L-polylactic acid microspheres as well as preparation method and application thereof

Abstract

The invention provides a poly-L-lactic acid microsphere. The poly-L-lactic acid microsphere is obtained by performing membrane emulsification on a poly-L-lactic acid-polyethylene glycol copolymer synthesized by taking lactide and polyethylene glycol as raw materials. According to the L-polylactic acid microspheres disclosed by the invention, the uniformity, the stability and the biocompatibility of the microspheres are improved, and meanwhile, the synthesis of skin collagen is promoted.

Description

TECHNICAL FIELD

[0001] The present disclosure belongs to the field of biomedical materials, and particularly relates to a left-handed polylactic acid microsphere and a preparation method and application thereof. BACKGROUND

[0002] The left-handed polylactic acid has a wide application prospect in the fields of medical aesthetics, tissue engineering and skin repair due to its excellent biocompatibility and biodegradability. In recent years, the left-handed polylactic acid microsphere is widely used for soft tissue filling and anti-aging treatment due to its slow degradation in vivo and continuous stimulation of collagen production, and becomes an important material in medical injection preparations.

[0003] However, the commercially available left-handed polylactic acid microsphere preparation still has problems such as uneven particle size distribution, poor dispersibility, solvent residue and the like in the preparation process, which seriously affects the stability and clinical effect of the preparation. Traditional emulsification methods such as high-energy emulsification or mechanical shear emulsification often lead to difficulty in accurately controlling the particle size of the microsphere, thereby affecting the uniformity and batch consistency of the product.

[0004] Therefore, it is of great significance to develop a left-handed polylactic acid microsphere material with controllable particle size, good dispersibility, high biological safety and collagen production promotion effect for improving its clinical application value in the fields of medical aesthetics and tissue engineering. SUMMARY

[0005] In view of the deficiencies in the prior art, the present disclosure provides a left-handed polylactic acid microsphere, which overcomes the problems of uneven particle size distribution, poor dispersibility, solvent residue and the like of the microsphere, improves the uniformity, stability and biocompatibility of the microsphere, and enhances the application effect of the microsphere in the fields of medical aesthetics, tissue engineering and skin repair.

[0006] According to one aspect of the present disclosure, a left-handed polylactic acid microsphere is provided, wherein the average particle size of the left-handed polylactic acid microsphere is 20-120 μm, and the particle size distribution coefficient is 10-60%.

[0007] In some embodiments, the average particle size of the left-handed polylactic acid microsphere is 40-100 μm, preferably 40-70 μm, and more preferably 40-55 μm. In some embodiments, the average particle size of the left-handed polylactic acid microsphere is 40 μm, 45 μm, 49 μm, 50 μm, 53 μm, 55 μm, 57 μm, 60 μm, 65 μm, 70 μm, 75 μm, 76 μm, 79 μm, 80 μm, 85 μm, 90 μm, 95 μm, 100 μm or any value therebetween.

[0008] In some embodiments, the poly-L-lactic acid microspheres have a particle size distribution coefficient of 15-50%, preferably 20-30%, more preferably 20-25%. In some embodiments, the poly-L-lactic acid microspheres have a particle size distribution coefficient of 15%, 17%, 20%, 22%, 24%, 25%, 28%, 30%, 32%, 35%, 37%, 40%, 41%, 45%, 47%, 49%, 50%, or any value therebetween.

[0009] In some embodiments, the poly-L-lactic acid microspheres are obtained by film emulsification of a poly-L-lactic acid-polyethylene glycol (PLLA-PEG) copolymer synthesized from lactide and polyethylene glycol.

[0010] According to another aspect of the present disclosure, a method for preparing poly-L-lactic acid microspheres is provided, the method comprising the following steps:

[0011] S1: ring-opening polymerization of lactide and polyethylene glycol in the presence of a catalyst to obtain a poly-L-lactic acid-polyethylene glycol copolymer;

[0012] S2: film emulsification of a polymer solution containing the poly-L-lactic acid-polyethylene glycol copolymer to obtain the poly-L-lactic acid microspheres.

[0013] In some embodiments, in step S1, the ring-opening polymerization is performed at a temperature of 100-200°C for 0.5-8h. In some embodiments, the ring-opening polymerization is performed at a temperature of 100°C, 120°C, 140°C, 160°C, 180°C, 200°C, or any value therebetween. In some embodiments, the ring-opening polymerization is performed for a time of 0.5h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, or any value therebetween.

[0014] In some embodiments, the catalyst comprises a metal organic compound.

[0015] In some embodiments, the catalyst comprises one or more of stannous octoate, tin octoate, lanthanum trifluoride, cobalt acetylacetonate, aluminum acetylacetonate, or diethyl zinc.

[0016] In some embodiments, in step S1, the polyethylene glycol has a molecular weight of 400-2000, for example, 400, 600, 800, 1000, 2000, or any value therebetween.

[0017] In some embodiments, in step S1, the molar ratio of the lactide and the polyethylene glycol is (100-1000): 1, for example, 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, or any value therebetween.

[0018] In some embodiments, in step S1, the molecular weight of the poly (L-lactic acid)-polyethylene glycol copolymer is 5-130,000 Daltons, for example, 50,000 Daltons, 60,000 Daltons, 70,000 Daltons, 80,000 Daltons, 90,000 Daltons, 100,000 Daltons, 110,000 Daltons, 120,000 Daltons, 130,000 Daltons, or any value therebetween.

[0019] In some embodiments, in step S2, the mass concentration of the poly (L-lactic acid)-polyethylene glycol copolymer in the polymer solution is 5%-20%, preferably 16-20%. In some embodiments, in step S2, the mass concentration of the poly (L-lactic acid)-polyethylene glycol copolymer in the polymer solution is 5%, 8%, 10%, 12%, 14%, 15%, 15.6%, 16%, 16.7%, 17%, 17.2%, 18%, 19%, 20%, or any value therebetween.

[0020] In some embodiments, in step S2, the polymer solution further comprises an organic solvent.

[0021] In some embodiments, the organic solvent is selected from one or more of dichloromethane, chloroform, ethyl acetate, or tetrahydrofuran.

[0022] In some embodiments, in step S2, the pore size of the microporous membrane used in the membrane emulsification treatment is 5-20 μm, preferably 8-14 μm. In some embodiments, in step S2, the pore size of the microporous membrane used in the membrane emulsification treatment is 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 9.9 μm, 10 μm, 12 μm, 13 μm, 13.1 μm, 14 μm, 16 μm, 18 μm, 20 μm, or any value therebetween.

[0023] In some embodiments, in step S2, the transmembrane pressure of the membrane emulsification treatment is 5-30 kPa, preferably 10-13 kPa. In some embodiments, in step S2, the transmembrane pressure of the membrane emulsification treatment is 5 kPa, 7 kPa, 10 kPa, 11 kPa, 12 kPa, 13 kPa, 14 kPa, 15 kPa, 17 kPa, 20 kPa, 25 kPa, 30 kPa, or any value therebetween.

[0024] In some embodiments, the stirring rate of the membrane emulsification treatment in step S2 is 100-400 rpm, preferably 250-300 rpm. In some embodiments, the stirring rate of the membrane emulsification treatment in step S2 is 100 rpm, 150 rpm, 200 rpm, 250 rpm, 280 rpm, 300 rpm, 350 rpm, 400 rpm, or any value therebetween.

[0025] In some embodiments, step S2 further comprises dispersing the emulsion droplets obtained from the membrane emulsification treatment in a water phase containing a surfactant, and then performing organic solvent removal, washing, and drying treatment to obtain the poly-L-lactic acid microspheres.

[0026] In some embodiments, the water phase contains polyvinyl alcohol and sodium dodecyl sulfate.

[0027] In some embodiments, the mass concentration of polyvinyl alcohol in the water phase is 1-5%, and the mass concentration of sodium dodecyl sulfate in the water phase is 0.01-1%. In some embodiments, the mass concentration of polyvinyl alcohol in the water phase can be 1%, 2%, 3%, 4%, 5%, or any value therebetween, and the mass concentration of sodium dodecyl sulfate in the water phase is 0.01%, 0.05%, 0.1%, 0.2%, 0.5%, 0.8%, 1%, or any value therebetween.

[0028] According to yet another aspect of the present disclosure, a poly-L-lactic acid microsphere preparation is provided, which comprises the poly-L-lactic acid microspheres of the present disclosure or the poly-L-lactic acid microspheres obtained by the preparation method of the present disclosure.

[0029] In some embodiments, the poly-L-lactic acid microsphere preparation further comprises one or more of a lyoprotectant, a thickening agent, and physiological saline.

[0030] In some embodiments, the poly-L-lactic acid microsphere preparation comprises a lyophilized poly-L-lactic acid microsphere powder or a poly-L-lactic acid microsphere injection.

[0031] In some embodiments, the thickening agent comprises one or more of sodium carboxymethyl cellulose, lysine, glutamic acid, sodium hyaluronate, polyvinylpyrrolidone, or xanthan gum.

[0032] In some embodiments, the lyoprotectant comprises mannitol.

[0033] According to yet another aspect of the present disclosure, a preparation method of a lyophilized poly-L-lactic acid microsphere powder is provided, which comprises the following steps: dispersing and freeze-drying a mixture containing the poly-L-lactic acid microspheres of the present disclosure or the poly-L-lactic acid microspheres obtained by the preparation method of the present disclosure, a thickening agent, and a lyoprotectant.

[0034] In some embodiments, the thickening agent comprises one or more of sodium carboxymethylcellulose, lysine, glutamic acid, sodium hyaluronate, polyvinylpyrrolidone, or xanthan gum, preferably sodium carboxymethylcellulose and / or glutamic acid.

[0035] In some embodiments, the lyoprotectant comprises mannitol.

[0036] In some embodiments, the mixture comprises, by weight, 1 part of the PLLA microspheres, 0.9-1.0 parts of the lyoprotectant, and 0.15-0.2 parts of the thickening agent. In some embodiments, the mixture comprises, by weight, 1 part of the PLLA microspheres, 0.9, 0.92, 0.94, 0.95, 0.96, 0.98, 1.0, or any value therebetween parts of the lyoprotectant, and 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, or any value therebetween parts of the thickening agent.

[0037] In some embodiments, the dispersion treatment comprises a stirring treatment and / or an ultrasonic treatment.

[0038] In some embodiments, the stirring rate of the stirring treatment is 200-600 rpm, for example, 200 rpm, 300 rpm, 400 rpm, 500 rpm, 600 rpm, or any value therebetween.

[0039] In some embodiments, the ultrasonic power of the ultrasonic treatment is 100-500 W, for example, 100 W, 200 W, 300 W, 400 W, 500 W, or any value therebetween.

[0040] In some embodiments, the preparation method further comprises an irradiation sterilization treatment on the freeze-dried powder after the freeze-drying treatment.

[0041] In some embodiments, the irradiation dose of the irradiation sterilization treatment is 25-30 kGy, for example, 25 kGy, 26 kGy, 27 kGy, 28 kGy, 29 kGy, 30 kGy, or any value therebetween.

[0042] According to yet another aspect of the present disclosure, there is provided a preparation method of a PLLA microsphere injection, the preparation method comprising dispersing the PLLA microspheres as described in the present disclosure, the PLLA microspheres obtained by the preparation method as described in the present disclosure, or the freeze-dried powder of the PLLA microspheres obtained by the preparation method as described in the present disclosure in a buffer solution.

[0043] In some embodiments, the buffer solution comprises physiological saline.

[0044] In some embodiments, the mass concentration of the PLLA microspheres in the injection solution is 2-100 mg / mL, for example, 2 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 80 mg / mL, 100 mg / mL, or any value therebetween.

[0045] According to yet another aspect of the present disclosure, there is provided use of the PLLA microspheres, the PLLA microspheres prepared by the preparation method, the PLLA microspheres preparation, the PLLA microspheres lyophilized powder, or the PLLA microspheres injection solution in the present disclosure in the preparation of a product, which includes a medical aesthetic material, a tissue engineering material, or a skin repair material.

[0046] In some embodiments, the PLLA microspheres, the PLLA microspheres preparation, the PLLA microspheres lyophilized powder, or the PLLA microspheres injection solution are used to promote collagen production, increase collagen thickness, and / or increase collagen density of the skin.

[0047] The PLLA microspheres of the present disclosure have uniform particle size and good dispersibility. Moreover, the membrane emulsification method is used to prepare the microspheres, which can accurately control the particle size of the emulsion droplets, obtain PLLA microspheres with uniform particle size and good dispersibility, and improve the stability of the product. Meanwhile, the introduction of the PLLA-PEG copolymer enhances the hydrophilicity of the polymer, reduces the aggregation and sedimentation of the microspheres in water, and also improves the water dispersion of the PLLA microspheres and the uniformity of the preparation. In addition, the present disclosure also optimizes the excipient system of the PLLA microspheres preparation, improves the biocompatibility: innovatively uses glutamic acid as a thickening agent to improve the uniform dispersibility of the microspheres, promote collagen synthesis, and improve the medical aesthetic application effect, which has high industrial application value and can be widely applied in the fields of medical aesthetics, tissue engineering, and skin repair. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 The scanning electron microscope photos of the PLLA microspheres prepared in Example 1 are shown.

[0049] Figure 2 The scanning electron microscope photos of the PLLA microspheres prepared in Example 2 are shown.

[0050] Figure 3 The scanning electron microscope photos of the PLLA microspheres prepared in Example 3 are shown.

[0051] Figure 4 The scanning electron microscope photos of the PLLA microspheres prepared in Example 4 are shown.

[0052] Figure 5The image shown is a scanning electron microscope (SEM) image of the PLLA microspheres prepared in Example 5.

[0053] Figure 6 The image shown is a scanning electron microscope (SEM) image of the PLLA microspheres prepared in Example 6.

[0054] Figure 7 The image shown is a scanning electron microscope image of the PLLA microspheres prepared in Example 7.

[0055] Figure 8 The image shown is a scanning electron microscope (SEM) image of the PLLA microspheres prepared in Example 8.

[0056] Figure 9 The reconstitution effect of the PLLA lyophilized powders in Examples 9-14 is shown.

[0057] Figure 10 The results of HE staining of skin sections from a mouse skin aging model at different concentrations of PLLA microsphere injection at weeks 8 and 20 are shown. The magnification of the figure is 20x and the scale bar is 100 μm.

[0058] Figure 11 Masson staining results of skin sections from a mouse skin aging model at different concentrations of PLLA microsphere injection at weeks 8 and 20 are shown. The magnification of the figure is 20x and the scale bar is 100 μm.

[0059] Figure 12 The dermal thickness of mouse skin treated with different concentrations of PLLA microspheres at weeks 8 and 20 is shown.

[0060] Figure 13 The results show the collagen density in mouse skin treated with different concentrations of PLLA microsphere injection at weeks 8 and 20. Detailed Implementation

[0061] Polylactic acid (PLA) is a hydrophobic polymer that is prone to aggregation and sedimentation in aqueous systems, making it difficult to form a uniform dispersion when used as an injectable material. This affects injection smoothness and in vivo efficacy. In this disclosure, the introduction of polyethylene glycol (PEG) significantly improves the polymer's hydrophilicity, reduces microsphere aggregation in the aqueous phase, and enhances its dispersion stability and biocompatibility.

[0062] In this disclosure, membrane emulsification technology is used in the preparation of L-polylactic acid microspheres. Compared with traditional emulsification technology, membrane emulsification can effectively control the microsphere particle size and distribution, improving product uniformity; the emulsification process is gentle, avoiding damage to the polymer structure caused by high shear; and less organic solvent is required, further enhancing the biocompatibility of the formulation.

[0063] Glutamic acid is introduced in the present disclosure as a thickening adjuvant to further improve the dispersibility and stability of the L-polylactic acid microsphere preparation during the dissolution process. Glutamic acid can effectively reduce the aggregation of microspheres in solution, forming a more stable and uniform dispersion system; at the same time, as a naturally occurring amino acid in the human body, glutamic acid has good tissue compatibility and participates in the cell metabolism process, which can provide certain nutritional support for skin repair.

[0064] Based on the above, the present disclosure provides an L-polylactic acid microsphere preparation, and the preparation method thereof includes the following steps: (1) synthesizing block L-polylactic acid with L-lactide and polyethylene glycol as raw materials to improve the hydrophilicity of PLLA; (2) preparing microspheres by membrane emulsification method, and obtaining microspheres with good dispersibility and an average particle size of 20-100 μm by optimizing the emulsification process, thereby avoiding the residual dissolution problem of traditional microsphere preparation; (3) mixing the obtained microspheres with ultrapure water, mannitol as a freeze-drying protective agent, and a thickening agent in a certain proportion, and the present disclosure uses a new amino acid thickening agent to further improve the dispersibility and stability of the PLLA microsphere preparation during the reconstitution process, and its tissue repair ability; (4) preparing a microsphere lyophilized powder by lyophilizing the solution prepared in step (3); and (5) irradiation sterilization treatment of the lyophilized powder obtained in step (4) to ensure the sterility and stability of the preparation. The PLLA preparation prepared by the method can be uniformly dispersed when reconstituted, has good injection property, and the L-polylactic acid microsphere preparation significantly promotes the generation of collagen in mouse animal experiments, showing good tissue repair and regeneration ability. The preparation of the present disclosure can be prepared on a large scale and is suitable for medical and aesthetic and tissue engineering fields, and has a broad clinical application prospect.

[0065] In some embodiments, the preparation method includes the following steps:

[0066] (1) synthesizing L-polylactic acid (PLLA) with L-lactide and polyethylene glycol (PEG) as raw materials;

[0067] (2) preparing L-polylactic acid microspheres with the L-polylactic acid of (1) as raw material by membrane emulsification method, and obtaining microspheres with uniform particle size and good dispersibility by optimizing the emulsification process, and the particle size range is 20-100 μm;

[0068] (3) uniformly mixing the microspheres of (2) with ultrapure water, mannitol and a thickening agent, lyophilizing to obtain a lyophilized powder, and sterilizing by irradiation to ensure the safety and stability of the preparation.

[0069] In some embodiments, the preparation method includes the following steps:

[0070] Step one: synthesis of PLLA-PEG copolymer

[0071] The ring-opening polymerization reaction is carried out under the protection of inert gas, using L-lactide and polyethylene glycol (PEG) as monomers. The reaction temperature is controlled at 100-200°C, and the reaction time is 0.5-8 hours. The catalyst can be selected from metal organic compounds, such as stannous octoate, tin octoate, lanthanum trifluoride, cobalt acetylacetonate, aluminum acetylacetonate or diethyl zinc, etc., and preferably stannous octoate or tin octoate. After the reaction, the PLLA-PEG copolymer with a molecular weight of 50-130 kDa is obtained through dissolution, purification by precipitation, filtration and drying. The introduction of PEG significantly improves the hydrophilicity of the left-handed polylactic acid, improves the dispersibility in the aqueous system, and reduces the risk of aggregation and sedimentation of the microspheres.

[0072] Step two: preparation of left-handed polylactic acid microspheres

[0073] The PLLA-PEG copolymer is dissolved in an organic solvent (such as dichloromethane, chloroform, ethyl acetate or tetrahydrofuran, preferably dichloromethane) to form a solution with a mass concentration of 5%-20%. The membrane emulsification method is used, and the emulsion is emulsified by a microporous membrane with a pore size of 5-20 μm. The transmembrane pressure (5-30 kPa) and stirring rate (100-400 rpm) are adjusted to uniformly disperse the emulsion droplets into an aqueous phase containing a surfactant (such as 1-5 wt.% polyvinyl alcohol PVA and 0.01-1 wt.% sodium dodecyl sulfate SDS). Under the conditions of stirring (200-600 rpm) and ultrasonic treatment (100-500 W), the organic solvent is volatilized to solidify and form the microspheres. Finally, the left-handed polylactic acid microspheres with uniform particle size and good dispersibility are obtained after separation, washing and drying. Compared with the traditional mechanical emulsification, the membrane emulsification method has the advantages of precise control of emulsion droplet size, less solvent residue and mild process, which helps to improve the safety and consistency of the preparation.

[0074] Step three: preparation of left-handed polylactic acid microsphere lyophilized powder

[0075] The obtained microspheres are mixed with a lyophilization protectant (such as mannitol) and a thickening agent (such as glutamic acid (Glu), sodium carboxymethyl cellulose (CMC), sodium hyaluronate, polyvinylpyrrolidone or xanthan gum, etc., preferably sodium carboxymethyl cellulose and glutamic acid), and an appropriate amount of physiological saline is added. After stirring and ultrasonic dispersion, the left-handed polylactic acid microsphere lyophilized powder is prepared by freeze-drying. Glutamic acid, as a new type of thickening excipient, has good water solubility, which can significantly improve the uniformity of the microspheres in the aqueous phase. Due to its good biocompatibility and metabolic activity in the body, it helps to enhance the tissue repair effect of the microspheres. The present application also uses irradiation sterilization method to ensure the sterility and long-term stability of the final preparation.

[0076] In some embodiments, the PLLA microspheres, mannitol, thickening agent include or are made of the following parts by weight: 1 part of PLLA, 0.9-1.0 parts of mannitol, and 0.15-0.2 parts of thickening agent.

[0077] In some embodiments, the irradiation sterilization has an irradiation dose of 25 kGy.

[0078] Step four: reconstitution of the injection solution

[0079] The lyophilized powder is reconstituted with normal saline to an appropriate concentration of 5-100 mg / mL to obtain the PLLA microsphere injection solution before use.

[0080] In some embodiments, the ratio of the microsphere lyophilized powder and normal saline is 5 mg / mL-100 mg / mL.

[0081] To make the purpose, technical solutions and advantages of the present disclosure more clear, the present disclosure is further described in detail below in combination with the drawings and examples. The specific examples described herein are only used to explain the present disclosure and do not constitute any limitation on the present disclosure. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present disclosure. Such structures and technologies are also described in many publications.

[0082] In the following examples, a scanning electron microscope (Regulus 8100, Japan HITACHI) is used to observe the diameter and surface morphology of the PLLA microspheres. The particle size distribution coefficient (CV value) of the microspheres is calculated according to the following formula:

[0083]

[0084] wherein σ is the standard deviation, D P is the average particle size obtained from the SEM image.

[0085] Example 1: Preparation of PLLA microspheres

[0086] Step one: synthesis of PLLA-PEG copolymer.

[0087] The purified L-lactide and PEG800 are placed in a dry reaction kettle in a molar ratio of 862.5:1 under nitrogen protection; the L-lactide and PEG are completely melted to be clear; at an internal temperature of 90°C, the water pump is degassed for 10 minutes, when the temperature reaches 135°C, stannous octoate (mass is one thousandth of the total mass of L-lactide and PEG800) is added; the reaction is continued to 150°C for 5 hours to obtain the PLLA-PEG copolymer; the product is dissolved in dichloromethane (DCM), precipitated with ethanol, filtered and dried for use.

[0088] Step two: Preparation of PLLA-PEG microspheres

[0089] PLLA-PEG microspheres were prepared by using membrane emulsification equipment. The SPG membrane with a pore size of 13.1 μm was used. PLLA and DCM were mixed according to the ratio in Table 1 to prepare an oil phase solution containing 17.4 wt.% PLLA-PEG. A pressure of 13 kPa was applied to make the oil phase pass through the membrane to form droplets and enter the water phase. The water phase contained 1 wt.% polyvinyl alcohol (PVA) and 0.045 wt.% sodium dodecyl sulfate (SDS), and the stirring rate was set to 200 rpm. The stirring was continued at room temperature for 48 hours to evaporate DCM, and then the formed PLLA-PEG microspheres were collected by centrifugation, washed several times with deionized water, and dried under vacuum for 48 hours for standby use.

[0090] Example 2-8: Preparation of PLLA microspheres

[0091] The synthesis of PLLA-PEG copolymer was the same as in Example 1. The experimental conditions for the preparation of polylactic acid microspheres are shown in Table 1. Examples 1 to 7 were prepared using an SPG membrane with a pore size of 13.1 μm, and Example 8 used an SPG membrane with a pore size of 9.9 μm.

[0092] Table 1: Preparation process of PLLA-PEG microspheres under different experimental conditions

[0093]

[0094]

[0095] Example 9: Preparation of PLLA microsphere lyophilized powder

[0096] The synthesis of PLLA-PEG copolymer and the preparation process of microspheres were the same as in Example 2. 300 mg of PLLA microspheres, 290 mg of mannitol as a lyophilization protective agent, and 45 mg of glutamic acid were mixed. An appropriate amount of normal saline was added, and the microspheres were stirred and ultrasonically dispersed. Freeze-drying was performed to finally obtain PLLA microsphere lyophilized powder.

[0097] Example 10: Preparation of PLLA microsphere lyophilized powder

[0098] The synthesis of PLLA-PEG copolymer and the preparation process of microspheres were the same as in Example 2. 300 mg of PLLA microspheres, 290 mg of mannitol as a lyophilization protective agent, and 45 mg of glutamic acid were mixed. An appropriate amount of normal saline was added, and the microspheres were stirred and ultrasonically dispersed. Freeze-drying was performed to finally obtain PLLA microsphere lyophilized powder.

[0099] Example 11: Preparation of PLLA microsphere lyophilized powder

[0100] The synthesis of PLLA-PEG copolymer and the preparation of microspheres were the same as those in Example 2. PLLA microspheres 300 mg, freeze-drying protective agent mannitol 290 mg, glutamic acid 55 mg were mixed. Appropriate amount of normal saline was added, and the microspheres were stirred and ultrasonically dispersed. Freeze-drying was carried out, and finally PLLA microsphere freeze-dried powder was obtained.

[0101] Example 12: Preparation of PLLA microsphere freeze-dried powder

[0102] The synthesis of PLLA-PEG copolymer and the preparation of microspheres were the same as those in Example 2. PLLA microspheres 300 mg, freeze-drying protective agent mannitol 290 mg, glutamic acid 55 mg were mixed. Appropriate amount of normal saline was added, and the microspheres were stirred and ultrasonically dispersed. Freeze-drying was carried out, and finally PLLA microsphere freeze-dried powder was obtained.

[0103] Example 13: Preparation of PLLA microsphere freeze-dried powder

[0104] The synthesis of PLLA-PEG copolymer and the preparation of microspheres were the same as those in Example 2. PLLA microspheres 300 mg, freeze-drying protective agent mannitol 290 mg, glutamic acid 55 mg were mixed. Appropriate amount of normal saline was added, and the microspheres were stirred and ultrasonically dispersed. Freeze-drying was carried out, and finally PLLA microsphere freeze-dried powder was obtained.

[0105] Example 14: Preparation of PLLA microsphere freeze-dried powder

[0106] The synthesis of PLLA-PEG copolymer and the preparation of microspheres were the same as those in Example 2. PLLA microspheres 300 mg, freeze-drying protective agent mannitol 290 mg, glutamic acid 55 mg were mixed. Appropriate amount of normal saline was added, and the microspheres were stirred and ultrasonically dispersed. Freeze-drying was carried out, and finally PLLA microsphere freeze-dried powder was obtained.

[0107] Comparative Example 1: Commercially available hyaluronic acid gel (HA).

[0108] Test Example 1. Evaluation of PLLA microsphere particle size uniformity

[0109] The PLLA microspheres prepared in Examples 1-8 were observed for morphology.

[0110] The PLLA microspheres prepared in Examples 1-8 were observed for morphology, and their particle size uniformity was systematically evaluated. The PLLA microsphere samples in different examples were observed by scanning electron microscopy, Figures 1-8 The results show that the particle size of the microspheres in Example 2 and Example 8 is the most uniform, the particle size distribution is consistent, and no obvious agglomeration phenomenon is observed.

[0111] The microporous membrane of Example 2 has a pore size of 13.1 microns, a PLLA mass fraction of 17.4%, a transmembrane pressure of 13 kPa, and a stirring rate of 250 rpm. Under these optimized conditions, the microspheres exhibit good uniformity in particle size, which is suitable for applications requiring high stability and consistency.

[0112] The microporous membrane of Example 8 has a pore size of 9.9 microns, a PLLA mass fraction of 15.2%, a transmembrane pressure of 13 kPa, and a stirring rate of 250 rpm. Adjustment of these parameters also effectively improves the uniformity of the microspheres, with a more consistent particle size distribution, indicating better process control.

[0113] Test Example 2: Evaluation of Reconstitution of Freeze-dried Powder

[0114] The reconstitution of freeze-dried powder is an important indicator for evaluating whether it can be quickly and uniformly dissolved in actual applications. Therefore, the reconstitution of the freeze-dried powder of Examples 9-14 was evaluated, and the injection solution was observed after standing for 30 minutes after reconstitution. The reconstitution effect is shown in Figure 9 .

[0115] From the results of Figure 9 , it can be seen that Example 9 shows obvious stratification, and the reconstitution of the freeze-dried powder of Examples 10, 11, and 12, 13 is good, with a smooth reconstitution process and no obvious residual particles, good solution uniformity, and excellent reconstitution. The reconstitution of Example 14 is not good, and there are a small amount of lumps in the liquid.

[0116] Test Example 3: Subcutaneous Implantation Experiment

[0117] To evaluate the repair effect of PLLA microsphere injection solutions of different concentrations in a mouse skin aging model, this experiment used galactose and ultraviolet light to create a mouse skin aging model, and the specific modeling method is as follows:

[0118] The experimental animals used in this study were female ICR mice weighing 22-25 grams, purchased from the Experimental Animal Center of Kunming Medical University (License No. SCXK (Yunnan) K2020-0004). All mice were raised in a specific pathogen-free (SPF) level experimental animal facility with environmental conditions controlled at room temperature 20-25°C, relative humidity 40-70%, and a 12-hour alternating light cycle. Before the experiment, all animals were acclimated to the experimental environment for 7 days, during which they could freely access drinking water and standard feed.

[0119] All animal experiments were performed in accordance with the National Regulations on Laboratory Animals and the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The experimental protocol was approved by the Yunnan University Animal Care and Use Committee (Approval No. YNU20240884, Approval Date: March 16, 2024) and strictly followed the principles of "Reduction, Replacement, and Optimization (3Rs)". All surgeries were performed under anesthesia to minimize animal suffering and discomfort.

[0120] To establish a skin aging animal model, the dorsal neck of all mice was depilated before the experiment, and regular depilation was performed during the experiment to ensure skin exposure. Except for the blank group, the mice in the other groups were subcutaneously injected with D-galactose (dose 1000 mg / kg, injection volume 100 μL) on the dorsal neck every day for 5 consecutive weeks, while receiving ultraviolet B (UVB) irradiation with an intensity of 0.12-0.20 mW / cm 2 , a light source perpendicular to the skin at a distance of 30-40 cm, and an irradiation time of 30-40 minutes per day for 5 consecutive weeks.

[0121] The PLLA microspheres lyophilized powder of Example 10 and Example 13 were subjected to irradiation sterilization treatment (irradiation dose of 25 kGy), and physiological saline was used to prepare injection solutions with concentrations of 12 mg / mL and 68 mg / mL. During the experiment, different concentrations of PLLA microsphere injection solutions (50 μL / time / mouse) were injected subcutaneously into the skin of mice every day, and hyaluronic acid was used as a control group (50 μL / time / mouse). At 8 weeks and 20 weeks after stopping injection, the dorsal skin tissue of the mice was taken for pathological examination to observe the inflammatory response and collagen production.

[0122] HE staining and Masson staining were performed on paraffin-embedded skin sections. HE staining was used to evaluate the epidermal thickness, dermal structure, and inflammatory cell infiltration; Masson staining was used to observe the distribution and reconstruction of collagen fibers. After the sections were routinely deparaffinized and rehydrated, the staining, dehydration, and mounting operations were completed according to the kit instructions. After taking images under a microscope, the Masson staining images were analyzed using ImageJ software to quantitatively calculate the thickness and collagen density (collagen area ratio) of the collagen layer.

[0123] After subcutaneous injection, no adverse reactions such as redness, swelling, and subcutaneous nodules were observed at the implantation sites of the skin in all experimental groups, indicating that the injection solutions had good biocompatibility.

[0124] The results of HE staining and Masson staining experiments are shown in Figures 10-13

[0125] ​The eighth week observation: the aging model was successfully established. The dermal layer thickness and collagen density of the model group were significantly lower than those of the blank group, indicating that the aging model was successfully established. The dermal layer thickness of the hyaluronic acid group at the eighth week was not significantly different from that of the model group, but the collagen density was significantly higher than that of the model group. In contrast, the glutamic acid and CMC thickener groups showed obvious effects better than the model group and the control group, especially in terms of dermal layer thickness. The glutamic acid 68mg / ml group showed the best performance, with a significant immediate filling effect.

[0126] The twentieth week observation: At this time, the model group and the blank group showed no significant changes, indicating that the skin had self-repairing ability. The dermal layer thickness of the glutamic acid 68mg / mL group, the CMC 68mg / mL group, and the glutamic acid 12mg / mL group, and the CMC 12mg / mL group were significantly better than those of the model group and the hyaluronic acid group. Among them, the collagen density of the CMC 68mg / mL group and the glutamic acid 12mg / mL group was the most outstanding.

[0127] The PLLA microsphere injection based on glutamic acid and CMC thickener can effectively promote the repair of mouse skin and significantly improve the collagen density, showing excellent immediate repair effect. Therefore, the injection has good application potential in the field of skin aging repair and tissue engineering.

[0128] The technical solutions of the present disclosure are not limited to the above specific embodiments, and any technical variations made according to the technical solutions of the present disclosure fall within the scope of protection of the present disclosure.

Claims

1. A microsphere of poly-L-lactic acid, wherein, The average particle size of the left-handed polylactic acid microspheres is 20-120 μm, and the particle size distribution coefficient is 10-60%.

2. The polylactic acid microsphere of claim 1, wherein, The average particle size of the left-handed polylactic acid microspheres is 40-100 μm, preferably 40-70 μm, more preferably 40-55 μm; and / or The particle size distribution coefficient of the left-handed polylactic acid microspheres is 15-50%, preferably 20-30%, more preferably 20-25%.

3. The polylactic acid microsphere of claim 1 or 2, wherein the polylactic acid microsphere is a polylactic acid microsphere having a particle size of 1 to 100 μm. The left-handed polylactic acid microspheres are obtained by film emulsification of a left-handed polylactic acid-polyethylene glycol copolymer synthesized from L-lactide and polyethylene glycol.

4. A method for preparing left-handed polylactic acid microspheres, comprising the following steps: S1: ring-opening polymerization of L-lactide and polyethylene glycol in the presence of a catalyst to obtain a left-handed polylactic acid-polyethylene glycol copolymer; S2: film emulsification treatment of a polymer solution containing the left-handed polylactic acid-polyethylene glycol copolymer to obtain the left-handed polylactic acid microspheres.

5. The preparation method according to claim 4, characterized in that, In step S1, the temperature of the ring-opening polymerization is 100-200°C, and the time is 0.5-8 h; and / or In step S1, the catalyst comprises a metal organic compound, preferably one or more of stannous octoate, tin octoate, lanthanum trifluoride, cobalt acetylacetonate, aluminum acetylacetonate, or diethyl zinc; and / or In step S1, the molecular weight of the polyethylene glycol is 400-2000; and / or In step S1, the molar ratio of the lactide to the polyethylene glycol is (100-1000):1; and / or In step S1, the molecular weight of the left-handed polylactic acid-polyethylene glycol copolymer is 5-130,000 Daltons; and / or In step S2, the mass concentration of the left-handed polylactic acid-polyethylene glycol copolymer in the polymer solution is 5%-20%, preferably 16-20%; and / or In step S2, the polymer solution further comprises an organic solvent, preferably one or more of dichloromethane, chloroform, ethyl acetate, or tetrahydrofuran; and / or In step S2, the pore size of the microporous membrane used in the film emulsification treatment is 5-20 μm, preferably 8-14 μm; and / or In step S2, the transmembrane pressure of the film emulsification treatment is 5-30 kPa, preferably 10-13 kPa; and / or In step S2, the stirring rate of the film emulsification treatment is 100-400 rpm, preferably 250-300 rpm; and / or Step S2 further comprises dispersing the emulsion droplets obtained from the film emulsification treatment in an aqueous phase containing a surfactant, followed by organic solvent removal, washing, and drying treatment to obtain the left-handed polylactic acid microspheres; Preferably, the aqueous phase contains polyvinyl alcohol and sodium dodecyl sulfate; more preferably, the mass concentration of the polyvinyl alcohol in the aqueous phase is 1-5%, and the mass concentration of the sodium dodecyl sulfate is 0.01-1%.

6. A left-handed polylactic acid microsphere preparation, comprising the left-handed polylactic acid microspheres of any one of claims 1-3 or obtained by the preparation method of claim 4 or 5.

7. The preparation of the L-polylactic acid microspheres according to claim 6, wherein, The left-handed polylactic acid microsphere preparation further comprises one or more of a lyoprotectant, a thickening agent, and physiological saline; Preferably, the L-polylactic acid microsphere preparation comprises a L-polylactic acid microsphere lyophilized powder or a L-polylactic acid microsphere injection solution. Preferably, the thickening agent comprises one or more of sodium carboxymethyl cellulose, lysine, glutamic acid, sodium hyaluronate, polyvinylpyrrolidone or xanthan gum. Preferably, the lyophilization protective agent comprises mannitol.

8. A preparation method of a L-polylactic acid microsphere lyophilized powder, comprising the following steps: dispersing and freeze-drying a mixed solution containing the L-polylactic acid microspheres according to any one of claims 1-3 or obtained by the preparation method of claim 4 or 5, a thickening agent and a lyophilization protective agent. Preferably, the thickening agent comprises one or more of sodium carboxymethyl cellulose, lysine, glutamic acid, sodium hyaluronate, polyvinylpyrrolidone or xanthan gum, preferably sodium carboxymethyl cellulose and / or glutamic acid. Preferably, the lyophilization protective agent comprises mannitol. Preferably, the mixed solution comprises 1 part of L-polylactic acid microspheres, 0.9-1.0 parts of lyophilization protective agent and 0.15-0.2 parts of thickening agent by weight. Preferably, the dispersing treatment comprises stirring treatment and / or ultrasonic treatment; more preferably, the stirring rate of the stirring treatment is 200-600 rpm; more preferably, the ultrasonic power of the ultrasonic treatment is 100-500 W. Preferably, the preparation method further comprises irradiation sterilization treatment of the lyophilized powder after the freeze-drying treatment; more preferably, the irradiation dose of the irradiation sterilization treatment is 25-30 kGy.

9. A preparation method of a L-polylactic acid microsphere injection solution, comprising dispersing the L-polylactic acid microspheres according to any one of claims 1-3, the L-polylactic acid microspheres obtained by the preparation method of claim 4 or 5, or the L-polylactic acid microsphere lyophilized powder obtained by the preparation method of claim 8 in a buffer solution. Preferably, the buffer solution comprises physiological saline. Preferably, the mass concentration of the L-polylactic acid microspheres in the injection solution is 2-100 mg / mL.

10. Use of the L-polylactic acid microspheres according to any one of claims 1-3, the L-polylactic acid microspheres obtained by the preparation method of claim 4 or 5, the L-polylactic acid microsphere preparation of claim 6 or 7, the L-polylactic acid microsphere lyophilized powder of claim 8 or the L-polylactic acid microsphere injection solution of claim 9 in the preparation of a product, wherein the product comprises a medical aesthetic material, a tissue engineering material or a skin repair material; and the product is preferably used for promoting skin collagen production, increasing skin collagen thickness and / or increasing skin collagen density.

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