Pha combination microspheres and preparation method and application thereof

Abstract

The application relates to the technical field of biomaterials, and particularly discloses a PHA combined microsphere as well as a preparation method and application thereof. The PHA combined microsphere provided by the application contains 10-40 wt% of PHA microspheres with a weight average molecular weight of less than 30000 Da and 1-40 wt% of PHA microspheres with a weight average molecular weight of more than 100000 Da, with the PHA being selected from one or more of poly-beta-hydroxybutyric acid, a copolymer of 3-hydroxybutyric acid ester and 3-hydroxyvaleric acid ester, a copolyester of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid and poly(3-hydroxybutyric acid-co-4-hydroxybutyric acid). After the PHA combined microsphere is filled in the skin, on one hand, the PHA combined microsphere can be rapidly degraded in an early stage and promote the reproduction of fibroblasts and endothelial cells in a short time; on the other hand, the PHA combined microsphere can be continuously degraded within 21 weeks and cannot cause inflammation, so that the effect of persistent filling is achieved.

Description

Technical Field

[0001] This application relates to the technical field of biomaterials, specifically to a PHA composite microsphere, its preparation method, and its application. Background Technology

[0002] As we age or are affected by certain diseases, the muscles and collagen tissues in the human body undergo varying degrees of functional degeneration, leading to problems such as skin depressions. Over the years, scientists have invented various biological fillers, such as hyaluronic acid, bovine collagen, and biodegradable polyester materials, to fill in sunken skin or stimulate muscle and collagen regeneration. However, the filling effects of hyaluronic acid and bovine collagen are relatively short-lived, often requiring frequent re-injections to maintain their effect, and they are prone to adverse reactions such as swelling and bruising. In contrast, biodegradable polyester materials have advantages such as good biocompatibility, a long degradation cycle, and excellent long-lasting filling effects, making them a very ideal dermal filler.

[0003] Polyhydroxyalkanoates (PHA) are polyester materials used as dermal fillers. They possess good biocompatibility, low toxicity, and immunogenicity, and can completely degrade under physiological conditions, with the main degradation product being 3HB. Related studies have found that 3HB is a cellular energy substance, and maintaining a 3HB concentration of 30-100 mg / L can effectively promote the proliferation of fibroblasts and endothelial cells. However, existing PHA microspheres exhibit excessively slow early degradation rates, with early 3HB concentrations <30 mg / L, making it difficult to rapidly promote cell proliferation and collagen regeneration in a short period. Degradation typically takes at least one year. Furthermore, due to the long degradation cycle of PHA microspheres, accelerated degradation in later stages leads to acid accumulation, which can easily trigger acute inflammation and granuloma formation, causing additional harm to the body.

[0004] Therefore, there is an urgent need to provide a PHA microsphere that can rapidly degrade in the early stages and continuously degrade over a long period without inducing inflammation. Summary of the Invention

[0005] To achieve rapid early degradation and long-term sustainable and stable degradation of PHA microspheres, this application provides a PHA composite microsphere, its preparation method, and its application.

[0006] In the first aspect, this application provides a PHA composite microsphere, which adopts the following technical solution:

[0007] A PHA composite microsphere, wherein the PHA composite microsphere comprises 10-40 wt% PHA microspheres with a weight average molecular weight of less than 30,000 Da and 1-40 wt% PHA microspheres with a weight average molecular weight of more than 100,000 Da.

[0008] The PHA composite microspheres provided in this application are prepared by mixing PHA microspheres of different molecular weights. By controlling the content of each molecular weight of PHA microsphere in the PHA composite microspheres within the above-mentioned range, the degradation cycle of the obtained PHA composite microspheres is controlled at about 21 weeks (18-22 weeks). This cycle can fully meet the needs of fibroblast and endothelial cell proliferation and collagen regeneration, achieving a long-lasting filling effect; on the other hand, it can basically achieve complete degradation.

[0009] Furthermore, PHA microspheres with different molecular weights exhibit different degradation behaviors. By controlling the molecular weight distribution within the PHA composite microspheres, a gradient in the degradation behavior of the PHA composite microspheres can be created. Specifically, the PHA composite microspheres of this application exhibit the following degradation behaviors: within a 1-week degradation period, the concentration of 3HB is 30 mg / L or higher; within a degradation period of 2-18 weeks, the concentration of 3HB is 30-100 mg / L; and within a degradation period of 20-22 weeks, the concentration of 3HB is 30 mg / L or lower.

[0010] The concentration of the degradation product 3HB of PHA in this application remains stable in the range of 30-100 mg / L during the degradation cycle of 2-18 weeks. It can continuously and effectively promote the proliferation of fibroblasts and endothelial cells and the regeneration of collagen, and does not cause acid accumulation in the later stage of degradation, thus effectively avoiding the occurrence of inflammation.

[0011] In the PHA microspheres provided in this application, the low molecular weight PHA microspheres can rapidly degrade within one week, and the degradation product 3HB, as a cellular energy substance, can rapidly promote the proliferation of fibroblasts and endothelial cells and the regeneration of collagen in the early stages; the high molecular weight PHA microspheres will degrade slowly over time, thereby achieving a long-lasting filling effect.

[0012] Preferably, based on 100wt% of the PHA composite microspheres, the PHA composite microspheres contain 10-30wt% of PHA microspheres with a weight average molecular weight of less than 30,000 Da and 5-30wt% of PHA microspheres with a weight average molecular weight of more than 100,000 Da.

[0013] In some embodiments, the PHA composite microspheres, based on 100wt%, comprise 10-40wt% of PHA microspheres with a weight-average molecular weight of 10,000 Da, 20-70wt% of PHA with a weight-average molecular weight of 60,000 Da, and 1-40wt% of PHA microspheres with a weight-average molecular weight of 130,000 Da.

[0014] In this application, the PHA is selected from one or more of poly-β-hydroxybutyric acid, copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate, copolyesters of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid, and poly(3-hydroxybutyrate-co-4-hydroxybutyrate).

[0015] Preferably, the PHA is poly(3-hydroxybutyrate-co-4-hydroxybutyrate), and the content of 4-hydroxybutyrate in the poly(3-hydroxybutyrate-co-4-hydroxybutyrate) is 8-10 mol%.

[0016] In this application, poly(3-hydroxybutyrate-co-4-hydroxybutyrate) is polymerized from 4-hydroxybutyrate and 3-hydroxybutyrate monomers. 3-hydroxybutyrate is a hard material with high brittleness, while 4-hydroxybutyrate has good flexibility. Therefore, this application uses 4-hydroxybutyrate to modify 3-hydroxybutyrate. By controlling the content of 4-hydroxybutyrate within the above-mentioned range, the brittleness of 3-hydroxybutyrate can be reduced, so that the prepared PHA microspheres have good elasticity and can still recover to their original shape after pressure, which is convenient for subsequent injection filling.

[0017] In one specific embodiment, the content of 4-hydroxybutyrate in the poly(3-hydroxybutyrate-co-4-hydroxybutyrate) can be 6 mol%, 8 mol%, 9 mol%, or 10 mol%.

[0018] Preferably, the particle size of the PHA microspheres is 30-50 μm.

[0019] In this application, when the particle size of PHA microspheres is too small, they are easily phagocytosed by cells; when the particle size of PHA microspheres is too large, they are not conducive to injection. Therefore, this application controls the particle size of PHA microspheres within the above-mentioned range, so that the resulting PHA microspheres can be injected smoothly and stably exert their promoting effects on cell proliferation and collagen regeneration.

[0020] Secondly, this application provides a method for preparing PHA composite microspheres.

[0021] A method for preparing PHA composite microspheres includes the following steps: preparing PHA microspheres with different weight-average molecular weights respectively, and then mixing the PHA microspheres with different weight-average molecular weights evenly to obtain PHA composite microspheres.

[0022] The preparation method of the PHA microspheres is as follows: PHA powder is dissolved in an organic solvent, and then a polyvinyl alcohol aqueous solution with a concentration of 0.5-1.5 wt% is added. After stirring for 3-6 hours, the mixture is heated, washed, filtered, and freeze-dried to obtain PHA microspheres.

[0023] Preferably, the organic solvent is selected from one or more of dichloromethane, N-methylpyrrolidone, trichloromethane, and acetonitrile.

[0024] Preferably, the volume ratio of the organic solvent to the polyvinyl alcohol aqueous solution is 1:(3-200).

[0025] Thirdly, the application of the PHA combined microspheres provided in this application in the preparation of tissue fillers or medical aesthetic products.

[0026] The PHA microspheres provided in this application can be used to prepare dermal fillers. When the dermal filler is injected into human skin tissue, fibroblasts in the skin tissue form connective tissue (fibrotic collagen capsules) that encapsulates the PHA microspheres. The connective tissue is then gradually replaced by fibroblasts, generating a large number of collagen fibers that are deposited on each PHA microsphere. Ultimately, each PHA microsphere is separated by collagen fibers, which promotes the formation of a regular network of regenerated collagen fibers around the PHA microspheres to fix them in place. This achieves a long-lasting filling effect through collagen regeneration and deposition, while the PHA microspheres inside completely degrade over time, eliminating the need for a second surgical removal.

[0027] In summary, this application has the following beneficial effects:

[0028] 1. The PHA composite microspheres provided in this application can form a gradient degradation during the degradation cycle according to the different molecular weights of the PHA microspheres, so that the concentration of the PHA degradation product 3HB can be kept stable in the range of 30-100 mg / L. This can continuously and effectively promote the proliferation of fibroblasts and endothelial cells and the regeneration of collagen, and will not cause acid accumulation in the later stage of degradation, thus effectively avoiding the occurrence of inflammation.

[0029] 2. This application, through degradation experiments, found that by further selecting poly(3-hydroxybutyrate-co-4-hydroxybutyrate) as the PHA raw material, the concentration of the degradation product 3HB in the obtained PHA composite microspheres can be maintained between 50-80 mg / L within 4-18 weeks, and the pH is also maintained within the range of 4.9-5.5. This slightly acidic environment is similar to human skin, therefore the PHA composite microspheres will not cause inflammatory reactions. Furthermore, this application controls the 4-hydroxybutyrate content in poly(3-hydroxybutyrate-co-4-hydroxybutyrate) to within the range of 8-10 mol%, which enables the prepared PHA composite microspheres to have good elasticity and can still recover their original shape after pressure, facilitating subsequent injection filling. Attached Figure Description

[0030] Figure 1 This is a scanning electron microscope image of the PHA combined microspheres provided in Example 1 at 300x magnification.

[0031] Figure 2 This is a scanning electron microscope image of the PHA combined microspheres provided in Example 1 at 500x magnification.

[0032] Figure 3 This is a scanning electron microscope image of the PHA combined microspheres provided in Example 1 at 1000x magnification.

[0033] Figure 4 This is a graph showing the concentration change of 3HB, the degradation product of PHA combined microspheres provided in Examples 1 and 9-11. Detailed Implementation

[0034] This application provides a PHA composite microsphere, wherein, based on 100 wt% of the PHA composite microsphere, the PHA composite microsphere comprises 10-40 wt% of PHA microspheres with a weight average molecular weight below 30,000 Da and 1-40 wt% of PHA microspheres with a weight average molecular weight above 100,000 Da. Further, based on 100 wt% of the PHA composite microsphere, the PHA composite microsphere comprises 10-30 wt% of PHA microspheres with a weight average molecular weight below 30,000 Da and 5-30 wt% of PHA microspheres with a weight average molecular weight above 100,000 Da.

[0035] The PHA is selected from one or more of poly-β-hydroxybutyrate, copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate, copolyesters of 3-hydroxybutyrate and 3-hydroxyhexanoate, and poly(3-hydroxybutyrate-co-4-hydroxybutyrate). Preferably, the PHA is poly(3-hydroxybutyrate-co-4-hydroxybutyrate), and the content of 4-hydroxybutyrate in the poly(3-hydroxybutyrate-co-4-hydroxybutyrate) is 8-10 mol%.

[0036] This application provides a method for preparing PHA composite microspheres, comprising the following steps:

[0037] (1) Dissolve PHA powder in an organic solvent at 50-70℃ for 20-40 min. Then add a 0.5-1.5 wt% polyvinyl alcohol aqueous solution to the system, stir for 3-6 h, heat to remove the organic solvent, and then wash with purified water 3-5 times to remove the polyvinyl alcohol. Filter and collect the filter cake, and freeze-dry the filter cake for 20-30 h. After sieving, obtain PHA microspheres with a particle size of 30-50 μm. The amount of organic solvent used is calculated based on the solubility of PHA in the corresponding organic solvent. The solubility of PHA in dichloromethane is approximately 35-45 g / L. The volume ratio of organic solvent to polyvinyl alcohol aqueous solution is 1:(3-200). Prepare PHA microspheres with different weight-average molecular weights according to the above method.

[0038] (2) Mix the PHA microspheres of each weight average molecular weight evenly to obtain the PHA composite microspheres;

[0039] In this application, all PHA powders were purchased from Zhuhai Medbio Biotechnology Co., Ltd.; other reagents and solvents were all commercially available.

[0040] The present application will be further described in detail below with reference to embodiments, performance testing tests and accompanying drawings.

[0041] Examples 1-5

[0042] Examples 1-5 each provide a PHA composite microsphere.

[0043] The difference in the above embodiments is that the amount of each weight-average molecular weight PHA microsphere added to the PHA composite microspheres is shown in Table 1.

[0044] In the above embodiments, PHA is poly(3-hydroxybutyrate-co-4-hydroxybutyrate); wherein, the content of 4-hydroxybutyrate is 9wt%.

[0045] The preparation method of the PHA composite microspheres provided in Examples 1-5 is as follows:

[0046] (1) Dissolve 5g of PHA powder with a weight average molecular weight of 10000 Da in 125mL of dichloromethane at 60℃ for 30min. Then add 1wt% polyvinyl alcohol aqueous solution to the system, stir for 4h, heat to remove dichloromethane, wash three times with purified water to remove polyvinyl alcohol, filter and collect the filter cake, freeze-dry the filter cake for 24h; after sieving, obtain PHA microspheres with a particle size of 30-50μm and a weight average molecular weight of 10000 Da. Prepare PHA microspheres with a weight average molecular weight of 60000 Da and PHA microspheres with a weight average molecular weight of 130000 Da according to the above method.

[0047] (2) PHA microspheres with a weight average molecular weight of 10,000 Da, PHA microspheres with a weight average molecular weight of 60,000 Da, and PHA microspheres with a weight average molecular weight of 130,000 Da are mixed evenly according to the addition amounts in Table 1 to obtain PHA composite microspheres.

[0048] Table 1. Amounts of PHA microspheres of various molecular weights added in the PHA composite microspheres provided in Examples 1-5

[0049]

[0050] Examples 6-8

[0051] Examples 6-8 each provide a PHA composite microsphere.

[0052] Examples 6-8 were carried out according to the method of Example 1, except that the molecular weight of the PHA microspheres in the PHA composite microspheres is shown in Table 2.

[0053] PHA microspheres with a weight average molecular weight of 3000 Da, 30000 Da, 100000 Da, and 200000 Da were prepared according to the preparation method of PHA microspheres with a weight average molecular weight of 10000 Da in Example 1.

[0054] Table 2. Molecular weight and dosage of PHA microspheres in the PHA composite microspheres provided in Examples 1 and 6-8.

[0055]

[0056] Examples 9-11

[0057] Examples 9-11 each provide a PHA composite microsphere.

[0058] Examples 9-11 were carried out according to the method of Example 1, except that the type of PHA in the PHA composite microspheres was as shown in Table 3.

[0059] Table 3. Types of PHA in the PHA composite microspheres provided in Examples 1 and 9-11

[0060]

[0061] Examples 12-14

[0062] Examples 12-14 each provide a PHA composite microsphere.

[0063] Examples 12-14 were carried out according to the method of Example 1, except that the content of 4-hydroxybutyrate in poly(3-hydroxybutyrate-co-4-hydroxybutyrate) was as shown in Table 4.

[0064] Table 4. 4-Hydroxybutyrate content in medium-polymer (3-hydroxybutyrate-co-4-hydroxybutyrate) in Examples 1, 12-14

[0065]

[0066] Comparative Examples 1-6

[0067] Comparative Examples 1-6 each provide a PHA composite microsphere.

[0068] Comparative Examples 1-6 were carried out according to the method of Example 1, except that the PHA microspheres in the PHA composite microspheres and the amount added are shown in Table 5.

[0069] Table 5. PHA microspheres and their dosage in the PHA composite microspheres provided in Examples 1-6

[0070]

[0071] Performance testing

[0072] The PHA composite microspheres obtained in Examples 1-14 and Comparative Examples 1-6 were subjected to simulated degradation experiments, as detailed below:

[0073] 1. Experimental method: Accurately weigh 1g of each of the above-mentioned microsphere combinations and place them in 15ml of physiological saline containing 1mol / L lipase. Seal and shake at a constant temperature of 37℃. Replace the physiological saline once after the first week and the second week to obtain the extract. Replace the physiological saline once every 2 weeks thereafter for 22 weeks.

[0074] 2. Detection method: The concentration of 3HB and pH value of the replaced extract were measured.

[0075] (1) Detection method of 3HB concentration: The 3HB concentration was detected using an LC-20 high performance liquid chromatograph (Shimadzu Laboratory Equipment Co., Ltd., Japan). The detector was a RID-10A refractive index differential detector (Shimadzu Laboratory Equipment Co., Ltd., Japan). The chromatographic column was an Aminex HPX-87H sugar detection column (Bio-Ray Biotech Co., Ltd., USA). The mobile phase was 5 mM sulfuric acid solution, the flow rate was 0.5 mL / min, the column temperature was 55 ℃, and the sample loading volume was 30 μL. The results of 3HB concentration detection are shown in Table 6.

[0076] (2) pH value: pH value was measured using a pH meter. The pH value test results are shown in Table 7.

[0077] Table 6. Concentration of 3HB in the extracts of Examples 1-14 and Comparative Examples 1-6

[0078]

[0079] Table 7. pH values ​​of the extracts from Examples 1-14 and Comparative Examples 1-6

[0080]

[0081] According to the test results in Tables 6 and 7, the PHA microspheres prepared in Examples 1-14 of this application have already degraded in the second week. The 3HB concentration in the extract in the first week has reached 30 mg / L or above, and the pH is 6.5 or below, indicating that the above-mentioned PHA microspheres have a relatively fast early degradation rate. When the PHA microspheres are injected into the skin, they can take effect and play a role within the first week, rapidly promoting the proliferation of fibroblasts and endothelial cells.

[0082] The degradation of the PHA combination microspheres in Comparative Example 1 mainly occurred in the early and middle stages. At 16 weeks, the 3HB concentration was already below 30 mg / L, indicating that the PHA combination microspheres had been almost completely degraded. Therefore, the durable filling effect of the PHA combination microspheres was poor.

[0083] The degradation of the PHA combination microspheres in Comparative Example 2 mainly occurred in the middle and late stages. At 22 weeks, the concentration of 3HB from degradation was still as high as 64 mg / L, indicating that although the PHA combination microspheres had good persistent filling effect, the degradation cycle was too long and it was difficult to achieve complete degradation in about 21 weeks.

[0084] The degradation of the PHA combination microspheres in Comparative Example 3 mainly occurred in the middle and late stages. The 3HB concentration of degradation in the first week was only 15 mg / L, indicating that the degradation rate of the PHA combination microspheres in the early stage was relatively slow. Therefore, when the PHA combination microspheres were injected into the skin, they had a slow onset of action and were difficult to exert their effects quickly.

[0085] In Comparative Example 4, the concentration of 3HB in the extract during weeks 6-8 exceeded 100 mg / L, and the pH was < 4.2, indicating that the degradation rate of the PHA combination microspheres in Comparative Example 4 was too fast during weeks 6-8, which easily caused acid accumulation in the skin. When the above PHA combination microspheres were injected into the skin, they were very likely to cause inflammation during weeks 6-8.

[0086] In Comparative Example 5, the concentration of 3HB in the extract during weeks 8-10 exceeded 100 mg / L, and the pH was < 4.2, indicating that the degradation rate of the PHA combination microspheres in Comparative Example 5 was too fast during weeks 8-10, which easily caused acid accumulation in the skin. Injecting the above PHA combination microspheres into the skin could easily cause inflammation during weeks 8-10.

[0087] In Comparative Example 6, the concentration of 3HB in the extract at week 18 exceeded 100 mg / L, and the pH was < 4.2, indicating that the degradation rate of the PHA combination microspheres in Comparative Example 6 was too fast at week 18, which easily caused acid accumulation in the skin; and at week 22, the concentration of 3HB from degradation was still as high as 75 mg / L, indicating that the degradation cycle of the PHA combination microspheres was too long and it was difficult to achieve complete degradation at around week 21.

[0088] Detection results of Examples 1 and 9-11 Figure 4 It can be seen that in Examples 9-11, which used copolymers of poly-β-hydroxybutyrate, 3-hydroxybutyrate, and 3-hydroxyvalerate, or copolyesters of 3-hydroxybutyrate and 3-hydroxyhexanoic acid as raw materials, the concentration of the degradation product 3HB in the obtained PHA composite microspheres was between 40-90 mg / L over 4-18 weeks, and the degradation rate fluctuated, indicating a relatively unstable degradation rate. In contrast, Example 1, which used poly(3-hydroxybutyrate-co-4-hydroxybutyrate) as a raw material, maintained a concentration of the degradation product 3HB in the obtained PHA composite microspheres between 50-80 mg / L over 4-18 weeks, and the pH remained stable within the range of 4.9-5.5. This indicates that the degradation rate of PHA in the mid-stage of the composite was more stable, which is more conducive to cell proliferation and collagen regeneration.

[0089] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A PHA composite microsphere, characterized in that, Based on 100wt% of PHA composite microspheres, the PHA composite microspheres comprise 10-40wt% of PHA microspheres with a weight average molecular weight of 10,000 Da, 20-70wt% of PHA with a weight average molecular weight of 60,000 Da, and 1-40wt% of PHA microspheres with a weight average molecular weight of 130,000 Da. The PHA is poly(3-hydroxybutyrate-co-4-hydroxybutyrate), and the content of 4-hydroxybutyrate in the poly(3-hydroxybutyrate-co-4-hydroxybutyrate) is 8-10 mol%; The PHA composite microspheres exhibit the following degradation behavior: during the 1-week degradation period, the concentration of the degradation product 3HB is 30 mg / L or higher; during the 2-18 week degradation period, the concentration of 3HB is 30-100 mg / L; during the 20-22 week degradation period, the concentration of 3HB is 30 mg / L or lower; and during the 4-18 week degradation period, the concentration of the degradation product 3HB can be maintained between 50-80 mg / L.

2. The PHA composite microspheres according to claim 1, characterized in that, The PHA microspheres have a particle size of 30-50 μm.

3. The method for preparing PHA composite microspheres according to any one of claims 1-2, characterized in that, Includes the following steps: PHA microspheres with different weight-average molecular weights were prepared separately, and then the PHA microspheres with different weight-average molecular weights were mixed evenly to obtain PHA composite microspheres.

4. The method for preparing PHA composite microspheres according to claim 3, characterized in that, The preparation method of the PHA microspheres is as follows: PHA powder is dissolved in an organic solvent, and then a polyvinyl alcohol aqueous solution with a concentration of 0.5-1.5wt% is added. After stirring for 3-6 hours, the mixture is heated, washed, filtered, and freeze-dried to obtain PHA microspheres.

5. The method for preparing PHA composite microspheres according to claim 4, characterized in that, The organic solvent is selected from one or more of dichloromethane, N-methylpyrrolidone, trichloromethane, and acetonitrile.

6. The method for preparing PHA composite microspheres according to claim 4, characterized in that, The volume ratio of the organic solvent to the polyvinyl alcohol aqueous solution is 1:(3-200).

7. The use of PHA composite microspheres as described in any one of claims 1-2 in the preparation of tissue fillers or medical aesthetic products.

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