Polysaccharide microspheres containing endogenous polycarboxylic acid as well as preparation method and application of polysaccharide microspheres

Abstract

The invention belongs to the technical field of beauty filler, and particularly relates to polysaccharide microspheres containing endogenous polycarboxylic acid as well as a preparation method and application of the polysaccharide microspheres. The preparation method comprises the following steps: dissolving polysaccharide and endogenous polycarboxylic acid in water to obtain a water phase; dissolving an emulsifier in liquid paraffin to obtain an oil phase; adding the water phase into the oil phase, stirring to form an emulsion, firstly heating to 80-95 DEG C for the first time, preserving heat for 12-72 hours, then heating to 100-120 DEG C for the second time, preserving heat for 6-24 hours, and finally cooling, drying and sieving to obtain the water-based paint. The polysaccharide microsphere is a biomedical material capable of degrading and slowly releasing endogenous carboxylic acid, and the endogenous carboxylic acid can promote collagen regeneration and has no obvious inflammatory reaction; in the degradation process, the slow release rate of endogenous carboxylic acid is stable, the lasting time is long, collagen regeneration can be effectively stimulated for a long time after implantation, and inflammation is reduced; and complete degradation can be realized within 12 months, so that long-term adverse reaction can be avoided.

Description

Technical Field

[0001] The invention belongs to the technical field of cosmetic fillers, and in particular relates to polysaccharide microspheres containing endogenous polycarboxylic acid, and a preparation method and application thereof. Background Art

[0002] The main function of injectable fillers is to treat congenital defects and acquired defects. In recent years, they are mostly used in the field of medical cosmetology, such as filling and repairing skin wrinkles and depressions. Ideal injectable fillers have the following characteristics: ① safe, less damaging, good biocompatibility, not easy to be infected or induce foreign body granulomas; ② good stability, not easy to move, can maintain a fixed volume and flexibility; ③ will not be swallowed by phagocytes; ④ effectively promote collagen regeneration, naturally and continuously change facial contours, and enhance the elasticity and moisture of the filled skin.

[0003] Among the many medical beauty injection products, microsphere or microparticle injection fillers can stimulate collagen production and are considered to have a regenerative effect. The regenerative mechanism of this type of product is mainly to use the foreign body stimulation of the microspheres to cause an inflammatory response in the tissue, thereby causing the cells to secrete collagen. There is always an inflammatory response at the injection site before the microspheres are completely degraded, and when the inflammation is uncontrollable, it causes greater adverse reactions such as nodules and granulomas. The materials of microspheres or microparticles are mainly 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 and its preparation method and application. The filler is polycaprolactone microparticles, which are spherical particles with a particle size of 20-100 μm. The polycaprolactone microparticles have a rough surface or a non-smooth surface. The polycaprolactone microspheres in the invention have a rough surface or a non-smooth surface, which increases the contact area with cells, improves the adhesion ability and retention time of cells, improves the affinity of polycaprolactone microspheres, and can stimulate and accelerate the growth of collagen.

[0005] Another Chinese invention patent publication number CN115414530A discloses an injectable filler for accelerating the induction of collagen regeneration and its preparation and application. The preparation method comprises: dissolving collagen in an acid 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 the induction of collagen regeneration. The invention promotes the rapid secretion of ECM by compounding collagen with biodegradable polyester microspheres and utilizing the good affinity of collagen for fibroblasts, thereby significantly improving the speed of inducing collagen regeneration in the body and maintaining the regeneration and filling effect.

[0006] However, although the polycaprolactone used in the above invention patent is a degradable material, it can generally remain in the tissue for more than 18 months, which means long-term safety risks. In view of this, the art urgently needs to provide a biomedical material that is degradable and promotes collagen regeneration without obvious inflammatory response. Summary of the invention

[0007] The present invention aims at the problems existing in the prior art and provides a polysaccharide microsphere containing endogenous polycarboxylic acid and a preparation method and application thereof.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is as follows: A method for preparing polysaccharide microspheres containing endogenous polycarboxylic acid comprises the following steps: (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 h. Then, heat it to 100-120°C for a second time and keep it warm for 6-24 h. Finally, cool it down, dry it, and sieve it to obtain the product.

[0009] Preferably, the polysaccharide in step (1) is carboxymethyl cellulose, dextran, hydroxyethyl cellulose, hydroxypropyl cellulose or carboxymethyl chitosan.

[0010] Preferably, the endogenous polycarboxylic acid in step (1) is citric acid, succinic acid or malic acid.

[0011] Preferably, the mass ratio of the polysaccharide to the endogenous polycarboxylic acid is 1:0.1-0.4.

[0012] More preferably, the mass ratio of the polysaccharide to the endogenous polycarboxylic acid is 1:0.2-0.4.

[0013] Preferably, the mass fraction of the polysaccharide in the aqueous phase is 1-10%.

[0014] More preferably, the mass fraction of the polysaccharide in the aqueous phase is 3-10%.

[0015] Further preferably, the mass fraction of the polysaccharide in the aqueous phase is 5-8%.

[0016] Preferably, the emulsifier in step (2) is Span 80 or Span 60.

[0017] Preferably, the mass fraction of the emulsifier in the oil phase is 0.5-2%.

[0018] More preferably, the mass fraction of the emulsifier in the oil phase is 1-2%.

[0019] Preferably, 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.

[0020] Preferably, 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 h to obtain dry microspheres.

[0021] Preferably, the sieving process in step (3) comprises placing the dry microspheres in water to swell, and sieving to obtain polysaccharide microspheres with a particle size of 20-60 μm; after the sieving, the polysaccharide microspheres are washed with ethanol, and then dried at 40-60° C. for 45-50 h.

[0022] Preferably, the organic solvent is petroleum ether, n-pentane, n-hexane or n-heptane.

[0023] The present invention also provides polysaccharide microspheres containing endogenous polycarboxylic acid prepared by the above preparation method.

[0024] The present invention also provides the use of the polysaccharide microspheres in preparing an injectable filler for promoting collagen regeneration.

[0025] Compared with the prior art, the present invention has the following beneficial effects: (1) The polysaccharide microspheres containing endogenous polycarboxylic acid provided by the present invention are biomedical materials that are degradable and slowly release endogenous carboxylic acid, which can promote collagen regeneration without obvious inflammatory response. The polysaccharide microspheres slowly release endogenous carboxylic acid at a stable rate and for a long time during the degradation process. After implantation, they can effectively stimulate collagen regeneration and reduce the occurrence of inflammation. The microspheres can be completely degraded within 12 months, which can avoid long-term adverse reactions.

[0026] (2) The polysaccharide microspheres containing endogenous polycarboxylic acid provided by the present invention are prepared by the O / W emulsion method through two heating steps based on the dehydration condensation reaction between the carboxyl group of the endogenous polycarboxylic acid and the hydroxyl group of the polysaccharide. The preparation method is simple to operate, the obtained spheres are regular, the particle size qualification rate is high, and mass production is possible. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a microscope picture of the polysaccharide microspheres prepared in Example 1 after they have absorbed water and swelled completely.

[0028] Figure 2 This is a microscope picture of the polysaccharide microspheres prepared in Comparative Example 1.

[0029] Figure 3 This is a microscope picture of the polysaccharide microspheres prepared in Comparative Example 2.

[0030] Figure 4 In vitro degradation curve.

[0031] Figure 5 In vitro carboxylic acid release curve.

[0032] Figure 6 This is a HE staining picture of the polysaccharide microspheres prepared in Example 1 after being implanted subcutaneously in rats for 4 weeks.

[0033] Figure 7 This is a Masson staining picture of the polysaccharide microspheres prepared in Example 1 after subcutaneous implantation in rats for 12 weeks. DETAILED DESCRIPTION

[0034] It is worth noting that the raw materials used in the present invention are all common commercially available products.

[0035] Example 1 A method for preparing polysaccharide microspheres containing endogenous polycarboxylic acid, comprising the following steps: (1) Weigh 24 g of carboxymethyl cellulose (molecular weight 90 kDa) and 4.8 g of citric acid, add water to 600 g, and stir to completely dissolve the carboxymethyl cellulose and citric acid. This is the aqueous phase.

[0036] (2) Weigh 809 g of Span and add it to 891 g of liquid paraffin. Stir until completely dissolved. This is the oil phase.

[0037] (3) Add the oil phase to a 3 L round-bottom flask, start stirring at a speed of 300 rpm, add the water phase to the oil phase, continue stirring for 30 min to form an emulsion, heat it to 95 °C once, and keep it warm for 12 h. Then heat it to 120 °C for a second time and keep it warm for 6 h. Cool it to 25 °C, 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 filtration to collect the microspheres. Wash the microspheres with petroleum ether, place them in a vacuum drying oven at 60 °C for 48 h, and obtain dry microspheres. Then disperse them in water and sieve them using a sieving machine equipped with 60 μm and 20 μm aperture sieves to obtain polysaccharide microspheres with a particle size of 20-60 μm. Wash the polysaccharide microspheres with ethanol, place them in a vacuum drying oven at 60 °C for 48 h, and finally obtain 17.8 g of polysaccharide microspheres containing endogenous polycarboxylic acids, with a yield of 61.8%.

[0038] Take a small amount of polysaccharide microspheres containing endogenous polycarboxylic acid, add physiological saline to swell completely, and then observe under a microscope. Figure 1 shown.

[0039] Example 2 A method for preparing polysaccharide microspheres containing endogenous polycarboxylic acid, comprising the following steps: (1) Weigh 24 g of carboxyethyl cellulose (molecular weight 90 kDa) and 9.6 g of succinic acid, add water to 300 g, and stir to completely dissolve the carboxyethyl cellulose and succinic acid. This is the aqueous phase.

[0040] (2) Weigh 30 g of Span 80 and add it to 1500 g of liquid paraffin. Stir until it is completely dissolved. This is the oil phase.

[0041] (3) Add the oil phase to a 3 L round-bottom flask, start stirring at a speed of 200 rpm, add the water phase to the oil phase, continue stirring for 30 min to form an emulsion, heat it to 80 °C once, and keep it warm for 72 h. Then heat it to 110 °C for the second time and keep it warm for 12 h. Cool it to 25 °C, 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 filtration to collect the microspheres. Wash the microspheres with n-heptane, place them in a vacuum drying oven at 60 °C for 48 h, and obtain dry microspheres. Then disperse them in water and sieve them using a sieving machine equipped with 60 μm and 20 μm aperture sieves to obtain polysaccharide microspheres with a particle size of 20-60 μm. Wash the polysaccharide microspheres with ethanol, place them in a vacuum drying oven at 60 °C for 48 h, and finally obtain 17.2 g of polysaccharide microspheres containing endogenous polycarboxylic acids, with a yield of 51.2%.

[0042] Example 3 A method for preparing polysaccharide microspheres containing endogenous polycarboxylic acid, comprising the following steps: (1) First weigh 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. This is the aqueous phase.

[0043] (2) Weigh 604 g of Span and add it to 396 g of liquid paraffin. Stir until completely dissolved. This is the oil phase.

[0044] (3) Add the oil phase to a 1 L round-bottom flask, start stirring at a speed of 200 rpm, add the water phase to the oil phase, continue stirring for 30 min to form an emulsion, heat it to 90 °C once, and keep it warm for 24 h. Then heat it to 100 °C for the second time and keep it warm for 24 h. Cool it to 25 °C, 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 filtration to collect the microspheres. Wash the microspheres with n-hexane, place them in a vacuum drying oven at 40 °C for 48 h, and obtain dry microspheres. Then disperse them in water and sieve them using a sieving machine equipped with 60 μm and 20 μm aperture sieves to obtain polysaccharide microspheres with a particle size of 20-60 μm. Wash the polysaccharide microspheres with ethanol, place them in a vacuum drying oven at 40 °C for 48 h, and finally obtain 6.8 g of polysaccharide microspheres containing endogenous polycarboxylic acids with a yield of 59.1%.

[0045] Example 4 A method for preparing polysaccharide microspheres containing endogenous polycarboxylic acid, comprising the following steps: (1) First weigh 20 g of hydroxypropyl cellulose (molecular weight 80k Da) and 4.0 g of citric acid, add water to 200 g, and stir to completely dissolve the hydroxypropyl cellulose and citric acid as the aqueous phase.

[0046] (2) Weigh 806 g of Span and add it to 294 g of liquid paraffin. Stir until completely dissolved. This is the oil phase.

[0047] (3) Add the oil phase to a 1 L round-bottom flask, start stirring at a speed of 200 rpm, add the water phase to the oil phase, continue stirring for 30 min to form an emulsion, heat it to 85 °C once, and keep it warm for 36 h. Then heat it to 120 °C for the second time and keep it warm for 6 h. Cool it to 25 °C, 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 filtration to collect the microspheres. Wash the microspheres with n-pentane, place them in a vacuum drying oven at 40 °C for 48 h, and obtain dry microspheres. Then disperse them in water and sieve them using a sieving machine equipped with 60 μm and 20 μm aperture sieves to obtain polysaccharide microspheres with a particle size of 20-60 μm. Wash the polysaccharide microspheres with ethanol, place them in a vacuum drying oven at 40 °C for 48 h, and finally obtain 12.5 g of polysaccharide microspheres containing endogenous polycarboxylic acids, with a yield of 52.1%.

[0048] Example 5 A method for preparing polysaccharide microspheres containing endogenous polycarboxylic acid, comprising the following steps: (1) First 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 to form the aqueous phase.

[0049] (2) Weigh 1.5 g of Span 80 and add it to 298.5 g of liquid paraffin. Stir until completely dissolved. This is the oil phase.

[0050] (3) Add the oil phase to a 1 L round-bottom flask, start stirring at a speed of 600 rpm, add the water phase to the oil phase, continue stirring for 30 min to form an emulsion, heat it to 90°C once, and keep it warm for 24 h. Then heat it to 110°C for a second time and keep it warm for 8 h. Cool it to 25°C, 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 filtration to collect the microspheres. Wash the microspheres with petroleum ether, place them in a vacuum drying oven at 60°C for 48 h, and obtain dry microspheres. Then disperse them in water and sieve them using a sieving machine equipped with 60 μm and 20 μm aperture sieves to obtain polysaccharide microspheres with a particle size of 20-60 μm. Wash the polysaccharide microspheres with ethanol, place them in a vacuum drying oven at 60°C for 48 h, and finally obtain 3.9 g of polysaccharide microspheres containing endogenous polycarboxylic acids, with a yield of 59.1%. Comparative Example 1 The same as Example 1, except that the temperature of the first heating is 70° C. and the holding time is 72 h.

[0051] A method for preparing polysaccharide microspheres, comprising the following steps: (1) Weigh 24 g of carboxymethyl cellulose (molecular weight 90 kDa) and 4.8 g of citric acid, add water to 600 g, and stir to completely dissolve the carboxymethyl cellulose and citric acid. This is the aqueous phase.

[0052] (2) Weigh 809 g of Span and add it to 891 g of liquid paraffin. Stir until completely dissolved. This is the oil phase.

[0053] (3) Add the oil phase to a 3 L round-bottom flask, start stirring at 300 rpm, add the water phase to the oil phase, continue stirring for 30 min to form an emulsion, heat to 70 °C once, and keep warm for 12 h. Then heat to 120 °C for a second time and keep warm for 6 h. Cool to 25 °C, 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 filtration to collect the microspheres. Wash the microspheres with petroleum ether and place them in a vacuum drying oven at 60 °C for 48 h to obtain irregularly shaped, clumping polysaccharide microspheres.

[0054] Take a small amount of polysaccharide microspheres, add physiological saline to swell completely, and then observe under a microscope. Figure 2 As shown, it can be seen that the polysaccharide microspheres obtained under this operation will adhere and aggregate into a mass.

[0055] Comparative Example 2 The same as Example 1, the only difference is that the temperature of the first heating is 100° C., maintained for 24 h, and no second heating is performed.

[0056] A method for preparing polysaccharide microspheres, comprising the following steps: (1) Weigh 24 g of carboxymethyl cellulose (molecular weight 90 kDa) and 4.8 g of citric acid, add water to 600 g, and stir to completely dissolve the carboxymethyl cellulose and citric acid. This is the aqueous phase.

[0057] (2) Weigh 809 g of Span and add it to 891 g of liquid paraffin. Stir until completely dissolved. This is the oil phase.

[0058] (3) Add the oil phase to a 3 L round-bottom flask, start stirring at 300 rpm, add the water phase to the oil phase, continue stirring for 30 min to form an emulsion, heat to 100 °C, and keep warm for 24 h. Cool to 25 °C, 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 filtration to collect the microspheres. Wash the microspheres with petroleum ether and place them in a vacuum drying oven at 60 °C for 48 h to obtain irregularly shaped, clumping polysaccharide microspheres.

[0059] Take a small amount of polysaccharide microspheres, add physiological saline to swell completely, and then observe under a microscope. Figure 3As shown, it can be seen that a large number of irregularly shaped substances and polysaccharide microspheres are adhered together under this operation.

[0060] Comparative Example 3 The same as Example 1, the only difference is that the temperature of the second heating is 98° C. and the holding time is 24 h.

[0061] A method for preparing polysaccharide microspheres containing endogenous polycarboxylic acid, comprising the following steps: (1) Weigh 24 g of carboxymethyl cellulose (molecular weight 90 kDa) and 4.8 g of citric acid, add water to 600 g, and stir to completely dissolve the carboxymethyl cellulose and citric acid. This is the aqueous phase.

[0062] (2) Weigh 809 g of Span and add it to 891 g of liquid paraffin. Stir until completely dissolved. This is the oil phase.

[0063] (3) Add the oil phase to a 3 L round-bottom flask, start stirring at 300 rpm, add the water phase to the oil phase, continue stirring for 30 min to form an emulsion, heat to 95 °C once, and keep warm for 12 h. Then heat to 98 °C for the second time and keep warm for 24 h. Cool to 25 °C, 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 filtration to collect the microspheres. Wash the microspheres with petroleum ether, place them in a vacuum oven at 60 °C for 48 h, and obtain dry microspheres. Then disperse them in water and sieve them using a sieving machine equipped with 60 μm and 20 μm pore size sieves to obtain polysaccharide microspheres with a particle size of 20-60 μm. Wash the polysaccharide microspheres with ethanol, place them in a vacuum oven at 60 °C for 48 h, and finally obtain 13.6 g of polysaccharide microspheres containing endogenous polycarboxylic acids, with a yield of 47.2%.

[0064] Comparative Example 4 The same as Example 1, the only difference is that the ratio of carboxymethyl cellulose and citric acid is different.

[0065] A method for preparing polysaccharide microspheres, comprising the following steps: (1) First weigh 24 g of carboxymethyl cellulose (molecular weight 90 kDa) and 1.2 g of citric acid, add water to 600 g, and stir to completely dissolve the carboxymethyl cellulose and citric acid. This is the aqueous phase.

[0066] (2) Weigh 809 g of Span and add it to 891 g of liquid paraffin. Stir until completely dissolved. This is the oil phase.

[0067] (3) Add the oil phase to a 3 L round-bottom flask, start stirring at 300 rpm, add the water phase to the oil phase, continue stirring for 30 min to form an emulsion, heat to 95 °C once, and keep warm for 12 h. Then heat to 120 °C for the second time and keep warm for 6 h. Cool to 25 °C, 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 filtration to collect the microspheres. Wash the microspheres with petroleum ether, place them in a vacuum drying oven at 60 °C for 48 h, and obtain dry microspheres. Then disperse them in water and sieve them using a sieving machine equipped with 60 μm and 20 μm pore size sieves to obtain polysaccharide microspheres with a particle size of 20-60 μm. Wash the polysaccharide microspheres with ethanol, place them in a vacuum drying oven at 60 °C for 48 h, and finally obtain 7.9 g of polysaccharide microspheres containing endogenous polycarboxylic acids with a yield of 31.3%.

[0068] Test Example 1 In vitro degradation experiment of microspheres: Take the polysaccharide microspheres prepared in Example 1, Example 2, Comparative Example 3 and Comparative Example 4 respectively, add them to physiological saline to swell them to saturation, take 1.0 g of the saturated polysaccharide microspheres and poly-L-lactic acid (PLLA) microspheres (molecular weight 120k Da, purchased from Shandong Liwanmin Biotechnology Co., Ltd.), add them to 5 mL of physiological saline respectively, seal them and place them in a 37°C constant temperature oscillator to continuously oscillate at 30 rpm. Prepare 13 groups of each sample, with 3 parallel samples in each group. Take samples from one group of each sample every 2 weeks, filter and collect the microspheres and filtrate, and replace the physiological saline for the remaining groups of samples. At the 26th week, the sampled microspheres are dried and weighed, and the percentage is plotted with the initial dry weight of the microspheres to draw a degradation curve as shown in the figure. Figure 4 shown.

[0069] from Figure 4 It can be seen that the polysaccharide microspheres of Example 1, Example 2 and Comparative Example 3 degrade faster than PLLA microspheres. Based on the data of the in vitro degradation experiment, it is estimated that the microspheres obtained in Example 1, Example 2 and Comparative Example 3 will degrade completely in vivo in 6-12 months, which is enough for the microspheres to stimulate collagen without causing adverse reactions in the long term. The degradation time of Comparative Example 4 in vivo is estimated to be about 3 months, which is generally not enough to stimulate collagen in such a short time.

[0070] Test Example 2 Endogenous carboxylic acid release experiment: Take the filtrate collected every two weeks in Example 1, Example 2 and Comparative Example 3 in Test Example 1, and use high performance liquid chromatography (Agilent 1100) to determine the content of citric acid or succinic acid in the filtrate. Chromatographic conditions: Use a C18 reverse phase column, the mobile phase is 0.1% phosphoric acid: methanol 75:25 (v / v), the column temperature is 40°C, and the flow rate is 1.0 mL / min.

[0071] The carboxylic acid release curve was drawn based on the measured results. Figure 5 As shown. It can be seen that the polysaccharide microspheres of Example 1 and Example 2 can continuously release carboxylic acid, and the release rate is relatively stable. However, the polysaccharide microspheres of Comparative Example 3 show a phenomenon of rapid release of carboxylic acid in the early stage, and the release rate cannot be kept stable. The rapid release of carboxylic acid will make the local acid concentration of the tissue too high, which is easy to cause adverse reactions.

[0072] Test Example 3 Rat implantation experiment: The polysaccharide microspheres prepared in Example 1 were added to physiological saline to swell to saturation, and the saturated polysaccharide microspheres were sterilized and mixed evenly with a sterilized sodium hyaluronate solution with a concentration of 10 mg / mL at a mass ratio of 3:7 to prepare the test samples for injection.

[0073] SD rats weighing 200-300 g were used as experimental animals for the experiment. The surgical field on the back of the experimental rats was shaved and treated with depilatory cream, and then routinely disinfected with iodine and alcohol. After weighing, 10% chloral hydrate was injected intraperitoneally at a dose of 0.3 mL / 100 g, and subcutaneous injection was performed after anesthesia. To ensure that the injection site of each rat was the same, 0.5 mL of the test sample was subcutaneously injected at the center of the back scapula.

[0074] Four weeks after implantation, tissues around the implantation site were removed, fixed with paraformaldehyde, and stained with HE for pathological observation. Figure 6 It can be seen that 4 weeks after implantation, the microspheres still basically maintained their spherical shape, and there was no obvious inflammatory reaction around the microspheres.

[0075] Twelve weeks after implantation, tissues around the implantation site were removed, fixed with paraformaldehyde, and then stained with Masson staining to observe the new collagen formation. Figure 7 As shown in Figure 2, it can be seen that the fiber density around the microspheres is higher than that in other areas, indicating that the microspheres have the effect of stimulating collagen production.

[0076] At 52 weeks after implantation, tissues around the implantation site were removed, fixed with paraformaldehyde and stained with HE for pathological observation. No residual microspheres were found, indicating that the microspheres had been completely degraded.

[0077] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate 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 h. Then, heat it to 100-120°C for a second time and keep it warm for 6-24 h. Finally, cool it down, dry it, and sieve it to obtain the product.

2. The preparation method according to claim 1, characterized in that: In step (1), the polysaccharide is carboxymethyl cellulose, dextran, hydroxyethyl cellulose, hydroxypropyl cellulose or carboxymethyl chitosan, and the endogenous polycarboxylic acid is citric acid, succinic acid or malic acid.

3. The preparation method according to claim 1, characterized in that: The mass ratio of the polysaccharide to the endogenous polycarboxylic acid is 1:0.1-0.

4.

4. The preparation method according to claim 3, characterized in that: The mass fraction of the polysaccharide in the aqueous phase is 1-10%.

5. 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%.

6. 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.

7. 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.

8. The preparation method according to claim 7, characterized in that: The organic solvent is petroleum ether, n-pentane, n-hexane or n-heptane.

9. Polysaccharide microspheres containing endogenous polycarboxylic acid prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the polysaccharide microspheres according to claim 9 in the preparation of an injectable filler for promoting collagen regeneration.

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