Polysaccharide microspheres containing endogenous polycarboxylic acid and preparation method and application thereof

Abstract

The present invention belongs to the technical field of cosmetic fillers, and specifically relates to a polysaccharide microsphere containing endogenous polycarboxylic acid, and a preparation method and application thereof. The preparation method comprises the following steps: first dissolving polysaccharide and endogenous polycarboxylic acid in water to obtain an aqueous phase; then dissolving an emulsifier in liquid paraffin to obtain an oil phase; adding the aqueous phase to the oil phase, stirring to form an emulsion, first heating to 80-95°C, keeping warm for 12-72 h, then heating to 100-120°C for a second time, keeping warm for 6-24 h, finally cooling, drying, sieving, and obtaining. The polysaccharide microsphere of the present invention is a biomedical material that is degradable and releases endogenous carboxylic acid slowly, and endogenous carboxylic acid can promote collagen regeneration without obvious inflammatory response; the rate of slow release of endogenous carboxylic acid during degradation is stable and long-lasting, and after implantation, collagen regeneration can be effectively stimulated for a long time and inflammation is reduced; and it is completely degraded within 12 months to avoid long-term adverse reactions.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic filler technology, specifically relating to a polysaccharide microsphere containing endogenous polycarboxylic acids, its preparation method, and its application. Background Technology

[0002] Injectable fillers are primarily used to treat congenital defects and acquired injuries. In recent years, they have been widely used in the field of cosmetic medicine, such as filling and repairing skin wrinkles and depressions. Ideal injectable fillers possess the following characteristics: ① Safe, minimally invasive, with good biocompatibility, and low risk of infection or inducing allogeneic granulomas; ② High stability, not easily migrated, maintaining a fixed volume and flexibility; ③ Not phagocytosed by phagocytes; ④ Effectively promotes collagen regeneration, naturally and continuously altering facial contours, and enhancing the elasticity and hydration of the filled skin.

[0003] Among numerous cosmetic injection products, microspheres or microparticles are considered to have regenerative effects because they can stimulate collagen production. The regenerative mechanism of this type of product mainly utilizes the foreign body stimulation of the microspheres to induce an inflammatory response in the tissue, thereby causing cells to secrete collagen. An inflammatory response persists at the injection site until the microspheres are completely degraded. When the inflammation becomes uncontrollable, it can cause significant adverse reactions such as nodules and granulomas. The materials used for microspheres or microparticles mainly include polymethyl methacrylate (PMMA), polyvinyl alcohol (PVA), poly-L-lactic acid (PLLA), polycaprolactone (PCL), and hydroxyapatite (HAP).

[0004] For example, Chinese invention patent publication number CN119236172A discloses an injectable filler, its preparation method, and its application. The filler is polycaprolactone microparticles, which are spherical particles with a particle size of 20-100 μm. The polycaprolactone microparticles have a rough or non-smooth surface. The rough or non-smooth surface of the polycaprolactone microspheres in this invention increases the contact area with cells, improves the cell adhesion and retention time, and enhances the affinity of the polycaprolactone microspheres, which can stimulate and accelerate collagen growth.

[0005] Another Chinese invention patent publication number CN115414530A discloses an injectable filler for accelerating collagen regeneration and its preparation and application. The preparation method includes: dissolving collagen in an acidic solution to obtain a collagen solution; mixing microspheres with a particle size of 20-100 μm with the obtained collagen solution under stirring at 500-2000 r / min, and adjusting the pH to obtain a mixed solution; freezing and drying the obtained mixed solution to obtain a lyophilized powder; mixing the lyophilized powder with a water-soluble polymer solution under stirring at 1000-5000 r / min, and lyophilizing to obtain an injectable filler for accelerating collagen regeneration. This invention, through the composite of collagen and biodegradable polyester microspheres, utilizes the good affinity of collagen for fibroblasts to promote the rapid secretion of ECM, significantly improves the speed of in vivo collagen regeneration, and can maintain the regeneration filling effect.

[0006] However, although the polycaprolactone used in the aforementioned patent is a biodegradable material, it can generally remain in tissues for more than 18 months, which implies long-term safety risks. Therefore, there is an urgent need in the field for a biodegradable biomedical material that promotes collagen regeneration and has no significant inflammatory response. Summary of the Invention

[0007] This invention addresses the problems existing in the prior art by providing a polysaccharide microsphere containing endogenous polycarboxylic acids, its preparation method, and its application.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] A method for preparing polysaccharide microspheres containing endogenous polycarboxylic acids includes the following steps:

[0010] (1) First, dissolve the polysaccharide and endogenous polycarboxylic acid in water to obtain an aqueous phase;

[0011] (2) The emulsifier is then dissolved in liquid paraffin to obtain the oil phase;

[0012] (3) Add the aqueous phase to the oil phase and stir to form an emulsion. First, heat the emulsion to 80-95℃ and keep it at that temperature for 12-72 h. Then, heat the emulsion to 100-120℃ and keep it at that temperature for 6-24 h. Finally, cool the emulsion, dry it, and sieve it to obtain the final product.

[0013] Preferably, the polysaccharide in step (1) is carboxymethyl cellulose, dextran, hydroxyethyl cellulose, hydroxypropyl cellulose or carboxymethyl chitosan.

[0014] Preferably, the endogenous polycarboxylic acid in step (1) is citric acid, succinic acid, or malic acid.

[0015] Preferably, the mass ratio of the polysaccharide to the endogenous polycarboxylic acid is 1:0.1-0.4.

[0016] More preferably, the mass ratio of the polysaccharide to the endogenous polycarboxylic acid is 1:0.2-0.4.

[0017] Preferably, the polysaccharide has a mass fraction of 1-10% in the aqueous phase.

[0018] More preferably, the mass fraction of the polysaccharide in the aqueous phase is 3-10%.

[0019] More preferably, the polysaccharide has a mass fraction of 5-8% in the aqueous phase.

[0020] Preferably, the emulsifier in step (2) is Span 80 or Span 60.

[0021] Preferably, the mass fraction of the emulsifier in the oil phase is 0.5-2%.

[0022] More preferably, the mass fraction of the emulsifier in the oil phase is 1-2%.

[0023] Preferably, in step (3), the volume ratio of the aqueous phase to the oil phase in the emulsion is 1:5-1:2, the stirring speed is 150-600 rpm, and the stirring time is 20-40 min.

[0024] Preferably, the cooling in step (3) needs to be reduced to 20-35℃, and water needs to be added after cooling to make the microspheres swell, and the microspheres are collected by filtration; the drying process includes first washing the microspheres with an organic solvent, and then drying them at 40-60℃ for 45-50 h to obtain dry microspheres.

[0025] Preferably, the sieving process in step (3) includes swelling the dry microspheres in water and sieving them to obtain polysaccharide microspheres with a particle size of 20-60 μm; after sieving, the polysaccharide microspheres are washed with ethanol and then dried at 40-60℃ for 45-50 h.

[0026] Preferably, the organic solvent is petroleum ether, n-pentane, n-hexane, or n-heptane.

[0027] The present invention also provides polysaccharide microspheres containing endogenous polycarboxylic acids prepared by the above preparation method.

[0028] This invention also provides the application of the above-mentioned polysaccharide microspheres in the preparation of injectable fillers that promote collagen regeneration.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) The polysaccharide microspheres containing endogenous polycarboxylic acids provided by this invention are a biomedical material that can degrade and sustainably release endogenous carboxylic acids. Endogenous carboxylic acids can promote collagen regeneration without significant inflammatory reactions. During the degradation process, the polysaccharide microspheres exhibit a stable and long-lasting rate of sustained release of endogenous carboxylic acids. After implantation, they can effectively stimulate collagen regeneration and reduce inflammation. The microspheres can be completely degraded within 12 months, thus avoiding long-term adverse reactions.

[0031] (2) The polysaccharide microspheres containing endogenous polycarboxylic acids provided by the present invention are prepared by a two-stage heating process using an O / W emulsion method based on the dehydration condensation reaction of the carboxyl groups of the endogenous polycarboxylic acids and the hydroxyl groups of the polysaccharides. This preparation method is simple to operate, and the resulting spheres are regular in shape and have a high particle size qualification rate, which can be used for mass production. Attached Figure Description

[0032] Figure 1 This is a microscope image of the polysaccharide microspheres prepared in Example 1 after they have fully swelled by absorbing water.

[0033] Figure 2 Microscopic images of polysaccharide microspheres prepared for Comparative Example 1.

[0034] Figure 3 Microscopic images of the polysaccharide microspheres prepared for Comparative Example 2.

[0035] Figure 4 The curves show the in vitro degradation profile.

[0036] Figure 5 This is the in vitro carboxylic acid release curve.

[0037] Figure 6 HE staining image of the polysaccharide microspheres prepared in Example 1 after subcutaneous implantation in rats for 4 weeks.

[0038] Figure 7 Masson staining image of the polysaccharide microspheres prepared in Example 1 after subcutaneous implantation in rats for 12 weeks. Detailed Implementation

[0039] It is worth noting that the raw materials used in this invention are all commercially available products.

[0040] Example 1

[0041] A method for preparing polysaccharide microspheres containing endogenous polycarboxylic acids, comprising the following steps:

[0042] (1) Weigh 24 g of carboxymethyl cellulose (molecular weight 90kDa) and 4.8 g of citric acid, add water to 600 g, and stir to completely dissolve the carboxymethyl cellulose and citric acid to form the aqueous phase.

[0043] (2) Weigh out 80.9 g of Span and add it to 891 g of liquid paraffin. Stir until completely dissolved to form the oil phase.

[0044] (3) Add the oil phase to a 3 L round-bottom flask, turn on the stirrer to 300 rpm, add the aqueous phase to the oil phase, and continue stirring for 30 min to form an emulsion. Heat to 95℃ once and keep warm for 12 h. Then heat to 120℃ a second time and keep warm for 6 h. Cool down to 25℃, add 900 g of water to the round-bottom flask, and after 10 min, pour the contents of the flask into a Buchner funnel for vacuum filtration to collect the microspheres. Wash the microspheres with petroleum ether and place them in a vacuum drying oven at 60℃ for 48 h to obtain dry microspheres. Then disperse them in water and sieve them using a sieve with 60 μm and 20 μm pore sizes to obtain polysaccharide microspheres with a particle size of 20-60 μm. Wash the polysaccharide microspheres with ethanol and place them in a vacuum drying oven at 60℃ for 48 h to finally obtain 17.8 g of polysaccharide microspheres containing endogenous polycarboxylic acids, with a yield of 61.8%.

[0045] Take a small amount of polysaccharide microspheres containing endogenous polycarboxylic acids, add physiological saline to completely swell them, and observe them under a microscope. Figure 1 As shown.

[0046] Example 2

[0047] A method for preparing polysaccharide microspheres containing endogenous polycarboxylic acids, comprising the following steps:

[0048] (1) Weigh 24 g of carboxyethyl cellulose (molecular weight 90kDa) and 9.6 g of succinic acid, add water to 300 g, and stir to completely dissolve the carboxyethyl cellulose and succinic acid to form the aqueous phase.

[0049] (2) Weigh out 30 g of Span 80 and add it to 1500 g of liquid paraffin. Stir until completely dissolved to form the oil phase.

[0050] (3) The oil phase was added to a 3 L round-bottom flask, and the stirring was started at 200 rpm. The aqueous phase was added to the oil phase, and the stirring was continued for 30 min to form an emulsion. The temperature was raised to 80℃ once and kept at that temperature for 72 h. Then the temperature was raised to 110℃ a second time and kept at that temperature for 12 h. The temperature was lowered to 25℃, and 900 g of water was added to the round-bottom flask. After 10 min, the contents of the flask were poured into a Buchner funnel for filtration and the microspheres were collected. The microspheres were washed with n-heptane and placed in a vacuum drying oven at 60℃ for 48 h to obtain dry microspheres. Then they were dispersed in water and sieved using a sieve with 60 μm and 20 μm pore sizes to obtain polysaccharide microspheres with a particle size of 20-60 μm. The polysaccharide microspheres were washed with ethanol and placed in a vacuum drying oven at 60℃ for 48 h to finally obtain 17.2 g of polysaccharide microspheres containing endogenous polycarboxylic acids, with a yield of 51.2%.

[0051] Example 3

[0052] A method for preparing polysaccharide microspheres containing endogenous polycarboxylic acids, comprising the following steps:

[0053] (1) Weigh out 10 g of dextran (molecular weight 70 kDa) and 1.5 g of malic acid, add water to 200 g, and stir to completely dissolve the dextran and malic acid to form the aqueous phase.

[0054] (2) Weigh out 60.4 g of Span and add it to 396 g of liquid paraffin. Stir until completely dissolved to form the oil phase.

[0055] (3) Add the oil phase to a 1 L round-bottom flask, turn on the stirrer to 200 rpm, add the aqueous phase to the oil phase, and continue stirring for 30 min to form an emulsion. Heat to 90℃ once and keep warm for 24 h. Then heat to 100℃ a second time and keep warm for 24 h. Cool down to 25℃, add 300 g of water to the round-bottom flask, and after 10 min, pour the contents of the flask into a Buchner funnel for vacuum filtration to collect the microspheres. Wash the microspheres with n-hexane and place them in a vacuum drying oven at 40℃ for 48 h to obtain dry microspheres. Then disperse them in water and sieve them using a sieve with 60 μm and 20 μm pore sizes to obtain polysaccharide microspheres with a particle size of 20-60 μm. Wash the polysaccharide microspheres with ethanol and place them in a vacuum drying oven at 40℃ for 48 h to finally obtain 6.8 g of polysaccharide microspheres containing endogenous polycarboxylic acids, with a yield of 59.1%.

[0056] Example 4

[0057] A method for preparing polysaccharide microspheres containing endogenous polycarboxylic acids, comprising the following steps:

[0058] (1) Weigh 20 g of hydroxypropyl cellulose (molecular weight 80 kDa) and 4.0 g of citric acid, add water to 200 g, and stir to completely dissolve the hydroxypropyl cellulose and citric acid to form the aqueous phase.

[0059] (2) Weigh out 80.6 g of Span and add it to 294 g of liquid paraffin. Stir until completely dissolved to form the oil phase.

[0060] (3) Add the oil phase to a 1 L round-bottom flask, turn on the stirrer to 200 rpm, add the aqueous phase to the oil phase, and continue stirring for 30 min to form an emulsion. Heat to 85℃ once and keep warm for 36 h. Then heat to 120℃ a second time and keep warm for 6 h. Cool down to 25℃, add 300 g of water to the round-bottom flask, and after 10 min, pour the contents of the flask into a Buchner funnel for vacuum filtration to collect the microspheres. Wash the microspheres with n-pentane and place them in a vacuum drying oven at 40℃ for 48 h to obtain dry microspheres. Then disperse them in water and sieve them using a sieve with 60 μm and 20 μm pore sizes to obtain polysaccharide microspheres with a particle size of 20-60 μm. Wash the polysaccharide microspheres with ethanol and place them in a vacuum drying oven at 40℃ for 48 h to finally obtain 12.5 g of polysaccharide microspheres containing endogenous polycarboxylic acids, with a yield of 52.1%.

[0061] Example 5

[0062] A method for preparing polysaccharide microspheres containing endogenous polycarboxylic acids, comprising the following steps:

[0063] (1) Weigh 6 g of carboxymethyl chitosan (molecular weight 110 kDa) and 0.6 g of citric acid, add water to 200 g, and stir to completely dissolve the carboxymethyl chitosan and citric acid as the aqueous phase.

[0064] (2) Weigh out 1.5 g of Span 80 and add it to 298.5 g of liquid paraffin. Stir until completely dissolved to form the oil phase.

[0065] (3) The oil phase was added to a 1 L round-bottom flask, and the stirring was turned on to 600 rpm. The aqueous phase was added to the oil phase, and the stirring was continued for 30 min to form an emulsion. The temperature was raised to 90℃ once and kept at that temperature for 24 h. Then the temperature was raised to 110℃ a second time and kept at that temperature for 8 h. The temperature was lowered to 25℃, and 300 g of water was added to the round-bottom flask. After 10 min, the contents of the flask were poured into a Buchner funnel for vacuum filtration to collect the microspheres. The microspheres were washed with petroleum ether and placed in a vacuum drying oven at 60℃ for 48 h to obtain dry microspheres. Then they were dispersed in water and sieved using a sieve with 60 μm and 20 μm pore sizes to obtain polysaccharide microspheres with a particle size of 20-60 μm. The polysaccharide microspheres were washed with ethanol and placed in a vacuum drying oven at 60℃ for 48 h to finally obtain 3.9 g of polysaccharide microspheres containing endogenous polycarboxylic acids, with a yield of 59.1%.

[0066] Comparative Example 1

[0067] Same as Example 1, except that the temperature for the first heating is 70°C and the holding time is 72 h.

[0068] A method for preparing polysaccharide microspheres, comprising the following steps:

[0069] (1) Weigh 24 g of carboxymethyl cellulose (molecular weight 90kDa) and 4.8 g of citric acid, add water to 600 g, and stir to completely dissolve the carboxymethyl cellulose and citric acid to form the aqueous phase.

[0070] (2) Weigh out 80.9 g of Span and add it to 891 g of liquid paraffin. Stir until completely dissolved to form the oil phase.

[0071] (3) Add the oil phase to a 3 L round-bottom flask, turn on the stirrer to 300 rpm, add the aqueous phase to the oil phase, and continue stirring for 30 min to form an emulsion. Raise the temperature to 70℃ once and keep it at that temperature for 12 h. Then raise the temperature to 120℃ a second time and keep it at that temperature for 6 h. Cool down to 25℃, add 900 g of water to the round-bottom flask, and after 10 min, pour the contents of the flask into a Buchner funnel for vacuum filtration to collect the microspheres. Wash the microspheres with petroleum ether and place them in a vacuum drying oven at 60℃ for 48 h to obtain irregularly shaped, aggregated polysaccharide microspheres.

[0072] Take a small amount of polysaccharide microspheres, add physiological saline to dissolve them completely, and then observe them under a microscope. Figure 2 As shown in the figure, the polysaccharide microspheres obtained under this operation will stick together and aggregate into a clump.

[0073] Comparative Example 2

[0074] Similar to Example 1, the only difference is that the temperature for the first heating is 100°C, which is maintained for 24 hours, and no second heating is performed.

[0075] A method for preparing polysaccharide microspheres, comprising the following steps:

[0076] (1) Weigh 24 g of carboxymethyl cellulose (molecular weight 90kDa) and 4.8 g of citric acid, add water to 600 g, and stir to completely dissolve the carboxymethyl cellulose and citric acid to form the aqueous phase.

[0077] (2) Weigh out 80.9 g of Span and add it to 891 g of liquid paraffin. Stir until completely dissolved to form the oil phase.

[0078] (3) Add the oil phase to a 3 L round-bottom flask, turn on the stirrer to 300 rpm, add the aqueous phase to the oil phase, and continue stirring for 30 min to form an emulsion. Heat to 100℃ and keep warm for 24 h. Cool down to 25℃, add 900 g of water to the round-bottom flask, and after 10 min, pour the contents of the flask into a Buchner funnel for vacuum filtration to collect the microspheres. Wash the microspheres with petroleum ether and place them in a vacuum drying oven at 60℃ for 48 h to obtain irregularly shaped, aggregated polysaccharide microspheres.

[0079] Take a small amount of polysaccharide microspheres, add physiological saline to dissolve them completely, and then observe them under a microscope. Figure 3 As shown in the figure, this operation yields a large number of irregularly shaped substances and polysaccharide microspheres that adhere together.

[0080] Comparative Example 3

[0081] Same as Example 1, except that the temperature for the second heating is 98°C and the holding time is 24 h.

[0082] A method for preparing polysaccharide microspheres containing endogenous polycarboxylic acids, comprising the following steps:

[0083] (1) Weigh 24 g of carboxymethyl cellulose (molecular weight 90kDa) and 4.8 g of citric acid, add water to 600 g, and stir to completely dissolve the carboxymethyl cellulose and citric acid to form the aqueous phase.

[0084] (2) Weigh out 80.9 g of Span and add it to 891 g of liquid paraffin. Stir until completely dissolved to form the oil phase.

[0085] (3) Add the oil phase to a 3 L round-bottom flask, turn on the stirrer to 300 rpm, add the aqueous phase to the oil phase, and continue stirring for 30 min to form an emulsion. Heat to 95℃ once and keep warm for 12 h. Then heat to 98℃ a second time and keep warm for 24 h. Cool down to 25℃, add 900 g of water to the round-bottom flask, and after 10 min, pour the contents of the flask into a Buchner funnel for vacuum filtration to collect the microspheres. Wash the microspheres with petroleum ether and place them in a vacuum drying oven at 60℃ for 48 h to obtain dry microspheres. Then disperse them in water and sieve them using a sieve with 60 μm and 20 μm pore sizes to obtain polysaccharide microspheres with a particle size of 20-60 μm. Wash the polysaccharide microspheres with ethanol and place them in a vacuum drying oven at 60℃ for 48 h to finally obtain 13.6 g of polysaccharide microspheres containing endogenous polycarboxylic acids, with a yield of 47.2%.

[0086] Comparative Example 4

[0087] Same as Example 1, except that the ratio of carboxymethyl cellulose and citric acid is different.

[0088] A method for preparing polysaccharide microspheres, comprising the following steps:

[0089] (1) Weigh 24 g of carboxymethyl cellulose (molecular weight 90kDa) and 1.2 g of citric acid, add water to 600 g, and stir to completely dissolve the carboxymethyl cellulose and citric acid to form the aqueous phase.

[0090] (2) Weigh out 80.9 g of Span and add it to 891 g of liquid paraffin. Stir until completely dissolved to form the oil phase.

[0091] (3) Add the oil phase to a 3 L round-bottom flask, turn on the stirrer to 300 rpm, add the aqueous phase to the oil phase, and continue stirring for 30 min to form an emulsion. Heat to 95℃ once and keep warm for 12 h. Then heat to 120℃ a second time and keep warm for 6 h. Cool down to 25℃, add 900 g of water to the round-bottom flask, and after 10 min, pour the contents of the flask into a Buchner funnel for vacuum filtration to collect the microspheres. Wash the microspheres with petroleum ether and place them in a vacuum drying oven at 60℃ for 48 h to obtain dry microspheres. Then disperse them in water and sieve them using a sieve with 60 μm and 20 μm pore sizes to obtain polysaccharide microspheres with a particle size of 20-60 μm. Wash the polysaccharide microspheres with ethanol and place them in a vacuum drying oven at 60℃ for 48 h to finally obtain 7.9 g of polysaccharide microspheres containing endogenous polycarboxylic acids, with a yield of 31.3%.

[0092] Test Example 1

[0093] In vitro degradation experiment of microspheres: Polysaccharide microspheres prepared in Examples 1, 2, 3, and 4 were added to physiological saline and swelled until saturated. 1.0 g each of the saturated polysaccharide microspheres and poly-L-lactic acid (PLLA) microspheres (molecular weight 120 kDa, purchased from Shandong Liwanmin Biotechnology Co., Ltd.) were added to 5 mL of physiological saline, sealed, and placed in a 37℃ constant-temperature shaker at 30 rpm. Thirteen groups of each sample were prepared, with three replicates per group. Every two weeks, one group of each sample was sampled, and the microspheres and filtrate were collected by filtration. The saline solution for the remaining groups was replaced. At week 26, the collected microspheres were dried and weighed, and the percentage was calculated by comparing the initial dry weight of the microspheres. Degradation curves were plotted as shown in the figure. Figure 4 As shown.

[0094] from Figure 4 It can be seen that the polysaccharide microspheres of Examples 1, 2, and 3 degrade faster than the PLLA microspheres. Based on the data from the in vitro degradation experiment, it is estimated that the microspheres obtained in Examples 1, 2, and 3 will completely degrade in vivo in 6-12 months. This degradation time is sufficient for the microspheres to exert their collagen-stimulating effect without easily causing long-term adverse reactions. In contrast, the degradation time of Comparative Example 4 in vivo is estimated to be around 3 months, which is generally insufficient to exert its collagen-stimulating effect.

[0095] Test Example 2

[0096] Endogenous carboxylic acid release experiment: filtrates collected every two weeks from Examples 1, 2, and Comparative Example 3 in Test Example 1 were analyzed using high performance liquid chromatography (Agilent 1100) to determine the content of citric acid or succinic acid in the filtrates. Chromatographic conditions: C18 reversed-phase column, mobile phase 0.1% phosphoric acid:methanol 75:25 (v / v), column temperature 40℃, flow rate 1.0 mL / min.

[0097] Based on the measured results, a carboxylic acid release curve was plotted as follows: Figure 5 As shown, the polysaccharide microspheres of Examples 1 and 2 can continuously release carboxylic acids, and the release rate is relatively stable. However, the polysaccharide microspheres of Comparative Example 3 exhibited rapid release of carboxylic acids in the initial stage, and the release rate could not remain stable. Rapid release of carboxylic acids can lead to excessively high local acid concentrations in tissues, easily causing adverse reactions.

[0098] Test Example 3

[0099] Rat implantation experiment: The polysaccharide microspheres prepared in Example 1 were added to physiological saline and swollen to saturation. The swollen and saturated polysaccharide microspheres were sterilized and mixed evenly with sterilized sodium hyaluronate solution with a concentration of 10 mg / mL at a mass ratio of 3:7. This mixture was then used as the test sample for injection.

[0100] SD rats weighing 200-300 g were used as experimental animals. The surgical field on the back of the rats was shaved and treated with depilatory cream, then routinely disinfected with iodine and alcohol. After weighing, 10% chloral hydrate was injected intraperitoneally at a dose of 0.3 mL / 100 g. Subcutaneous injection was then performed after anesthesia. To ensure that the injection site was identical for each rat, 0.5 mL of the experimental sample was subcutaneously injected into the center of the scapula on the back.

[0101] Four weeks after implantation, tissue was harvested from the area surrounding the implantation site, fixed in paraformaldehyde, stained with hematoxylin and eosin (HE), and examined pathologically. Figure 6 As shown in the image, four weeks after implantation, the microspheres remained largely spherical, with no obvious inflammatory reaction around them.

[0102] Twelve weeks after implantation, tissue was harvested from the area surrounding the implantation site, fixed with paraformaldehyde, and stained with Masson's stain to observe collagen regeneration. Figure 7 As shown in the image, the fiber density around the microspheres is higher than in other areas, indicating that the microspheres have a stimulating effect on collagen production.

[0103] Tissue around the implantation site was harvested 52 weeks after implantation, fixed with paraformaldehyde, stained with hematoxylin and eosin (HE), and examined pathologically. No microsphere residue was found, indicating that the microspheres had been completely degraded.

[0104] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing polysaccharide microspheres containing endogenous polycarboxylic acid, characterized in that: The steps include: (1) First, dissolve the polysaccharide and endogenous polycarboxylic acid in water to obtain an aqueous phase; (2) dissolving the emulsifier in liquid paraffin to obtain an oil phase; (3) Add the water phase to the oil phase and stir to form an emulsion. First, heat it to 80-95°C and keep it warm for 12-72 hours. Then heat it to 100-120°C for a second time and keep it warm for 6-24 hours. Finally, cool it down, dry it, and sieve it to obtain the product. The mass ratio of the polysaccharide to the endogenous polycarboxylic acid is 1:0.2-0.4, the polysaccharide is carboxymethyl cellulose, dextran, hydroxyethyl cellulose, hydroxypropyl cellulose or carboxymethyl chitosan, the endogenous polycarboxylic acid is citric acid, succinic acid or malic acid, and the mass fraction of the polysaccharide in the aqueous phase is 1-10%.

2. The preparation method according to claim 1, characterized in that: The emulsifier in step (2) is Span 80 or Span 60, and the mass fraction of the emulsifier in the oil phase is 0.5-2%.

3. The preparation method according to claim 1, characterized in that: In step (3), the volume ratio of the water phase to the oil phase in the emulsion is 1:5-1:2, the stirring speed is 150-600 rpm, and the stirring time is 20-40 min.

4. The preparation method according to claim 1, characterized in that: The cooling in step (3) needs to be reduced to 20-35°C, and after cooling, water needs to be added to swell the microspheres, and the microspheres are collected by filtration; the drying process includes first washing the microspheres with an organic solvent, and then drying at 40-60°C for 45-50 hours to obtain dry microspheres; the screening process includes placing the dry microspheres in water to swell, and screening to obtain polysaccharide microspheres with a particle size of 20-60 μm; after the screening is completed, the polysaccharide microspheres are washed with ethanol, and then dried at 40-60°C for 45-50 hours.

5. The preparation method according to claim 4, characterized in that: The organic solvent is petroleum ether, n-pentane, n-hexane or n-heptane.

6. Polysaccharide microspheres containing endogenous polycarboxylic acid prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the polysaccharide microspheres as claimed in claim 6 in the preparation of an injectable filler for promoting collagen regeneration.

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