A polyhydroxyalkanoate microsphere composite gel and its preparation method and use

Abstract

The present invention discloses a polyhydroxyalkanoate microsphere composite gel and a preparation method and use thereof. The preparation method of the present invention comprises: adding polyhydroxyalkanoate to an organic solvent to prepare an oil phase; mixing at least two dispersants with water to prepare an aqueous phase; mixing the oil phase and the aqueous phase for emulsification, and then solidifying to prepare microspheres; after mixing CMC gel with water, adding microspheres to keep them suspended to obtain a composite gel. The polyhydroxyalkanoate microsphere composite gel and preparation method of the present invention use PHA with good biocompatibility and biodegradability, which is non-toxic and has no rejection reaction to the human body. In addition, the membrane emulsification preparation method of the present invention can obtain microspheres with controllable particle size uniformity and sphericity, further improving the safety performance of the product, and at the same time, the batch repeatability is better.

Description

Technical Field

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

[0002] With the improvement of material living standards, more and more people will pay more attention to their quality of life and pursuit of beauty. In addition, due to the influence of certain diseases, the muscles and collagen tissues in the human body have different degrees of functional degradation, resulting in skin depression and other problems. This has gradually entered a stage of rapid development in my country's cosmetic surgery industry. At present, there are two main types of injectable beauty products on the market. One is direct injection filling with sodium hyaluronate fillers, and the other is the use of polycaprolactone or poly-lactic acid microspheres as injectable fillers to stimulate muscle and collagen regrowth to achieve regeneration and repair effects.

[0003] However, these materials have some disadvantages during use. For example, sodium hyaluronate degrades quickly and requires frequent injections to maintain its filling effect. In addition, the use of sodium hyaluronate is more likely to cause the following side effects: local skin unevenness, swelling and stiffness, congestion, allergies, infection, etc. When using existing polycaprolactone microsphere beauty products, some patients have experienced inflammation at the injection site and adverse reactions such as facial nerve paralysis. In addition, there is also stimulation of excessive collagen proliferation, resulting in the production of more collagen in a short period of time, and the appearance of nodules or swelling. The degradation intermediate product of polylactic acid microsphere beauty products is lactic acid (strongly acidic, with a dissociation constant of pKa = 3.86 at 25°C), which can cause acidosis when the concentration is too high.

[0004] In order to achieve long-term filling effects, people have tried to use slow-degrading or non-degradable materials to make microspheres as fillers, such as polyvinyl alcohol, polymethyl methacrylate, etc. Although the filling effect of such materials can be significantly prolonged, these materials will also be corroded or degraded over time in the body, and release harmful substances, causing more serious side effects that endanger human health.

[0005] Therefore, there is an urgent need to provide corresponding solutions to the above problems existing in the current human body filling beauty products on the market, as well as the problems existing in the conventional preparation methods of existing biomedical friendly materials (such as polyhydroxyalkanoates), so as to provide a safe and reliable product for the medical beauty field.

[0006] The information in the background technology is only for illustrating the general background of the present invention and should not be regarded as admitting or suggesting in any form that such information constitutes the prior art known to a person skilled in the art. Summary of the invention

[0007] In order to solve at least some of the technical problems in the prior art, the present invention provides a polyhydroxyalkanoate microsphere composite gel and a preparation method and use thereof. Specifically, the present invention includes the following contents.

[0008] One aspect of the present invention provides a polyhydroxyalkanoate microsphere composite gel, which comprises a CMC gel and polyhydroxyalkanoate microspheres dispersed in the gel.

[0009] In certain embodiments, according to the polyhydroxyalkanoate microsphere composite gel of the present invention, the polyhydroxyalkanoate is selected from polyhydroxybutyrate, polyhydroxyvalerate, polyhydroxyhexanoate, poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate), poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), poly(3-hydroxybutyrate-co-4-hydroxybutyrate), and poly(3-hydroxybutyrate-co-3-hydroxyvalerate).

[0010] In certain embodiments, according to the polyhydroxyalkanoate microsphere composite gel of the present invention, the weight average molecular weight of the polyhydroxyalkanoate is 50,000-1,000,000 Daltons.

[0011] The second aspect of the present invention provides a method for preparing a polyhydroxyalkanoate microsphere composite gel, which comprises the following steps:

[0012] (1) adding polyhydroxyalkanoate into an organic solvent to prepare an oil phase;

[0013] (2) mixing at least two dispersants with water to prepare an aqueous phase;

[0014] (3) mixing the oil phase and the water phase for emulsification, and then solidifying to form microspheres;

[0015] (4) After mixing a swellable carrier with water, the microspheres are added to keep them suspended to obtain the composite gel, wherein the swellable carrier is preferably CMC gel.

[0016] In certain embodiments, according to the method for preparing the polyhydroxyalkanoate microsphere composite gel of the present invention, the organic solvent is selected from at least one of N-methylpyrrolidone, dichloromethane, chloroform, dichloroethane, and acetonitrile.

[0017] In certain embodiments, according to the method for preparing the polyhydroxyalkanoate microsphere composite gel of the present invention, in the oil phase, the concentration of the polyhydroxyalkanoate in the organic solvent is 1-250 mg / mL.

[0018] In certain embodiments, according to the method for preparing the polyhydroxyalkanoate microsphere composite gel of the present invention, the at least two dispersants include polyvinyl alcohol and soluble celluloses, and the soluble celluloses are selected from at least one of methyl hydroxyethyl cellulose, hydroxyethyl cellulose, methyl hydroxypropyl cellulose, and hydroxypropyl cellulose.

[0019] In certain embodiments, according to the method for preparing the polyhydroxyalkanoate microsphere composite gel of the present invention, the at least two dispersants include polyvinyl alcohol and gelatin.

[0020] In certain embodiments, according to the method for preparing the polyhydroxyalkanoate microsphere composite gel of the present invention, the particle size of the microsphere is 20-60 μm.

[0021] The third aspect of the present invention provides use of the polyhydroxyalkanoate microsphere composite gel described herein in the preparation of a tissue filler.

[0022] The polyhydroxyalkanoate microsphere composite gel and preparation method of the present invention adopt PHA with good biocompatibility and biodegradability, which is non-toxic and has no rejection reaction to the human body, and can be gradually degraded and excreted from the body with the metabolism of the human body. At the same time, its main degradation product 3-hydroxybutyric acid is weakly acidic (its dissociation constant pKa=4.70 under 25°C, which is lower than lactic acid in acidity), and has no obvious stimulation to the human body. In addition, 3-hydroxybutyric acid is an endogenous substance in the human body and a natural energy source for human cells. It can promote the growth of fibroblasts and the regeneration of collagen, making the regeneration and repair effect more obvious.

[0023] At the same time, the degradation time of PHA materials can be controlled by adjusting parameters such as the molecular weight of the materials, such as from one month to several years. And the membrane emulsification preparation method of the present invention can obtain microspheres with controllable particle size uniformity and sphericity, further improving the safety performance of the product, and at the same time, the batch repeatability is better.

[0024] The polyhydroxyalkanoate microsphere composite gel and preparation method of the present invention also use sodium carboxymethylcellulose (sometimes referred to herein as CMC) as a gel carrier, which has good viscosity and elasticity. After being injected into the surface layer of the skin, wrinkles will be immediately improved. CMC is an ingredient certified by the U.S. Food and Drug Administration (FDA), and its final products are carbon dioxide, water and glucose, which can be fully absorbed and metabolized by the human body, and are safe and have no residue.

[0025] In addition, the existing membrane emulsification preparation method (SPG) uses a single dispersant aqueous solution as the water phase. Not only does it take a long time to solidify at room temperature, but the prepared microspheres are often mixed with flocs, which seriously affects the safety of tissue filling products. The microspheres prepared by the existing mechanical stirring method not only have poor particle size uniformity, but also need to be screened to obtain microspheres in the target particle size range, but after screening, there are still many microspheres with poor sphericity. Different from the existing methods, the preparation method of this civilization has a short curing time, and more importantly, it can reduce the amount of flocs, or even completely eliminate flocs, and the particle size of the particles in the gel is uniform. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Optical microscope image of the microspheres prepared in Preparation Example 1 of the present invention.

[0027] Figure 2 Particle size distribution diagram of the microspheres prepared in Preparation Example 1 of the present invention.

[0028] Figure 3 Optical microscope image of the microspheres prepared in Preparation Example 2 of the present invention.

[0029] Figure 4 Particle size distribution diagram of the microspheres prepared in Preparation Example 2 of the present invention.

[0030] Figure 5 Optical microscope image of the microspheres prepared in Comparative Example 1 of the present invention.

[0031] Figure 6 Particle size distribution diagram of the microspheres prepared in Comparative Example 1 of the present invention.

[0032] Figure 7 Optical microscope image of the microspheres prepared in Comparative Example 2 of the present invention.

[0033] Figure 8 Particle size distribution diagram of the microspheres prepared in Comparative Example 2 of the present invention.

[0034] Fig. 9 Optical microscope image of the microspheres prepared in Comparative Example 3 of the present invention.

[0035] Fig.10 Particle size distribution diagram of the microspheres prepared in Comparative Example 3 of the present invention. DETAILED DESCRIPTION

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0038] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0039] Polyhydroxyalkanoate microsphere composite gel

[0040] One aspect of the present invention provides an injectable polyhydroxyalkanoate microsphere composite gel, which includes CMC gel and polyhydroxyalkanoate microspheres dispersed in the gel. The polyhydroxyalkanoate microspheres refer to microspheres obtained by emulsifying an oil phase containing polyhydroxyalkanoate and an aqueous phase containing at least two dispersants. The present invention finds that the composite gel obtained by mixing such microspheres with a specific carrier (such as CMC gel) has significantly improved properties.

[0041] In the present invention, the type of polyhydroxyalkanoate is not particularly limited, and examples thereof include, but are not limited to, polyhydroxybutyrate, polyhydroxyvalerate, polyhydroxycaproate, poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxycaproate), poly(3-hydroxybutyrate-co-3-hydroxycaproate), poly(3-hydroxybutyrate-co-4-hydroxybutyrate), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), and one or more of the above ingredients may be used in the present invention. In the case of using multiple ingredients, the dosage ratio between the ingredients is not particularly limited, and can be freely proportioned by those skilled in the art as needed.

[0042] The molecular weight of the internal polyester compound in the present invention is not particularly limited, but is generally controlled between 50,000 and 1,000,000 daltons, preferably between 50,000 and 900,000 daltons, and more preferably between 100,000 and 800,000 daltons, for example, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, etc.

[0043] The particle size of the microspheres in the present invention needs to be controlled between 20-60 μm, preferably between 30-50 μm. A particle size of 20-60 μm is necessary for the purpose of the present invention. If the diameter of the microspheres is below 20 μm, they may be swallowed by human cells. On the other hand, if the microspheres are too large, it is not conducive to injection, and in severe cases, it may clog the needle or even cause skin rupture. Although the particle size of the microspheres can vary within the above range, the microspheres of the present invention have a more uniform particle size distribution.

[0044] When the gel used is CMC gel, the viscosity thereof is preferably in the range of 100-6500 MPa·s (1% aqueous solution), more preferably 1000-3000 MPa·s, still more preferably 1000-2000 MPa·s, and most preferably 1200 MPa·s.

[0045] In the composite gel of the present invention, the weight ratio of PHA microspheres to CMC gel is 20-40:60-80, for example, 20:80, 30:70, 40:60, or any weight ratio within the above range. If the weight ratio of PHA microspheres to CMC gel is lower than the above range, for example, the amount of microspheres is too low, the stimulation of collagen regeneration is not obvious. If the weight ratio of PHA microspheres to CMC gel is higher than the above range, for example, the amount of microspheres is too high, the injection process may cause discomfort to the patient. It is understood that as a composite gel, it can optionally include additional carriers or excipients. A non-limiting example of an additional carrier or excipient is mannitol. In the case of mannitol in the composite gel, the ratio of PHA microspheres, CMC gel and mannitol is preferably 25-55:35-75:1-10, and preferably 30-40:50-70:2-8.

[0046] It should be noted that the PHA microspheres in the composite gel of the present invention are prepared by a specific emulsification method rather than a conventional stirring method, which is described in detail below. In addition, the present invention also found that the polyhydroxyalkanoate and at least two dispersants in the PHA microspheres affect the properties of the final microspheres.

[0047] Preparation method

[0048] Another aspect of the present invention provides a method for preparing a polyhydroxyalkanoate microsphere composite gel, which includes but is not limited to the following steps (1)-(4):

[0049] (1) adding polyhydroxyalkanoate into an organic solvent to prepare an oil phase;

[0050] (2) mixing at least two dispersants with water to prepare an aqueous phase;

[0051] (3) mixing the oil phase and the water phase for emulsification, and then solidifying to form microspheres;

[0052] (4) After mixing the CMC gel with water, the microspheres are added to keep them suspended to obtain the composite gel.

[0053] Those skilled in the art will appreciate that the numbers (1), (2), etc. representing the steps are only for the purpose of distinguishing different steps, and do not indicate the order of the steps. As long as the purpose of the present invention can be achieved, the order of the above steps is not particularly limited. In addition, those skilled in the art will also appreciate that other steps or operations may be included between the steps, such as before and after steps (1)-(4), or between these arbitrary steps, for example, to further optimize and / or improve the method described in the present invention.

[0054] In the present invention, step (1) is a step of preparing an oil phase, and step (1) comprises adding polyhydroxyalkanoate to an organic solvent to prepare the oil phase, wherein the concentration of the polyhydroxyalkanoate in the organic solvent is 1-250 mg / mL, preferably 10-100 mg / mL, further preferably 10-60 mg / mL, further preferably 10-30 mg / mL, and most preferably 15 mg / mL. Examples of organic solvents include, but are not limited to, N-methylpyrrolidone, dichloromethane, dichloroethane, chloroform and acetonitrile, and the present invention may use one or more of the above solvents. When using multiple solvents, the amount ratio of each solvent is not particularly limited and can be freely set by a technician as needed.

[0055] The polyhydroxyalkanoate used in the present invention is of medical grade with a purity of 99.99% or more and a weight average molecular weight of 50,000 to 1,000,000 Daltons, preferably 500,000 to 500,000 Daltons, for example, 50,000 to 500,000 Daltons.

[0056] In the present invention, step (2) is a step of preparing an aqueous phase, and step (2) includes mixing at least two dispersants with water to prepare an aqueous phase. Unlike conventional emulsification techniques, the present invention uses at least two dispersants instead of a single dispersant. The inventors found in experiments that when a single dispersant is used for preparation, floccules appear in the prepared microspheres, while the use of at least two dispersants can avoid the influence of floccules. Preferably, the at least two dispersants include polyvinyl alcohol and soluble cellulose or gelatin, wherein the soluble cellulose is selected from at least one of methyl hydroxyethyl cellulose, hydroxyethyl cellulose, methyl hydroxypropyl cellulose, and hydroxypropyl cellulose. Most preferably, the at least two dispersants include polyvinyl alcohol and gelatin.

[0057] In step (2) of the present invention, when one dispersant is polyvinyl alcohol and the other dispersant is gelatin, the concentration of polyvinyl alcohol in the aqueous phase may be 0.5wt%-5wt%, preferably 1wt%-3wt%, and more preferably 1.5%. The concentration of gelatin in the aqueous phase may be 0.5wt%-1.5wt%, preferably 0.8wt%-1.2wt%, and more preferably 1wt%.

[0058] In step (2) of the present invention, the water in the mixed aqueous solution may be, for example, purified water or distilled water, etc., and is not particularly limited.

[0059] In the present invention, step (3) is a step of preparing microspheres by mixing two phases. The volume of the water phase needs to be greater than the volume of the oil phase, and the volume ratio of the oil phase to the water phase is generally 1:2-1:20, preferably 1:5-1:15, and more preferably 1:10.

[0060] In the present invention, the mixing of the oil phase and the aqueous phase can be carried out by a membrane emulsifier. When using a membrane emulsifier for mixing, the emulsification pressure is controlled between 0.01-0.1MPa, preferably between 0.02-0.05MPa. The pore size of the membrane tube can be selected from 5-50μm, preferably 10-20μm, and more preferably 15μm; the mixing and emulsification time is generally between 30-180 minutes, preferably between 30-90 minutes, and more preferably between 60-90 minutes. The stirring speed during mixing and emulsification can be controlled at 120-500rpm, preferably 200-400rpm, and more preferably 200-350r / min. After the emulsification is completed, it can be further solidified.

[0061] In step (3) of the present invention, the curing temperature is 20-85° C., preferably 30-60° C., more preferably 40° C. The curing time is preferably 3-10 hours, preferably 5 hours.

[0062] Step (4) of the present invention is a gel preparation step, wherein a swellable carrier is mixed with water and fully and evenly swelled, and then mannitol and the microspheres of step (3) are added, stirred, and the microspheres are kept in a suspended state. Preferably, the swellable carrier is a gel. Also preferably, the gel is selected from sodium hyaluronate, collagen, CMC, chitosan, sodium alginate, or a combination thereof. Further preferably, the gel used is CMC, and most preferably pharmaceutical grade CMC.

[0063] In step (4) of the present invention, the viscosity of CMC is 100-6500 mPa·s (1% aqueous solution), preferably 1000-3000 mPa·s, more preferably 1000-2000 mPa·s, and most preferably 1200 mPa·s.

[0064] In step (4) of the present invention, the ratio of the microspheres, CMC and mannitol to be mixed is 25-55:35-75:0-10, preferably 30-40:50-70:0-10.

[0065] use

[0066] The present invention also provides the use of injectable polyhydroxyalkanoate microsphere composite gel, preferably for preparing tissue fillers. The microspheres of the present invention have good biocompatibility and biodegradability, and have good cell compatibility with cells in vivo. The polyhydroxyalkanoate microspheres prepared by the present invention are regular and uniform in particle size, and in particular, no flocs are generated, thereby avoiding the influence of flocs on the injection effect of the finished product.

[0067] Preparation Example 1

[0068] This preparation example is the preparation of microspheres, which is specifically as follows:

[0069] 0.6 g of poly (3-hydroxybutyrate-co-4-hydroxybutyrate) white powder (medical grade, purity 99.99%; molecular weight Mw≈250 kDa, Mw / Mn=1.37, Zhuhai Medfa Biotechnology Co., Ltd.) was dissolved in 40 mL of dichloromethane to obtain the oil phase.

[0070] Add 210 mL of purified water to a 500 mL beaker, add 6.5 g of PVA (pharmaceutical grade) under stirring, and quickly stir at 60°C to dissolve into a clear solution. Then, add 210 mL of 2 wt% gelatin solution, stir for 30 minutes, and mix well to obtain the aqueous phase. Cool to room temperature and set aside.

[0071] The oil phase was emulsified into the rotating water phase through a membrane emulsifier, and the volume ratio of the oil phase to the water phase was controlled to be 1:5, the emulsification pressure was 0.04MPa, the membrane tube pore size was 15μm, the emulsification time was 90min, the magnetic length was 5cm, and the emulsification stirring speed was 300rpm. After the emulsification was completed, the oil phase was transferred to a 1000mL beaker, stirred with a 10cm stirring paddle, and then 200mL of water phase was added, and the stirring speed was 120r / min.

[0072] Heat to 40℃, stir at 120r / min and solidify for 5h. Filter, remove the water phase, and wash the balls with 60℃ injection water, wash the balls 6 times, each time for 30min. Take samples and observe the morphology of the microspheres with an optical microscope, such as Figure 1 As shown. Figure 1 It can be seen that no flocs or irregular microspheres were found.

[0073] The obtained microspheres were freeze-dried to remove water using a freeze dryer to obtain about 0.50 g of PHA microspheres with an average particle size of 42.978 μm. The span value (dispersion of particle size distribution) was 0.647. The particle size distribution diagram is shown in Table 1 and Figure 2 As shown; the yield is 83.33%.

[0074] Table 1

[0075]

[0076] This process was repeated for three batches, and no floccules or irregular microspheres were found. The yields were 81.2%, 84.6% and 82%, respectively. The total amount of the four batches of products was 1.99 g.

[0077] Preparation Example 2

[0078] This preparation example is the preparation of microspheres, which is specifically as follows:

[0079] 0.6 g of poly (3-hydroxybutyrate-co-4-hydroxyhexanoate) white powder (medical grade, purity 99.93%; molecular weight Mw≈240 kDa, Mw / Mn=1.42, Zhuhai Medfa Biotechnology Co., Ltd.) was dissolved in 40 mL of dichloromethane to obtain the oil phase.

[0080] 210mL of purified water was added to a 500mL beaker. Under stirring conditions, 12.6g of PVA (pharmaceutical grade) was added. Under stirring conditions at 60°C, the mixture was dissolved into a clear solution. Then, 210mL of 1wt% gelatin solution was added. The mixture was stirred for 30 minutes and mixed evenly to obtain the aqueous phase. The mixture was cooled to room temperature and set aside.

[0081] The oil phase was emulsified into the rotating water phase through a membrane emulsifier, and the volume ratio of the oil phase to the water phase was controlled to be 1:5, the emulsification pressure was 0.04MPa, the membrane tube pore size was 15μm, the emulsification time was 90min, the magnetic length was 5cm, and the emulsification stirring speed was 300rpm. After the emulsification was completed, the oil phase was transferred to a 1000mL beaker, stirred with a 10cm stirring paddle, and then 200mL of water phase was added, and the stirring speed was 120r / min.

[0082] Heat to 40℃, stir at 120r / min and solidify for 5h. Filter, remove the water phase, and wash the balls with 60℃ injection water, wash the balls 6 times, each time for 30min. Take samples and observe the morphology of the microspheres with an optical microscope, such as Figure 3 As shown. Figure 3 It can be seen that no flocs or irregular microspheres were found.

[0083] The obtained microspheres were freeze-dried to remove moisture using a freeze dryer to obtain about 0.48 g of PHA microspheres with an average particle size of 43.036 μm, a span value of 0.653, and a particle size distribution diagram as shown in FIG. Figure 4 As shown in Table 2, the yield was 80.0%.

[0084] Table 2

[0085]

[0086] This process was repeated for three batches, and no floccules or irregular microspheres were found. The yields were 82.2%, 83.6% and 84.7%, respectively. The total amount of the four batches of products was 1.98 g.

[0087] Example 1

[0088] This example is the preparation of composite gel, which is specifically as follows:

[0089] Take 15 g of CMC (medical grade) and stir it thoroughly with sterile water for injection to make it swell evenly, and adjust the viscosity to 1200 mPa·s to obtain CMC gel.

[0090] Take 1g of the PHA microspheres prepared in Preparation Example 1, mix the PHA microspheres and CMC gel in a weight ratio of 30:70, and stir for 2 hours until a suspension is uniformly formed. Then, fill it into a vial or a prefilled needle and sterilize it with ethylene oxide.

[0091] Pushing force test: Use a 1 ml syringe to extract the suspension after standing, add a 26G needle, and use a microcomputer-controlled electronic universal testing machine to test the pushing force of the skin filler. The pushing force is maintained at 10N-30N. It can be seen that the skin filler of this embodiment can be pushed smoothly.

[0092] The composite PHA microsphere gel of this embodiment did not show aggregation and precipitation, and could still maintain a suspension state after being placed for two weeks. Using a push-pull force tester to test, the pushing force could be maintained at 10-30N, and the injection and pushing were smooth.

[0093] Example 2

[0094] Take 15 g of CMC (medical grade) and stir it thoroughly with sterile water for injection to make it swell evenly, and adjust the viscosity to 1200 mPa·s to obtain CMC gel.

[0095] Take 1g of the PHA microspheres prepared in Preparation Example 1, mix the PHA microspheres, CMC gel and mannitol in a weight ratio of 30:60:10, and stir for 2 hours until a suspension is uniformly formed. Then, fill it into a vial or a pre-filled needle and sterilize it with ethylene oxide.

[0096] Pushing force test: Use a 1 ml syringe to extract the suspension after standing, add a 26G needle, and use a microcomputer-controlled electronic universal testing machine to test the pushing force of the skin filler. The pushing force is kept in the range of 10N-30N. It can be seen that the skin filler of this embodiment can be pushed smoothly.

[0097] The composite PHA microsphere gel of this embodiment did not show aggregation and precipitation, and could still maintain a suspension state after being placed for two weeks. Using a push-pull force tester to test, the pushing force could still be maintained in the range of 10-30N, and the injection and pushing were smooth.

[0098] Example 3

[0099] Take 15 g of CMC (medical grade) and stir it thoroughly with sterile water for injection to make it swell evenly, and adjust the viscosity to 1200 mPa·s to obtain CMC gel.

[0100] Take 1g of the PHA microspheres prepared in Preparation Example 2, mix the PHA microspheres and CMC gel in a weight ratio of 30:70, and stir for 2 hours until a suspension is uniformly formed. Then, fill it into a vial or a pre-filled needle and sterilize it with ethylene oxide.

[0101] Pushing force test: Use a 1 ml syringe to extract the suspension after standing, add a 26G needle, and use a microcomputer-controlled electronic universal testing machine to test the pushing force of the skin filler. The pushing force is maintained at 10N-30N. It can be seen that the skin filler of this embodiment can be pushed smoothly.

[0102] The composite PHA microsphere gel of this embodiment can still maintain a suspension state after being placed for two weeks, and no aggregation and precipitation occurs. Using a push-pull force tester to test, the pushing force can be maintained at 10-30N, and the injection and pushing are smooth.

[0103] Example 4

[0104] Take 15 g of CMC (medical grade) and stir it thoroughly with sterile water for injection to make it swell evenly, and adjust the viscosity to 1200 mPa·s to obtain CMC gel.

[0105] Take 1g of the PHA microspheres prepared in Preparation Example 2, mix them in a weight ratio of 30:60:10 for PHA microspheres, CMC gel and mannitol, and stir for 2h until a suspension is formed. Then, fill them into vials or prefilled needles and sterilize them with ethylene oxide.

[0106] Pushing force test: Use a 1 ml syringe to extract the suspension after standing, add a 26G needle, and use a microcomputer-controlled electronic universal testing machine to test the pushing force of the skin filler. The pushing force is kept in the range of 10N-30N. It can be seen that the skin filler of this embodiment can be pushed smoothly.

[0107] The composite PHA microsphere gel of this embodiment can still maintain a suspension state after being placed for two weeks, and no aggregation and precipitation occurs. The pushing force can still be maintained in the range of 10-30N when tested by a push-pull force tester, and the injection and pushing are smooth.

[0108] Comparative Example 1

[0109] This comparative example is the preparation of microspheres. The difference from Preparation Example 1 is that only one dispersant PVA is used in the preparation process of the aqueous phase, as shown below:

[0110] 0.6 g of poly (3-hydroxybutyrate-co-4-hydroxybutyrate) white powder (medical grade, purity 99.99%; molecular weight Mw≈250 kDa, Mw / Mn=1.37, Zhuhai Medfa Biotechnology Co., Ltd.) was dissolved in 40 mL of dichloromethane to obtain the oil phase.

[0111] Add 420 mL of purified water to a 500 mL beaker, add 6.5 g of PVA (pharmaceutical grade) under stirring, and quickly stir at 60°C to dissolve into a clear solution. Cool to room temperature and set aside.

[0112] The oil phase was emulsified into the rotating water phase through a membrane emulsifier, and the volume ratio of the oil phase to the water phase was controlled to be 1:5, the emulsification pressure was 0.04MPa, the membrane tube pore size was 15μm, and the emulsification time was 90min; the magnetic length was 5cm, and the emulsification stirring speed was 300rpm. After the emulsification was completed, the oil phase was transferred to a 1000mL beaker, stirred with a 10cm stirring paddle, and then 200mL of water phase was added, the stirring speed was 120r / min, and stirred at room temperature for 24h.

[0113] Filter the water phase and wash the microspheres with 60℃ injection water for 6 times, 30 minutes each time. Take samples and observe the microsphere morphology with an optical microscope. Figure 5 As shown. Figure 5 It can be seen that no irregular microspheres were found, but floccules were produced. The produced floccules may affect the injection effect of the finished product and block the needle.

[0114] The obtained microspheres were freeze-dried to remove moisture using a freeze dryer to obtain about 0.45 g of PHA microspheres with an average particle size of 52.568 μm (D (4,3) value), a span value of 0.681, and a particle size distribution diagram as shown in FIG. Figure 6 As shown in Table 3, the yield was 75.0%. In addition, due to the generation of floccules, the average particle size detected was relatively larger.

[0115] Table 3

[0116]

[0117] Comparative Example 2

[0118] This comparative example is the preparation of microspheres. The difference from Preparation Example 1 is that the amount of gelatin added during the preparation of the aqueous phase is different, as shown below:

[0119] 0.6 g of poly (3-hydroxybutyrate-co-4-hydroxybutyrate) white powder (medical grade, purity 99.99%; molecular weight Mw≈250 kDa, Mw / Mn=1.37, Zhuhai Medfa Biotechnology Co., Ltd.) was dissolved in 40 mL of dichloromethane to obtain the oil phase.

[0120] Add 210 mL of purified water to a 500 mL beaker, add 6.5 g of PVA (pharmaceutical grade) under stirring, and quickly stir at 60°C to dissolve into a clear solution. Then, add 210 mL of 4 wt% gelatin solution, stir for 30 minutes, and mix well to obtain the aqueous phase. Cool to room temperature and set aside.

[0121] The oil phase was emulsified into the rotating water phase through a membrane emulsifier, and the volume ratio of the oil phase to the water phase was controlled to be 1:5, the emulsification pressure was 0.04MPa, the membrane tube pore size was 15μm, the emulsification time was 90min, the magnetic length was 5cm, and the emulsification stirring speed was 300rpm. After the emulsification was completed, the oil phase was transferred to a 1000mL beaker, stirred with a 10cm stirring paddle, and then 200mL of water phase was added, and the stirring speed was 120r / min.

[0122] Heat to 40℃, stir at 120r / min and solidify for 5h. Filter, remove the water phase, and wash the balls with 60℃ injection water, wash the balls 6 times, each time for 30min. Take samples and observe the morphology of the microspheres with an optical microscope, such as Figure 7 As shown. Figure 7 It can be seen that no flocs or irregular microspheres were found, but some small-sized microspheres appeared.

[0123] The obtained microspheres were freeze-dried to remove moisture using a freeze dryer to obtain about 0.47 g of PHA microspheres with an average particle size of 28.680 μm (D (4,3) value) and a span value of 0.885. The particle size distribution diagram is shown in FIG. Figure 8 As shown in the table below, the yield is 79.0%. A higher span value makes the particle size distribution of the microspheres in this comparative example wider.

[0124] Table 4

[0125]

[0126] Comparative Example 3

[0127] This comparative example is the preparation of microspheres. The difference from Preparation Example 1 is that the microspheres are subsequently prepared by a conventional stirring method, as shown below:

[0128] 0.6 g of poly (3-hydroxybutyrate-co-4-hydroxybutyrate) white powder (medical grade, purity 99.99%; molecular weight Mw≈250 kDa, Mw / Mn=1.37, Zhuhai Medfa Biotechnology Co., Ltd.) was dissolved in 40 mL of dichloromethane to obtain the oil phase.

[0129] Add 210 mL of purified water to a 500 mL beaker, add 6.5 g of PVA (pharmaceutical grade) under stirring, and quickly stir at 60°C to dissolve into a clear solution. Then, add 210 mL of 2 wt% gelatin solution, stir for 30 minutes, and mix well to obtain the aqueous phase. Cool to room temperature and set aside.

[0130] According to the ratio of oil phase to water phase of 1:5, after adding the oil phase to the water phase, stir at 350rpm for 10 minutes using the traditional stirring method to disperse the oil phase into oil droplets with suitable and uniform particle size to form a stable emulsion. Maintain the speed of 250rpm and continue stirring at room temperature for 24 hours. Filter and filter out the water phase, and wash the balls with 60℃ injection water, washing the balls 6 times, each time for 30 minutes. Sieve with stainless steel sieves with pore sizes of 20μm and 60μm, and take products with a particle size range of 20μm-60μm. After screening, use an optical microscope to observe the morphology of the microspheres, such as Fig. 9 As shown. Fig. 9 It can be seen that even after screening, there are still a lot of irregular microspheres remaining in the product.

[0131] The microspheres were sieved using stainless steel sieves with pore sizes of 60 μm and 20 μm, and freeze-dried using a freeze dryer to remove moisture, obtaining 0.24 g of PHA microspheres with a particle size range of 20-60 μm, with a yield of 40.0%. The particle size distribution test results are shown in Fig.10 and as shown in the table below.

[0132]

[0133] Comparative Example 4

[0134] This comparative example is the preparation of the composite gel, which is specifically as follows:

[0135] Take 15 g of CMC (medical grade) and stir it thoroughly with sterile water for injection to make it swell evenly, and adjust the viscosity to 7000 mPa·s to obtain CMC gel.

[0136] Take 1g of the PHA microspheres prepared in Preparation Example 1, mix the PHA microspheres and CMC gel in a weight ratio of 30:70, and stir for 2 hours until a suspension is uniformly formed. Then, fill it into a vial or a prefilled needle and sterilize it with ethylene oxide.

[0137] Pushing force test: Use a 1 ml syringe to extract the static suspension, add a 26G needle, and use a microcomputer-controlled electronic universal testing machine to test the pushing force of the skin filler. The pushing force is 45N, which is difficult to inject.

[0138] Although the present invention has been described with reference to exemplary embodiments, it should be understood that the present invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments of the present specification without departing from the scope or spirit of the present invention. The scope of the claims should be based on the broadest interpretation to cover all modifications and equivalent structures and functions.

Claims

1. A polyhydroxyalkanoate microsphere composite gel, characterized in that: The invention comprises CMC gel and polyhydroxyalkanoate microspheres dispersed in the gel, wherein the polyhydroxyalkanoate microsphere composite gel is prepared by the following steps: (1) adding polyhydroxyalkanoate to an organic solvent to prepare an oil phase, wherein the weight average molecular weight of the polyhydroxyalkanoate is 150,000-1,000,000 daltons; (2) mixing at least two dispersants with water to prepare an aqueous phase, wherein the at least two dispersants include polyvinyl alcohol and gelatin, wherein the concentration of polyvinyl alcohol in the aqueous phase is 0.5-5wt%, and the concentration of gelatin in the aqueous phase is 0.5-1.5wt%; (3) mixing the oil phase and the water phase for emulsification, and then solidifying to form microspheres, wherein the mixing of the oil phase and the water phase is performed by a membrane emulsifier; (4) After CMC is mixed with water to obtain CMC gel, the microspheres are added to keep them suspended to obtain the composite gel, wherein the viscosity of the CMC gel in a 1% aqueous solution is 100-6500 mPa·s.

2. The polyhydroxyalkanoate microsphere composite gel according to claim 1, characterized in that: The polyhydroxyalkanoic acid ester is obtained by polymerizing at least one monomer selected from hydroxybutyric acid, hydroxyvaleric acid, hydroxycaproic acid, hydroxyheptanoic acid, and hydroxyoctanoic acid.

3. A method for preparing a polyhydroxyalkanoate microsphere composite gel, characterized in that: The following steps are involved: (1) adding polyhydroxyalkanoate to an organic solvent to prepare an oil phase, wherein the weight average molecular weight of the polyhydroxyalkanoate is 150,000-1,000,000 daltons; (2) mixing at least two dispersants with water to prepare an aqueous phase, wherein the at least two dispersants include polyvinyl alcohol and gelatin, wherein the concentration of polyvinyl alcohol in the aqueous phase is 0.5-5wt%, and the concentration of gelatin in the aqueous phase is 0.5-1.5wt%; (3) mixing the oil phase and the water phase for emulsification, and then solidifying to form microspheres, wherein the mixing of the oil phase and the water phase is performed by a membrane emulsifier; (4) After CMC is mixed with water to obtain CMC gel, the microspheres are added to keep them suspended to obtain the composite gel, wherein the viscosity of the CMC gel in a 1% aqueous solution is 100-6500 mPa·s.

4. The method for preparing the polyhydroxyalkanoate microsphere composite gel according to claim 3, characterized in that: The organic solvent is selected from at least one of N-methylpyrrolidone, dichloromethane, chloroform, dichloroethane and acetonitrile.

5. The method for preparing the polyhydroxyalkanoate microsphere composite gel according to claim 3, characterized in that: The concentration of the polyhydroxyalkanoate in the organic solvent is 1-250 mg / mL.

6. The method for preparing the polyhydroxyalkanoate microsphere composite gel according to claim 3, characterized in that: The weight ratio of the microspheres to the CMC gel is 20-40:60-80.

7. The method for preparing the polyhydroxyalkanoate microsphere composite gel according to claim 3, characterized in that: The particle size of the microspheres is 20-60 μm.

8. Use of the polyhydroxyalkanoate microsphere composite gel according to claim 1 or 2 in the preparation of a tissue filler.

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