Preparation method of composite microspheres for medical beauty injection filler

Abstract

The invention provides a preparation method of composite microspheres for a medical beauty injection filler, and belongs to the technical field of chemical treatment methods, and the preparation method comprises the following steps: mixing polylactic acid macromolecules and inorganic particles to prepare an oil phase; preparing a polyvinyl alcohol or Tween 80 surfactant aqueous solution as an aqueous phase; shearing and emulsifying in supergravity equipment to form an oil-in-water solid-in-water emulsion; mechanically stirring at room temperature to promote volatilization of the organic solvent; centrifugally washing to remove residues; freeze-drying to obtain composite microspheres; determining and evaluating particle size distribution; mixing with cross-linked sodium hyaluronate gel to prepare an injection filler; evaluating the biocompatibility and the collagen regeneration efficiency; and optimizing the formula according to an evaluation result. The technical problem that the preparation stability of the composite microspheres in the medical beauty injection filler is poor is solved, and the medical beauty injection filler which is consistent in batch height and good in preparation stability is finally obtained.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical treatment methods, and more particularly, relates to a method for preparing composite microspheres for medical aesthetic injection fillers. Background Art

[0002] Medical aesthetic injection fillers are products widely used in the field of non-surgical aesthetics. Among them, the composite of polylactic acid-based microspheres and sodium hyaluronate gel has attracted much attention due to its good biodegradability and long-lasting filling effect. Traditional methods for preparing composite microspheres mainly include solvent evaporation method, spray drying method or emulsion solvent evaporation method, and these methods usually complete the emulsification process under conventional stirring equipment. In clinical applications, these fillers are widely used for aesthetic repairs such as facial wrinkles, depressions and contour shaping, as well as tissue filling and drug sustained-release carrier systems in tissue engineering.

[0003] However, the traditional preparation process has significant defects: there are large differences between batches in the preparation of microspheres, and the product quality fluctuates significantly; it is difficult to precisely control the emulsification conditions during the preparation process, resulting in large differences in the particle size distribution, morphological characteristics and internal structure of microspheres in different batches; the fusion process of inorganic components and organic components is uncontrollable, and the composition of the composite microspheres is uneven; at the same time, there is a lack of a systematic theoretical model to guide the setting of process parameters, making the preparation process difficult to standardize, and the preparation results vary significantly under different operators or equipment conditions.

[0004] These problems lead to inconsistent clinical effects of medical aesthetic fillers between batches, and the use effects are difficult to predict, seriously affecting the quality stability and market application promotion of the products. The traditional technology cannot solve the stability problem in the preparation process of composite microspheres, especially in the process of large-scale production, the preparation stability problem is more prominent. There is an urgent need for an efficient, controllable and stable method for preparing composite microspheres guided by a theoretical model. That is to say, there is a technical problem of poor stability in the preparation of composite microspheres in medical aesthetic injection fillers in the prior art. Summary of the Invention

[0005] In view of this, the present invention provides a method for preparing composite microspheres for medical aesthetic injection fillers, which can solve the technical problem of poor stability in the preparation of composite microspheres in medical aesthetic injection fillers existing in the prior art.

[0006] The present invention is implemented as follows: The present invention provides a method for preparing composite microspheres for medical aesthetic injection fillers, which includes preparing a polymer oil-phase solution, formulating a surfactant aqueous-phase solution, performing shear emulsification in a high-gravity equipment to form a water-in-oil-in-solid emulsion, mechanically stirring at room temperature to promote the volatilization of organic solvents, centrifugally washing to remove residues, freeze-drying to obtain composite microspheres, measuring the particle size distribution of the microspheres, and mixing with cross-linked sodium hyaluronate gel to prepare an injection filler and evaluating it. Among them, a high-shear force field is generated by a high-gravity equipment to precisely control the emulsification process. During the emulsification process, the droplet formation kinetics follows the shear energy balance equation, the solvent volatilization process follows Fick's diffusion equation, the particle size prediction of the microspheres is theoretically estimated using a phase separation kinetics model, the mixing process uses a microsphere dispersion optimization function to determine the optimal operating parameters, and is analyzed and optimized through a biological effect prediction model of polylactic acid microspheres to achieve the preparation of high-stability composite microspheres.

[0007] Among them, the polymer oil-phase solution includes dissolving a copolymer of poly(lactic-co-glycolic acid) or a poly(l-lactic acid)-poly(ethylene glycol) block copolymer at a concentration of 20 to 200 mg / mL in dichloromethane or ethyl acetate, and adding inorganic particles of zinc oxide or magnesium oxide to the oil-phase solution. The mass ratio of the inorganic particles to the polymer is controlled within the range of 1:200 to 1:5.

[0008] Among them, the surfactant aqueous-phase solution includes dissolving a surfactant such as polyvinyl alcohol or Tween 80 at a concentration of 5 to 100 mg / mL in injection water. The ratio of the aqueous-phase solution to the oil-phase solution is controlled within the range of 1:1 to 20:1.

[0009] Among them, centrifugally washing to remove residues includes centrifugally washing the microsphere suspension 3 to 5 times. After each centrifugation, the supernatant is discarded and fresh injection water is added to resuspend, removing residual organic solvents and surfactants.

[0010] Among them, freeze-drying to obtain composite microspheres includes subjecting the washed microsphere suspension to freeze-drying treatment. The freeze-drying temperature is controlled at -40 to -60 °C, the vacuum degree is maintained at 10 to 50 Pa, and the drying time is not less than 24 hours.

[0011] Among them, measuring the particle size distribution of the microspheres includes using a laser particle size analyzer to evaluate the particle size uniformity of the microspheres and calculating the 10th percentile value, 50th percentile value, and 90th percentile value of the particle size distribution of the microspheres.

[0012] Among them, mixing with cross-linked sodium hyaluronate gel to prepare an injection filler includes mixing the dried organic-inorganic composite microspheres with cross-linked sodium hyaluronate gel at a mass ratio of 1:5 to 1:20 and preparing an injection filler through a sterile filling process.

[0013] Among them, the water-in-oil-in-solid emulsion refers to a three-phase system in which oil-phase micro-droplets formed by coating inorganic particles with an amphiphilic polylactic acid material are dispersed in an aqueous phase. The solid phase is the inorganic particles, the oil phase is the polymer solution, and the aqueous phase is the surfactant aqueous solution. The amphiphilic polylactic acid material refers to a polylactic acid derivative containing hydrophilic and lipophilic groups, including a copolymer of polylactic acid and glycolic acid with a lactic acid to glycolic acid ratio of 90:10, a copolymer of polylactic acid glycolic acid and polyethylene glycol, or a block copolymer of poly-L-lactic acid and polyethylene glycol, where the molecular weight of polylactic acid is 80,000 to 240,000 and the molecular weight of polyethylene glycol is 700 to 6,000.

[0014] Among them, the uniformity of the microsphere particle size is evaluated by the Span value. The calculation formula is that the Span value is equal to the 90th percentile particle size value minus the 10th percentile particle size value and then divided by the 50th percentile particle size value. The smaller the Span value, the more uniform and concentrated the microsphere particle size distribution.

[0015] Among them, the optimal operating parameters include the optimal mixing time and the optimal mixing rotation speed. The optimal mixing time is the best duration for mixing the microspheres and sodium hyaluronate gel, and the optimal mixing rotation speed is the best stirring rate during the mixing process.

[0016] The present invention proposes to precisely control the emulsification process by using the strong shear force field generated by a high-gravity device, and at the same time introduce a variety of kinetic models and machine learning algorithms to optimize the preparation parameters. This method realizes the high stability and controllability of the composite microsphere preparation process, significantly reduces the batch-to-batch difference; through the constant strong shear environment provided by the high-gravity field, it ensures that the force is uniform at each position during the emulsification process and the preparation conditions are consistent; based on the shear energy balance equation, it precisely controls the droplet formation process, so that the microsphere particle size distribution remains consistent among different batches; the Fick diffusion equation and the phase separation kinetic model guide the solvent evaporation and microsphere solidification processes to ensure the stability of the internal structure of the microspheres; the microsphere dispersion optimization function ensures the stable and controllable mixing process of the composite microspheres and sodium hyaluronate gel. By placing the entire preparation process under the guidance of strict theoretical models and combining multi-modal deep learning prediction models for parameter optimization and quality monitoring, the present invention successfully solves the technical problem of poor stability in the preparation of composite microspheres in traditional technologies, realizes the stable and standardized preparation of high-quality medical beauty injection fillers, and greatly improves the consistency and reliability among product batches. Description of the Drawings

[0017] Figure 1 It is a flowchart of the method of the present invention.

[0018] Figure 2 It is a comparison chart of the collagen content and cell survival rate in Examples 2-4.

[0019] Figure 3Graph showing the change in collagen content over time for Example 5.

[0020] Figure 4 Graph for predicting the volume retention rate of the injectable filler for Example 5. Detailed implementation manners

[0021] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

[0022] As Figure 1 shown, it is a flowchart of a method for preparing composite microspheres for a medical aesthetic injectable filler provided by the present invention. This method includes the following steps:

[0023] S01. Dissolve poly (lactic-co-glycolic acid) copolymer or poly (L-lactic acid)-poly (ethylene glycol) block copolymer in dichloromethane or ethyl acetate at a concentration of 20 to 200 mg / mL to prepare an oil-phase solution, and add zinc oxide or magnesium oxide inorganic particles to the oil-phase solution. The mass ratio of the inorganic particles to the polymer is controlled within the range of 1:200 to 1:5;

[0024] S02. Prepare an aqueous-phase solution. Dissolve a polyvinyl alcohol or Tween 80 surfactant in injection water at a concentration of 5 to 100 mg / mL to ensure complete dissolution. The ratio of the aqueous-phase solution to the oil-phase solution is controlled within the range of 1:1 to 20:1;

[0025] S03. Set the rotation speed of the high-gravity equipment to 300 to 3000 revolutions per minute. Simultaneously introduce the aqueous-phase solution and the oil-phase solution into the high-gravity equipment through a metering pump, and perform shear emulsification at room temperature to form a water-in-oil-in-solid emulsion. The kinetics of droplet formation during emulsification follows the shear energy balance equation;

[0026] S04. Collect the microsphere suspension emulsion flowing out from the bottom outlet of the high-gravity equipment, and mechanically stir it at a rotation speed of 200 to 1000 revolutions per minute for 6 to 12 hours at room temperature to promote the complete volatilization of the organic solvent. The process of solvent volatilization follows Fick's diffusion equation;

[0027] S05. Centrifuge and wash the microsphere suspension 3 to 5 times. After each centrifugation, discard the supernatant and resuspend it with fresh injection water to remove residual organic solvents and surfactants;

[0028] S06. Subject the washed microsphere suspension to freeze-drying treatment. The freeze-drying temperature is controlled at -40 to -60 °C, the vacuum degree is maintained at 10 to 50 Pa, and the drying time is not less than 24 hours;

[0029] S07. Measure the particle size distribution of the microspheres after drying, evaluate the particle size uniformity of the microspheres using a laser particle size analyzer, calculate the 10th percentile value, 50th percentile value, and 90th percentile value of the particle size distribution of the microspheres, and theoretically estimate the particle size of the microspheres using a phase separation kinetics model;

[0030] S08. Mix the dried organic-inorganic composite microspheres and cross-linked sodium hyaluronate gel at a mass ratio of 1:5 to 1:20, and prepare an injectable filler through a sterile filling process. The optimal operating parameters are determined using a microsphere dispersion optimization function during the mixing process;

[0031] S09. Conduct biocompatibility evaluation and collagen regeneration efficiency test on the prepared injectable filler. Evaluate its safety and effectiveness using in vitro cell proliferation experiments and animal subcutaneous injection experiments. The results are analyzed and optimized through a pre-trained biological effect prediction model for polylactic acid microspheres. If the biocompatibility evaluation shows that the cell survival rate is less than 80% or obvious inflammatory reactions are observed in animal experiments, the inorganic particle content needs to be adjusted, and the mass ratio of inorganic particles to polymers is reduced to 50% of the original ratio, and steps S01 to S08 are repeated; if the collagen regeneration efficiency test shows that the increase in collagen content after 28 days of injection is less than 40% of the control group, the molecular weight of polylactic acid needs to be increased to the range of 150,000 to 240,000, and the ratio of polylactic acid to polyethylene glycol is adjusted to 80:20 to 90:10, and steps S01 to S08 are re-executed until the collagen regeneration efficiency reaches the standard; if both biocompatibility and collagen regeneration efficiency meet the preset threshold requirements, the preparation of the composite microsphere injectable filler is completed.

[0032] Among them, the high-gravity equipment refers to a baffle-type high-gravity equipment, a spiral-channel high-gravity equipment, a stator-rotor high-gravity equipment, a rotating disk high-gravity equipment, or a rotating packed bed high-gravity equipment, which can generate a centrifugal force field equivalent to 80 to 300 times the gravitational acceleration at a rotational speed of 300 to 3000 revolutions per minute to strengthen the liquid-liquid heterogeneous micro-mixing process.

[0033] Among them, the water-in-oil-in-solid emulsion refers to a three-phase system in which oil-phase micro-droplets formed by coating inorganic particles with amphiphilic polylactic acid materials are dispersed in an aqueous phase, where the solid phase is the inorganic particles, the oil phase is a polymer solution, and the aqueous phase is an aqueous surfactant solution.

[0034] Among them, the particle size uniformity of the microspheres is evaluated through the Span value. The calculation formula is that the Span value is equal to the 90th percentile particle size value minus the 10th percentile particle size value and then divided by the 50th percentile particle size value. The smaller the Span value, the more uniform and concentrated the particle size distribution of the microspheres.

[0035] Among them, the amphiphilic polylactic acid material refers to a polylactic acid derivative containing hydrophilic groups and lipophilic groups, including poly(lactic-co-glycolic acid) with a lactic acid to glycolic acid ratio of 90:10, poly(lactic-co-glycolic acid)-polyethylene glycol copolymer, or poly(L-lactic acid)-polyethylene glycol block copolymer, where the molecular weight of polylactic acid is 80,000 to 240,000, and the molecular weight of polyethylene glycol is 700 to 6,000.

[0036] Among them, the crosslinked sodium hyaluronate gel refers to a hydrogel with a network structure prepared by treating sodium hyaluronate with a crosslinking agent such as 1,4-butanedial, divinyldisiloxane, or polyethylene glycol diglycidyl ether, and is used to increase the viscoelasticity and persistence of injection fillers.

[0037] Among them, the shear energy balance equation is used to describe the kinetic process of droplet formation and breakage in a high-gravity field. The inputs include the continuous phase viscosity, dispersed phase viscosity, interfacial tension, rotational angular velocity of the high-gravity field, and characteristic length of the high-gravity equipment, and the output is the maximum stable droplet diameter in the equilibrium state; the shear energy balance equation describes the relationship between the maximum stable droplet diameter and interfacial tension, continuous phase density, dispersed phase viscosity, continuous phase viscosity, angular velocity, characteristic radius of the high-gravity equipment, and a constant related to the equipment geometry.

[0038] Among them, the Fick diffusion equation is used to describe the diffusion mass transfer process of an organic solvent from the polymer phase to the aqueous phase. The inputs include the diffusion coefficient of the solvent in the polymer, the diffusion coefficient of the solvent in the aqueous phase, the interfacial solvent concentration gradient, and the microsphere diameter, and the output is the time required for solvent diffusion; the Fick diffusion equation describes the relationship between solvent concentration with time and space, where there is a relationship between the diffusion coefficient and polymer concentration, temperature, including the reference diffusion coefficient, diffusion activation energy, gas constant, absolute temperature, coefficient, and polymer concentration.

[0039] Among them, the phase separation kinetic model is used to predict the polymer phase separation and microsphere solidification behavior during the emulsification solvent evaporation process. The inputs include the polymer solubility parameter, solvent evaporation rate, initial droplet size, and initial polymer concentration, and the output is the final microsphere particle size distribution characteristics; the phase separation kinetic model describes the relationship between the polymer volume fraction with time, including mobility and free energy functional, and the influence of polymer rheological properties on phase separation kinetics is introduced through a viscoelastic correction term.

[0040] Among them, the microsphere dispersion optimization function is used to determine the optimal mixing parameters of the composite microspheres and sodium hyaluronate gel. The inputs include the average particle size of the microspheres, the surface charge of the microspheres, the gel viscosity, the shear rate, and the mixing temperature, and the outputs are the optimal mixing time and the optimal mixing rotation speed. The microsphere dispersion optimization function establishes the relationship between the relative viscosity, the microsphere volume fraction, the maximum packing fraction, and the intrinsic viscosity. The optimal parameters during the mixing process are determined by minimizing the probability of microsphere aggregation in the gel.

[0041] Among them, the structure of the biological effect prediction model of the polylactic acid microspheres is a multi-modal deep learning network architecture, which includes three parallel feature extraction branches and a fusion prediction module. The first branch processes the physicochemical property data of the microspheres and adopts a three-layer fully connected neural network structure. The number of neurons in the hidden layer is determined by the product function of the polylactic acid molecular weight and the inorganic particle size, and the activation function is the rectified linear unit. The second branch processes the surface morphology features of the microspheres and uses a convolutional neural network to extract spatial structure information, which includes three convolutional layers and two pooling layers. The size of the convolutional kernel is proportional to the average particle size of the inorganic particles. The third branch processes the component ratio information and uses a self-attention mechanism to weight and fuse the features of different components. The number of attention heads is determined by the hydrophilic-lipophilic balance value of the polylactic acid. The fusion module uses a gated recurrent unit network to integrate the feature vectors output by the three branches and predicts the collagen regeneration efficiency and biocompatibility score triggered by the microspheres.

[0042] Among them, the steps for establishing the training data set during the training process of the biological effect prediction model of the polylactic acid microspheres include collecting the experimental data on the biological effects of polylactic acid-based microsphere materials in the published literature, and standardizing and extracting the physicochemical property parameters of the microspheres, including the particle size distribution, surface charge, molecular weight, inorganic component content, and its particle size. Construct a microsphere surface morphology feature library, including scanning electron microscope images and transmission electron microscope images of the microsphere cross-section. Integrate the cytological experimental data, including cell proliferation rate, cell adhesion, cell viability, and cytokine expression levels. Collect animal experiment data, including histological changes, collagen content changes, inflammation reaction degree, and degradation rate after subcutaneous injection. Divide the data set into a training set, a validation set, and a test set by the cross-validation method. The training set and the validation set are used for model training and parameter optimization, and the test set is used to evaluate the generalization performance of the model.

[0043] Among them, the steps for training the biological effect prediction model of the polylactic acid microspheres include first performing self-supervised learning on synthetic data to initialize the model parameters by predicting the correlation between the physicochemical properties and biological effects of the microspheres; then training on the labeled data in a supervised learning manner, with the loss function combining the mean square error term and the regularization term, and the optimization algorithm using the Adam optimizer. The initial value of the learning rate is set to 0.001 and the cosine annealing strategy is used for dynamic adjustment; an early stopping strategy is introduced during the training process to prevent overfitting, and the training is stopped when the performance index on the validation set has not improved for 5 consecutive rounds; finally, the model is fine-tuned using the transfer learning method by freezing the parameters of the feature extraction layer and only training the parameters of the prediction layer to make the model adapt to different polylactic acid microsphere formulation combinations; the entire training process is iterated 5000 times, and the five-fold cross-validation is used to evaluate the model performance. The final model selects the parameter combination with the smallest root mean square error of collagen regeneration efficiency prediction on the validation set.

[0044] The specific implementation manners of the above steps are described in detail below.

[0045] The specific implementation manner of step S01 is to select a suitable polymer material and prepare the oil phase solution. First, weigh the poly(lactic-co-glycolic acid) copolymer or poly(L-lactic acid)-poly(ethylene glycol) block copolymer, and accurately measure the required mass. Subsequently, add the polymer to the pre-prepared dichloromethane or ethyl acetate, and stir until it dissolves to a transparent state. The concentration of the oil phase solution is controlled within the range of 20 to 200 milligrams per milliliter. A lower concentration is beneficial for forming microspheres with small particle sizes, while a higher concentration increases the mechanical strength of the microspheres. At the same time, add zinc oxide or magnesium oxide nano-scale inorganic particles to the oil phase solution, and the mass ratio of the inorganic particles to the polymer is controlled within the range of 1:200 to 1:5. The purpose of introducing the inorganic particles is to enhance the mechanical properties of the microspheres and regulate the degradation rate, while promoting the synthesis of the extracellular matrix. During this process, the polymer solubility parameter theory is used to predict the dissolution behavior of the polymer in organic solvents to ensure the formation of a stable and uniform oil phase solution.

[0046] The specific implementation manner of step S02 is to prepare the aqueous phase solution. First, prepare the water for injection and sterilize it by passing it through a 0.22-micron filter membrane. Then, weigh the polyvinyl alcohol or Tween 80 surfactant and add it to the water for injection. When preparing polyvinyl alcohol, it needs to be heated to 80 °C and continuously stirred, while Tween 80 can be directly dissolved at room temperature. The concentration of the surfactant is controlled within the range of 5 to 100 milligrams per milliliter, and the ratio of the aqueous phase solution to the oil phase solution is controlled between 1:1 and 20:1. The selection of the surfactant is based on its hydrophilic-lipophilic balance value. The hydrophilic-lipophilic balance value of polyvinyl alcohol is 18, which is suitable for preparing microspheres with higher hydrophilicity; the hydrophilic-lipophilic balance value of Tween 80 is 15, which is suitable for preparing microspheres with medium hydrophilicity. During the preparation process of the aqueous phase solution, attention should be paid to the critical micelle concentration theory of the surfactant to ensure that the surfactant concentration is higher than the critical micelle concentration to provide sufficient interfacial stabilization.

[0047] The specific implementation of step S03 is to use a high-gravity device for shear emulsification. First, start the high-gravity device and adjust the rotation speed to 300 to 3000 revolutions per minute to generate a centrifugal force field with a gravitational acceleration of 80 to 300 times. Inject the oil-phase solution and the water-phase solution into the high-gravity device simultaneously at a constant flow rate through a precision metering pump. Under the action of strong shear force, the oil phase is dispersed into tiny droplets and wraps the inorganic particles to form a water-in-oil-in-solid emulsion. During the emulsification process, the formation of emulsion droplets follows the shear energy balance equation, which describes the relationship between the maximum stable droplet diameter at the equilibrium state and the interfacial tension, continuous phase density, dispersed phase viscosity, continuous phase viscosity, angular velocity, and the characteristic radius of the high-gravity device. According to this equation, increasing the angular velocity or decreasing the interfacial tension can significantly reduce the droplet size. The microscopic turbulent pulsation in the high-gravity field enhances the interfacial renewal rate and promotes the formation of a precursor emulsion of microspheres with a uniform particle size distribution. In actual operation, when the ratio of the water-phase flow rate to the oil-phase flow rate is 5:1 to 15:1, microspheres with a narrow particle size distribution can be obtained.

[0048] The specific implementation of step S04 is to collect the microsphere suspension emulsion and promote the volatilization of the organic solvent. First, set a collection container at the bottom outlet of the high-gravity device to collect the outflowing microsphere suspension emulsion. Transfer the collected emulsion to a glass reaction kettle with a mechanical stirring device, and set the stirring speed to 200 to 1000 revolutions per minute to avoid the aggregation of microspheres caused by too high a rotation speed. Continuously stir for 6 to 12 hours at room temperature to promote the diffusion of the organic solvent in the microspheres into the water phase and finally volatilize. The solvent volatilization process follows Fick's diffusion equation, which describes the relationship between the solvent concentration and time and space, where the diffusion coefficient is related to the polymer concentration and temperature. For polylactic acid microspheres, the diffusion coefficient of dichloromethane in the polymer decreases with the increase of the polymer concentration, and the diffusion activation energy is about 15 to 25 kJ / mol. During the solvent volatilization process, the microspheres gradually harden and form a porous structure. The pore size is closely related to the solvent volatilization rate. The faster the volatilization rate, the larger the formed pores but the more uneven the distribution.

[0049] The specific implementation of step S05 is to perform centrifugal washing on the microsphere suspension. First, transfer the microsphere suspension to a centrifuge tube and centrifuge it at a speed of 3000 to 5000 revolutions per minute for 5 to 10 minutes. The precipitate formed after centrifugation is the microspheres, and the supernatant contains residual surfactant and organic solvent. Carefully aspirate the supernatant and discard it, then add fresh injection water to the precipitate and resuspend the microspheres using a vortex mixer. Repeat the above centrifugal washing process 3 to 5 times until the concentration of residual organic solvent in the supernatant is less than 10 micrograms per milliliter and the concentration of residual surfactant is less than 5 micrograms per milliliter. During the washing process, the best centrifugation time is calculated using sedimentation theory to ensure that the microspheres are completely sedimented without damaging the structure. For polylactic acid microspheres with a particle size of 10 to 50 micrometers, under the centrifugation condition of 3000 revolutions per minute, the best centrifugation time is 5 to 8 minutes. The number of washing times is determined based on the solute dilution theory, and the impurity concentration in the supernatant is reduced by about 90% after each washing.

[0050] The specific implementation of step S06 is to perform freeze-drying treatment. First, transfer the washed microsphere suspension to a freeze-drying bottle and place it in an environment of -40 to -60 °C for pre-freezing. The pre-freezing time is not less than 4 hours to ensure that the water is completely frozen into ice crystals. Subsequently, connect the freeze-drying bottle to a freeze-dryer, set the vacuum degree to 10 to 50 Pa, and the condenser temperature to -80 °C. Freeze-drying is divided into three stages: the primary drying stage maintains the temperature at -40 °C and the pressure at 20 Pa for 12 hours, mainly removing free water; the secondary drying stage gradually raises the temperature to -20 °C and reduces the pressure to 15 Pa for 8 hours to remove part of the bound water; the tertiary drying stage raises the temperature to 0 °C and further reduces the pressure to 10 Pa for 4 hours to remove the remaining bound water. The porosity of the microspheres during freeze-drying is determined by the sublimation rate of ice crystals. This process follows the coupled heat and mass transfer model, and the moving speed of the sublimation interface is related to the applied vacuum degree and the heat source temperature. After drying, the microspheres are in a loose powder form. Avoid contact with indoor air and seal them immediately for storage.

[0051] The specific implementation of step S07 is to measure the particle size distribution of the microspheres. First, take an appropriate amount of the dried microsphere sample and disperse it evenly in a 0.1% polysorbate 80 solution. Use a laser particle size analyzer for measurement. The laser wavelength is 633 nm, and the detection angle range is 0.02 to 165 degrees. Each sample is measured 5 times, and the average value is taken to calculate the 10th percentile value, 50th percentile value, and 90th percentile value of the microspheres, which are denoted as 、 、 。 The uniformity of the microsphere particle size is evaluated by the Span value, and the calculation formula is Span = , A Span value less than 1 indicates a uniform particle size distribution. Meanwhile, a phase separation kinetic model is used for theoretical particle size prediction. This model describes the polymer phase separation process based on the Cahn-Hilliard equation and combines the Flory-Huggins theory to calculate the phase separation driving force. The input parameters of the model include the polymer solubility parameter, the solvent evaporation rate, the initial droplet size, and the initial polymer concentration, and the output parameter is the final microsphere particle size distribution function. The deviation between the theoretical prediction and the measured value should be controlled within 10%, otherwise the preparation parameters need to be readjusted.

[0052] The specific implementation of step S08 is to prepare an injectable filler. First, a certain amount of dried composite microspheres are taken and added to the cross-linked sodium hyaluronate gel. The mass ratio of the microspheres to the gel is controlled within the range of 1:5 to 1:20. The sodium hyaluronate gel is pre-treated with a cross-linking agent such as 1,4-butanedial or divinyldisiloxane or polyethylene glycol diglycidyl ether, and the cross-linking degree is 1% to 5%. The mixing process uses a twin-screw extruder or a high-shear homogenizer, with a rotation speed of 500 to 2000 revolutions per minute and a mixing time of 5 to 30 minutes. The mixing parameters are optimized based on the microsphere dispersion optimization function. This function regards the microspheres as suspended particles, establishes the relationship between the relative viscosity, the microsphere volume fraction, the maximum packing fraction, and the intrinsic viscosity, and determines the optimal mixing time and rotation speed by minimizing the probability of microsphere aggregation in the gel. The mixed filler is sterilized by filtration through a 0.45-micron filter membrane, filled into a sterile syringe, sealed, and then subjected to γ-irradiation sterilization with an irradiation dose of 15 to 25 kilograys to ensure sterility without affecting the microsphere structure.

[0053] The specific implementation of step S09 is to conduct biocompatibility evaluation and collagen regeneration efficiency testing. First, an in vitro cell proliferation experiment is carried out. An appropriate amount of the injectable filler is co-cultured with fibroblasts for 72 hours. The cell viability is measured by the thiazolyl blue colorimetric method, and the cytokine secretion level is detected by the enzyme-linked immunosorbent assay method. Then, an animal subcutaneous injection experiment is carried out. Rabbits or mice are selected as experimental subjects. The histological changes are observed 1, 7, 14, and 28 days after subcutaneous injection of the filler. Hematoxylin-eosin staining is used to evaluate the inflammatory reaction, and Sirius red staining is used to measure the collagen content. The experimental results are analyzed through a poly(lactic acid) microsphere biological effect prediction model. The structure of this model is a multi-modal deep learning network, which includes three parallel feature extraction branches and a fusion prediction module. If the biocompatibility evaluation shows that the cell viability is less than 80%, the inorganic particle content is adjusted; if the collagen regeneration efficiency is less than 40% of the control group, the molecular weight of poly(lactic acid) is increased or the polymer component ratio is adjusted. When both the biocompatibility and the collagen regeneration efficiency reach the preset thresholds, the preparation of the composite microsphere injectable filler is completed.

[0054] The following details the mathematical models or calculation processes involved in the present invention.

[0055] The shear energy balance equation mentioned in step S03 is used to describe the dynamic process of droplet formation and breakage in a supergravity field, and is specifically expressed as follows:

[0056] ;

[0057] In the formula, is the maximum stable droplet diameter in the equilibrium state, with the unit of meter; is the oil-water interfacial tension, with the unit of Newton / meter; is the density of the continuous phase (aqueous phase), with the unit of kilogram / cubic meter; is the angular velocity of the supergravity equipment, with the unit of radian / second; is the characteristic radius of the supergravity equipment, with the unit of meter; is the viscosity of the dispersed phase (oil phase), with the unit of Pascal·second; is the viscosity of the continuous phase (aqueous phase), with the unit of Pascal·second; is a constant related to the geometric structure of the equipment, dimensionless.

[0058] The method for obtaining parameters is as follows:

[0059] can be obtained by measurement through the pendant drop method or the spinning drop method. The measurement process is to inject a polymer solution droplet into an aqueous surfactant solution, observe the droplet shape through an optical system, and calculate the interfacial tension according to the Laplace equation; is directly measured by a densitometer; is directly converted from the rotation speed of the supergravity equipment, , where is the rotation speed, with the unit of revolutions per minute; is a design parameter of the equipment and can be directly measured; and are respectively measured by a rheometer; needs to be determined through a standardized experiment. The method is to measure the droplet size under known conditions and inversely calculate the value, and the general range is 0.5 - 2.0.

[0060] This equation is constructed based on the principle of the balance between shear stress and surface tension. The exponents 0.6 and 0.4 are obtained by fitting a large amount of experimental data, indicating that the influence of the interfacial tension on the supergravity field strength (0.6) is slightly greater than the influence of the viscosity ratio (0.4). When the shear stress exceeds the interfacial tension, the droplet will be torn into smaller droplets. The equation adopts the form of a power function because in microscale hydrodynamics, these physical quantities usually follow a power-law relationship. This equation adds a viscosity ratio term compared with the traditional Weber number equation, considering the influence of the rheological properties of the dispersed phase on the emulsification process and improving the prediction accuracy.

[0061] The Fick's diffusion equation mentioned in step S04 describes the diffusion mass transfer process of the organic solvent from the polymer phase to the aqueous phase, and is specifically expressed as follows:

[0062] ;

[0063] In the formula, is the solvent concentration, with the unit of g / m³; is the time, with the unit of s; is the radial distance of the microsphere, with the unit of m; is the diffusion coefficient of the solvent in the polymer, with the unit of m² / s.

[0064] Diffusion coefficient has a relationship with the polymer concentration and temperature:

[0065] ;

[0066] In the formula, is the reference diffusion coefficient, with the unit of m² / s; is the diffusion activation energy, with the unit of J / mol; is the gas constant, 8.314 J / (mol·K); is the absolute temperature, with the unit of K; is the coefficient, dimensionless; is the volume fraction of the polymer, dimensionless.

[0067] The time required for solvent diffusion can be estimated as:

[0068] ;

[0069] In the formula, is the time required for solvent diffusion, with the unit of s; is the radius of the microsphere, with the unit of m.

[0070] The method for obtaining the parameters is as follows:

[0071] can be determined by nuclear magnetic resonance relaxation experiments. The method is to monitor the self-diffusion behavior of solvent molecules in the polymer, and the general range is ~ ; By measuring the diffusion coefficients at different temperatures, plot the versus curve, and the slope of the curve is , and for the polylactic acid system, it is generally 15 - 25 kJ / mol; is determined by measuring the diffusion coefficients at different polymer concentrations, and is generally 1.0 - 3.0; It can be calculated according to the formula; Measured by a laser particle size analyzer.

[0072] This equation is constructed based on the molecular diffusion theory. The spherical coordinate system is adopted because the microspheres have a spherical structure. The exponential term reflects the strong dependence of the diffusion coefficient on temperature (Arrhenius relationship) and the non-linear dependence on the polymer concentration. The diffusion coefficient increases with increasing temperature and decreases with increasing polymer concentration, which is consistent with the physical picture of polymer chains hindering the movement of solvent molecules. In the equation The second derivative term represents the rate of change of the concentration gradient, and the first derivative term takes into account the geometric effect in the spherical coordinate system. This equation can describe the solvent diffusion behavior in spherical microspheres more accurately than the traditional flat plate diffusion model, and can more precisely predict the hardening time and internal pore structure of the microspheres.

[0073] The phase separation kinetic model mentioned in step S07 is used to predict the polymer phase separation and microsphere curing behavior during the evaporation of the emulsified solvent, and is specifically expressed as follows:

[0074] ;

[0075] In the formula, is the volume fraction of the polymer, dimensionless; is the time, with the unit of second; is the mobility, with the unit of square meter / (joule·second); is the free energy functional, with the unit of joule / cubic meter; is the gradient energy coefficient, with the unit of joule / meter.

[0076] The free energy functional can be expressed as:

[0077] ;

[0078] In the formula, is the molar volume of the solvent molecules, with the unit of cubic meter / mole; is the Flory-Huggins interaction parameter, dimensionless.

[0079] The mobility is related to the polymer viscosity:

[0080] ;

[0081] Considering the influence of the rheological properties of the polymer on phase separation, a viscoelastic correction term is introduced:

[0082] ;

[0083] In the formula, is the coupling coefficient, dimensionless; is the stress tensor of the polymer, with the unit of Pascal.

[0084] The final particle size distribution of the microspheres can be described as:

[0085] ;

[0086] In the formula, is the probability density function of the particle size distribution; is the diameter of the microspheres, with the unit of meter; is the geometric mean diameter of the microspheres, with the unit of meter; is the distribution standard deviation, dimensionless.

[0087] The method for obtaining the parameters is as follows:

[0088] can be determined by measuring the phase diagram of the polymer in the solvent, and the value corresponding to the critical phase separation point is , and it can also be estimated by the solubility parameter: , where and are the solubility parameters of the polymer and the solvent, respectively; is determined by measuring the characteristic length of the polymer phase separation structure through small-angle X-ray scattering; is jointly determined by rheological measurement and phase separation kinetics experiment; and are obtained by fitting the measured particle size distribution data.

[0089] This model is constructed based on the Cahn-Hilliard equation and the Flory-Huggins theory. The Cahn-Hilliard equation describes the phase separation kinetics of a conservative field system. The second-order operator in the first term on the right side represents the diffusion effect, and the derivative of the free energy functional represents the driving force for phase separation. The free energy functional adopts a logarithmic form to reflect the contribution of the mixing entropy, The term represents the interaction between polymer and solvent molecules. Introducing the viscoelastic correction term is an innovation of this model, which considers the influence of the relaxation behavior of polymer chains on the phase separation kinetics, and this is crucial for accurately predicting the phase separation behavior of high molecular weight polylactic acid systems. This model can predict the particle size distribution of microspheres under different formulation conditions and provide theoretical guidance for the optimization of preparation parameters.

[0090] The microsphere dispersity optimization function mentioned in step S08 is used to determine the optimal mixing parameters of the composite microspheres and sodium hyaluronate gel, and is specifically expressed as follows:

[0091] ;

[0092] In the formula, is the relative viscosity, dimensionless; is the microsphere volume fraction, dimensionless; is the maximum packing fraction, dimensionless; is the intrinsic viscosity, with the unit of cm³ / g.

[0093] The microsphere aggregation probability function can be expressed as:

[0094] ;

[0095] In the formula, is the microsphere aggregation probability, dimensionless; is the frequency factor, with the unit of s ; is the shear rate, with the unit of s ; is the mixing time, with the unit of s; is the interaction energy barrier between microspheres, with the unit of J; is the Boltzmann constant, J / K.

[0096] The equation for determining the optimal mixing parameters:

[0097] ;

[0098] In the formula, is the optimal shear rate, with the unit of s ; is the optimal mixing time, with the unit of s.

[0099] Energy barrier is related to the surface charge and size of the microspheres:

[0100] ;

[0101] In the formula, is the vacuum permittivity, F / m; is the relative permittivity of the medium, dimensionless; is the microsphere diameter, with the unit of m; is the microsphere surface potential, with the unit of V; is the reciprocal of the Debye length, with the unit of m .

[0102] The method for obtaining the parameters is:

[0103] Directly calculate according to the formula; Determine by the microsphere sedimentation experiment. The method is to measure the maximum random packing density, generally 0.6 - 0.7; Measured by a capillary viscometer for dilute solutions and then extrapolated to zero concentration; Determined by studying the microsphere aggregation kinetics under different shear conditions; Directly converted from the rotational speed of the mixing equipment, where is the equipment shear coefficient; Measured by a Zeta potentiometer; Can be calculated according to the ionic strength, where is Avogadro's constant, is the elementary charge, is the ionic strength.

[0104] The optimization function is constructed based on rheology and colloid stability theory. The equation for relative viscosity uses the Krieger-Dougherty model, which describes the non-linear relationship between the viscosity of the suspension system and the particle concentration. The introduction of the intrinsic viscosity in the exponent is to consider the influence of particle shape and surface properties on rheological behavior. The aggregation probability function refers to the Smoluchowski coagulation kinetics and the DLVO theory. The energy barrier term in the exponent indicates the influence of temperature and surface charge on the stability of microspheres. The determination of the optimal mixing parameters adopts the principle of minimizing the aggregation probability. This method can automatically adjust the mixing conditions in actual production, avoid the fragmentation of microspheres under high shear or the aggregation under low shear, and improve the uniformity and stability of the injection filler.

[0105] Specifically, the principle of the present invention is: The core principle of the method of the present invention is based on the theory of enhanced phase separation and structure formation in a high-gravity field, combined with a multi-modal deep learning prediction model to achieve precise control of process parameters. First, its working principle stems from the stable shear environment provided by the high-gravity field. Compared with conventional stirring, the high-gravity equipment can generate a centrifugal force field of 80-300 times the gravitational acceleration, forming a uniform shear force distribution throughout the reaction area, eliminating the dead zones and local high-shear zones existing in traditional stirring equipment, and making the emulsification process tend to be uniform in space. Under this uniform shear environment, the microdroplet formation kinetics strictly follows the shear energy balance equation, the time for the microdroplet size to reach the equilibrium state is greatly shortened, and the batch-to-batch difference is significantly reduced.

[0106] Secondly, the process of organic solvent volatilization is a key factor affecting the stability of microsphere formation. The present invention uses Fick's diffusion equation to accurately describe the diffusion mass transfer process of the solvent from the polymer phase to the aqueous phase. By controlling parameters such as the environmental temperature and stirring rate, precise regulation of the solvent volatilization rate is achieved, avoiding differences in the internal structure of microspheres caused by uneven volatilization rates. At the same time, the phase separation kinetics model guides the phase change behavior of the polymer during the solvent volatilization process, ensuring that different batches of microspheres maintain a similar internal structure evolution path during the formation process.

[0107] Third, the composite mechanism of amphiphilic polylactic acid materials and inorganic particles is based on the balance of interfacial forces. In the environment of a high-gravity field, the distribution of interfacial forces is more uniform. The hydrophilic groups in the amphiphilic polymer molecules form stable interfacial interactions with the surface of inorganic particles, and the lipophilic groups maintain their affinity with the organic phase, realizing the uniform and stable coating of inorganic particles. This precisely controlled interfacial interaction ensures the consistency of the inorganic component distribution in different batches of composite microspheres.

[0108] Finally, the biological effect prediction model of polylactic acid microspheres is based on a multi-modal deep learning network architecture. By integrating the physicochemical properties, surface morphology characteristics, and component ratio information of the microspheres, it can monitor in real time the impact of fluctuations in the preparation process parameters on the performance of the final product and adjust the parameters to maintain the stability of the product quality. This model is trained with a large amount of historical data and can predict the performance fluctuations caused by small changes in process parameters, providing closed-loop feedback control for the preparation process and fundamentally ensuring the preparation stability.

[0109] A specific Example 1 of the present invention is provided below, and the specific implementation manners of each step in this Example 1 are described in detail as follows.

[0110] The specific implementation manner of step S01 is to select a suitable polymer material and prepare an oil-phase solution. First, weigh the copolymer of polylactic acid and glycolic acid or the block copolymer of poly-L-lactic acid and polyethylene glycol, and accurately measure the required mass. Subsequently, add the polymer to the pre-prepared dichloromethane or ethyl acetate and stir until it is dissolved to a transparent state. The concentration of the oil-phase solution is controlled within the range of 20 to 200 milligrams per milliliter. A lower concentration is beneficial for forming microspheres with small particle sizes, while a higher concentration increases the mechanical strength of the microspheres. At the same time, add zinc oxide or magnesium oxide nano-scale inorganic particles to the oil-phase solution, and the mass ratio of the inorganic particles to the polymer is controlled within the range of 1:200 to 1:5. The purpose of introducing the inorganic particles is to enhance the mechanical properties of the microspheres and regulate the degradation rate, while promoting the synthesis of the extracellular matrix. In this process, the polymer solubility parameter theory is used to predict the dissolution behavior of the polymer in organic solvents to ensure the formation of a stable and uniform oil-phase solution. The polymer solubility parameter can be calculated by the group contribution method: , where is the polymer solubility parameter, with the unit of ; is the molar attraction constant of the i-th group in the molecular structure, with the unit of ; is the molar volume of the i-th group in the molecular structure, with the unit of . The solubility parameter of polylactic acid is generally 19 - 21 , the solubility parameter of dichloromethane is 20.2 , and the solubility parameter of ethyl acetate is 18.1 , when the difference in solubility parameters between the polymer and the solvent is less than 2 , the polymer is easily soluble.

[0111] The specific implementation of step S02 is to prepare an aqueous solution. First, prepare water for injection and filter and sterilize it through a 0.22-micron filter membrane. Then weigh polyvinyl alcohol or Tween 80 surfactant and add it to the water for injection. When preparing polyvinyl alcohol, it needs to be heated to 80 °C and continuously stirred, while Tween 80 can be directly dissolved at room temperature. The surfactant concentration is controlled within the range of 5 to 100 mg / mL, and the ratio of the aqueous solution to the oil phase solution is controlled between 1:1 and 20:1. The selection of the surfactant is based on its hydrophilic-lipophilic balance value. The hydrophilic-lipophilic balance value of polyvinyl alcohol is 18, which is suitable for preparing microspheres with higher hydrophilicity; the hydrophilic-lipophilic balance value of Tween 80 is 15, which is suitable for preparing microspheres with medium hydrophilicity. During the preparation of the aqueous solution, attention should be paid to the theory of the critical micelle concentration of the surfactant to ensure that the surfactant concentration is higher than the critical micelle concentration to provide sufficient interfacial stabilization. The critical micelle concentration can be determined by the surface tension method: , where is the surface tension, with the unit of ; is the surface tension of pure water, with the unit of ; is the gas constant, 8.314 ; is the absolute temperature, with the unit of ; is the maximum surface adsorption amount, with the unit of ; is the surfactant concentration, with the unit of ; is a constant, dimensionless. The critical micelle concentration is the concentration value corresponding to the inflection point of the surface tension-concentration curve. The critical micelle concentration of polyvinyl alcohol is about 0.03 - 0.05 , and the critical micelle concentration of Tween 80 is about 0.01 - 0.02 .

[0112] The specific implementation of step S03 is to carry out shear emulsification using a high-gravity device. First, start the high-gravity device and adjust the rotation speed to 300 to 3000 revolutions per minute to generate a centrifugal force field with a gravitational acceleration of 80 to 300 times. Inject the oil phase solution and the aqueous solution into the high-gravity device simultaneously at a constant flow rate through a precision metering pump. Under the action of strong shear force, the oil phase is dispersed into tiny droplets and wraps the inorganic particles to form an oil-in-water-in-solid emulsion. During the emulsification process, the formation of emulsion droplets follows the shear energy balance equation, which describes the relationship between the maximum stable droplet diameter at the equilibrium state and the interfacial tension, continuous phase density, dispersed phase viscosity, continuous phase viscosity, angular velocity, and characteristic radius of the high-gravity device. The shear energy balance equation is specifically expressed as: , where is the maximum stable droplet diameter at equilibrium, in meters; is the oil-water interfacial tension, in Newtons per meter; is the density of the continuous phase (aqueous phase), in kilograms per cubic meter; is the angular velocity of the high-gravity equipment, in radians per second; is the characteristic radius of the high-gravity equipment, in meters; is the viscosity of the dispersed phase (oil phase), in Pascal-seconds; is the viscosity of the continuous phase (aqueous phase), in Pascal-seconds; is a constant related to the geometric structure of the equipment, dimensionless, generally in the range of 0.5 to 2.0. The microscopic turbulent pulsation in the high-gravity field enhances the interfacial renewal rate and promotes the formation of a microsphere precursor emulsion with a uniform particle size distribution. In actual operation, when the ratio of the aqueous phase flow rate to the oil phase flow rate is 5:1 to 15:1, a microsphere with a narrow particle size distribution can be obtained.

[0113] The specific implementation of step S04 is to collect the microsphere suspension emulsion and promote the volatilization of the organic solvent. First, a collection container is set at the bottom outlet of the high-gravity equipment to collect the outflowing microsphere suspension emulsion. The collected emulsion is transferred to a glass reaction kettle with a mechanical stirring device, and the stirring speed is set to 200 to 1000 revolutions per minute to avoid microsphere aggregation caused by too high a rotation speed. Stir continuously for 6 to 12 hours at room temperature to promote the diffusion of the organic solvent in the microspheres into the aqueous phase and finally volatilize. The solvent volatilization process follows Fick's diffusion equation, which describes the relationship between the solvent concentration and time and space. Fick's diffusion equation in spherical coordinates is expressed as: , where is the solvent concentration, in grams per cubic meter; is the time, in seconds; is the radial distance of the microsphere, in meters; is the diffusion coefficient of the solvent in the polymer, in square meters per second. The diffusion coefficient is related to the temperature and polymer concentration: , where is the reference diffusion coefficient, in square meters per second, generally in the range of ; is the diffusion activation energy, in joules per mole, generally 15 to 25 for the polylactic acid system ; is the gas constant, 8.314 joules / (mole·Kelvin); is the absolute temperature, in Kelvin; is a coefficient, dimensionless, generally 1.0 to 3.0; is the polymer volume fraction, dimensionless. During the solvent evaporation process, the microspheres gradually harden and form a porous structure. The pore size is closely related to the solvent evaporation rate. The faster the evaporation rate, the larger the formed pores but the more uneven the distribution. The time required for solvent diffusion can be estimated as: , where is the time required for solvent diffusion, in seconds; is the radius of the microsphere, in meters.

[0114] The specific implementation of step S05 is to centrifuge and wash the microsphere suspension. First, transfer the microsphere suspension to a centrifuge tube and centrifuge at a speed of 3000 to 5000 revolutions per minute for 5 to 10 minutes. The precipitate formed after centrifugation is the microsphere, and the supernatant contains residual surfactant and organic solvent. Carefully aspirate the supernatant and discard it, then add fresh injection water to the precipitate and resuspend the microspheres using a vortex mixer. Repeat the above centrifugation and washing process 3 to 5 times until the concentration of residual organic solvent in the supernatant is lower than 10 micrograms per milliliter and the concentration of residual surfactant is lower than 5 micrograms per milliliter. The Stokes sedimentation theory is used to calculate the optimal centrifugation time during the washing process: , where is the centrifugation time, in seconds; is the viscosity of the continuous phase, in Pascal·seconds; is the inner diameter of the centrifuge rotor, in meters; is the outer diameter of the centrifuge rotor, in meters; is the angular velocity, in radians / second; is the density of the microsphere, in kilograms per cubic meter; is the density of the continuous phase, in kilograms per cubic meter; is the diameter of the microsphere, in meters. For polylactic acid microspheres with a particle size of 10 to 50 microns, under the condition of centrifugation at 3000 revolutions per minute, the optimal centrifugation time is 5 to 8 minutes. The number of washing times is determined based on the solute dilution theory, and the impurity concentration in the supernatant is reduced by about 90% after each washing. The concentration of residual solvent can be detected by gas chromatography, and the concentration of residual surfactant can be detected by ultraviolet spectrophotometry.

[0115] The specific implementation of step S06 is freeze-drying treatment. First, transfer the washed microsphere suspension to a freeze-drying bottle, and place it in an environment of -40 to -60 °C for pre-freezing. The pre-freezing time is not less than 4 hours to ensure that the water is completely frozen into ice crystals. Then connect the freeze-drying bottle to a freeze-dryer, set the vacuum degree to 10 to 50 Pa, and the condenser temperature to -80 °C. The freeze-drying is divided into three stages: the primary drying stage maintains the temperature at -40 °C and the pressure at 20 Pa for 12 hours to mainly remove free water; the secondary drying stage gradually raises the temperature to -20 °C and reduces the pressure to 15 Pa for 8 hours to remove some bound water; the tertiary drying stage raises the temperature to 0 °C and further reduces the pressure to 10 Pa for 4 hours to remove the remaining bound water. The porosity of the microspheres during the freeze-drying process is determined by the sublimation rate of the ice crystals, and this process follows the coupled heat and mass transfer model: , where is the sublimation rate, with the unit of kg / s; is the sublimation interface area, with the unit of m²; is the diffusion coefficient of water vapor in the porous layer, with the unit of m² / s; is the molar mass of water, 0.018 kg / mol; is the water vapor pressure at the ice interface, with the unit of Pa; is the water vapor pressure on the condenser surface, with the unit of Pa; is the gas constant, 8.314 J / (mol·K); is the absolute temperature, with the unit of K; is the thickness of the porous layer, with the unit of m. The faster the sublimation rate of the ice crystals, the higher the porosity of the formed microspheres, but the mechanical strength is relatively low. After drying, the microspheres are in a loose powder form. Avoid contact with indoor air and immediately seal and store them.

[0116] The specific implementation of step S07 is to measure the particle size distribution of the microspheres. First, take an appropriate amount of the dried microsphere sample and disperse it evenly in a 0.1% polysorbate 80 solution. Use a laser particle size analyzer for measurement. The laser wavelength is 633 nm, and the detection angle range is 0.02 to 165 degrees. Each sample is measured 5 times, and the average value is taken to calculate the 10th percentile value, 50th percentile value, and 90th percentile value of the microspheres, which are denoted as , , . The uniformity of the microsphere particle size is evaluated by the Span value, and the calculation formula is . A Span value less than 1 indicates a uniform particle size distribution. At the same time, a phase separation kinetic model is used for theoretical particle size prediction. This model describes the polymer phase separation process based on the Cahn-Hilliard equation: , where is the polymer volume fraction, dimensionless; is time, with the unit of second; is the mobility, with the unit of square meter / (joule·second); is the free energy functional, with the unit of joule / cubic meter; is the gradient energy coefficient, with the unit of joule / meter. The free energy functional is expressed based on the Flory-Huggins theory as: , where is the molar volume of solvent molecules, with the unit of cubic meter / mole; is the Flory-Huggins interaction parameter, dimensionless. Considering the influence of the rheological properties of polymers on phase separation, a viscoelastic correction term is introduced: , where is the coupling coefficient, dimensionless; is the polymer corresponding stress tensor, with the unit of pascal. The final particle size distribution of the microspheres can be described as a log-normal distribution: , where is the particle size distribution probability density function; is the diameter of the microspheres, with the unit of meter; is the geometric mean diameter of the microspheres, with the unit of meter; is the distribution standard deviation, dimensionless. The deviation between the theoretical prediction and the measured value should be controlled within 10%, otherwise the preparation parameters need to be readjusted.

[0117] The specific implementation of step S08 is to prepare an injectable filler. First, take a certain amount of dried composite microspheres and add them to the cross-linked sodium hyaluronate gel. The mass ratio of the microspheres to the gel is controlled within the range of 1:5 to 1:20. The sodium hyaluronate gel is pre-treated with a cross-linking agent such as 1,4-butanedial or divinyldisiloxane or polyethylene glycol diglycidyl ether, and the cross-linking degree is 1% to 5%. The mixing process uses a twin-screw extruder or a high-shear homogenizer, with a rotation speed of 500 to 2000 revolutions per minute and a mixing time of 5 to 30 minutes. The mixing parameters are optimized based on the microsphere dispersion optimization function, which regards the microspheres as suspended particles and establishes the relationship between relative viscosity, microsphere volume fraction, maximum packing fraction, and intrinsic viscosity: , where is the relative viscosity, dimensionless; is the microsphere volume fraction, dimensionless; is the maximum packing fraction, dimensionless, generally 0.6 - 0.7; is the intrinsic viscosity, with the unit of cubic centimeter / gram. The microsphere aggregation probability function can be expressed as: , where is the microsphere aggregation probability, dimensionless; is the frequency factor, with the unit of second ; is the shear rate, with the unit of second ; is the mixing time, with the unit of second; is the interaction energy barrier between microspheres, with the unit of joule; is the Boltzmann constant, joule / Kelvin. The equation for determining the optimal mixing parameters: , where is the optimal shear rate, with the unit of second ; is the optimal mixing time, with the unit of second. The filled agent after mixing is sterilized by filtration through a 0.45-μm filter membrane and filled into a sterile syringe. After sealing, it is sterilized by γ-irradiation, and the irradiation dose is 15 to 25 kGy to ensure sterility without affecting the microsphere structure.

[0118] The specific implementation of step S09 is to conduct biocompatibility evaluation and collagen regeneration efficiency test. First, an in vitro cell proliferation experiment is carried out. An appropriate amount of the injectable filler is co-cultured with fibroblasts for 72 hours. The cell viability is measured by the thiazolyl blue colorimetric method, and the cytokine secretion level is detected by the enzyme-linked immunosorbent assay. Then, an animal subcutaneous injection experiment is conducted. Rabbits or mice are selected as the experimental subjects. The histological changes are observed 1, 7, 14, and 28 days after subcutaneous injection of the filler. Hematoxylin-eosin staining is used to evaluate the inflammatory response, and Sirius red staining is used to measure the collagen content. The experimental results are analyzed by the biological effect prediction model of poly(lactic acid) microspheres. The structure of this model is a multi-modal deep learning network, which includes three parallel feature extraction branches and a fusion prediction module. The evaluation indexes predicted by the model include the fitting result of the cell growth curve: , where is the cell number at time t; is the initial cell number; is the cell growth rate constant, with the unit of hour ; is the maximum cell capacity. The evaluation of the collagen content uses a quantitative index: , where is the collagen growth index, dimensionless; is the collagen content in the sample group, with the unit of μg / mg tissue; is the collagen content in the control group, with the unit of μg / mg tissue. If the biocompatibility evaluation shows that the cell viability is lower than 80%, the inorganic particle content is adjusted; if the collagen regeneration efficiency is less than 40% of the control group, the molecular weight of poly(lactic acid) is increased or the polymer component ratio is adjusted. When both the biocompatibility and the collagen regeneration efficiency reach the preset thresholds, the preparation of the composite microsphere injectable filler is completed.

[0119] To better understand and implement the present invention, Example 2 of a specific application scenario of the present invention is provided below: Researchers prepared microspheres composed of a poly (L-lactic acid)-poly (ethylene glycol) block copolymer and zinc oxide by a high-gravity emulsification process for use as a medical aesthetic injection filler. An amphiphilic poly(lactic acid) organic material and inorganic particles were emulsified using a surfactant and high-gravity equipment to form an S / O / W-coated microsphere suspension emulsion, which was then separated and freeze-dried to obtain the organic-inorganic composite microspheres.

[0120] The specific preparation method is as follows: First, prepare the aqueous solution. Add 1.8 g of polyvinyl alcohol to 180 ml of deionized water, heat and stir at 80 °C for 1 h, and then naturally cool to room temperature. Then prepare the oil-phase solution. Add 0.72 g of PEG-PLLA with a molecular weight of 180,000 and 0.018 g of zinc oxide particles with a particle size of 15 nm to 36 ml of dichloromethane and dissolve them thoroughly. Set the rotation speed of the high-gravity equipment to 1200 r / min through a frequency modulation instrument, and pump the aqueous and oil-phase solutions into the liquid inlet respectively. Set the aqueous-phase feeding speed to 1500 ml / min and the oil-phase feeding speed to 300 ml / min. Turn on the motor and the pump to achieve the feeding of the aqueous and oil-phase solutions and high-gravity emulsification, and collect the S / O / W emulsion from the liquid outlet at the bottom of the high-gravity equipment.

[0121] At room temperature, mechanically stir the emulsion after high-gravity emulsification at a rotation speed of 450 r / min for 8 h to remove the dichloromethane solvent. Centrifuge and wash the microsphere suspension after solvent removal and curing. The centrifugation speed is 5000 r / min, and 40 ml of deionized water is used for each washing. Centrifuge and wash 4 times in total, and finally perform freeze-drying treatment to obtain the PEG-PLLA@ZnO composite microspheres.

[0122] The measurement results of the particle size distribution of the prepared PEG-PLLA@ZnO composite microspheres are shown in Table 1:

[0123] Table 1 Particle size distribution of PEG-PLLA@ZnO composite microspheres

[0124]

[0125] Mix the prepared PEG-PLLA@ZnO composite microspheres and crosslinked sodium hyaluronate gel at a mass ratio of 1:15, and use them as a medical aesthetic injection filler after sterilization. Conduct in vitro cell experiments and mouse subcutaneous injection experiments to evaluate their biological properties. The results show that the composite microsphere filler has good biocompatibility. In the in vitro cell proliferation experiment, the cell survival rate reaches 95.3%, which is higher than 87.2% of the pure sodium hyaluronate gel. Sixteen weeks after subcutaneous injection in mice, the collagen content at the injection site increased by 68.7%, which is significantly higher than 23.4% of the simple sodium hyaluronate injection group and 42.1% of the PEG-PLLA microsphere group without zinc oxide.

[0126] Through the analysis of the expression of genes related to collagen biosynthesis, the expression level of COL1A1 in the PEG-PLLA@ZnO / HA group was up-regulated by 3.6 times compared with the control group and 1.8 times compared with the PEG-PLLA / HA group. At the same time, the expression of inflammation-related genes was reduced by 42% compared with the PEG-PLLA / HA group, and the pH value was maintained within the range of 7.2 - 7.4, indicating that the addition of zinc oxide effectively neutralized the acidic substances generated during the degradation of polylactic acid and alleviated the inflammatory response.

[0127] A specific Example 3 is provided below: Researchers used the high gravity emulsification technology to develop a microsphere composed of PLLA and magnesium oxide for medical injection fillers. This Example 3 explored the effect of the rotation speed of the high gravity equipment on the morphology and particle size distribution of the microspheres.

[0128] The specific preparation process is as follows: Prepare the aqueous solution. Add 1.5 g of polyvinyl alcohol to 150 ml of deionized water, heat and stir at 80 °C for 1 h, then cool to room temperature naturally, and add 25 ml of glycerol solution and stir evenly. The preparation of the oil phase solution is to dissolve 0.06 g of magnesium oxide particles with a particle size of 40 nm and 0.6 g of PLLA with a molecular weight of 120,000 in 15 ml of dichloromethane.

[0129] Set the rotation speeds of the high gravity equipment to 800 r / min, 1000 r / min, and 1200 r / min respectively for comparative experiments. The feeding speed of the aqueous solution is uniformly set to 1000 ml / min, and the feeding speed of the oil phase solution is set to 100 ml / min. Turn on the motor and pump to realize the feeding of the water and oil phase solutions and high gravity emulsification, and collect the primary emulsion from the liquid outlet at the bottom of the high gravity equipment. Stir the primary emulsion at room temperature and 400 r / min for 12 h to remove the dichloromethane solvent. Then, centrifuge and wash the microsphere suspension. The centrifugation speed is 5000 r / min, and 30 ml of deionized water is used for each washing. Centrifuge and wash 3 times in total. Finally, freeze-dry to obtain the PLLA@MgO composite microspheres.

[0130] The particle size distributions of the PLLA@MgO composite microspheres prepared under different rotation speed conditions are shown in Table 2:

[0131] Table 2 Particle size distributions of PLLA@MgO composite microspheres under different high gravity equipment rotation speeds

[0132]

[0133] As can be seen from Table 2, with the increase of the rotation speed of the high gravity equipment, the particle size of the prepared PLLA@MgO composite microspheres gradually decreases, and the particle size distribution becomes more concentrated. This is because the increase in rotation speed leads to an increase in shear force, causing the oil phase to be dispersed into smaller droplets. From the shear energy balance equation It can be seen that the angular velocity is inversely proportional to the maximum stable droplet diameter , which is consistent with the experimental results.

[0134] The PLLA@MgO composite microspheres prepared under the condition of a rotational speed of 1000 r / min were selected and mixed with cross-linked sodium hyaluronate gel at a mass ratio of 1:10 to prepare an injection filler. A biological performance evaluation experiment was carried out under the same conditions as in Example 2. The results showed that in the in vitro cell proliferation experiment of the PLLA@MgO / HA group, the cell activity increased by 27.8% compared with the control group. In the subcutaneous injection experiment in mice, the collagen content increased by 54.3% after 8 weeks of injection and 71.2% after 16 weeks. The pH value detection showed that the pH value of the PLLA@MgO / HA group was maintained within the range of 7.3 - 7.6, which was significantly higher than that of the PLLA / HA group (6.2 - 6.8), indicating that magnesium oxide effectively neutralized the acidic substances produced by the degradation of polylactic acid.

[0135] The following provides a specific Example 4: Researchers studied the effects of different inorganic particles on the properties of polylactic acid-based microspheres and designed a preparation method for a composite microsphere of combined inorganic particles. PLGA was used as the organic material, and multiple inorganic particles were compounded to improve the biological properties of the microspheres.

[0136] The specific preparation method is as follows: Weigh polyvinyl alcohol at a concentration of 40 mg / ml and add it to deionized water. Heat and stir at 80 °C for 1 h and then naturally cool to room temperature to obtain an aqueous solution. Weigh PLGA with a molecular weight of 150,000 and LA / GA = 80:20 at a concentration of 12 mg / ml, and add different proportions of inorganic particles to prepare three oil-phase solutions: ① Add zinc oxide particles at a concentration of 1.5 mg / ml; ② Add magnesium oxide particles at a concentration of 1.5 mg / ml; ③ Add a mixture of zinc oxide / magnesium oxide / magnesium carbonate with a total concentration of 1.5 mg / ml and a mass ratio of 2:2:1. All oil phases use ethyl acetate as the solvent, and the volume ratio of water to oil is 5:1.

[0137] Adjust the temperature of the jacket circulating water of the high-gravity equipment to 8 °C, set the rotational speed of the high-gravity equipment to 2000 r / min, and pump the water and oil phase solutions into the liquid inlet respectively. The water phase feeding speed is 500 ml / min, and the oil phase feeding speed is 100 ml / min. Turn on the motor and the pump to realize the feeding of the water and oil phase solutions and high-gravity emulsification, and collect the S / O / W emulsion from the liquid outlet at the bottom of the high-gravity equipment. Stir the high-gravity emulsion mechanically at 600 r / min at room temperature for 6 h to remove the ethyl acetate solvent. Centrifuge and wash the microsphere suspension after solvent removal and curing. The centrifugation speed is 5000 r / min, 40 ml of deionized water is used for each washing, and the centrifugation and washing are carried out 4 times, and then freeze-drying treatment is performed to obtain three kinds of PLGA-based composite microspheres.

[0138] The particle size distributions of three kinds of PLGA-based composite microspheres are shown in Table 3:

[0139] Table 3 Particle size distributions of PLGA-based composite microspheres with different inorganic particles

[0140]

[0141] Three kinds of microspheres were mixed with crosslinked sodium hyaluronate gel at a mass ratio of 1:12 to prepare an injectable filler for biological performance evaluation. In in vitro cell experiments, the cell survival rate of the PLGA@ZnO / MgO / MgCO3 / HA group was the highest, reaching 97.1%, while those of the PLGA@ZnO / HA and PLGA@MgO / HA groups were 94.3% and 95.2% respectively. The test of inflammatory-related gene expression showed that the expression level of inflammatory factors in the PLGA@ZnO / MgO / MgCO3 / HA group was the lowest, which was 58.6% lower than that of the control group. This is due to the synergistic effect of multiple inorganic particles, which can not only neutralize the acidic substances produced by the degradation of polylactic acid, but also slowly release metal ions to regulate the cell microenvironment.

[0142] After 16 weeks of subcutaneous injection in mice, the collagen content at the injection sites of the three fillers increased significantly. Among them, the increase in the PLGA@ZnO / MgO / MgCO3 / HA group was the most obvious, reaching 82.5%, while those of the PLGA@ZnO / HA and PLGA@MgO / HA groups were 73.6% and 76.2% respectively. The detection of the expression level of the collagen biosynthesis-related gene COL1A1 showed that the expression level in the PLGA@ZnO / MgO / MgCO3 / HA group was up-regulated by 4.2 times compared with the control group, which was significantly higher than the other two groups.

[0143] Conventionally, the preparation of polylactic acid-based microspheres usually adopts the mechanical stirring emulsification method or the membrane emulsification method. The microspheres prepared by the former have a wide particle size distribution, and the Span value is usually in the range of 1.5 - 3.0, and screening treatment is required to be used for medical injection; although the latter has a narrow particle size distribution, the equipment is complex and the production efficiency is low. The present invention adopts the high gravity emulsification technology, and uses the strong shear force generated by the high gravity field to disperse the oil phase into tiny droplets. The prepared microspheres have a narrow particle size distribution (the Span value is generally less than 1.2), and can be directly used for medical injection without screening, greatly improving the production efficiency and reducing the production cost.

[0144] In addition, traditional polylactic acid-based fillers are prone to produce a local acidic environment during the in vivo degradation process, inducing inflammatory reactions and resulting in low patient satisfaction. In the present invention, inorganic particles such as zinc oxide, magnesium oxide, and magnesium carbonate are introduced into the polylactic acid-based microspheres, effectively neutralizing the acidic substances generated by the degradation of polylactic acid, significantly reducing the inflammatory reaction. At the same time, these metal ions can promote collagen synthesis and enhance the filling effect. Especially when multiple inorganic particles are used in combination, a synergistic effect is exhibited, greatly improving the biocompatibility and effect of the filler. Experimental results show that the composite microsphere filler containing inorganic particles is superior to traditional products in terms of biocompatibility and collagen regeneration ability, providing a new direction for the development of medical aesthetic injection fillers.

[0145] Figure 2 Figure 4 shows the comparison results of the percentage increase in collagen content and cell viability of different composite microsphere fillers in Examples 2-4. The chart shows the percentage increase in collagen content (blue bars) in the form of a bar chart, and at the same time shows the cell viability (red line) in the form of a line chart. It can be clearly seen from the figure that with the introduction and optimization of inorganic particles in the composite microspheres, the increase in collagen content shows an obvious upward trend. The increase in collagen in the group injected with pure sodium hyaluronate was the lowest (23.4%), while the effect of the PLGA@ZnO / MgO / MgCO3 / HA group (Example 4) was the best (82.5%). At the same time, the cell viability data shows that the composite microsphere filler containing inorganic particles has better biocompatibility, and the cell viability of all examples is higher than that of the pure sodium hyaluronate group. Among them, the cell viability of the PLGA@ZnO / MgO / MgCO3 / HA group was the highest, reaching 97.1%. This chart intuitively demonstrates the superior performance of different examples in the present invention, especially the synergistic effect exhibited when multiple inorganic particles are combined.

[0146] To better understand and implement the present invention, Example 5 of a specific application scenario of the present invention is provided below: Researchers prepared a composite microsphere injection filler using poly(L-lactic acid)-poly(ethylene glycol) block copolymer and zinc oxide nanoparticles. First, 8.0 grams of poly(L-lactic acid)-poly(ethylene glycol) block copolymer (PLLA-PEG, PLLA molecular weight 180,000, PEG molecular weight 2,000, PLLA:PEG mass ratio 85:15) was accurately weighed and dissolved in 80 milliliters of ethyl acetate to prepare an oil-phase solution with a concentration of 100 milligrams per milliliter. The solution was stirred at 25 °C for 4 hours until completely transparent. Subsequently, 0.4 grams of zinc oxide nanoparticles (average particle size 52 nanometers) was added to the oil-phase solution, such that the mass ratio of zinc oxide nanoparticles to the polymer was 1:20, and ultrasonic dispersion was carried out for 30 minutes to ensure that the inorganic particles were uniformly dispersed in the oil phase.

[0147] The aqueous solution was prepared by weighing 12 g of polyvinyl alcohol (molecular weight 23,000, degree of hydrolysis 88%) and dissolving it in 400 mL of water for injection. It was stirred in a water bath at 80 °C for 2 hours until completely dissolved. After cooling to room temperature, an aqueous solution with a concentration of 30 mg / mL was obtained. The volume ratio of the aqueous phase to the oil phase was controlled at 5:1.

[0148] Emulsification was carried out using a rotating disk high gravity equipment. The rotation speed was set at 1500 revolutions per minute, which was equivalent to about 150 times the gravitational acceleration. The oil phase solution was injected into the high gravity equipment at a flow rate of 12 mL / min, and the aqueous phase solution was injected at a flow rate of 60 mL / min through a metering pump. An oil-in-water-in-solid emulsion was formed under the action of shear force, and the microsphere suspension emulsion flowing out from the bottom outlet of the equipment was collected. The collected emulsion was transferred to a 500 mL reaction kettle and mechanically stirred at a rotation speed of 500 revolutions per minute for 8 hours to promote the volatilization of the organic solvent.

[0149] The microsphere suspension was centrifuged and washed at 4000 revolutions per minute for 8 minutes. After discarding the supernatant, 200 mL of fresh water for injection was added to resuspend. The centrifugation and washing were repeated 4 times until the residual concentration of ethyl acetate in the supernatant was lower than 8 μg / mL (detected by gas chromatography) and the residual concentration of polyvinyl alcohol was lower than 3 μg / mL (detected by ultraviolet spectrophotometry).

[0150] The washed microsphere suspension was transferred to a freeze-drying bottle and pre-frozen at °C for 6 hours, then connected to a freeze-dryer. The temperature of the condenser was set at °C, and the vacuum degree was 15 Pa. Three-stage drying was carried out according to the program: primary drying at °C for 12 hours; secondary drying at °C for 8 hours; tertiary drying at 0 °C for 6 hours. After drying, 6.8 g of white porous powdery composite microspheres were obtained, and the yield was 85%.

[0151] The particle size distribution of the microspheres was measured using a laser particle size analyzer, and the results are shown in Table 4:

[0152] Table 4 Particle size distribution parameters of composite microspheres

[0153]

[0154] The Span value of the particle size uniformity of the microspheres was calculated to be 0.97, indicating that the particle size distribution was relatively uniform. The morphology of the microspheres was observed by scanning electron microscopy, and it was found that the microspheres were spherical, with slightly porous surfaces, and zinc oxide nanoparticles were uniformly distributed inside the microspheres. By thermal analysis, the glass transition temperature of the composite microspheres was 56 °C, the melting point was 172 °C, and the thermal degradation temperature was 235 °C. The thermal analysis results are shown in Table 5:

[0155] Table 5 Thermal analysis results of composite microspheres

[0156]

[0157] Mix 5 g of the dried composite microspheres with 75 g of sodium hyaluronate cross-linked gel (molecular weight 2.0× , cross-linking degree 3%, gel viscosity 3500 mPa·s, cross-linked by 1,4-butanedial) at a mass ratio of 1:15. The physical and chemical properties of the sodium hyaluronate gel are shown in Table 6:

[0158] Table 6 Physical and Chemical Properties of Cross-linked Sodium Hyaluronate Gel

[0159]

[0160] Mix using a twin-screw extruder at a rotational speed of 1200 revolutions per minute for 15 minutes. According to the calculation of the microsphere dispersion optimization function, the optimal mixing parameters are a shear rate of 1050 , mixing time of 14.8 minutes, and a theoretical microsphere agglomeration probability of 0.032. The mixed filler is filtered through a 0.45-μm filter membrane and filled into a 2-ml sterile syringe, and sterilized by γ-irradiation at a dose of 20 kGy.

[0161] Evaluate the biocompatibility of the prepared injectable filler. After co-culturing human skin fibroblasts (HSF) with the filler for 72 hours, the cell survival rate is determined to be 92.5% by the thiazolyl blue colorimetric method, indicating that the filler has good biocompatibility. The cytokine detection results are shown in Table 7:

[0162] Table 7 Cytokine Levels in the Culture Supernatant of Fibroblasts

[0163]

[0164] Observe histological changes after subcutaneous injection of the filler in rabbits, and detect the collagen content by Sirius red staining. The changes in collagen content at different time points after injection are shown in Table 8:

[0165] Table 8 Changes in Collagen Content at Different Time Points after Injection

[0166]

[0167] Research data show that 28 days after injection, the collagen content increases by 63.8% compared with the control group, far higher than the preset threshold of 40%, indicating that the composite microsphere injectable filler has good collagen regeneration effect. Histological analysis did not observe obvious inflammatory reactions, and cell proliferation was active in the injection area, and new blood vessel formation was good. Figure 3It shows the change trend of collagen content at different time points (1 day, 7 days, 14 days, and 28 days) after injecting the filler in Example 5. The chart shows the growth of collagen content over time through scatter points and fitted curves, and marks the growth rate percentage relative to the control group at each time point. It can be seen from the figure that the collagen content increases significantly with the extension of the injection time, showing a non-linear growth trend. At the beginning (1 day after injection), the collagen content is 45.2 μg / mg of tissue, and the growth rate is only 5.4%; by 28 days after injection, the collagen content reaches 92.1 μg / mg of tissue, and the growth rate is as high as 63.8%. The red dashed line in the figure marks the critical collagen level (65 μg / mg), and it can be seen that this level is reached approximately 8-9 days after injection, indicating that this filler has the effect of rapidly promoting collagen regeneration.

[0168] The experimental data was analyzed using the biological effect prediction model of polylactic acid microspheres. It is predicted that the degradation period of the filler in vivo is 10-12 months, and the duration of collagen regeneration is 12-16 months. The volume retention rate curve after injection is shown in Table 9:

[0169] Table 9 Prediction of the volume retention rate of the injection filler

[0170]

[0171] Figure 4Shows the prediction model for the volume retention rate of the injectable filler developed in Example 5. The graph shows the volume retention of the filler in the body for up to 18 months through scatter points and an exponential fitting curve, and is compared with traditional fillers (blue dashed line). The time axis in the graph is divided into three stages: the initial filling period (0 - 6 months, green area), the maintenance period (6 - 12 months, yellow area), and the degradation period (12 - 20 months, red area). It can be seen from the data that the filler of the present invention still maintains 95.2% of its volume 1 month after injection, 72.3% after 6 months, and still has a volume retention rate of 42.8% after 12 months, significantly better than the performance of traditional fillers at the same time points (92.1%, 60.2%, and 25.8% respectively). The graph marks the half-life of the filler to be approximately 10.5 months, at which time the volume retention rate of the filler is 50%. This graph intuitively demonstrates the long-term performance advantages of the filler of the present invention and provides an important prediction basis for clinical applications. The preparation methods of traditional medical aesthetic injectable fillers mainly use conventional emulsification-solvent evaporation methods, such as stirring methods, homogenization methods, etc., which have the advantages of simple processes and low equipment requirements, but have disadvantages such as a wide microsphere particle size distribution (the Span value is usually between 1.5 and 3.0), low preparation efficiency, and poor batch-to-batch consistency. In addition, the polylactic acid microspheres prepared by traditional methods often cause strong inflammatory reactions after injection, have low collagen regeneration efficiency (usually less than 30% of the control group), and poor volume retention stability, affecting the durability and naturalness of the aesthetic effect.

[0172] Compared with traditional methods, the present invention uses a high-gravity device for shear emulsification, significantly improving the uniformity of microsphere particle size (the Span value is reduced to less than 1.0) and increasing the preparation efficiency (the yield is increased by 15% - 25%). By introducing inorganic particles to form organic-inorganic composite microspheres, the degradation rate of microspheres is effectively regulated, the inflammatory reaction is reduced, and the collagen regeneration efficiency is increased (reaching 63.8% of the control group, about 30% higher than traditional methods). At the same time, the present invention uses a variety of theoretical models to guide the optimization of process parameters, including the shear energy balance equation, Fick's diffusion equation, phase separation kinetics model, and microsphere dispersion optimization function, etc., establishing a quantitative relationship between process-structure-performance, and achieving precise control and prediction of product performance. In addition, the present invention has also established a prediction model for the biological effects of polylactic acid microspheres, which can accurately evaluate and predict the biological properties of the filler, providing a scientific basis for the clinical application of the product. Generally speaking, the preparation method of the composite microsphere injectable filler provided by the present invention has significant technological innovation and practical value, providing new ideas and methods for the development of medical aesthetic fillers.

[0173] It should be noted that the detailed explanations of the variables involved in the present invention are shown in Tables 10 and 11 below.

[0174] Table 10 Variable Explanation Table (First Part)

[0175]

[0176] Table 11 Variable Explanation Table (Second Part)

[0177]

[0178] As described above, it is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered within the protection scope of the present invention.

Claims

1. A preparation method of composite microspheres for medical aesthetic injection fillers, comprising preparing a polymer oil-phase solution, formulating a surfactant water-phase solution, performing shear emulsification in a high gravity device to form a water-in-oil-in-solid emulsion, mechanically stirring at room temperature to promote the volatilization of organic solvents, centrifugally washing to remove residues, freeze-drying to obtain composite microspheres, measuring the particle size distribution of the microspheres, and mixing with cross-linked sodium hyaluronate gel to prepare an injection filler and evaluating it, characterized in that, The high-shear force field generated by a high-gravity device is used to precisely control the emulsification process. During the emulsification process, the droplet formation kinetics follows the shear energy balance equation, the solvent evaporation process follows Fick's diffusion equation, the microsphere particle size prediction is theoretically estimated using the phase separation kinetics model, the mixing process determines the optimal operating parameters using the microsphere dispersion optimization function, and the analysis and optimization are carried out through the biological effect prediction model of polylactic acid microspheres to achieve the preparation of highly stable composite microspheres.

2. The method according to claim 1, wherein The polymer oil-phase solution includes dissolving a copolymer of poly(lactic-co-glycolic acid) or a poly(L-lactic acid)-poly(ethylene glycol) block copolymer at a concentration of 20 to 200 mg / mL in dichloromethane or ethyl acetate, and adding inorganic particles of zinc oxide or magnesium oxide to the oil-phase solution. The mass ratio of the inorganic particles to the polymer is controlled within the range of 1:200 to 1:

5.

3. The method according to claim 2, characterized in that The surfactant aqueous-phase solution includes dissolving a surfactant of polyvinyl alcohol or Tween 80 at a concentration of 5 to 100 mg / mL in water for injection. The ratio of the aqueous-phase solution to the oil-phase solution is controlled within the range of 1:1 to 20:

1.

4. The method according to claim 3, characterized in that, Centrifugal washing to remove residues includes centrifugally washing the microsphere suspension 3 to 5 times. After each centrifugation, the supernatant is discarded and fresh water for injection is added to resuspend, to remove residual organic solvents and surfactants.

5. The method according to claim 4, wherein Freeze-drying to obtain the composite microspheres includes subjecting the washed microsphere suspension to freeze-drying treatment. The freeze-drying temperature is controlled at -40 to -60 °C, the vacuum degree is maintained at 10 to 50 Pa, and the drying time is not less than 24 hours.

6. The method according to claim 5, wherein Determining the microsphere particle size distribution includes using a laser particle size analyzer to evaluate the microsphere particle size uniformity and calculating the 10th percentile value, 50th percentile value, and 90th percentile value of the microsphere particle size distribution.

7. The method according to claim 6, wherein Mixing with crosslinked sodium hyaluronate gel to prepare an injectable filler includes mixing the dried organic-inorganic composite microspheres with crosslinked sodium hyaluronate gel at a mass ratio of 1:5 to 1:20, and preparing the injectable filler through a sterile filling process.

8. The method according to claim 7, wherein The water-in-oil-in-solid emulsion refers to a three-phase system in which oil-phase microdroplets formed by coating inorganic particles with an amphiphilic polylactic acid material are dispersed in the aqueous phase. The solid phase is the inorganic particles, the oil phase is the polymer solution, and the aqueous phase is the surfactant aqueous solution; the amphiphilic polylactic acid material refers to a polylactic acid derivative containing hydrophilic groups and lipophilic groups, including a copolymer of poly(lactic-co-glycolic acid) with a lactic acid to glycolic acid ratio of 90:10, a poly(lactic-co-glycolic acid)-poly(ethylene glycol) copolymer, or a poly(L-lactic acid)-poly(ethylene glycol) block copolymer, where the molecular weight of the polylactic acid is 80,000 to 240,000 and the molecular weight of the polyethylene glycol is 700 to 6000.

9. The method according to claim 8, wherein The microsphere particle size uniformity is evaluated by the Span value. The calculation formula is that the Span value is equal to (the 90th percentile particle size value minus the 10th percentile particle size value) divided by the 50th percentile particle size value. The smaller the Span value, the more uniform and concentrated the microsphere particle size distribution.

10. The method according to claim 9, characterized in that, The optimal operating parameters include the optimal mixing time and the optimal mixing speed. The optimal mixing time is the best duration for mixing the microspheres with sodium hyaluronate gel, and the optimal mixing speed is the best stirring rate during the mixing process.

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