A kind of polyhydroxyalkanoate porous microsphere and preparation method thereof

Abstract

The present invention relates to a method for preparing a porous polyhydroxyalkanoate microsphere, comprising the following steps: (S1) preparing an oil phase; (S2) preparing an aqueous phase; (S3) mixing the oil phase and the aqueous phase; and (S4) preparing the porous microsphere. The porous microsphere of the present invention has a relatively regular spherical structure, a smooth surface and uniformly distributed pores, a suitable particle size and pore size range, is particularly suitable for use as a filling material in the field of medical cosmetology, and has good needle permeability when used for injection.

Description

Technical Field

[0001] The invention relates to the field of biomedical materials, and in particular to a polyhydroxyalkanoate porous microsphere and a preparation method thereof. Background Art

[0002] Due to aging or disease, as people age, substances such as collagen in human skin and other tissues will continue to be lost, causing the skin to lose moisture and elasticity. Therefore, scientists have discovered that porous microspheres can be filled in the skin or subcutaneous tissue to modify the contour and improve the surface texture of the skin. The principle is that after being injected, the porous microspheres stay stably in the tissue and will not migrate or degrade immediately. They can stimulate the tissue to produce collagen and promote the formation of new blood vessels, thereby achieving the effect of medical beauty.

[0003] Polyhydroxyalkanoates, also known as polyhydroxyalkanoates, or PHA for short, are a class of high molecular weight polyester materials. Compared with polycaprolactone (PCL), PHA has strong adjustability in degradation performance and mechanical strength. Compared with polylactic acid (PLA, PLLA, PDLLA), PHA degradation products have a high pKa (4.7), are milder to skin tissue, and are gradually degraded into CO2 and H2O and excreted from the body as the body metabolizes. They have good biocompatibility and are suitable for the preparation of medical microspheres.

[0004] It is known that the surface morphology of microspheres used in the field of medical aesthetics plays an important role in their efficacy. Parameters such as particle size, roundness, surface smoothness, porous morphology and porosity of porous microspheres will affect the degradation cycle of the material, its ability to promote collagen production, and side effects at the injection site, such as nodules, granulomas and inflammation.

[0005] However, when the surface of the microspheres has a porous structure, it is conducive to the attachment and growth of tissue cells on the surface of the microspheres. The porous structure can serve as a scaffold to provide growth space for collagen, reducing the risk of nodules and granulomas. On the other hand, when the microspheres are smooth, they can reduce the stimulation to human tissues, reduce inflammatory responses, and improve injection patency. Since porosity and smoothness are generally considered to be incompatible, it is difficult to prepare porous and smooth microspheres due to the particularity of PHA's chemical structure. Therefore, PHA microspheres prepared by the prior art often have problems such as small pores, few pores, surface wrinkles, and poor roundness of the microspheres.

[0006] In view of this, how to prepare round PHA microspheres with porous and smooth surfaces is an urgent problem to be solved. Summary of the invention

[0007] In order to solve the above problems, the present invention provides a polyhydroxyalkanoate porous microsphere and a preparation method thereof, which has a relatively regular spherical structure, a smooth surface and uniformly distributed pores, a suitable particle size and pore size range, can be used to prepare biomedical materials, and is particularly suitable for use in the field of medical cosmetology as a filling material, and has good needle permeability when used for injection.

[0008] In a first aspect, the present invention provides a method for preparing polyhydroxyalkanoate porous microspheres, comprising the following steps:

[0009] (S1) dissolving polyhydroxyalkanoate in a combined organic solvent to obtain an oil phase;

[0010] (S2) mixing the dispersant and water to obtain an aqueous phase;

[0011] (S3) mixing the oil phase and the water phase to obtain an emulsion reaction system; and,

[0012] (S4) removing the organic solvent from the reaction system to obtain porous microspheres;

[0013] in:

[0014] The combined organic solvent in S1 includes organic solvent 1 and organic solvent 2; the density of organic solvent 1 at 25° C. is greater than that of water, and the density of organic solvent 2 at 25° C. is less than that of water;

[0015] The volume ratio of the oil phase to the water phase in S3 is Voil:Vwater=1:5~1:1000.

[0016] Preferably, in the method for preparing the polyhydroxyalkanoate porous microspheres: the density of the organic solvent 1 at 25°C is 1.001-1.6 g / mL; and / or the density of the organic solvent 2 at 25°C is 0.5-0.98 g / mL.

[0017] Preferably, in the method for preparing the polyhydroxyalkanoate porous microspheres: the density of the organic solvent 1 at 25°C is 1.02-1.5 g / mL; and / or the density of the organic solvent 2 at 25°C is 0.6-0.91 g / mL.

[0018] Preferably, in the preparation method of the polyhydroxyalkanoate porous microspheres: the organic solvent 1 includes one or more of dichloromethane, chloroform, dimethyl sulfoxide or N-methylpyrrolidone; and / or the organic solvent 2 includes one or more of methanol, ethanol, acetone, isopropanol, ethyl acetate, acetonitrile, tetrahydrofuran, n-pentane, n-hexane, n-heptane, or n-octane.

[0019] Preferably, in the method for preparing the polyhydroxyalkanoate porous microspheres: the mass volume ratio of the polyhydroxyalkanoate to the organic solvent 1 is 1:2-1:1000 (g:mL); and / or the mass volume ratio of the polyhydroxyalkanoate to the organic solvent 2 is 1:0.01-1:10 (g:mL).

[0020] Preferably, in the method for preparing the polyhydroxyalkanoate porous microspheres: the mass volume ratio of the polyhydroxyalkanoate to the organic solvent 1 is 1:3-1:500 (g:mL); and / or the mass volume ratio of the polyhydroxyalkanoate to the organic solvent 2 is 1:0.1-1:5 (g:mL).

[0021] Preferably, in the method for preparing the polyhydroxyalkanoate porous microspheres: in the oil phase, the concentration of the polyhydroxyalkanoate is 1-500 mg / ml.

[0022] Preferably, in the method for preparing the polyhydroxyalkanoate porous microspheres, the concentration of the polyhydroxyalkanoate in the oil phase is 5-400 mg / ml.

[0023] Preferably, in the method for preparing the polyhydroxyalkanoate porous microspheres, the volume ratio of the organic solvent 1 to the organic solvent 2 is 1000:1 to 1:1.

[0024] Preferably, in the method for preparing the polyhydroxyalkanoate porous microspheres, the volume ratio of the organic solvent 1 to the organic solvent 2 is 500:1 to 1:1.

[0025] Preferably, in the method for preparing the polyhydroxyalkanoate porous microspheres, the organic solvent 1 and the organic solvent 2 are miscible with each other.

[0026] Preferably, in the method for preparing the polyhydroxyalkanoate porous microspheres, the organic solvent 1 and the organic solvent 2 are volatile organic solvents.

[0027] Preferably, in the method for preparing the polyhydroxyalkanoate porous microspheres: the volume ratio of the oil phase to the water phase in S3 is Voil:Vwater=1:10-1:500.

[0028] Preferably, in the method for preparing the polyhydroxyalkanoate porous microspheres, the volume ratio of the oil phase to the water phase in S3 is V oil: V water = 1:50 to 1:500.

[0029] Preferably, in the method for preparing the polyhydroxyalkanoate porous microspheres, at least 50 wt% of the chain segments in the polyhydroxyalkanoate are derived from the polymerization product of one or more monomers of hydroxybutyric acid, hydroxyvaleric acid, hydroxyhexanoic acid, hydroxyheptanoic acid or hydroxyoctanoic acid.

[0030] Preferably, in the method for preparing the polyhydroxyalkanoate porous microspheres, the polyhydroxyalkanoate is a polymerization product of one or more monomers of hydroxybutyric acid, hydroxyvaleric acid, hydroxycaproic acid, hydroxyheptanoic acid or hydroxyoctanoic acid.

[0031] Preferably, in the method for preparing the polyhydroxyalkanoate porous microspheres, the dispersant includes one or more of polyvinyl alcohol, Tween, carboxymethyl cellulose and its salts, mannitol, gelatin, hyaluronic acid and its salts, or collagen.

[0032] Preferably, in the method for preparing the polyhydroxyalkanoate porous microspheres: the concentration of the dispersant in the aqueous phase is 0.01-10 wt %.

[0033] In a second aspect, the present invention provides a polyhydroxyalkanoate porous microsphere.

[0034] Preferably, the polyhydroxyalkanoate porous microspheres are prepared by any of the methods described above.

[0035] Preferably, in the polyhydroxyalkanoate porous microspheres, the pore size of the microspheres ranges from 1 to 10 μm, and the average particle size of the microspheres ranges from 10 to 100 μm.

[0036] Preferably, in the polyhydroxyalkanoate porous microspheres, the Mw of the polyhydroxyalkanoate is 30-300 kDa.

[0037] Preferably, in the polyhydroxyalkanoate porous microspheres, the polyhydroxyalkanoate is selected from one or more of a copolymer of 3-hydroxybutyric acid and 4-hydroxybutyric acid, a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid, or a homopolymer of 4-hydroxybutyric acid.

[0038] In a third aspect, the present invention provides any of the aforementioned methods or use of polyhydroxyalkanoate porous microspheres prepared by any of the aforementioned methods in the preparation of biomedical materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 : SEM photos of microspheres in Example 1: (a) 500X; (b) 2000X; (c) internal (cross-section) structure of microspheres.

[0040] Figure 2 : SEM photos of microspheres in Example 2: (a) 500X; (b) 2000X; (c) internal (cross-section) structure of microspheres.

[0041] Figure 3 : SEM photos of microspheres in Example 3: (a) 500X; (b) 2000X.

[0042] Figure 4: SEM photos of microspheres in Example 4: (a) 200X; (b) 2000X.

[0043] Figure 5 : SEM photos of microspheres in Example 5: (a) 200X; (b) 2000X.

[0044] Figure 6 : SEM photos of microspheres in Example 6: (a) 200X; (b) 1000X.

[0045] Figure 7 : SEM photos of microspheres in Example 7: (a) 200X; (b) 1000X.

[0046] Figure 8 : SEM photos of microspheres in Example 8: (a) 500X; (b) 2000X; (c) internal (cross-section) structure of microspheres.

[0047] Fig. 9 : SEM photos of microspheres in Example 9: (a) 500X; (b) 2000X.

[0048] Fig.10 : SEM photos of microspheres in Example 10: (a) 500X; (b) 2000X.

[0049] Fig.11 : SEM photos of microspheres in Example 11: (a) 500X; (b) 2000X.

[0050] Fig.12 : SEM photos of microspheres in Example 12: (a) 500X; (b) 2000X.

[0051] Fig.13 : SEM photos of microspheres of Comparative Example 1: (a) 200X; (b) 1000X.

[0052] Fig.14 : SEM photos of microspheres of Comparative Example 2: (a) 200X; (b) 1000X.

[0053] Fig.15 : SEM photos of microspheres of Comparative Example 3: (a) 1000X; (b) 2000X.

[0054] Fig.16 : SEM photos of microspheres of Comparative Example 4: (a) 1000X; (b) 2000X.

[0055] Fig.17 : SEM photograph of microspheres of Comparative Example 5 (500X). DETAILED DESCRIPTION

[0056] In the following description, a large number of specific details are provided to provide a more thorough understanding of the present invention. However, it is apparent to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some technical features well known in the art are not described.

[0057] The implementation of the present invention is described in detail below in conjunction with the definitions of terms:

[0058] I Preparation method of polyhydroxyalkanoate porous microspheres

[0059] The method for preparing porous microspheres of the present invention comprises the following steps:

[0060] (S1) dissolving polyhydroxyalkanoate in a combined organic solvent to obtain an oil phase;

[0061] (S2) mixing the dispersant and water to obtain an aqueous phase;

[0062] (S3) mixing the oil phase and the water phase to obtain an emulsion reaction system; and,

[0063] (S4) removing the organic solvent from the reaction system to obtain porous microspheres.

[0064] (i) Polyhydroxyalkanoates

[0065] The polyhydroxyalkanoate (PHA) of the present invention, also known as polyhydroxyalkanoate, is a polymer of hydroxyalkyl carboxylic acid or its ester. For example, a typical PHA may include the following structure:

[0066] Formula (I).

[0067] In formula (I), p ≥ 0, n is 10-100,000, represents the average degree of polymerization, R and R ’ may be independently selected from hydrogen or C1-C 30 of alkyl.

[0068] In formula (I), p, n, R and R ’ The carbon atom numbers in the table represent number averages.

[0069] In some embodiments, in formula (I), p=1-20, preferably 1-12, and more preferably 1-6.

[0070] In some embodiments, in formula (I), n=50-10000, preferably 100-5000, more preferably 200-3000.

[0071] In some embodiments, R and R in formula (I) ’ may be independently selected from hydrogen or C1-C 10The alkyl group is preferably selected from hydrogen or a C1-C4 alkyl group, more preferably selected from hydrogen, methyl or ethyl.

[0072] In some embodiments, in formula (I), p=1-20, n=50-10000, R and R ’ may be independently selected from hydrogen or C1-C 10 of alkyl.

[0073] In some embodiments, in formula (I), p=1-12, n=100-5000, R and R ’ They may be independently selected from hydrogen or C1-C4 alkyl.

[0074] In some embodiments, in formula (I), p=1-6, n=200-3000, R and R ’ May be independently selected from hydrogen, methyl or ethyl.

[0075] The PHA of the present invention can be obtained from nature or synthesized artificially, for example, it can be obtained from microorganisms such as bacteria in physiological activities, or it can be obtained by polymerizing monomers of hydroxy fatty acids or esters thereof.

[0076] The PHA of the present invention may be a homopolymer, a copolymer (such as a random copolymer or a block copolymer), a modified (or functionally modified) polymer, and a blend (a blend refers to a mixture of two or more homopolymers, copolymers, modified polymers, or functionally modified polymers of PHA).

[0077] In some embodiments, the monomers from which PHAs can be prepared include fatty carboxylic acids containing hydroxyl groups, such as C4-C 10 The hydroxy fatty carboxylic acid may specifically be one or more of hydroxybutyric acid, hydroxyvaleric acid, hydroxycaproic acid, hydroxyheptanoic acid or hydroxyoctanoic acid.

[0078] The PHA of the present invention may include a homopolymer of one of the aforementioned monomers, a copolymer of at least two of the aforementioned monomers, or a blend of at least one of the aforementioned monomers (a blend refers to a mixture of homopolymers, copolymers, modified polymers or functionally modified polymers of two or more PHAs). Further, the PHA segment may also include a polymer segment derived from other functional monomers. In some embodiments, at least 50wt%, 60wt%, 70wt%, 80wt% or more than 90wt% or 100wt% of the segments in the PHA of the present invention are derived from the polymerization product of hydroxy fatty acids or their ester monomers. In some embodiments, at least 50wt%, 60wt%, 70wt%, 80wt% or more than 90wt% or 100wt% of the segments in the PHA of the present invention are derived from the polymerization product of one or more monomers of hydroxybutyric acid, hydroxyvaleric acid, hydroxyhexanoic acid, hydroxyheptanoic acid or hydroxyoctanoic acid. In some embodiments, the PHA of the present invention is a copolymer of 3-hydroxybutyric acid and 4-hydroxybutyric acid or a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid or a homopolymer of 4-hydroxybutyric acid.

[0079] In some embodiments, the PHA of the present invention has a conventional weight average molecular weight such as 30-2000 kDa, preferably such as 40-1000 kDa, such as 50-800 kDa, 60-600 kDa, 70-500 kDa, 80-300 kDa; 90-200 kDa, 100-180 kDa, 120-150 kDa.

[0080] In some embodiments, the concentration of PHA of the present invention in the oil phase is 1-500 mg / ml, 2-450 mg / mL or 5-400 mg / ml, for example, it can be 10 mg / ml, 20 mg / ml, 40 mg / ml, 80 mg / ml, 100 mg / ml, 150 mg / ml, 200 mg / ml, 250 mg / ml, 300 mg / ml, 400 mg / ml or 500 mg / ml, etc.

[0081] (ii) Combined organic solvents

[0082] The combined organic solvent of the present invention comprises at least an organic solvent 1 and an organic solvent 2.

[0083] The density described in the present invention is the density of the substance at 25° C. and 1 atm.

[0084] Organic solvent 1

[0085] The density of the organic solvent 1 of the present invention is greater than that of water. In some embodiments, the density of the organic solvent 1 of the present invention is 1.001-1.6 g / mL, 1.01-1.55 g / mL or 1.02-1.5 g / mL, for example, it may include one or more of dichloromethane, chloroform, dimethyl sulfoxide, and N-methylpyrrolidone, preferably dichloromethane or chloroform.

[0086] In some embodiments, the mass volume ratio of the PHA and the organic solvent 1 in the oil phase is 1:2-1:1000 (g:mL), 1:3-1:500 (g:mL) or 1:3.3-1:100 (g:mL), for example, it can be (g:mL): 1:5, 1:10, 1:20, 1:30, 1:40, 1:50, 1:75, 1:100, 1:200 or 1:300, etc.

[0087] In some embodiments, the organic solvent 1 of the present invention is a volatile organic solvent.

[0088] Organic solvent 2

[0089] The density of the organic solvent 2 of the present invention is less than that of water. In some embodiments, the density of the organic solvent 2 of the present invention is 0.5-0.98 g / mL, 0.55-0.95 g / mL or 0.6-0.91 g / mL, for example, it may include one or more of methanol, ethanol, acetone, isopropanol, ethyl acetate, acetonitrile, tetrahydrofuran, n-pentane, n-hexane, n-heptane, or n-octane, preferably, the organic solvent 2 is one or more of methanol, ethanol or n-pentane.

[0090] In some embodiments, the mass volume ratio of the PHA and the organic solvent 2 in the oil phase is 1:0.01-1:10 (g:mL), 1:0.1-1:5 (g:mL) or 1:0.2-1:3 (g:mL), for example, it can be (g:mL): 1:0.05, 1:0.15, 1:0.25, 1:0.5, 1:1, 1:1.5, 1:2, 1:3, 1:5 or 1:8, etc.

[0091] In some embodiments, the organic solvent 2 of the present invention is a volatile organic solvent.

[0092] The volatile organic solvent described in the present invention refers to an organic compound having a saturated vapor pressure greater than 133.32 Pa (ie, 1 mmHg) and a boiling point lower than 260°C at room temperature (usually 25°C).

[0093] In some embodiments, in the combined organic solvent of the present invention, the volume ratio of the organic solvent 1 to the organic solvent 2 is 1000:1-1:1, 500:1-1:1 or 300:1-2:1, for example, it can be 250:1, 200:1, 150:1, 100:1, 80:1, 50:1, 30:1, 10:1, 5:1, etc.

[0094] In some embodiments, the organic solvent 1 and the organic solvent 2 of the present invention are miscible.

[0095] The mutual solubility of the present invention means that at 25° C., the two liquids can be mixed in any proportion to form a uniform and stable solution without stratification.

[0096] The advantages of the combined organic solvent selected in the present invention are as follows: the density of the organic solvent 1 is greater than that of water. During the curing process of the microspheres, the organic solvent 1 is not easy to float to the water surface after being separated from the microspheres, and its volatilization speed is slow. The density of the organic solvent 2 is less than that of water, so it is easy to float on the water surface, and it is easier to volatilize from the water, and its volatilization speed is fast. The two solvents with different regional concentrations and different volatilization speeds are used together, so that the microspheres can form a better spherical structure during the curing process, and the surface is smooth and porous.

[0097] (iii) Dispersants

[0098] The dispersant used in the present invention is a dispersant known in the art, for example, may include a cationic, anionic or nonionic dispersant or surfactant. In some embodiments, the dispersant used in the present invention may be a natural polymer or synthetic polymer dispersant, or a small molecule dispersant.

[0099] In some embodiments, the dispersant of the present invention may include polyvinyl alcohol, Tween (such as Tween 80, etc.), carboxymethyl cellulose (such as sodium carboxymethyl cellulose), mannitol, gelatin, hyaluronic acid (such as sodium hyaluronate, etc.) or collagen, etc. Preferably, the dispersant contains polyvinyl alcohol or the dispersant is polyvinyl alcohol.

[0100] In some embodiments, the concentration of the dispersant in the aqueous phase is 0.01-10 wt %, 0.1-5 wt % or 0.5-2 wt %, etc.

[0101] (iv) Emulsion reaction system

[0102] The aforementioned oil phase and water phase of the present invention are mixed to obtain an emulsion reaction system. In some embodiments, in the emulsion reaction system, the volume ratio of the oil phase to the water phase is V oil:V water = 1:5 to 1:1000, 1:10 to 1:500 or 1:10 to 1:250, for example, it can be 1:5, 1:10, 1:20, 1:30, 1:40, 1:50, 1:75, 1:100, 1:150, 1:200, 1:300, 1:500 or 1:800, etc.

[0103] The above oil-water ratio can be used to adjust the kinetic process of removing the organic solvent, and by combining the selection of two organic solvents, round and smooth porous microspheres can be produced.

[0104] In some embodiments, the step of mixing the oil phase and the water phase of the present invention is to add the oil phase to the water phase for emulsification. In some embodiments, the emulsification time is 1-10 min or 1-5 min to form a stable and uniform emulsion.

[0105] The emulsion of the present invention is in the form of an O / W emulsion.

[0106] (v) Removal of organic solvents

[0107] The invention obtains porous microspheres by removing the organic solvent in the emulsion.

[0108] In some embodiments, the step of removing the organic solvent can be performed at room temperature or under heating. In some embodiments, the organic solvent is removed from the reaction system by applying an external mechanical force. The external mechanical force can be applied by stirring or the like, and the organic solvent can be removed from the reaction system by volatilization.

[0109] II Polyhydroxyalkanoate porous microspheres

[0110] The porous microspheres of the present invention can be prepared by any of the aforementioned methods.

[0111] The porous microspheres of the present invention have a suitable particle size. In some embodiments, for example, the particle size range may be 10 to 100 μm or the average particle size may be in the range of 10 to 100 μm.

[0112] The porous microspheres of the present invention have a suitable pore size. In some embodiments, for example, the pore size range may be 1 to 10 μm.

[0113] III Application

[0114] The aforementioned method of the present invention and the polyhydroxyalkanoate porous microspheres prepared therefrom can be used to prepare biomedical materials, for example, as filling materials in the field of medical cosmetology, and have good needle permeability when used for injection, and can be made into injection, powder injection and other dosage forms.

[0115] The present invention is further described in detail below through specific examples.

[0116] raw material

[0117] PHA1: a copolymer of 3-hydroxybutyric acid and 4-hydroxybutyric acid, with a Mw of 30-300 kDa. Except for Example 3 and Example 6, the remaining examples used PHA1.

[0118] PHA2: copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid, Mw is 50~150 kDa. PHA2 is used in Example 6.

[0119] PHA3: 4-hydroxybutyric acid monomer, Mw is 50~250kDa, Example 3 uses PHA3.

[0120] The remaining reagents were used directly without purification.

[0121] instrument

[0122] Scanning electron microscope (SEM): JEOL, JSM-IT500.

[0123] Example 1-14 Preparation of porous microspheres of the present invention

[0124] The porous microspheres were prepared according to the types and contents of the raw materials in Table 1.

[0125] (1) dissolving PHA in a mixed solvent of solvent 1 and solvent 2 to prepare an oil phase with a certain concentration;

[0126] (2) dissolving the dispersant in a certain amount of water to prepare an aqueous phase;

[0127] (3) The oil phase of step (1) was added to the water phase according to the oil-water ratio and emulsified for 5 min. The stirring was continued until the organic solvent evaporated to solidify the microspheres. The microspheres were obtained after drying. The SEM photos of Examples 1-12 are shown in FIG. Figure 1-12 As shown, Examples 1-14 observed the roundness of the microspheres, whether the surface was smooth, whether there were holes, and whether the hole distribution was uniform, and the results are shown in Table 1. It can be seen that the microspheres in the embodiments of the present invention have a smooth surface, good spherical roundness, and the formed pores are evenly distributed, the pore size range is basically 1-10 μm, and the average particle size of the microspheres is basically 10-100 μm, which is suitable for use in the field of medical cosmetology.

[0128] Comparative Examples 1-6

[0129] Comparative Example 1

[0130] (1) Dissolve 1 g of PHA material in 20 ml of dichloromethane to prepare an oil phase with a concentration of 50 mg / ml;

[0131] (2) Dissolve 45 g of PVA in 3000 ml of pure water to prepare a 1.5 wt% aqueous phase;

[0132] (3) Dissolve 7.5 g of ammonium bicarbonate in 50 ml of pure water to obtain a pore-forming agent.

[0133] (4) Weigh 0.1 g of Tween 80; add 20 ml of the oil phase and 4 ml of the pore-forming agent into a 25 ml beaker, place in an ice-water bath for 5 min, and emulsify at 8000 rpm for 2 min to obtain a primary emulsion.

[0134] (5) Immediately pour the primary emulsion into 800 ml of 1.5% PVA aqueous solution and cure at 40°C and 150 rpm for 5 hours to obtain microspheres. Take SEM photos as shown below: Fig.13 As shown, it can be seen that the obtained microspheres have poor spherical roundness, the surface of the microspheres is not smooth, and the pores of the obtained microspheres are small.

[0135] Comparative Example 2

[0136] (1) Dissolve 0.3 g of PHA material in 20 ml of dichloromethane to prepare an oil phase with a concentration of 15 mg / ml;

[0137] (2) Take 1.5L of 1.5% PVA aqueous solution, add 3L of pure water, stir evenly, and dilute to 0.5% PVA aqueous solution;

[0138] (3) Dissolve 1.5 g of ammonium bicarbonate in 10 ml of pure water to obtain a pore-forming agent.

[0139] (4) Take 20 ml of the oil phase and add 4 ml of the pore-forming agent into a 25 ml small beaker. Let it stand in an ice-water bath for 5 min. Use an overhead homogenizer to stir in an ice-water bath for pre-emulsification at a speed of 20,000 r / min for 2 min to obtain a primary emulsion.

[0140] (5) Immediately inject the primary emulsion into 1100 ml of PVA aqueous solution and cure at 35°C and 150 rpm for 5 hours to obtain microspheres. Take SEM photos as shown below: Fig.14 As shown, it can be seen that the obtained microspheres have poor spherical roundness, the surface of the microspheres is not smooth, there are many wrinkles, and the pores of the obtained microspheres are small.

[0141] The formulations and microsphere morphology results of Comparative Examples 1-2 are shown in Table 2.

[0142] Comparative Examples 3-6

[0143] Microspheres were prepared in the same manner as in Example 1 according to the types and amounts of raw materials in Table 3, and SEM photos were taken. The microspheres prepared in Comparative Examples 3-5 were as follows: Figure 15-17As shown, the product of Comparative Example 6 did not form a ball and could not be photographed by SEM. The microsphere morphology results are shown in Table 3.

[0144] It can be seen that compared with the comparative example, the preparation method of the present invention has no internal water phase, and by adjusting the process parameters (such as the selection of the combined solvent and the oil-water ratio, etc.), round, smooth, porous and uniform microspheres with even very uniform internal pores are prepared.

[0145] Application Example 1 Injection Experiment

[0146] SD rats were used as experimental animals (12W adult rats, 4 rats in each comparative example group and 2 rats in each example group). The microspheres of Examples 1-2, 4-7, 12 and Comparative Examples 1-5 were prepared into injection aqueous solutions of different concentrations (30 mg / ml as the highest concentration, 0.5 ml of normal saline was added each time for dilution) and then injected into the dermis of rat skin. The maximum concentration of each group of microspheres at which 0.2 mL could be smoothly injected was recorded, and the results are shown in Table 4. The results show that the microspheres of the examples of the present invention have higher needle permeability than the comparative examples. When the pores on the surface of the PHA microspheres become larger, at the same mass concentration, the relative volume concentration becomes larger, and the injection resistance increases, but they can still be smoothly injected into the skin tissue at an injection concentration of 20 mg / ml.

[0147] Application Example 2 Mouse Fibroblast Proliferation Experiment

[0148] (1) L-929 cells were cultured in MEM medium containing 10% fetal bovine serum and antibiotics (penicillin 100 IU / mL, streptomycin 100 µg / mL) in a 37°C, 5% CO2 incubator. The cells were digested with 0.25% trypsin (containing EDTA) to prepare a single-cell suspension, the cell suspension was centrifuged (200 g, 5 min), and the cells were redispersed in the culture medium to adjust the cell density to 1×10 5 The cell suspension was inoculated into a 24-well culture plate with 1 mL per well, and the plate was placed in an incubator (5% CO2, 37°C) for 24 h.

[0149] (2) Discard the original culture medium, add 1 mL of MEM complete medium to each well of the 24-well plate, and then add 1 mL of test samples of different concentrations (the final concentration of the high dose is 0.5 mg / mL, the final concentration of the medium dose is 0.25 mg / mL, and the final concentration of the low dose is 0.125 mg / mL) to the cell chamber according to the grouping in Table 5, and culture for 72 hours at 37°C and 5% CO2. Perform three parallel experiments in each group.

[0150] (3) After 72 h, remove the cell chamber and observe the cell morphology first. Then aspirate the original culture medium and add 500 µL of MTT (1 mg / mL) to each well. Incubate for 2 h, discard the supernatant, and add 500 µL of isopropanol to each well to dissolve the crystals.

[0151] (4) Pipette 100 µL of the liquid in each well of the 24-well plate into a 96-well plate and measure the absorbance on a microplate reader with 570 nm as the main absorption wavelength and 650 nm as the reference wavelength.

[0152] Under the experimental conditions, the results of the cell proliferation test are shown in Table 6. Compared with the negative group, the latex control group (latex extract: using sterile latex gloves, according to 6cm 2 / mL ratio added to complete cell culture medium and extracted at 37℃ for 72h) cell proliferation rate was significantly inhibited (p<0.05), and the proliferation rate of the positive promotion group was significantly increased (p<0.05), proving that the experimental system is effective. The cell proliferation rate of each batch increased with the decrease of sample concentration, and was significantly reduced compared with the negative group (p<0.05). Among the samples in the 0.5mg / mL dosage group, the highest cell proliferation rate was the sample in Example 5, which was 95.07%, followed by the sample in Example 8, which was 85.08%, and the lowest was the sample in Comparative Example 5, which was 73.36%. Among the samples in the 0.125 mg / mL dosage group, the sample with the highest cell proliferation rate was Example 5, which was 118.54%, followed by Example 7, which was 98.36%, and the lowest batch was Comparative Example 5, which was 79.63%. It shows that the porous microspheres of the present invention can effectively promote cell proliferation.

[0153] Application Example 3 Experiment on the effect of type I collagen on mouse fibroblasts

[0154] (1) L-929 cells were cultured in MEM medium containing 10% fetal bovine serum and antibiotics (penicillin 100 IU / mL, streptomycin 100 µg / mL) in a 37°C, 5% CO2 incubator. The cells were digested with 0.25% trypsin (containing EDTA) to prepare a single-cell suspension, the cell suspension was centrifuged (200 g, 5 min), and the cells were redispersed in the culture medium to adjust the cell density to 1×10 5 Inoculate the above cell suspension into a 24-well culture plate with 1 mL per well and culture in an incubator (5% CO2, 37°C) for 24 h.

[0155] (2) Discard the original culture medium, add 1 mL of MEM complete medium to each well of a 24-well plate, and then group the samples according to Table 5 (there is no blank group in this example), add 1 mL of test samples of different concentrations (the final concentration of the high dose is 0.5 mg / mL, the final concentration of the medium dose is 0.25 mg / mL, and the final concentration of the low dose is 0.125 mg / mL) to the cell chamber, and culture at 37°C and 5% CO2 for 7 days. Perform three parallel experiments in each group.

[0156] (3) On the third day, the cell chambers in each well were picked up, the supernatant was removed with a pipette, and 1 mL of the corresponding fresh culture medium components was added again. After 7 days, the supernatant and cells of each group were collected. The cells were broken with RIPA lysis buffer, centrifuged at 15000g for 10 minutes, and the cell lysate supernatant was collected. The intracellular type I collagen content in the culture medium supernatant and the cell lysate supernatant was detected respectively. 100 μL of the test solution was taken from each well and the mouse type I collagen detection kit was used to measure the absorbance OD value on the microplate reader with 450 nm as the main absorption wavelength and 630 nm as the reference wavelength. The gradient concentration of the mouse type I collagen standard was used to detect the standard concentration corresponding to the absorbance at 450 nm, and the standard curve was fitted. Then, the concentration of mouse type I collagen in each group was calculated based on the OD value, and the difference in the concentration of mouse type I collagen in each group of each sample was statistically analyzed.

[0157] Under the experimental conditions, the detailed results of the type I collagen content detection test are shown in Table 7. The collagen concentration of the positive promotion control group was 37.80 ng / mL, which was significantly higher than that of the negative control group (p<0.05), and the collagen concentration of the positive inhibition control group was 1.41 ng / mL, which was significantly lower than that of the negative control group (p<0.05), indicating that the experimental system was effective. Compared with the negative control group, the expression concentration of type I collagen in each sample group showed significant differences in different dose groups (p<0.05). Except for the sample of comparative example 4, the collagen expression concentration of the other 6 batches of samples increased with the increase of dosage concentration, among which the sample with the highest expression concentration was the 0.50 mg / mL dosage group of Example 7, which was 35.11 ng / mL; followed by the 0.50 mg / mL dosage groups of the samples of Example 5 and Example 8, which were 27.97 ng / mL and 27.79 ng / mL respectively; the sample with the lowest collagen expression was the 0.25 mg / mL dosage group of comparative example 4, which was 10.50 ng / mL. It shows that the porous microspheres of the present invention can effectively promote the growth of collagen.

[0158] In the above embodiments, all technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

[0159] Table 1

[0160]

[0161] Table 2

[0162]

[0163] Table 3

[0164]

[0165] Table 4

[0166]

[0167] Table 5

[0168]

[0169] Table 6 (Cell proliferation assay and results)

[0170]

[0171] Note: *: indicates that there is a significant difference compared with the negative group, p<0.05.

[0172] Table 7 (Collagen experiment and results)

[0173]

[0174] Note: *: Compared with the negative group, p<0.05, indicating a significant difference.

Claims

1. A method for preparing polyhydroxyalkanoate porous microspheres, comprising the following steps: (S1) dissolving polyhydroxyalkanoate in a combined organic solvent to obtain an oil phase; (S2) mixing the dispersant and water to obtain an aqueous phase; (S3) mixing the oil phase and the water phase to obtain an emulsion reaction system; and, (S4) removing the organic solvent from the reaction system to obtain porous microspheres; in: The combined organic solvent in S1 includes organic solvent 1 and organic solvent 2; the density of organic solvent 1 at 25° C. is greater than that of water, and the density of organic solvent 2 at 25° C. is less than that of water; The volume ratio of the oil phase to the water phase in S3 is Voil:Vwater=1:10-1:500; The polyhydroxyalkanoate porous microspheres are used as filling materials in the field of medical cosmetology; The density of the organic solvent 1 at 25° C. is 1.02-1.5 g / mL; the density of the organic solvent 2 at 25° C. is 0.6-0.91 g / mL; The organic solvent 1 includes one or more of dichloromethane, chloroform, dimethyl sulfoxide or N-methylpyrrolidone; the organic solvent 2 includes one or more of methanol, ethanol, acetone, isopropanol, ethyl acetate, acetonitrile, tetrahydrofuran, n-pentane, n-hexane, n-heptane or n-octane; The mass volume ratio of the polyhydroxyalkanoate to the organic solvent 1 is g:mL 1:2-1:75; the mass volume ratio of the polyhydroxyalkanoate to the organic solvent 2 is g:mL 1:0.2-1:3; In the oil phase, the concentration of polyhydroxyalkanoate is 10-500 mg / ml; The volume ratio of the organic solvent 1 to the organic solvent 2 is 250:1-1:

1.

2. The method according to claim 1, wherein: The organic solvent 1 and the organic solvent 2 are miscible with each other.

3. The method according to claim 1, wherein: The organic solvent 1 and the organic solvent 2 are volatile organic solvents.

4. The method according to claim 1, wherein: The volume ratio of the oil phase to the water phase in S3 is Voil:Vwater=1:50~1:

500.

5. The method according to claim 1, wherein: At least 50 wt % of the segments in the polyhydroxyalkanoate are derived from the polymerization product of one or more monomers of hydroxybutyric acid, hydroxyvaleric acid, hydroxycaproic acid, hydroxyheptanoic acid or hydroxyoctanoic acid.

6. The method according to claim 1, wherein: The polyhydroxyalkanoic acid ester is a polymerization product of one or more monomers of hydroxybutyric acid, hydroxyvaleric acid, hydroxycaproic acid, hydroxyheptanoic acid or hydroxyoctanoic acid.

7. The method according to claim 1, wherein: The polyhydroxyalkanoate is selected from one or more of a copolymer of 3-hydroxybutyric acid and 4-hydroxybutyric acid, a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid, or a homopolymer of 4-hydroxybutyric acid.

8. The method according to claim 1, wherein: The Mw of the polyhydroxyalkanoate is 30-300 kDa.

9. The method according to claim 1, wherein: The dispersant includes one or more of polyvinyl alcohol, Tween, carboxymethyl cellulose and its salts, mannitol, gelatin, hyaluronic acid and its salts or collagen.

10. The method of claim 1, wherein: The concentration of the dispersant in the water phase is 0.01-10 wt %.

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