微球分散液(微球干重为0.1g，记为M0)为实验样品。分别称取0.1g实施例1～5及对比例1～5中所制备的微球，分别分散于1mL蒸馏水中，放置于玻璃瓶中。快速混匀后，形成微球分散液。将分散液完全注入1mL注射器，并挤推注射器，收集注射出的分散液，并分别冷冻干燥注射出的分散液的微球质量(M2)，计算其占总微球质量(M0)的百分比，即为微球的注射率(M2/M0)×100％。
结果表明，实施例1～5中的PHBHHx微球、PHBVHHx微球、PHB微球、PHBV微球和P34HB微球的注射率分别为91％、92％、90％、90％和88％。对比例1～5中的PHBHHx微球、PHBVHHx微球、PHB微球、PHBV微球和P34HB微球的注射率分别为7％、11％、13％、8％和4％。可见，实施例1～5的微球的注射率远远高于对比例1～5。由此证明，相比于采用聚乙烯醇水溶液作为水相物质，本发明采用透明质酸水溶液作为水相物质，显著提升了微球的可注射性。
以上实验结果表明，本发明提供的制备方法具有以下有益效果：①微球收率高：微球收率在70％以上。②分散性好：本发明制得的PHA微球适当混合即可分散于水中，静置后，微球在水中表现出良好的分散性，未出现分层现象，既不浮在水上层、也不沉积水底，而是均匀分散在水体中，形成均匀分散液。将上述分散液风干后，观察微球形态，结果显示，微球形态完整、彼此独立，没有团聚现象，微球颗粒中全部或大部分颗粒粒径在60μm以下，能够满足注射需求。③可吸取性好：利用注射器抽吸上述分散液，微球的吸取率达到85％以上，表现出优异的可吸取性。④可注射性好：挤推注射器并收集注射出的分 散液，计算微球的注射率，结果表明，微球注射率达到88％以上，表现出优异的可注射性。
本文中应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其核心思想，包括最佳方式，并且也使得本领域的任何技术人员都能够实践本发明，包括制造和使用任何装置或系统，和实施任何结合的方法。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。本发明专利保护的范围通过权利要求来限定，并可包括本领域技术人员能够想到的其他实施例。如果这些其他实施例具有近似于权利要求文字表述的结构要素，或者如果它们包括与权利要求的文字表述无实质差异的等同结构要素，那么这些其他实施例也应包含在权利要求的范围内。

Claims (14)

  1. 一种聚羟基脂肪酸酯(PHA)微球的制备方法，其特征在于，包括以下步骤：
    a)将PHA材料溶于有机溶剂中，得到油相；
    所述PHA材料的分子量为10～100KDa；
    所述油相中PHA材料的质量浓度为2.5％～10％；
    b)将透明质酸溶于水中，得到水相；
    所述透明质酸的分子量为5～500KDa；
    所述水相中透明质酸的质量浓度为0.01％～1％；
    c)在搅拌条件下，将所述油相滴加到所述水相中，滴加完后，持续搅拌使有机溶剂挥发，之后，进行固液分离和干燥，得到PHA微球；
    所述步骤a)和步骤b)没有顺序限制。
  2. 根据权利要求1所述的制备方法，其特征在于，所述步骤c)中，所述油相与水相的体积比为1∶(10～200)。
  3. 根据权利要求1所述的制备方法，其特征在于，所述有机溶剂选自二氯甲烷、氯仿和乙酸乙酯中的一种或几种。
  4. 根据权利要求1所述的制备方法，其特征在于，所述PHA材料的分子量均值为15～80KDa。
  5. 根据权利要求1所述的制备方法，其特征在于，所述PHA材料为PHBHHx，分子量20～100KDa，分子量均值为50KDa；或者
    所述PHA材料为PHBVHHx，分子量20～100KDa，分子量均值为50KDa；或者
    所述PHA材料为PHB，分子量20～100KDa，分子量均值为80KDa；或者
    所述PHA材料为PHBV，分子量20～100KDa，分子量均值为40KDa；或者
    所述PHA材料为P34HB，分子量10～30KDa，分子量均值为15KDa。
  6. 根据权利要求1所述的制备方法，其特征在于，所述透明质酸的分子量均值为50～300KDa。
  7. 根据权利要求1所述的制备方法，其特征在于，所述透明质酸的分子量5～100KDa，分子量均值为50KDa；或者
    所述透明质酸的分子量10～200KDa，分子量均值为50KDa；或者
    所述透明质酸的分子量100～500KDa，分子量均值为300KDa。
  8. 根据权利要求1所述的制备方法，其特征在于，所述搅拌的速率为100～500rpm。
  9. 根据权利要求1所述的制备方法，其特征在于，所述搅拌为利用磁力搅拌器进行搅拌；
    滴加完油相后，持续搅拌的时间为≥4h。
  10. 根据权利要求9所述的制备方法，其特征在于，滴加完油相后，持续搅拌的时间为4～6h。
  11. 根据权利要求1所述的制备方法，其特征在于，所述搅拌的温度为10～37℃。
  12. 根据权利要求1所述的制备方法，其特征在于，所述干燥为先冷冻，再冷冻干燥；
    所述冷冻的温度为-80～-20℃，所述冷冻干燥的温度为-60～-40℃。
  13. 一种权利要求1～12中任一项所述的制备方法制得的聚羟基脂肪酸酯微球。
  14. 根据权利要求13所述的聚羟基脂肪酸酯微球，其特征在于，所述聚羟基脂肪酸酯微球的粒径在60μm以下；和/或
    所述聚羟基脂肪酸酯微球的吸取率在85％以上；和/或
    所述聚羟基脂肪酸酯微球的注射率在88％以上。
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ZHANG JUNYU; SHISHATSKAYA EKATERINA I.; VOLOVA TATIANA G.; DA SILVA LUIZIANA FERREIRA; CHEN GUO-QIANG: "Polyhydroxyalkanoates (PHA) for therapeutic applications", MATERIALS SCIENCE AND ENGINEERING C, ELSEVIER SCIENCE S.A., CH, vol. 86, 5 January 2018 (2018-01-05), CH , pages 144 - 150, XP085358296, ISSN: 0928-4931, DOI: 10.1016/j.msec.2017.12.035 *

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