Polylactic acid-carnosine microspheres, and preparation method and application thereof

Abstract

This invention belongs to the field of medical tissue engineering materials technology, and provides a polylactic acid-carnosine microsphere, its preparation method, and its application. Carnosine-grafted polylactic acid is dissolved in an organic solvent to obtain an oil phase; an aqueous phase containing polyvinyl alcohol is uniformly dispersed in the oil phase to obtain a mixture; the organic solvent in the mixture is removed to obtain a polylactic acid-carnosine microsphere suspension. The carnosine-grafted polylactic acid in the microspheres improves its hydrophilicity, making it an amphiphilic substance; simultaneously, carnosine can play an anti-aging role from the perspectives of anti-oxidation and anti-glycation. The polylactic acid-carnosine microspheres prepared from this polymer can also encapsulate drugs to achieve sustained-release drug delivery; these amphiphilic microspheres have good dispersibility and are not prone to aggregation; the microsphere size can be adjusted by adjusting the concentration of the oil phase to maximize the encapsulation of active ingredients and increase their content.

Description

Technical Field

[0001] This invention belongs to the field of medical tissue engineering materials technology, specifically relating to polylactic acid-carnosine microspheres and their application in skin filling. Background Technology

[0002] The information disclosed in this background section is intended to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Aging causes various changes in the skin, including wrinkles, atrophy, loss of elasticity, and subcutaneous volume loss. Dermal fillers can easily and simply correct facial volume loss. Therefore, the use of dermal fillers to revitalize the skin has attracted widespread interest from those seeking cosmetic enhancements and the medical aesthetics industry. Common filler materials are either naturally derived from animals or synthetic materials. Commonly used microsphere fillers contain raw materials including calcium hydroxyapatite (CaHA), polymethyl methacrylate (PMMA), polylactic acid (PLLA), and polycaprolactone (PCL). PLLA is a biodegradable polymer whose biocompatibility has been medically proven in various surgical materials, including sutures, staples, pins, screws, and surgical plates. PLLA fillers injected into the skin with a carrier solution immediately increase the volume at the injection site; as the carrier solution is absorbed by the tissue, the volume rapidly disappears. The remaining PLLA particles degrade into lactic acid, promoting collagen synthesis by fibroblasts and gradually increasing dermal thickness. Immune cells recognize PLLA as a foreign body; subclinical inflammation and foreign body reactions can induce PLLA-induced collagen synthesis. During the foreign body reaction, the extracellular matrix (ECM) attracts neutrophils, monocytes, and fibroblasts from the blood: activated neutrophils engulf small foreign bodies and induce edema to promote cell migration; monocytes can transform into macrophages to eliminate large foreign bodies and apoptotic neutrophils; fibroblasts secrete immature liquid gel type I collagen into the ECM, which then transforms into mature type III collagen, gradually forming a network structure and tightening to provide tissue support. In the entire tissue filling process, the core component is the microspheres, which act as foreign bodies to trigger the body's reaction, and the resulting collagen provides support. PCL, approved by the US FDA, is now widely used in long-term implants such as sutures and drug delivery devices. It is a biodegradable material, and its clinical applications are relatively widespread as orthopedic implants and surgical sutures. It is also currently the best material for thread lifting. Polycaprolactone (PCL) microspheres have good biocompatibility and are a biodegradable polyester. However, as filler products, their degradation time is generally at least two years or more, which increases the risk of inflammatory reactions in tissues. Furthermore, due to their low melting point, their preparation and transportation require strict environmental conditions. Additionally, the risk of microsphere morphology changes under high temperatures after facial filling limits their application. Hydroxyapatite microspheres and polymethyl methacrylate microspheres, as non-degradable filler materials, are rarely used in clinical facial filling due to the increased risk of granulomas and nodules. Summary of the Invention

[0004] To address the issues of slow onset of action and long degradation cycle of microspheres stimulating collagen, this invention provides a carnosine-grafted polylactic acid and its preparation method. This material is amphiphilic and can self-assemble into microspheres in solution, which can be used to encapsulate drugs.

[0005] Another objective of this invention is to provide polylactic acid-carnosine microspheres and a preparation method thereof. Using the above-mentioned carnosine-grafted polylactic acid as raw material, microspheres with different particle sizes and wall thicknesses can be obtained by adjusting the preparation process. These microspheres are suitable for loading different drugs and have good dispersibility and are not prone to aggregation.

[0006] Another object of the present invention is to provide a skin filler formed from the above-mentioned polylactic acid-carnosine microspheres, including glycine, alanine and proline. Carnosine has antioxidant and anti-glycation functions and can play an anti-aging role. The three amino acids provide raw materials for collagen synthesis and increase the efficiency of collagen synthesis.

[0007] To achieve the above objectives, the present invention adopts the following technical solution.

[0008] A method for preparing carnosine-grafted polylactic acid includes the following steps: (1) Mix polylactic acid and carnosine in water to obtain a mixture; (2) The mixture is subjected to radiation reaction under oxygen-free conditions to obtain a reaction solution; (3) After the reaction solution is separated into solid and liquid, the liquid is dried to obtain carnosine-grafted polylactic acid.

[0009] The polylactic acid is L-PLA (PLLA), D-PLA (PDLA), or racemic DL-PLA (PDLLA). L-PLA can be selected for better biocompatibility.

[0010] The molecular weight of polylactic acid (PLA) affects the degradation process and ease of reaction of carnosine-grafted PLA in vivo (the number / degree of carnosine grafting). For example, a higher molecular weight increases the difficulty of the reaction, reduces the number of grafts for the same amount of carnosine, and results in a longer degradation time when the reaction products enter the body. Therefore, a suitable molecular weight can be selected based on the specific application of carnosine-grafted PLA. Preferably, the molecular weight of PLA is 16 kDa to 80 kDa.

[0011] The radiation dose affects the yield and grafting rate of carnosine-grafted polylactic acid (PLA). Under the same conditions, increasing the dose increases both the yield and grafting rate of PLA. However, increasing the dose can lead to the decomposition of raw materials or products, thereby reducing the yield and grafting rate. For PLAs with higher molecular weights, the reaction rate can be increased by appropriately increasing the irradiation dose. Distance also ultimately affects the reaction by influencing the radiation dose; excessive distance reduces the irradiation amount, while an appropriate distance yields the optimal product yield and carnosine grafting rate. In some embodiments, the irradiation dose is 10 kGy to 30 kGy; the irradiation distance is 20 cm to 80 cm. Preferably, the irradiation dose is 15 kGy to 25 kGy; the irradiation distance is 30 cm to 50 cm.

[0012] The mass ratio of carnosine to polylactic acid (PLA) or the molar ratio of carnosine to PLA monomers also affects the specific grafting quantity. The number of carnosine grafts onto PLA can influence the hydrophilic / lipophilic properties of the final product, which can be characterized by parameters such as the hydrophilic-lipophilic balance (HLB) value and droplet contact angle. By adjusting the reaction conditions and parameters, PLA with different grafting quantities can be obtained, thereby adapting to the hydrophilicity / lipophilicity of different microsphere contents. In some embodiments, the mass ratio of PLA to carnosine is 0.2 to 2:1.

[0013] Since carnosine-grafted polylactic acid (PLA) is soluble in water, while PLA is water-insoluble, solid-liquid separation can be achieved through centrifugation, with the insoluble particles precipitating and being removed. The centrifugation speed of the reaction solution can be determined based on the molecular weight of PLA. The centrifugation speed can be calculated by centrifuging PLA of the same molecular weight under the same irradiation conditions to determine the separation rate. Generally, a centrifugation speed with a higher separation rate should be selected. However, since carnosine-grafted PLA is an amphiphilic polymer, vesicles will form in the solution when the critical micelle concentration is exceeded. Excessively high centrifugation speeds, such as exceeding 15,000 g, will also cause the vesicles to settle. In some embodiments, the centrifugation speed is 100 g to 3,000 g.

[0014] A carnosine-grafted polylactic acid obtained by the above preparation method. The grafting rate of the carnosine is 10%~60%.

[0015] A method for preparing polylactic acid-carnosine microspheres includes the following steps: (i) Carnosine-grafted polylactic acid is dissolved in an organic solvent to obtain an oil phase; (ii) Disperse the oil phase in an aqueous phase containing polyvinyl alcohol to obtain a mixture; (iii) Remove the organic solvent from the mixture to obtain polylactic acid-carnosine microspheres.

[0016] Organic solvents are good solvents for polylactic acid, such as chloroform, dichloromethane, acetone, ethyl acetate, N,N'-dimethylformamide, etc.

[0017] The volume ratio of the oil phase to the aqueous phase is 1:10~50. The polyvinyl alcohol is of type 1788 or 1799; its mass percentage concentration in the aqueous phase is 0.3%~3%.

[0018] The purpose of dispersion is to form small liquids from the oil phase in a continuous aqueous phase. Specifically, dispersion methods can include ultrasonic dispersion and shear dispersion.

[0019] The oil phase may also contain other components, which are encapsulated into the microspheres as solutes (for lipid-soluble components) or insoluble substances (for water-soluble components) to obtain polylactic acid-carnosine microspheres with contents. In some embodiments, the other components are a mixed solution of glycine, alanine, and proline in a mass ratio of (1~5):(1~5):(1~5); preferably, the ratio is 2:2:1. To use water-soluble components as contents of the polylactic acid-carnosine microspheres, the water-soluble component droplets can be dispersed in a mixture of the continuous oil phase as the oil phase in step (i). Preferably, the volume ratio of the total volume of the water-soluble component droplets to the continuous oil phase is 1:3~10. The obtained polylactic acid-carnosine microspheres can be used as a filler for skin filling.

[0020] When microspheres need to enter the bloodstream or lymphatic system for circulation, higher ultrasonic power or shear velocity can reduce the particle size to below 10 μm, preferably below 1 μm. When microspheres need to avoid entering the circulatory system, the particle size generally needs to be controlled above 20 μm. In some embodiments, the particle size of microspheres used for skin filling is 20 μm to 80 μm.

[0021] In some embodiments, the preparation method of polylactic acid-carnosine microspheres includes the following steps: S1. Glycine, alanine, and proline, with concentrations of 0.1 mg / mL to 0.5 mg / mL respectively, are mixed in a mass ratio of 2:2:1 to form the contents solution. S2. Dissolve carnosine-grafted polylactic acid in dichloromethane to obtain an oil phase solution with a mass concentration of 2%~20%; S3. Place the oil phase solution under a shear emulsification device, adjust the shearing speed to 500 rpm to 5000 rpm, inject the contents solution and continue shearing for 10 to 60 seconds. The volume ratio of the contents solution to the oil phase solution is 1:3-10 to obtain an oil phase containing the contents. S4. Place a 0.3-3% polyvinyl alcohol solution under a shear emulsification device, adjust the shear speed to 500 rpm to 5000 rpm, then inject the oil phase from S3, and shear for 10 s to 60 s to obtain an emulsion. The shear speed and shear time are directly proportional to the particle size of the microspheres obtained by shear emulsification. To obtain microspheres of appropriate particle size, appropriate shear speed and shear time are required.

[0022] S5. Stir the emulsion to accelerate the volatilization of dichloromethane and solidify the microspheres. Stir at 50 rpm to 300 rpm for 3 hours. Perform wet sieving on the obtained microsphere suspension to obtain polylactic acid-carnosine microspheres with a size of 20 μm to 80 μm that encapsulate glycine, alanine, and proline.

[0023] A polylactic acid-carnosine microsphere prepared by the above method.

[0024] The application of the above-mentioned carnosine-grafted polylactic acid and polylactic acid-carnosine microspheres in the preparation of injectable dermal fillers. The above-mentioned carnosine-grafted polylactic acid or polylactic acid-carnosine microspheres can be used directly as an active ingredient in dermal fillers, or mixed with other ingredients; the other ingredients can be pharmaceuticals or inert components; the inert components can be sugars, inorganic salts, or organic salts to maintain osmotic pressure, suspending agents to prevent aggregation and precipitation, or preservatives.

[0025] The present invention has the following advantages: This invention provides a carnosine-grafted polylactic acid (PLA) polymer. Carnosine grafting improves the hydrophilicity of PLA, making the polymer amphiphilic. Simultaneously, carnosine provides anti-aging benefits from antioxidant and anti-glycation perspectives. This polymer can be prepared into PLA-carnosine microspheres, which can also encapsulate drugs for sustained-release delivery. Especially when encapsulating amino acids such as glycine and proline, it can serve as a raw material for collagen, promoting collagen synthesis and resulting in better effects when used for skin filling. Under specific preparation conditions, these amphiphilic microspheres exhibit excellent dispersibility and are not prone to aggregation. The microsphere size can be adjusted by regulating the oil phase concentration to maximize the encapsulation of active ingredients and increase their content. Attached Figure Description

[0026] Figure 1 These are micrographs (a, b) and scanning electron microscope images (c, d) of the PLLA / L-carnosine microsphere suspension; where a is magnified 100 times, b is magnified 400 times, c is magnified 500 times, and d is magnified 5000 times. Figure 2 It is the distribution of microsphere particle size range; Figure 3 The results show the collagen content of type I (a) and type III (b) in rabbits after implantation at different times; Figure 4 This refers to the change in molecular weight over time after microspheres in the experimental and control groups were implanted into rabbits. Detailed Implementation

[0027] To obtain carnosine-grafted polylactic acid (PLA) that is more beneficial for skin filling, the preparation of carnosine-grafted PLA includes the following steps, using L-carnosine and poly-L-lactic acid as raw materials: (1) Add poly-L-lactic acid powder with a molecular weight of 16~80kDa to an aqueous solution of L-carnosine with a concentration of 25~100mg / mL, so that the mass ratio of poly-L-lactic acid to L-carnosine is 0.2~2:1, then transfer it to a polymerization tube, and seal the tube after repeated nitrogen purging and deoxygenation. (2) Irradiate under a high-energy electron beam at a dose of 10-25 kGy and a distance of 30-50 cm at an ambient temperature of 25℃. After the reaction, centrifuge at 2500 rpm for 5 min, discard the supernatant, rinse with deionized water, and centrifuge again. Repeat this process multiple times to remove unreacted monomers. Dry under reduced pressure at 0.05 mPa at room temperature to obtain L-carnosine-grafted modified poly-L-lactic acid. Calculate the grafting rate using the following formula: Grafting rate (%) = .

[0028] Under the above reaction conditions, the yield of carnosine-grafted polylactic acid is 10-60%.

[0029] The aforementioned carnosine-grafted polylactic acid can be used as a capsule wall material to prepare microspheres, which can then be used for skin filling. Therefore, this invention provides a method for preparing polylactic acid-carnosine microspheres: (1) Glycine, alanine and proline with concentrations of 0.1 mg / mL to 0.5 mg / mL, respectively, in a mass ratio of 2:2:1, are mixed to form the contents solution; (2) Dissolve carnosine-grafted polylactic acid in dichloromethane to obtain an oil phase solution with a mass concentration of 2% to 20%; (3) Place the oil phase solution under a shear emulsification device, adjust the shearing speed to 500 rpm to 5000 rpm, inject the contents solution and continue shearing for 10-60 s. The volume ratio of the contents solution to the oil phase solution is 1:3-10 to obtain an oil phase containing the contents. (4) Place a polyvinyl alcohol solution with a concentration of 0.3~3% under a shear emulsification device, adjust the shear speed to 500rpm~3500rpm, and then inject the oil phase of S2; (5) Place the aqueous phase solution under a shear emulsification device, adjust the shearing speed to 500 rpm to 5000 rpm, then inject the oil phase, and shear for 10 s to 60 s to obtain an emulsion; S6. Stir the emulsion to accelerate the volatilization of dichloromethane and solidify the microspheres. Stir at 50 rpm to 300 rpm for 3 hours. Perform wet sieving on the obtained microsphere suspension to obtain polylactic acid-carnosine microspheres with a size of 20 μm to 80 μm that encapsulate glycine, alanine, and proline.

[0030] The polylactic acid-carnosine microspheres encapsulating glycine, alanine, and proline prepared by the above method can be used as injectable dermal fillers. They can be mixed with a lyophilization medium (mannitol and sodium cellulose solution) and then lyophilized to form a lyophilized powder. Before use, they can be added to water for injection or physiological saline or compounded with lidocaine hydrochloride and injected into the desired area (nasolabial folds, neck wrinkles, etc.). The single dose of microspheres should not exceed 200 mg.

[0031] The present invention will be further described below with reference to the embodiments and accompanying drawings, but the present invention is not limited to the following embodiments.

[0032] Example 1: Preparation of carnosine-grafted polylactic acid Add 5g of poly-L-lactic acid powder (molecular weight 50kDa) to 30mL of L-carnosine aqueous solution with a concentration of 85mg / mL, transfer to a polymerization tube, repeatedly purge with nitrogen to remove oxygen, and then seal the tube.

[0033] The mixture was irradiated under a high-energy electron beam at a dose of 15-25 kGy and a distance of 30-70 cm at an ambient temperature of 25℃. After the reaction, the mixture was centrifuged at 2500 g for 5 min, the supernatant was discarded, and the mixture was rinsed with 40 mL of deionized water and centrifuged again. This process was repeated multiple times to remove unreacted monomers. The mixture was then dried under reduced pressure of 0.05 MPa at room temperature to obtain the modified poly(L-lactic acid / L-carnosine) polymer.

[0034] Table 1 Effect of irradiation dose on copolymer yield Table 2 Effect of irradiation distance on copolymer yield As shown in Tables 1 and 2, a high yield can be obtained when the irradiation distance is 30-50 cm and the irradiation dose is 20 kGy; the carnosine grafting rate at the highest yield is 58%.

[0035] Example 2 Preparation of polylactic acid-carnosine microspheres A 16% oil phase solution was prepared by dissolving the grafted polylactic acid / L-carnosine polymer in dichloromethane; glycine, alanine, and proline were prepared into solutions with concentrations of 0.5 mg / mL, 0.5 mg / mL, and 0.25 mg / mL, respectively; and polyvinyl alcohol was dissolved in water to prepare an aqueous phase solution with a concentration of 0.35%.

[0036] Polylactic acid-carnosine microspheres containing the contents were prepared according to the following method: The contents solution and the oil phase solution were measured in a volume ratio of 1:4. The oil phase solution was then placed under a shear emulsification device, the shearing speed was adjusted to 4200 rpm, the contents solution was injected, and shearing continued for 40 seconds to obtain an oil phase solution containing the contents. Two solutions were measured with a volume ratio of oil phase solution to aqueous phase solution of 1:25. The aqueous phase solution was placed under a shear emulsification device and the shearing speed was adjusted to 4000 rpm. Then the oil phase was injected and sheared for 18 seconds to obtain an emulsion. The emulsion was stirred to accelerate the volatilization of dichloromethane and solidify the microspheres. Stirring at 160 rpm for 3 hours yielded a microsphere suspension containing PLLA / L-carnosine microspheres encapsulated with three amino acids. After lyophilization, the microsphere powder was obtained. Microscopic and scanning electron microscope images are shown below. Figure 1 As shown, it presents a sphere with a relatively uniform diameter.

[0037] Add the microsphere powder to a 2% sodium dodecyl sulfate (SDS) solution to disperse it evenly; slowly add it to a beaker in the sample processing unit, and measure the microsphere particle size with the detector occlusion in the range of 2% to 20%. Stir at a speed of 1500 rpm (sonicate for about 30 seconds before starting the measurement), and measure three times consecutively. Take the average value as the microsphere particle size.

[0038] The results are as follows Figure 2 As shown, the microspheres have a D10 of 27.137 μm, a D50 of 37.284 μm, and a D90 of 53.691 μm. More than 80% of the microspheres have a particle size distribution between 25.179 μm and 50.238 μm, and the particle size range is uniform, which can reduce the immune response.

[0039] Comparative Example 1: Preparation of Polylactic Acid / Carnosine Microspheres Poly(L-lactic acid) (50 kDa) was dissolved in dichloromethane to obtain an oil phase solution with a concentration of 16%; glycine, alanine, and proline were prepared into solutions with concentrations of 0.5 mg / mL, 0.5 mg / mL, and 0.25 mg / mL, respectively; polyvinyl alcohol and carnosine were dissolved in water to obtain an aqueous phase solution, wherein the concentration of polyvinyl alcohol was 0.35% and the concentration of carnosine was 0.32%.

[0040] Polylactic acid / carnosine microspheres containing the contents were prepared according to the following method: The contents solution and the oil phase solution were measured in a volume ratio of 1:4. The oil phase solution was then placed under a shear emulsification device, the shearing speed was adjusted to 4200 rpm, the contents solution was injected, and shearing continued for 40 seconds to obtain an oil phase solution containing the contents. Two solutions were measured with a volume ratio of oil phase solution to aqueous phase solution of 1:25. The aqueous phase solution was placed under a shear emulsification device and the shearing speed was adjusted to 4000 rpm. Then the oil phase was injected and sheared for 18 seconds to obtain an emulsion. The emulsion was stirred to accelerate the volatilization of dichloromethane and solidify the microspheres. The mixture was stirred at 160 rpm for 3 hours to obtain a microsphere suspension containing PLLA microspheres encapsulated with three amino acids. After freeze-drying, the microspheres and carnosine powder were obtained.

[0041] Application example: Polylactic acid-carnosine microspheres used as injectable dermal fillers The microspheres obtained in Example 1 and Comparative Example 1 were used as experimental and control samples, respectively. New Zealand white rabbits were used as experimental subjects to simulate their effects on in vivo degradation and promotion of collagen synthesis. The fur on the back and sides of the spine of the rabbits was shaved to create a sufficient implantation area. The rabbits were anesthetized with sodium pentobarbital intravenously, and the surgical site was disinfected with povidone-iodine. Two implantation points were selected on each side of the rabbit's spine, 2.5 cm from the midline and parallel to the spine, and permanently marked. Implanted samples were spaced approximately 2.5 cm apart. Control samples were implanted on one side of the rabbit's subcutaneous tissue, and experimental samples were implanted on the other side. At 1, 3, 6, 9, 12, and 15 months post-implantation, rabbits were randomly euthanized. A harvest of tissue containing the implant and sufficient surrounding tissue was placed in pre-chilled PBS buffer. After washing away blood and impurities, the tissue was homogenized and added to pre-chilled PBS at a 1:5 ratio (1g tissue to 5mL). The homogenate was sonicated in an ice bath, centrifuged at 5000 rpm for 30 min, the supernatant was discarded, and the microspheres were collected and dried at 55°C for in vivo degradation studies. The microsphere powder was then placed in 20mL of… Dissolve the polystyrene in chloroform in a beaker to prepare a 9 mg / mL solution, shake, and let stand overnight at room temperature to prepare the test solution. Take polystyrene standards of different molecular weights, dilute them with chloroform to a certain concentration, shake, and let stand overnight at room temperature to prepare the reference solutions. The injection volume of both the test solution and the reference solution is 20 μL. Use a gel chromatography column with cross-linked polystyrene gel (5 μm) as the packing material, chloroform as the mobile phase, differential refractive index detector, column temperature 30℃, and the theoretical plate number calculated based on the polystyrene peak should not be less than 3000.

[0042] Depend on Figure 3 It can be seen that after microsphere implantation, both the experimental group and the control group mice were stimulated by the implant to promote the production of type I and type III collagen to varying degrees. Both groups showed that type III collagen was first accumulated and then gradually produced to provide network support. In particular, the experimental group, due to the encapsulation of glycine, alanine and proline, and their gradual release, continued to stimulate the production of type III collagen at 12 months, compared with the control group.

[0043] Tissue samples were obtained from the area surrounding the injection site in rabbits, and type I and type III collagen were immunofluorescently stained and processed using ImageJ for quantitative analysis.

[0044] Gel permeation chromatography (GPC) was used to determine the weight-average molecular weight of microspheres to infer their in vivo degradation. Figure 4 As shown: After implantation, the experimental group was completely degraded within one year.

[0045] Example 3 Preparation of polylactic acid-carnosine microspheres 1. Preparation of carnosine-grafted polylactic acid (1) Weigh 5g of poly-L-lactic acid powder with an average molecular weight of 20kDa and add it to 50mL of L-carnosine aqueous solution with a concentration of 100mg / mL. Then transfer it to a polymerization tube, repeatedly purge with nitrogen to remove oxygen, and then seal the tube. (2) Place it under a high-energy electron beam for irradiation. The irradiation dose is 15 kGy, the irradiation distance is 30 cm, and the ambient temperature is 25℃. After the reaction, centrifuge at 3000g for 5 min and discard the supernatant. Add deionized water to rinse and centrifuge again. Repeat this process multiple times to remove unreacted monomers. After drying under reduced pressure of 0.05 MPa at room temperature, L-carnosine-grafted modified poly-L-lactic acid is obtained with a grafting rate of 38%.

[0046] 2. Preparation of carnosine-grafted polylactic acid microspheres Carnosine-grafted polylactic acid was dissolved in dichloromethane to obtain a 20% oil phase solution; a 3% polyvinyl alcohol solution was prepared as the aqueous phase solution.

[0047] Two solutions were measured with a volume ratio of oil phase solution to aqueous phase solution of 1:15. The aqueous phase solution was placed under a shear emulsification device, and the shearing speed was adjusted to 4000 rpm. Then the oil phase solution was injected, and the shearing time was 18 s to obtain an emulsion. The emulsion was stirred to accelerate the volatilization of dichloromethane and solidify the microspheres. The mixture was stirred at 150 rpm for 3 hours to obtain a microsphere suspension. After freeze-drying, microsphere powder was obtained with a median diameter of 53 micrometers.

[0048] Example 4 Preparation of polylactic acid-carnosine microspheres 1. Preparation of carnosine-grafted polylactic acid (1) Weigh 1g of poly-L-lactic acid powder with an average molecular weight of 80kDa and add it to 50mL of L-carnosine aqueous solution with a concentration of 100mg / mL. Then transfer it to a polymerization tube, repeatedly purge with nitrogen to remove oxygen, and then seal the tube. (2) Place it under a high-energy electron beam for irradiation. The irradiation dose is 25 kGy, the irradiation distance is 30 cm, and the ambient temperature is 25℃. After the reaction, centrifuge at 1000g for 5 min and discard the supernatant. Add deionized water to rinse and centrifuge again. Repeat this process multiple times to remove unreacted monomers. After drying under reduced pressure of 0.05 MPa at room temperature, L-carnosine-grafted modified poly-L-lactic acid is obtained with a grafting rate of 27%.

[0049] 2. Preparation of carnosine-grafted polylactic acid microspheres Carnosine-grafted polylactic acid was dissolved in dichloromethane to obtain a 5% oil phase solution; a 1% polyvinyl alcohol solution was prepared as the aqueous phase solution.

[0050] Two solutions were measured with a volume ratio of oil phase solution to aqueous phase solution of 1:10. The aqueous phase solution was placed under a shear emulsification device, and the shearing speed was adjusted to 4000 rpm. Then the oil phase solution was injected, and the shearing time was 10 s to obtain an emulsion. The emulsion was stirred to accelerate the volatilization of dichloromethane and solidify the microspheres. The mixture was stirred at 150 rpm for 3 hours to obtain a microsphere suspension. After freeze-drying, microsphere powder was obtained with a median diameter of 41 micrometers.

[0051] Example 5 Preparation of polylactic acid-carnosine microspheres 1. Preparation of carnosine-grafted polylactic acid (1) Weigh 5g of poly-L-lactic acid powder with an average molecular weight of 50kDa and add it to 100mL of L-carnosine aqueous solution with a concentration of 100mg / mL. Then transfer it to a polymerization tube, repeatedly purge with nitrogen to remove oxygen, and then seal the tube. (2) Place it under a high-energy electron beam for irradiation. The irradiation dose is 15 kGy, the irradiation distance is 30 cm, and the ambient temperature is 25℃. After the reaction, centrifuge at 3000g for 5 min and discard the supernatant. Add deionized water to rinse and centrifuge again. Repeat this process multiple times to remove unreacted monomers. After drying under reduced pressure of 0.05 MPa at room temperature, L-carnosine-grafted modified poly-L-lactic acid is obtained with a grafting rate of 40%.

[0052] 2. Preparation of carnosine-grafted polylactic acid microspheres A 25% oil phase solution was prepared by dissolving the grafted polylactic acid / L-carnosine polymer in dichloromethane; a solution of glycine, alanine, and proline was prepared with concentrations of 0.5 mg / mL, 0.5 mg / mL, and 0.25 mg / mL, respectively; and a 2% aqueous phase solution was prepared by dissolving polyvinyl alcohol in water.

[0053] Polylactic acid-carnosine microspheres containing the contents were prepared according to the following method: The contents solution and the oil phase solution were measured in a volume ratio of 1:9. The oil phase solution was then placed under a shear emulsification device, the shearing speed was adjusted to 3500 rpm, the contents solution was injected, and shearing continued for 60 seconds to obtain an oil phase solution containing the contents. Two solutions were measured with a volume ratio of oil phase solution to aqueous phase solution of 1:15. The aqueous phase solution was placed under a shear emulsification device, and the shearing speed was adjusted to 4000 rpm. Then the oil phase was injected, and the shearing time was 20 s to obtain an emulsion. The emulsion was stirred to accelerate the volatilization of dichloromethane and solidify the microspheres. After stirring at 150 rpm for 3 hours, the microsphere powder was obtained by freeze drying, with a median diameter of 45 micrometers.

[0054] The above description is merely an embodiment of this application and is not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. A method for preparing carnosine-grafted polylactic acid, characterized in that, Includes the following steps: (1) Mix polylactic acid and carnosine in water to obtain a mixture; (2) The mixture is subjected to radiation reaction under oxygen-free conditions to obtain a reaction solution; (3) After the reaction solution is separated into solid and liquid, the liquid is dried to obtain carnosine-grafted polylactic acid; The molecular weight of polylactic acid is 16kDa~80kDa; The mass ratio of polylactic acid to carnosine is 0.2~2:1; The irradiation dose is 10kGy to 30kGy; the irradiation distance is 20cm to 80cm.

2. The preparation method according to claim 1, characterized in that, The polylactic acid is L-polylactic acid, D-polylactic acid, or racemic polylactic acid; The irradiation dose is 15kGy~25kGy; the irradiation distance is 30cm~50cm; The concentration of carnosine in water is 25~100 mg / mL.

3. A carnosine-grafted polylactic acid obtained by the preparation method according to claim 1 or 2, characterized in that, The grafting rate of the carnosine is 10% to 60%.

4. A method for preparing polylactic acid-carnosine microspheres, characterized in that, Includes the following steps: (i) Dissolve the carnosine-grafted polylactic acid according to claim 3 in an organic solvent to obtain an oil phase; (ii) Disperse the oil phase in an aqueous phase containing polyvinyl alcohol to obtain a mixture; (iii) Remove the organic solvent from the mixture to obtain polylactic acid-carnosine microspheres.

5. The preparation method according to claim 4, characterized in that, The organic solvent is selected from chloroform, dichloromethane, acetone, ethyl acetate, or N,N'-dimethylformamide; The volume ratio of the oil phase to the water phase is 1:10~50; The polyvinyl alcohol has a mass percentage concentration of 0.3% to 3% in the aqueous phase. The dispersion method is ultrasonic dispersion or shear dispersion. The oil phase also contains other components.

6. The preparation method according to claim 5, characterized in that, The polyvinyl alcohol is of type 1788 or 1799; the other components are a mixed solution of glycine, alanine and proline in a mass ratio of (1~5): (1~5): (1~5).

7. The preparation method according to claim 6, characterized in that, Includes the following steps: S1. Glycine, alanine and proline in a mass ratio of 2:2:1 and with concentrations of 0.1 mg / mL to 0.5 mg / mL are mixed to form the contents solution. S2. Dissolve carnosine-grafted polylactic acid in the organic solvent dichloromethane to obtain an oil phase solution with a mass concentration of 2%~20%; S3. Place the oil phase solution under a shear emulsification device, adjust the shearing speed to 500 rpm to 5000 rpm, inject the contents solution and continue shearing for 10 to 60 seconds. The volume ratio of the contents solution to the oil phase solution is 1:3 to 10 to obtain an oil phase containing the contents. S4. A polyvinyl alcohol solution with a concentration of 0.3-3% is used as the aqueous phase and placed under a shear emulsification device. The shearing speed is adjusted to 500 rpm to 5000 rpm. Then, the oil phase containing the contents of S3 is injected. The shearing time is 10 s to 60 s to obtain an emulsion. S5. Stir the emulsion to evaporate the organic solvent and solidify the microspheres. Stir at 50 rpm to 300 rpm for 3 hours. Perform wet sieving on the obtained microsphere suspension to obtain polylactic acid-carnosine microspheres with a size of 20 μm to 80 μm that encapsulate glycine, alanine, and proline.

8. A polylactic acid-carnosine microsphere obtained by the preparation method according to any one of claims 4-7.

9. The polylactic acid-carnosine microspheres according to claim 8, characterized in that, The particle size is 20μm~80μm.

10. The use of carnosine-grafted polylactic acid as described in claim 3 and polylactic acid-carnosine microspheres as described in claim 8 or 9 in the preparation of injectable dermal fillers.

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