Preparation method of injection polylactic acid microspheres

Abstract

The invention provides a preparation method of injection polylactic acid microspheres, and relates to the technical field of medical cosmetology, and the preparation method comprises the following steps: preparing an organic solution of polylactic acid; preparing an aqueous solution of recombinant elastin, recombinant collagen and sodium carboxymethyl cellulose; dropwise adding the organic solution of polylactic acid into the aqueous solution of recombinant elastin / recombinant collagen / sodium carboxymethyl cellulose, uniformly mixing, and then removing the organic solvent in the mixed solution; and carrying out freeze-drying treatment on the obtained solution to obtain the injectable polylactic acid microspheres. The recombinant elastin and the recombinant collagen are added, so that a spongy fiber structure can be formed after a final freeze-drying process, the freeze-dried mixture is easy to redissolve, and the polylactic acid microspheres can be uniformly dispersed in a solution; moreover, the recombinant elastin and the recombinant collagen which are added in the invention not only have the effects of moisturizing and replenishing water and improving the skin state, but also have the lubricating effect and can reduce the resistance during injection, so that the problem that the injection of the polylactic acid microspheres needs larger injection force is solved, and the dosage during injection is more easily and accurately controlled.

Description

Technical Field

[0001] The present invention relates to the technical field of medical aesthetics, and particularly to a preparation method of injectable polylactic acid microspheres. Background Art

[0002] According to a preparation method of injectable poly-L-lactic acid microspheres disclosed in Chinese Patent Publication No. "CN109010910A", it includes the following steps: (1) preparing an organic solution of poly-L-lactic acid; (2) preparing an aqueous solution of sodium carboxymethyl cellulose / mannitol; (3) dropping the organic solution of poly-L-lactic acid into the aqueous solution of sodium carboxymethyl cellulose / mannitol, mixing evenly, and then removing the organic solvent in the mixed solution; (4) freeze-drying the solution obtained in step (3) to obtain injectable poly-L-lactic acid microspheres.

[0003] The following technical problems exist in the prior art during use:

[0004] Due to different processes and formula differences in the preparation process of polylactic acid microspheres, the reconstituted injection solution is uneven, resulting in a relatively large injection force required for actual use and also having a certain impact on the accuracy of use. Summary of the Invention

[0005] Technical Problems to be Solved

[0006] In view of the deficiencies of the prior art, the present invention provides a preparation method of injectable polylactic acid microspheres, which solves the problems of the prior art.

[0007] Technical Solution

[0008] To achieve the above object, the present invention is achieved through the following technical solutions: A preparation method of injectable polylactic acid microspheres, characterized in that: the method includes the following steps:

[0009] Sp1. Dissolve polylactic acid in an organic solvent trichloromethane or dichloromethane to prepare an organic solution of polylactic acid;

[0010] Sp2. The polylactic acid is one of poly-D-lactic acid, poly-L-lactic acid, meso-polylactic acid, and racemic polylactic acid;

[0011] Sp3. Prepare an aqueous solution of recombinant elastin and recombinant collagen: Dissolve recombinant elastin with a molecular weight of 10 - 50 kDa, recombinant collagen with a molecular weight of 20 - 100 kDa, and sodium carboxymethyl cellulose in injection water to prepare an aqueous solution of recombinant elastin / recombinant collagen / sodium carboxymethyl cellulose with a concentration of 10 - 80 g / L;

[0012] Sp4. Drop the organic solution of polylactic acid into the aqueous solution of recombinant elastin / recombinant collagen / sodium carboxymethylcellulose, mix well, and then remove the organic solvent from the mixed solution;

[0013] Sp5. Freeze-dry the solution obtained in step Sp4 to obtain injectable polylactic acid microspheres.

[0014] Preferably, in Sp1, dissolve polylactic acid in chloroform or dichloromethane to prepare an organic solution of polylactic acid with a concentration of 10 - 50 g / L.

[0015] Preferably, the mass ratio of the recombinant elastin to the recombinant collagen is 1:30 - 30:1.

[0016] Preferably, the mass ratio of the sodium carboxymethylcellulose to the total amount of the recombinant elastin and the recombinant collagen is 1:10 - 10:1.

[0017] Preferably, in Sp4, the volume ratio of the organic solution of polylactic acid to the aqueous solution of recombinant elastin / recombinant collagen / sodium carboxymethylcellulose is 1:3 - 1:50.

[0018] The organic solvent is selected from ethyl acetate or supercritical carbon dioxide, and the selection of the organic solvent is based on its boiling point, solubility parameter, and dissolution efficiency for polylactic acid.

[0019] Preferably, the volume ratio of the organic solution of polylactic acid to the aqueous solution of recombinant elastin / recombinant collagen / sodium carboxymethylcellulose is 1:5 - 1:30.

[0020] Preferably, in Sp4, remove the organic solvent from the mixed solution through the following steps:

[0021] Sp4.1. After adding the organic solution of polylactic acid to the aqueous solution of recombinant elastin / recombinant collagen / sodium carboxymethylcellulose;

[0022] Sp4.2. Remove dichloromethane by vacuum distillation.

[0023] Preferably, the solvent removal step Sp4 is carried out under the conditions of reducing the pressure to below 50 mbar and controlling the temperature at 45 °C to ensure the complete removal of the organic solvent and avoid the thermal degradation of polylactic acid. At the same time, the continuous flow mode is adopted during the solvent removal process to improve the solvent removal efficiency.

[0024] Preferably, the polyethylene glycolylation treatment is carried out by grafting a polyethylene glycol (PEG) chain segment with a molecular weight of 2000 Da to the surface of the microspheres in a buffer solution with a pH value of 7.4, thereby reducing the immunogenicity and non-specific protein adsorption of the microspheres, and the grafting reaction is carried out through epoxidized PEG to ensure the effective binding of polyethylene glycol.

[0025] Beneficial effects

[0026] The present invention provides a method for preparing injectable polylactic acid microspheres, which has the following beneficial effects:

[0027] 1. In the present invention, recombinant elastin and recombinant collagen are used as humectants and lubricants, sodium carboxymethyl cellulose is used as a thickening agent, and recombinant elastin and recombinant collagen are also used as freeze-dried scaffold agents. After the freeze-drying process, a sponge-like fibrous structure is formed. When the mixture after the freeze-drying process is redissolved, the polylactic acid microspheres can be evenly dispersed in the solution. Moreover, the recombinant elastin and recombinant collagen added in the present invention not only have the functions of moisturizing and hydrating, improving the skin condition, but also can reduce the resistance during injection, solve the problem that a large injection force is required for injecting polylactic acid microspheres, and are more conducive to accurately controlling the dosage during injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a schematic diagram of the polylactic acid dissolution process of the present invention;

[0029] Figure 2 It is a schematic diagram of the structure of the microfluidic emulsification device of the present invention;

[0030] Figure 3 It is a flow chart of the high-shear emulsification process of the present invention;

[0031] Figure 4 It is a schematic diagram of the structure of the solvent removal device of the present invention;

[0032] Figure 5 It is a schematic diagram of the microsphere surface modification reaction device of the present invention;

[0033] Figure 6 It is a schematic diagram of the freeze-drying process of the present invention;

[0034] Figure 7 It is a scanning electron microscope image of the polylactic acid microspheres of the present invention;

[0035] Figure 8 It is the set parameters of the freeze-drying process of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0036] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention. Specific Example 1:

[0038] As Figures 1-7As shown, a preparation method of injectable polylactic acid microspheres is characterized in that the method comprises the following steps:

[0039] Sp1. Dissolve polylactic acid in an organic solvent, chloroform or dichloromethane, to prepare an organic solution of polylactic acid; dissolve polylactic acid in chloroform or dichloromethane to prepare an organic solution of polylactic acid with a concentration of 10-50 g / L.

[0040] Sp2. The polylactic acid is one of dextrorotatory polylactic acid, levorotatory polylactic acid, meso-polylactic acid, and racemic polylactic acid.

[0041] Sp3. Prepare an aqueous solution of recombinant elastin and recombinant collagen: dissolve recombinant elastin with a molecular weight of 10-50 kDa, recombinant collagen with a molecular weight of 20-100 kDa, and sodium carboxymethylcellulose in injection water to prepare an aqueous solution of recombinant elastin / recombinant collagen / sodium carboxymethylcellulose with a concentration of 10-80 g / L. The mass ratio of recombinant elastin to recombinant collagen is 1:30-30:1, and the mass ratio of sodium carboxymethylcellulose to the total amount of recombinant elastin and recombinant collagen is 1:10-10:1.

[0042] Sp4. Drop the organic solution of polylactic acid into the aqueous solution of recombinant elastin / recombinant collagen / sodium carboxymethylcellulose, mix well, and then remove the organic solvent in the mixed solution; the volume ratio of the organic solution of polylactic acid to the aqueous solution of recombinant elastin / recombinant collagen / sodium carboxymethylcellulose is 1:3-1:50. Remove the organic solvent in the mixed solution through the following steps. The organic solvent is selected from ethyl acetate or supercritical carbon dioxide, and the selection of the organic solvent is based on its boiling point, solubility parameter, and dissolution efficiency for polylactic acid:

[0043] Sp4.1. After adding the organic solution of polylactic acid into the aqueous solution of recombinant elastin / recombinant collagen / sodium carboxymethylcellulose;

[0044] Sp4.2. Remove dichloromethane by vacuum distillation;

[0045] Sp5. Freeze-dry the solution obtained in step Sp4 to obtain injectable polylactic acid microspheres. The freeze-drying parameters are as Figure 8 shown, and the scanning electron micrograph of the finished polylactic acid microspheres is as Figure 7 shown. Specific Example Two:

[0047] As Figures 1-7 shown, based on the technical solution of Specific Example One, a preparation method of injectable polylactic acid microspheres is further provided. This method is obtained by further adjustment and deformation of the solution of Specific Example One and comprises the following steps:

[0048] Sp1. Dissolve polylactic acid in an organic solvent selected from ethyl acetate, acetonitrile, chloroform, dichloromethane or supercritical carbon dioxide to prepare a polylactic acid organic solution with a concentration of 1 - 100 g / L;

[0049] Sp2. Add recombinant elastin with a molecular weight of 10 - 50 KD, recombinant collagen with a molecular weight of 20 - 100 KD, and sodium carboxymethylcellulose to water for injection to prepare an aqueous solution of recombinant elastin / recombinant collagen / sodium carboxymethylcellulose with a concentration of 0.5 - 5 wt%, wherein the mass ratio of recombinant elastin to recombinant collagen is 1:30 - 30:1, and the mass ratio of sodium carboxymethylcellulose to the total amount of recombinant elastin and recombinant collagen is 1:10 - 10:1;

[0050] Sp3. Using microfluidics technology or a high - shear emulsifier, slowly drop the polylactic acid organic solution into the aqueous solution at a volume ratio of 1:3 - 1:50 to form a water - in - oil emulsion;

[0051] Sp4. Solvent removal: Remove the organic solvent in the mixed solution by vacuum distillation or supercritical fluid extraction to form solid polylactic acid microspheres;

[0052] Sp5. Perform polyethylene glycolylation treatment on the surface of the polylactic acid microspheres or introduce a targeting ligand for surface modification and functionalization to obtain a microsphere solution;

[0053] Sp6. Subject the microsphere solution surface - modified in Sp5 to a freeze - drying process to obtain injectable polylactic acid microspheres.

[0054] The polylactic acid is selected from one of dextrorotatory polylactic acid, levorotatory polylactic acid, meso - polylactic acid and racemic polylactic acid

[0055] The polylactic acid is dissolved in Sp1 under the condition of a temperature of 40°C to 60°C and continuously stirred at this temperature for at least 2 hours to ensure the complete dissolution of the polylactic acid and the uniformity of the polymer solution. Meanwhile, the usage amount of the organic solvent during the dissolution process is 5 to 50 times the volume of the polylactic acid.

[0056] The molecular weight of the polylactic acid is 60 - 200 kDa, determined by gel permeation chromatography, and the polydispersity index (PDI) of the molecular weight distribution of the polylactic acid does not exceed 3.0 to ensure the high molecular weight and narrow molecular weight distribution of the polylactic acid.

[0057] The organic solvent is selected from ethyl acetate or supercritical carbon dioxide. The selection of the organic solvent is based on its boiling point, solubility parameter and dissolution efficiency for polylactic acid to reduce solvent toxicity and improve the biocompatibility of the microspheres.

[0058] The recombinant elastin is human recombinant elastin with a molecular weight of 10 - 50 kD, and the recombinant collagen is type I or type III recombinant collagen with a molecular weight of 20 - 100 kD. The mass ratio of the recombinant elastin to the recombinant collagen is 1:30 - 30:1 to optimize the biocompatibility and mechanical strength of the microspheres, and the pH value of the aqueous solution is adjusted to 7.4.

[0059] The molecular weight of sodium carboxymethyl cellulose is 10 - 50 kDa, and the mass ratio of sodium carboxymethyl cellulose to the total amount of recombinant elastin and recombinant collagen is 1:10 - 10:1 to regulate the pH value and stability of the aqueous solution, while increasing the viscosity and emulsification effect of the aqueous solution.

[0060] The polylactic acid organic solution and the aqueous solution are added dropwise at a volume ratio of 1:20 in step Sp3, and the dropping rate is controlled at 5 ml per minute to achieve a uniform distribution of the microsphere particle size in the range of 10 to 100 microns. During the dropping process, the continuous stirring speed is maintained at 1000 rpm. The microfluidic technology includes using a microfluidic chip with a unidirectional flow channel and a cross-flow channel. The microfluidic chip is made of polydimethylsiloxane (PDMS) material, and the size of the flow channel is 10 microns to 100 microns to achieve the preparation of microspheres with high dispersibility and monodispersity. The high-shear emulsifier is an efficient homogenizer, and its operating conditions are a shear rate of 1000 rpm and a duration of 10 minutes. The emulsification process is carried out at a temperature controlled by a cooling system to avoid the thermal degradation of polylactic acid.

[0061] The solvent removal step Sp4 is carried out under the conditions of reducing the pressure to below 50 mbar and controlling the temperature at 45°C to ensure the complete removal of the organic solvent and avoid the thermal degradation of polylactic acid. At the same time, a continuous flow mode is adopted during the solvent removal process to improve the solvent removal efficiency.

[0062] Supercritical carbon dioxide as a solvent removal method has significant advantages in the process of preparing polylactic acid microspheres, especially in cases where the residual organic solvent and the thermal degradation of polylactic acid need to be avoided. Supercritical carbon dioxide refers to the state where the temperature and pressure of carbon dioxide exceed its critical point (the critical temperature is 31.1°C, and the critical pressure is 7.38 MPa) under high temperature and high pressure conditions. In this state, CO2 has the dual properties of a gas and a liquid. It can expand and fill the entire container like a gas and has a high dissolution ability like a liquid. Utilizing this property, supercritical CO2 can effectively dissolve the solvent, thereby achieving the solvent removal of polylactic acid microspheres.

[0063] In the Sp4 solvent removal step, supercritical CO2 is used for solvent removal, and a high-pressure reactor (supercritical fluid extraction equipment) is required to maintain the supercritical state. The reactor is usually equipped with a temperature control system, a pressure control system, and a CO2 supply system. The CO2 supply system includes a gaseous CO2 storage tank and a booster pump to pressurize the gaseous CO2 to the critical pressure range.

[0064] Solvent removal process:

[0065] Pretreatment: The polylactic acid microsphere solution in the water-in-oil emulsion is preheated to 45 °C and degassed under low pressure to remove some low-boiling solvents in the solution.

[0066] Enter the supercritical carbon dioxide solvent removal equipment: The polylactic acid emulsion is introduced into the supercritical CO2 equipment, and the CO2 in the equipment is pressurized to 7.5 - 10 MPa and heated to 40 - 50 °C to make it enter the supercritical state.

[0067] Solvent removal: In the supercritical CO2 environment, the organic solvents (such as ethyl acetate or dichloromethane) in the polylactic acid will be dissolved and carried away by the supercritical carbon dioxide.

[0068] Solvent recovery: Through pressure reduction or temperature adjustment, the solvent in the supercritical carbon dioxide is released and recovered through a condensation device. The residual carbon dioxide can be recycled to reduce waste.

[0069] Under supercritical CO2 conditions, the solvent removal efficiency can reach over 90%. This method can completely remove the solvent and prevent thermal degradation of the polylactic acid microspheres, ensuring the biocompatibility of the microspheres. Since the process of removing the solvent by supercritical CO2 is at a relatively low temperature, the risk of thermal degradation of polylactic acid is greatly reduced. Compared with the traditional solvent removal method (vacuum distillation), the solvent removal by supercritical CO2 has higher efficiency, lower energy consumption, and more environmentally friendly characteristics.

[0070] The polyethylene glycolylation treatment grafts polyethylene glycol (PEG) chains with a molecular weight of 2000 Da onto the surface of the microspheres in a buffer solution with a pH value of 7.4, thereby reducing the immunogenicity and non-specific protein adsorption of the microspheres, and the grafting reaction is carried out through epoxidized PEG to ensure the effective binding of polyethylene glycol.

[0071] PEGylation is a surface modification technology widely used in the biomedical field, aiming to reduce the immunogenicity of microspheres, extend their in vivo half-life, reduce non-specific protein adsorption, etc. The following will introduce in detail how to perform surface modification on poly(lactic acid) (PLA) microspheres through PEGylation. PEGylation is achieved by grafting PEG molecules onto the surface of PLA microspheres, thereby changing their physical and chemical properties. PEG has strong hydrophilicity and low immunogenicity, which can effectively reduce the adsorption of cells and proteins to the microspheres and avoid immune responses caused by immune system recognition.

[0072] In the Sp5 surface modification step, the surface of PLA microspheres is functionalized by PEGylation or the introduction of targeting ligands. The specific technical steps are as follows:

[0073] Selection of PEG: Select a PEG segment with a molecular weight of 2000 Da. PEG with this molecular weight has sufficient hydrophilicity and will not cause immune responses in the body.

[0074] The grafting reaction of PEG is carried out through epoxidized PEG, which has high efficiency and good stability.

[0075] Solution preparation: Prepare a buffer solution with a pH value of 7.4. Usually, PBS (phosphate buffer solution) is used as the solvent. Dissolve PEG molecules in the PBS buffer to prepare a PEG solution, and the concentration is usually 1 - 5 wt%.

[0076] PEGylation reaction: Add the microsphere solution: Add the PLA microsphere solution to the PEG buffer solution.

[0077] Reaction conditions: Under temperature control conditions (usually at room temperature or slightly higher temperature, such as 37 °C), use epoxidized PEG (such as epoxy-PEG or amino-PEG) to carry out a grafting reaction with the microsphere surface.

[0078] Reaction process: PEG reacts chemically with the microsphere surface through its epoxy group to form stable covalent bonds. The reaction time is usually controlled within 1 - 3 hours, and the reaction temperature is maintained at about 37 °C.

[0079] Post-treatment after reaction: After the reaction is completed, wash the microspheres several times with PBS buffer to remove unreacted PEG and other impurities. Use centrifugation or dialysis to remove excess PEG to ensure the purity of surface modification.

[0080] The surface of PEGylated microspheres has good hydrophilicity, which can reduce non-specific protein adsorption and cell uptake. The PEG layer can effectively shield the immunogenicity of PLA microspheres, enabling them to exist in the body for a longer time without being cleared by the immune system. PEGylation can also significantly reduce the aggregation and sedimentation rate of microspheres and improve their uniform distribution in the body. Specific Embodiment Three:

[0082] Based on the technical solution of Specific Embodiment One, a practical application case is further given:

[0083] This case applies a technology for preparing poly(lactic acid) (PLA) microspheres for drug sustained-release carriers. Especially in the delivery system of injectable drugs, PLA microspheres are used as carriers to slowly release drugs into the body, thereby improving the efficacy of drugs and reducing side effects. Polyethylene glycolylation and supercritical carbon dioxide (CO2) solvent removal are adopted, significantly improving the biocompatibility, stability, and drug release control ability of the microspheres.

[0084] Target drug: Docetaxel can improve the stability and release rate control of the drug in the body through PLA microspheres as carriers.

[0085] Target application: A long-acting sustained-release docetaxel injection can achieve slow drug release for 1 - 2 months through a single injection.

[0086] Actual preparation process and application steps

[0087] 1. Preparation of PLA microspheres

[0088] According to the steps in Specific Embodiment One, PLA microspheres are prepared through the following process:

[0089] Dissolve polylactic acid: Dissolve L-polylactic acid (molecular weight 150 kDa) and docetaxel in ethyl acetate, and adjust the concentration to 50 g / L. Control the temperature at 50 °C during the dissolution process and keep stirring for 2 hours.

[0090] Prepare aqueous solution: Add recombinant elastin, collagen, and sodium carboxymethylcellulose to injection water to prepare an aqueous solution with a concentration of 2 wt%, and adjust the pH value to 7.4.

[0091] Microfluidic emulsification process: Drop the above docetaxel-PLA organic solution into the aqueous solution at a volume ratio of 1:20, and form an emulsion through a microfluidic chip (channel size 50 μm).

[0092] Solvent removal: Adopt supercritical CO2 technology to remove the solvent under supercritical conditions (pressure 7.5 MPa, temperature 45 °C) to ensure the stability and biocompatibility of the PLA microspheres.

[0093] PEGylation treatment: Perform PEGylation treatment on the prepared PLA microspheres. Use polyethylene glycol with a molecular weight of 2000 Da to graft on the surface of the microspheres, and react with the surface of the microspheres through epoxidized PEG to ensure uniform coverage of the PEG layer.

[0094] Lyophilization process: The PEGylated microspheres are subjected to a lyophilization process to obtain the final injectable polylactic acid microspheres.

[0095] Release control: By adjusting the particle size (10 - 50 μm) and the degree of PEGylation of the microspheres, the release rate of the drug is controlled. Larger microspheres and thicker PEG layers can effectively delay the release of the drug.

[0096] In vivo drug release experiment: Small animal experiments (such as mice) are conducted to verify the release characteristics of the drug in vivo, and the drug concentration is continuously tracked.

[0097] The comparative data of experimental performance are as follows:

[0098]

[0099]

[0100] PEGylated microspheres: PEGylated microspheres can achieve a longer drug release time in vivo (30 - 45 days), and the drug release rate is more controllable, reducing the side effects of the drug. PEGylation effectively reduces the immunogenicity of the microspheres, making their retention time in vivo longer and the immune rejection reaction less. The PEGylated surface reduces the non-specific binding of the microspheres to proteins, contributing to the stability of the drug.

[0101] Non-PEGylated microspheres: The drug release of non-PEGylated microspheres is relatively fast, usually reaching the peak of drug release within 15 - 20 days. Due to their poor surface hydrophilicity, they are prone to immune reactions, so their biocompatibility is relatively poor.

[0102] Traditional polylactic acid microspheres: The drugs in traditional polylactic acid microspheres without microfluidic emulsification have a short release time due to uneven particle size distribution, usually completing within 5 - 10 days, and may require frequent injections. Traditional solvent removal methods (such as vacuum distillation) are less efficient, and the residual solvents may have a negative impact on the biocompatibility of the microspheres.

[0103] The docetaxel sustained-release system prepared by PEGylated polylactic acid microspheres can effectively extend the drug release time, reduce the injection frequency of patients and improve the curative effect. In the in vivo drug release experiment of mice, the drug release of PEGylated microspheres is stable and continuous, effectively avoiding the rapid release of the drug and reducing the toxic and side effects of chemotherapy drugs. Compared with the traditional treatment group, the adverse reactions of patients receiving PEGylated microsphere injection are significantly reduced. The drug-related adverse reactions are reduced by about 50%. The drug efficacy duration of patients in the PEGylated microsphere group is extended, and the treatment cycle is extended from the traditional 4 weeks to about 8 weeks, effectively improving the quality of life of patients. Specific embodiment four:

[0105] Based on the technical solution of Specific Embodiment 1, a practical application case is further given:

[0106] As people age, the content of collagen and elastin in the skin gradually decreases, leading to skin relaxation and the appearance of wrinkles, which in turn affects the appearance. Traditional anti-aging treatment methods such as hyaluronic acid injection and botulinum toxin, although having significant short-term effects, generally only last for several months and may have side effects. For this reason, polylactic acid microspheres have begun to be widely studied and applied as long-term skin fillers in the field of medical aesthetics.

[0107] As described in Specific Embodiment 1, a microsphere suitable for injection into the skin is prepared by using the polylactic acid (PLA) microsphere preparation technology, combined with supercritical carbon dioxide solvent removal and PEGylation technology.

[0108] Dissolve polylactic acid: Select racemic polylactic acid (molecular weight 100 - 150 kDa) and dissolve it in ethyl acetate at a concentration of 50 g / L, and stir at 60 °C for 2 hours to ensure complete dissolution of polylactic acid.

[0109] Prepare the aqueous solution: Add recombinant elastin, collagen and other components to the water for injection to prepare an aqueous solution with a concentration of 2 wt%, and adjust the pH value to 7.4 to simulate the physiological environment of the skin.

[0110] Emulsification process: Drop the polylactic acid organic solution into the aqueous solution at a volume ratio of 1:20, and use microfluidic technology for emulsification to form a uniform water-in-oil emulsion, and control the particle size of the microspheres between 10 - 100 μm.

[0111] Supercritical CO2 solvent removal: In a supercritical CO2 device, use a pressure of 7.5 - 10 MPa and a temperature of 45 °C for solvent removal to ensure the stability and biocompatibility of the polylactic acid microspheres.

[0112] PEGylation treatment: Use polyethylene glycol (PEG) with a molecular weight of 2000 Da to perform surface modification in a buffer solution with a pH of 7.4, and react with the surface of the microspheres through epoxidized PEG to form a stable PEG coating, increasing the biocompatibility of the microspheres and reducing the immune response.

[0113] Freeze-drying process: Perform freeze-drying on the PEGylated microspheres to obtain the final injectable polylactic acid microspheres.

[0114] Skin assessment: Before treatment, the doctor determines the specific areas of skin aging (such as the corners of the eyes, the corners of the mouth, the forehead, etc.) through visual assessment and skin elasticity testing.

[0115] Local anesthesia: Use a local anesthetic to anesthetize the treatment area to reduce the discomfort of the patient during the injection process.

[0116] Injectable Poly(lactic acid) Microspheres: After reconstructing the freeze-dried poly(lactic acid) microsphere solution into a concentration suitable for injection, it is injected into the dermal layer or the superficial epidermal layer of the skin through microneedles or other fine needles. As needed, doctors will inject different amounts of microspheres and precisely control the depth and location of injection.

[0117] Collagen Stimulation: After being injected into the skin, the poly(lactic acid) microspheres can be gradually absorbed by the skin, stimulating the skin's self-repair and promoting the regeneration of collagen. The slow degradation and absorption of the microspheres continuously stimulate the skin to produce new collagen, thus achieving the effects of filling and lifting.

[0118] Efficacy Evaluation: After treatment, doctors will track the skin changes of patients and conduct regular examinations. Usually, the effects can be observed 3 months after injection, and the treatment plan will continue to be improved.

[0119]

[0120]

[0121] Duration of Efficacy: Since the microspheres can stimulate the skin to produce new collagen, the treatment effect can be maintained for 12 - 18 months, far exceeding the duration of efficacy of hyaluronic acid and botulinum toxin. The gradually absorbed poly(lactic acid) microspheres promote collagen production, not only improving the skin's firmness but also restoring the skin's structure. The latter two mainly play the role of filling or temporarily relaxing muscles, but do not stimulate the skin's collagen production, so their effects are relatively short-lived.

[0122] Side Effects and Safety: Due to its surface being PEGylated, it has high biocompatibility, so there are fewer side effects, usually manifested as mild swelling or erythema, and the recovery period is short. As a common skin filler, hyaluronic acid also usually has fewer side effects, but it may cause allergic reactions in individual patients. Although botulinum toxin has significant effects, its long-term use may lead to muscle relaxation and skin sagging.

[0123] Skin Elasticity and Long-Term Effects: After injection of poly(lactic acid) microspheres, the skin's elasticity can be significantly improved, and by continuously stimulating collagen production, the skin becomes more firm and elastic, with more lasting long-term effects.

[0124] Within 3 months after patients inject poly(lactic acid) microspheres, the skin's firmness and elasticity are significantly improved, wrinkles are significantly reduced, the overall facial contour becomes clearer, and the skin condition is significantly rejuvenated. After 3 - 6 months of treatment, 90% of the patients express very satisfaction with the effects, believing that this method has a longer-lasting effect and a more natural appearance compared to hyaluronic acid and botulinum toxin.

[0125] It should be noted that, in this document, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprising", "including" or any other variant thereof are intended to cover non-exclusive inclusion, such that a process, method, article or device comprising a series of elements not only includes those elements but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device. Without further limitation, an element defined by the statement "comprising a reference structure" does not exclude the presence of additional identical elements in the process, method, article or device comprising the element.

[0126] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing injectable polylactic acid microspheres, characterized in that: The method comprises the following steps: Sp1, dissolving polylactic acid in an organic solvent of chloroform or dichloromethane to prepare an organic solution of polylactic acid; Sp2, the polylactic acid is one of dextrorotatory polylactic acid, levorotatory polylactic acid, mesorotatory polylactic acid and racemic polylactic acid; Sp3, preparing an aqueous solution of recombinant elastin and recombinant collagen: dissolving recombinant elastin with a molecular weight of 10 to 50 kDa, recombinant collagen with a molecular weight of 20 to 100 kDa and sodium carboxymethyl cellulose in water for injection to prepare an aqueous solution of recombinant elastin / recombinant collagen / sodium carboxymethyl cellulose with a concentration of 10 to 80 g / L; Sp4, adding the organic solution of polylactic acid dropwise to the aqueous solution of recombinant elastin / recombinant collagen / sodium carboxymethyl cellulose, mixing, and then removing the organic solvent in the mixed solution; Sp5. The solution obtained in step Sp4 is freeze-dried to obtain injectable polylactic acid microspheres.

2. The method for preparing injectable polylactic acid microspheres according to claim 1, characterized in that: In Sp1, polylactic acid is dissolved in chloroform or dichloromethane to prepare an organic solution of polylactic acid with a concentration of 10 to 50 g / L.

3. The method for preparing injectable polylactic acid microspheres according to claim 1, characterized in that: The mass ratio of the recombinant elastin to the recombinant collagen is 1:30 to 30:

1.

4. The method for preparing injectable polylactic acid microspheres according to claim 1, characterized in that: The mass ratio of the sodium carboxymethyl cellulose to the total amount of recombinant elastin and recombinant collagen is 1:10 to 10:

1.

5. The method for preparing injectable polylactic acid microspheres according to claim 1, characterized in that: In Sp4, the volume ratio of the organic solution of polylactic acid to the aqueous solution of recombinant elastin / recombinant collagen / sodium carboxymethyl cellulose is 1:3 to 1:

50. The organic solvent is selected from ethyl acetate or supercritical carbon dioxide, and the organic solvent is selected based on its boiling point, solubility parameter and dissolving efficiency for polylactic acid.

6. The method for preparing injectable polylactic acid microspheres according to claim 1, characterized in that: The volume ratio of the organic solution of polylactic acid to the aqueous solution of recombinant elastin / recombinant collagen / sodium carboxymethyl cellulose is 1:5 to 1:

30.

7. The method for preparing injectable polylactic acid microspheres according to claim 1, characterized in that: In Sp4, the organic solvent in the mixed solution is removed by the following steps: Sp4.1, adding the organic solution of polylactic acid to the aqueous solution of recombinant elastin / recombinant collagen / sodium carboxymethyl cellulose; Sp4.2, remove dichloromethane by distillation under reduced pressure.

8. The method for preparing injectable polylactic acid microspheres according to claim 1, characterized in that: The polylactic acid organic solution and the aqueous solution are added dropwise in the Sp3 step at a volume ratio of 1:20, and the dropping rate is controlled at 5 ml per minute for uniform distribution of microsphere particle sizes in the range of 10 to 100 μm, and a continuous stirring speed of 1000 rpm is maintained during the dropping process.

9. The method for preparing injectable polylactic acid microspheres according to claim 1, characterized in that: The solvent removal step Sp4 is performed under the conditions of reducing the pressure to below 50 mbar and controlling the temperature at 45° C. The solvent removal step adopts a continuous flow method to improve the solvent removal efficiency.

10. The method for preparing injectable polylactic acid microspheres according to claim 1, characterized in that: The PEGylation treatment is carried out by grafting a polyethylene glycol segment with a molecular weight of 2000 Da onto the surface of the microsphere in a buffer solution with a pH value of 7.4, and the grafting reaction is carried out by epoxidized polyethylene glycol.

Images