A method for preparing composite microspheres for medical cosmetic injection fillers

Abstract

The application provides a composite microsphere preparation method for medical cosmetic injection fillers, and belongs to the technical field of chemical treatment methods.The application comprises the following steps: preparing an oil phase by mixing polylactic acid polymers and inorganic particles; preparing a polyvinyl alcohol or Tween 80 surfactant aqueous solution as a water phase; performing shearing emulsification in a supergravity equipment to form a solid-in-oil-in-water emulsion; promoting the volatilization of an organic solvent by mechanical stirring at room temperature; removing residues by centrifugal washing; obtaining the composite microspheres by freeze-drying; determining and evaluating the particle size distribution; mixing the composite microspheres with cross-linked sodium hyaluronate gel to prepare injection fillers; and evaluating the biocompatibility and collagen regeneration efficiency, and optimizing the formula according to the evaluation results. The technical problem of poor preparation stability of the composite microspheres in the medical cosmetic injection fillers is solved, and finally, the medical cosmetic injection fillers with high consistency between batches and good preparation stability are obtained.

Description

Technical Field

[0001] This invention belongs to the field of chemical processing technology, and more specifically, relates to a method for preparing composite microspheres for use as injectable fillers in medical aesthetics. Background Technology

[0002] Injectable fillers in medical aesthetics are widely used in the field of non-surgical cosmetic procedures. Among them, polylactic acid microspheres combined with sodium hyaluronate gel have attracted much attention due to their excellent biodegradability and long-lasting filling effect. Traditional methods for preparing composite microspheres mainly employ solvent evaporation, spray drying, or emulsification solvent evaporation methods. These methods typically complete the emulsification process under conventional stirring equipment. In clinical applications, these fillers are widely used for cosmetic repair of facial wrinkles, depressions, and contouring, as well as for tissue filling and drug delivery systems in tissue engineering.

[0003] However, traditional preparation processes have significant drawbacks: there are large batch-to-batch differences in microsphere preparation, resulting in significant fluctuations in product quality; the emulsification conditions are difficult to control precisely during the preparation process, leading to large differences in particle size distribution, morphology, and internal structure among different batches of microspheres; the fusion process of inorganic and organic components is uncontrollable, resulting in uneven composition of composite microspheres; at the same time, the lack of a systematic theoretical model to guide the setting of process parameters makes it difficult to standardize the preparation process, and the preparation results vary significantly under different operators or equipment conditions.

[0004] These issues lead to batch-to-batch inconsistencies in the clinical effects of medical aesthetic fillers, making it difficult to predict usage outcomes and severely impacting product quality stability and market application. Traditional technologies cannot solve the stability problems in the preparation process of composite microspheres, especially during scale-up production, where stability issues become even more pronounced. There is an urgent need for an efficient, controllable, and stable composite microsphere preparation method guided by theoretical models. In other words, existing technologies suffer from poor stability in the preparation of composite microspheres for medical aesthetic injectable fillers. Summary of the Invention

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

[0006] This invention is achieved as follows: It provides a method for preparing composite microspheres for medical aesthetic injectable fillers, including preparing a polymer oil phase solution, preparing a surfactant aqueous phase solution, performing shear emulsification in a hypergravity apparatus to form an oil-in-water emulsion, mechanically stirring at room temperature to promote organic solvent evaporation, centrifuging and washing to remove residues, freeze-drying to obtain composite microspheres, measuring the microsphere particle size distribution, and mixing with cross-linked sodium hyaluronate gel to prepare injectable fillers and evaluating the results. Specifically, a strong shear force field generated by a hypergravity apparatus is used to precisely control the emulsification process. During emulsification, droplet formation kinetics follow the shear energy balance equation, solvent evaporation follows the Fick diffusion equation, microsphere particle size is theoretically estimated using a phase separation kinetic model, and the optimal operating parameters for the mixing process are determined using a microsphere dispersion optimization function. Furthermore, the mixture is analyzed and optimized using a polylactic acid microsphere biological effect prediction model to achieve the preparation of highly stable composite microspheres.

[0007] The polymer oil phase solution comprises dissolving polylactic acid glycolate copolymer or poly-L-lactic acid polyethylene glycol block copolymer in dichloromethane or ethyl acetate at a concentration of 20 to 200 mg / mL, and adding zinc oxide or magnesium oxide inorganic particles to the oil phase solution, wherein the mass ratio of inorganic particles to polymer is controlled within the range of 1:200 to 1:5.

[0008] The surfactant aqueous solution comprises polyvinyl alcohol or Tween 80 surfactant dissolved in water for injection at a concentration of 5 to 100 mg / mL, and the ratio of aqueous solution to oil solution is controlled at 1:1 to 20:1.

[0009] The centrifugal washing process to remove residues includes centrifuging the microsphere suspension 3 to 5 times, discarding the supernatant after each centrifugation, adding fresh water for injection to resuspend the suspension, and removing residual organic solvents and surfactants.

[0010] The process of obtaining composite microspheres by freeze-drying includes freeze-drying a washed microsphere suspension, controlling the freeze-drying temperature at -40 to -60 degrees Celsius, maintaining the vacuum at 10 to 50 Pa, and drying for no less than 24 hours.

[0011] The determination of microsphere size distribution includes using a laser particle size analyzer to evaluate the uniformity of microsphere size and calculating the 10th, 50th, and 90th percentile values ​​of the microsphere size distribution.

[0012] The preparation of injectable fillers by mixing with cross-linked sodium hyaluronate gel includes mixing dried organic-inorganic composite microspheres with cross-linked sodium hyaluronate gel at a mass ratio of 1:5 to 1:20, and preparing injectable fillers through aseptic filling process.

[0013] The oil-in-water emulsion refers to a three-phase system in which inorganic particles are coated with amphiphilic polylactic acid (PLA) material to form oil droplets dispersed in an aqueous phase. The solid phase is the inorganic particles, the oil phase is a polymer solution, and the aqueous phase is an aqueous surfactant solution. The amphiphilic PLA material refers to a PLA derivative containing both hydrophilic and lipophilic groups, including PLA copolymers with a lactic acid to glycolic acid ratio of 90:10, PLA copolymers with glycolic acid, or PLA block copolymers with poly(L-lactic acid) and polyethylene glycol, wherein the PLA molecular weight is 80,000 to 240,000 and the polyethylene glycol molecular weight is 700 to 6,000.

[0014] Among them, the uniformity of microsphere 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 size distribution is.

[0015] The optimal operating parameters include the optimal mixing time and the optimal mixing speed. The optimal mixing time is the optimal duration for which the microspheres and sodium hyaluronate gel need to be mixed, and the optimal mixing speed is the optimal stirring rate during the mixing process.

[0016] This invention proposes using a strong shear force field generated by hypergravity equipment to precisely control the emulsification process, while introducing multiple kinetic models and machine learning algorithms to optimize preparation parameters. This method achieves high stability and controllability in the composite microsphere preparation process, significantly reducing batch-to-batch variations. The constant strong shear environment provided by the hypergravity field ensures uniform stress at all locations during emulsification, maintaining consistent preparation conditions. The droplet formation process is precisely controlled based on the shear energy balance equation, ensuring consistent microsphere particle size distribution across different batches. The Fick diffusion equation and phase separation kinetic model guide solvent evaporation and microsphere solidification, guaranteeing the stability of the microsphere's internal structure. A microsphere dispersion optimization function ensures stable and controllable mixing of the composite microspheres and sodium hyaluronate gel. By placing the entire preparation process under the guidance of a rigorous theoretical model, combined with multimodal deep learning prediction models for parameter optimization and quality monitoring, this invention successfully solves the technical problem of poor stability in composite microsphere preparation in traditional technologies, achieving stable and standardized preparation of high-quality medical aesthetic injectable fillers, and significantly improving batch-to-batch consistency and reliability. Attached Figure Description

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

[0018] Figure 2 The graph shows a comparison of collagen content and cell survival rate in Examples 2-4.

[0019] Figure 3This is a graph showing the change in collagen content over time in Example 5.

[0020] Figure 4 This is a prediction graph of the volume retention rate of the injected filler in Example 5. Detailed Implementation

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

[0022] like Figure 1 The diagram shows a flowchart of a method for preparing composite microspheres for medical aesthetic injectable fillers provided by the present invention. This method includes the following steps:

[0023] S01. Prepare an oil phase solution by dissolving polylactic acid glycolate copolymer or poly-L-lactic acid polyethylene glycol block copolymer in dichloromethane or ethyl acetate at a concentration of 20 to 200 mg / mL, and add zinc oxide or magnesium oxide inorganic particles to the oil phase solution, wherein the mass ratio of the inorganic particles to the polymer is controlled in the range of 1:200 to 1:5.

[0024] S02. Prepare an aqueous solution by dissolving polyvinyl alcohol or Tween 80 surfactant in water for injection at a concentration of 5 to 100 mg / mL, ensuring complete dissolution. The ratio of the aqueous solution to the oil solution is controlled at 1:1 to 20:1.

[0025] S03. Set the rotation speed of the hypergravity equipment to 300 to 3000 revolutions per minute. Simultaneously introduce the aqueous solution and the oil solution into the hypergravity equipment through a metering pump. Perform shear emulsification at room temperature to form a water-in-oil-in-solids emulsion. During the emulsification process, the droplet formation kinetics follow the shear energy balance equation.

[0026] S04. Collect the microsphere suspension emulsion flowing out of the bottom outlet of the supergravity equipment, and mechanically stir it at a speed of 200 to 1000 revolutions per minute for 6 to 12 hours at room temperature to promote the complete evaporation of organic solvents. The solvent evaporation process follows the Fick diffusion equation.

[0027] S05. The microsphere suspension is centrifuged and washed 3 to 5 times. After each centrifugation, the supernatant is discarded, and fresh water for injection is added to resuspend the suspension to remove residual organic solvents and surfactants.

[0028] S06. The washed microsphere suspension is freeze-dried at a temperature of -40 to -60 degrees Celsius, with a vacuum of 10 to 50 Pa and a drying time of not less than 24 hours.

[0029] S07. Determine the particle size distribution of the dried microspheres, evaluate the uniformity of the microsphere particle size using a laser particle size analyzer, and calculate the 10th percentile, 50th percentile, and 90th percentile of the microsphere particle size distribution. The microsphere particle size prediction is theoretically estimated using a phase separation kinetic model.

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

[0031] S09. The prepared injectable filler is subjected to biocompatibility evaluation and collagen regeneration efficiency testing. Its safety and efficacy are evaluated using in vitro cell proliferation experiments and animal subcutaneous injection experiments. The results are analyzed and optimized using a pre-trained polylactic acid microsphere biological effect prediction model. If the biocompatibility evaluation shows that the cell survival rate is less than 80% or obvious inflammatory response is observed in animal experiments, the inorganic particle content needs to be adjusted, reducing the inorganic particle to polymer mass ratio to 50% of the original ratio, and steps S01 to S08 are repeated. If the collagen regeneration efficiency test shows that the collagen content increases by less than 40% of the control group 28 days after injection, 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 needs to be adjusted to 80:20 to 90:10. Steps S01 to S08 are repeated until the collagen regeneration efficiency meets the target. If both biocompatibility and collagen regeneration efficiency meet the preset threshold requirements, the preparation of the composite microsphere injectable filler is completed.

[0032] The hypergravity equipment refers to baffle-type hypergravity equipment, spiral channel hypergravity equipment, stator-rotor hypergravity equipment, rotating disc hypergravity equipment, or rotating packed bed hypergravity equipment, which can generate a centrifugal force field equivalent to 80 to 300 times the gravitational acceleration at a rotation speed of 300 to 3000 revolutions per minute, thereby enhancing the micro-mixing process of heterogeneous liquid-liquid mixtures.

[0033] The water-in-oil-in-solid emulsion refers to a three-phase system in which inorganic particles are coated with amphiphilic polylactic acid material to form oil droplets dispersed in an aqueous phase, wherein the solid phase is the inorganic particles, the oil phase is a polymer solution, and the aqueous phase is an aqueous surfactant solution.

[0034] The uniformity of microsphere size is evaluated by the Span value, which is calculated as follows: 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 size distribution is.

[0035] The amphiphilic polylactic acid material refers to a polylactic acid derivative containing both hydrophilic and lipophilic groups, including polylactic acid-glycolic acid copolymers in which the ratio of lactic acid to glycolic acid is 90:10, polylactic acid-glycolic acid polyethylene glycol copolymers, or poly-L-lactic acid-polyethylene glycol block copolymers, wherein 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] The cross-linked sodium hyaluronate gel refers to a hydrogel with a network structure prepared by treating sodium hyaluronate with 1,4-butanedialdehyde, diene disiloxane, or polyethylene glycol diglycidyl ether cross-linking agent, which is used to increase the viscoelasticity and durability of injectable fillers.

[0037] The shear energy balance equation describes the dynamic process of droplet formation and breakage in a hypergravity field. The inputs include the viscosity of the continuous phase, the viscosity of the dispersed phase, the interfacial tension, the rotational angular velocity of the hypergravity field, and the characteristic length of the hypergravity equipment. The output is the maximum stable droplet diameter in equilibrium. The shear energy balance equation describes the relationship between the maximum stable droplet diameter and the interfacial tension, the density of the continuous phase, the viscosity of the dispersed phase, the angular velocity, the characteristic radius of the hypergravity equipment, and constants related to the geometry of the equipment.

[0038] The Fick diffusion equation describes the diffusion mass transfer process of 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. The output is the time required for solvent diffusion. The Fick diffusion equation describes the relationship between solvent concentration and time and space. The diffusion coefficient is related to polymer concentration and temperature. It includes a reference diffusion coefficient, diffusion activation energy, gas constant, absolute temperature, coefficient, and polymer concentration.

[0039] The phase separation kinetic model is used to predict the polymer phase separation and microsphere solidification behavior during the evaporation of emulsified solvent. The inputs include polymer solubility parameters, 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 polymer volume fraction and time, including mobility and free energy functionals. The influence of polymer rheological properties on phase separation kinetics is introduced through a viscoelastic correction term.

[0040] 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. The outputs are the optimal mixing time and the optimal mixing speed. The microsphere dispersion optimization function establishes the relationship between relative viscosity, microsphere volume fraction, maximum packing fraction, and intrinsic viscosity. The optimal parameters of the mixing process are determined by minimizing the probability of microsphere aggregation in the gel.

[0041] The polylactic acid (PLA) microsphere biological effect prediction model is structured as a multimodal deep learning network architecture, comprising three parallel feature extraction branches and a fusion prediction module. The first branch processes the physicochemical properties of the microspheres, employing a three-layer fully connected neural network structure. The number of neurons in the hidden layer is determined by the product function of the PLA molecular weight and the inorganic particle size, and the activation function is a linear rectified unit. The second branch processes the surface morphology features of the microspheres, using a convolutional neural network to extract spatial structure information. It includes three convolutional layers and two pooling layers, with the convolutional kernel size proportional to the average particle size of the inorganic particles. The third branch processes the component ratio information, employing a self-attention mechanism to weightedly fuse features of different components. The number of attention heads is determined by the PLA hydrophilic-lipophilic balance value. The fusion module uses a gated recurrent unit network to integrate the feature vectors output from the three branches, predicting the collagen regeneration efficiency and biocompatibility score induced by the microspheres.

[0042] The steps for establishing the training dataset during the training process of the polylactic acid microsphere biological effect prediction model include: collecting experimental data on the biological effects of polylactic acid-based microsphere materials from published literature; standardizing and extracting physicochemical property parameters of the microspheres, including particle size distribution, surface charge, molecular weight, inorganic component content, and particle size; constructing a microsphere surface morphology feature library, including scanning electron microscopy images and transmission electron microscopy images of microsphere cross-sections; integrating cell experimental data, including cell proliferation rate, cell adhesion, cell viability, and cytokine expression levels; collecting animal experimental data, including histological changes after subcutaneous injection, changes in collagen content, degree of inflammatory response, and degradation rate; and dividing the dataset into training, validation, and test sets using cross-validation methods. The training and validation sets are used for model training and parameter optimization, while the test set is used to evaluate the model's generalization performance.

[0043] The training steps of the polylactic acid microsphere biological effect prediction model include: first, performing self-supervised learning on synthetic data to initialize model parameters by predicting the correlation between the physicochemical properties of microspheres and biological effects; then, training on labeled data using supervised learning, with the loss function combining a mean squared error term and a regularization term, and the Adam optimizer used as the optimization algorithm, with the initial learning rate set to 0.001 and dynamically adjusted using a cosine annealing strategy; an early stopping strategy is introduced during training to prevent overfitting, stopping training when the performance index on the validation set fails to improve for 5 consecutive rounds; finally, a transfer learning method is used to fine-tune the model, freezing the feature extraction layer parameters and training only the prediction layer parameters to adapt the model to different polylactic acid microsphere formulation combinations; the entire training process is iterated 5000 times, and five-fold cross-validation is used to evaluate the model performance, ultimately selecting the parameter combination with the smallest root mean square error in collagen regeneration efficiency prediction on the validation set.

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

[0045] The specific implementation of step S01 involves selecting a suitable polymer material and preparing an oil phase solution. First, the polylactic acid-glycolic acid copolymer or poly(L-lactic acid-polyethylene glycol) block copolymer is weighed, and the required mass is accurately measured. Then, the polymer is added to pre-prepared dichloromethane or ethyl acetate and stirred until dissolved and transparent. The concentration of the oil phase solution is controlled within the range of 20 to 200 mg / mL; lower concentrations are beneficial for forming small-diameter microspheres, while higher concentrations increase the mechanical strength of the microspheres. Simultaneously, zinc oxide or magnesium oxide nanoscale inorganic particles are added to the oil phase solution, with the inorganic particle to polymer mass ratio controlled within the range of 1:200 to 1:5. The purpose of introducing inorganic particles is to enhance the mechanical properties of the microspheres and regulate the degradation rate, while simultaneously promoting extracellular matrix synthesis. During this process, polymer solubility parameter theory is used to predict the polymer's dissolution behavior in organic solvents, ensuring the formation of a stable and homogeneous oil phase solution.

[0046] The specific implementation of step S02 involves preparing an aqueous solution. First, prepare water for injection, filtering it through a 0.22-micron filter membrane for sterilization. Then, weigh either polyvinyl alcohol or Tween 80 surfactant and add it to the water for injection. Polyvinyl alcohol requires heating to 80°C and continuous stirring during preparation, while Tween 80 can dissolve directly 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 solution is controlled between 1:1 and 20:1. The surfactant is selected based on its hydrophilic-lipophilic balance (HLP). Polyvinyl alcohol has an HLP of 18, suitable for preparing microspheres with high hydrophilicity; Tween 80 has an HLP of 15, suitable for preparing moderately hydrophilic microspheres. During the preparation of the aqueous solution, attention should be paid to the critical micelle concentration (CMC) theory of surfactants, ensuring that the surfactant concentration is higher than the CMC to provide sufficient interfacial stabilization.

[0047] The specific implementation of step S03 involves using a hypergravity apparatus for shear emulsification. First, the hypergravity apparatus is started and its rotation speed is adjusted to 300 to 3000 revolutions per minute, generating a centrifugal force field with an acceleration of 80 to 300 times gravity. The oil phase solution and the aqueous phase solution are simultaneously injected into the hypergravity apparatus at a constant flow rate using a precision metering pump. Under strong shear force, the oil phase is dispersed into tiny droplets that encapsulate inorganic particles, forming an oil-in-water emulsion. During emulsification, droplet formation follows the shear energy balance equation, which describes the relationship between the maximum stable droplet diameter at equilibrium and interfacial tension, continuous phase density, dispersed phase viscosity, continuous phase viscosity, angular velocity, and the characteristic radius of the hypergravity apparatus. According to this equation, increasing the angular velocity or decreasing the interfacial tension can significantly reduce the droplet size. The microscopic turbulent pulsations in the hypergravity field enhance the interfacial renewal rate, promoting the formation of a microsphere precursor emulsion with a uniform particle size distribution. In practice, microspheres with a narrower particle size distribution can be obtained when the ratio of the aqueous phase flow rate to the oil phase flow rate is 5:1 to 15:1.

[0048] The specific implementation of step S04 involves collecting the microsphere suspension emulsion and promoting the evaporation of the organic solvent. First, a collection container is installed at the bottom outlet of the hypergravity equipment to collect the outflowing microsphere suspension emulsion. The collected emulsion is transferred to a glass reactor equipped with a mechanical stirrer, and the stirring speed is set to 200 to 1000 rpm to avoid excessive speed causing microsphere aggregation. Stirring is continued at room temperature for 6 to 12 hours to promote the diffusion of the organic solvent within the microspheres into the aqueous phase and its eventual evaporation. The solvent evaporation process follows the Fick diffusion equation, which describes the relationship between solvent concentration and time and space, where the diffusion coefficient is related to polymer concentration and temperature. For polylactic acid microspheres, the diffusion coefficient of dichloromethane in the polymer decreases with increasing polymer concentration, and the diffusion activation energy is approximately 15 to 25 kJ / mol. During solvent evaporation, 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 pores formed, but the more uneven their distribution.

[0049] The specific implementation of step S05 involves centrifuging and washing the microsphere suspension. First, the microsphere suspension is transferred to a centrifuge tube and centrifuged at 3000 to 5000 rpm for 5 to 10 minutes. The precipitate formed after centrifugation is the microspheres, and the supernatant contains residual surfactants and organic solvents. The supernatant is carefully aspirated and discarded, and fresh water for injection is added to the precipitate. The microspheres are resuspended using a vortex mixer. This centrifugation and washing process is repeated 3 to 5 times until the concentration of residual organic solvents in the supernatant is below 10 μg / mL and the concentration of residual surfactants is below 5 μg / mL. The optimal centrifugation time is calculated using sedimentation theory during the washing process to ensure complete sedimentation of the microspheres without damaging their structure. For polylactic acid microspheres with a particle size of 10 to 50 micrometers, the optimal centrifugation time at 3000 rpm is 5 to 8 minutes. The number of washes is determined based on solute dilution theory; after each wash, the impurity concentration in the supernatant decreases by approximately 90%.

[0050] The specific implementation of step S06 involves freeze-drying. First, the washed microsphere suspension is transferred to a freeze-drying flask and pre-frozen in an environment of -40 to -60°C for at least 4 hours to ensure complete freezing of water into ice crystals. Then, the freeze-drying flask is connected to a freeze dryer, with a vacuum level of 10 to 50 Pa and a condenser temperature of -80°C. Freeze-drying is divided into three stages: the first stage maintains a temperature of -40°C and a pressure of 20 Pa for 12 hours, primarily removing free water; the second stage gradually increases the temperature to -20°C and decreases the pressure to 15 Pa for 8 hours, removing some bound water; the third stage increases the temperature to 0°C and further decreases the pressure to 10 Pa for 4 hours, removing residual bound water. During freeze-drying, the porosity of the microspheres is determined by the ice crystal sublimation rate, which follows a heat-mass transfer coupling model. The sublimation interface migration speed is related to the applied vacuum level and the heat source temperature. After drying, the microspheres are in a loose powder form; to prevent contact with indoor air, they are immediately sealed and stored.

[0051] The specific implementation of step S07 involves determining the microsphere size distribution. First, a suitable amount of dried microsphere sample is taken and dispersed evenly in a 0.1% polysorbate 80 solution. Measurement is performed using a laser particle size analyzer with a laser wavelength of 633 nm and a detection angle range of 0.02 to 165 degrees. Each sample is measured five times, and the average value is used to calculate the 10th percentile, 50th percentile, and 90th percentile of the microspheres, which are recorded as follows: , , Microsphere particle size uniformity is evaluated using the Span value, calculated as Span = A Span value less than 1 indicates uniform particle size distribution. A phase separation kinetic model was used to predict the theoretical particle size. This model describes the polymer phase separation process based on the Cahn-Hilliard equation and calculates the driving force of phase separation using Flory-Huggins theory. The model input parameters include polymer solubility parameters, solvent evaporation rate, initial droplet size, and initial polymer concentration; 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 involves preparing an injectable filler. First, a certain amount of dried composite microspheres are taken and added to a cross-linked sodium hyaluronate gel, with the microsphere to gel mass ratio controlled within the range of 1:5 to 1:20. The sodium hyaluronate gel is pre-treated with a cross-linking agent of 1,4-butanedialdehyde, diene disiloxane, or polyethylene glycol diglycidyl ether, with a cross-linking degree of 1% to 5%. The mixing process uses a twin-screw extruder or a high-shear homogenizer at a speed of 500 to 2000 rpm for a mixing time of 5 to 30 minutes. The mixing parameters are optimized based on a microsphere dispersion optimization function, which treats the microspheres as suspended particles and establishes the relationship between relative viscosity, microsphere volume fraction, maximum packing fraction, and intrinsic viscosity. The optimal mixing time and speed are determined 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 sterilized by gamma irradiation with an irradiation dose of 15 to 25 kGrey to ensure sterility without affecting the microsphere structure.

[0053] The specific implementation of step S09 involves conducting biocompatibility evaluation and collagen regeneration efficiency testing. First, an in vitro cell proliferation experiment is performed. An appropriate amount of the injection filler is co-cultured with fibroblasts for 72 hours. Cell viability is determined using the thiazolyl blue colorimetric method, and cytokine secretion levels are detected using enzyme-linked immunosorbent assay (ELISA). Then, an animal subcutaneous injection experiment is conducted, using rabbits or mice as experimental subjects. Histological changes are observed at 1, 7, 14, and 28 days after subcutaneous injection of the filler. Inflammatory response is assessed using hematoxylin-eosin staining, and collagen content is determined using Sirius red staining. The experimental results are analyzed using a polylactic acid microsphere biological effect prediction model. This model is a multimodal deep learning network containing three parallel feature extraction branches and a fusion prediction module. If the biocompatibility evaluation shows a cell viability rate below 80%, the inorganic particle content is adjusted; if the collagen regeneration efficiency is less than 40% of the control group, the polylactic acid molecular weight is increased or the polymer component ratio is adjusted. When both biocompatibility and collagen regeneration efficiency reach preset thresholds, the preparation of the composite microsphere injection filler is complete.

[0054] The mathematical model or calculation process involved in this invention will be described in detail below.

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

[0056] ;

[0057] In the formula, The maximum stable droplet diameter under equilibrium conditions, expressed in meters; The oil-water interfacial tension is expressed in Newtons per meter (N / m). This is the density of the continuous phase (aqueous phase), expressed in kilograms per cubic meter. Angular velocity for hypergravity equipment, measured in radians per second; The characteristic radius of the hypergravity equipment is expressed in meters. The viscosity of the dispersed phase (oil phase) is expressed in Pascals per second. Viscosity of the continuous phase (aqueous phase), measured in Pascals per second; These are dimensionless constants related to the geometry of the equipment.

[0058] The parameter acquisition method is as follows:

[0059] It can be measured by the pendant drop method or the rotating drop method. The measurement process involves injecting the polymer solution into the surfactant aqueous solution, observing the drop shape through an optical system, and calculating the interfacial tension according to the Laplace equation. Measured directly using a densitometer; Directly converted from the rotational speed of the hypergravity equipment ,in Rotational speed, measured in revolutions per minute; These are equipment design parameters that can be directly measured; and Measured using a rheometer; It needs to be determined through standardized experiments, which involves measuring the droplet size under known conditions and then calculating it backwards. The value is generally in the range of 0.5 to 2.0.

[0060] This equation is based on the principle of shear stress and surface tension equilibrium. The exponents 0.6 and 0.4 are obtained by fitting a large amount of experimental data, indicating that the influence of interfacial tension and the strength of the hypergravity field (0.6) is slightly greater than the influence of the viscosity ratio (0.4). When the shear stress exceeds the interfacial tension, the emulsion droplets are torn into smaller droplets. The equation adopts a power function form because in microscale fluid dynamics, these physical quantities usually follow a power law relationship. Compared with the traditional Weber number equation, this equation adds a viscosity ratio term, considers the influence of the rheological properties of the dispersed phase on the emulsification process, and improves the prediction accuracy.

[0061] The Fick diffusion equation mentioned in step S04 describes the diffusion mass transfer process of organic solvents from the polymer phase to the aqueous phase, as shown below:

[0062] ;

[0063] In the formula, This is the solvent concentration, expressed in grams per cubic meter. Time, in seconds; This represents the radial distance of the microspheres, in meters. ν is the diffusion coefficient of the solvent in the polymer, expressed in square meters per second.

[0064] diffusion coefficient There is a relationship between polymer concentration and temperature:

[0065] ;

[0066] In the formula, The reference diffusion coefficient is expressed in square meters per second. The activation energy is for diffusion, expressed in joules per mole. The gas constant is 8.314 joules / (molar Kelvin); This is absolute temperature, measured in Kelvin. The coefficient is dimensionless. The value represents the polymer volume fraction, which is dimensionless.

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

[0068] ;

[0069] In the formula, The time required for solvent diffusion, in seconds; The radius of the microsphere is in meters.

[0070] The parameter acquisition method is as follows:

[0071] It can be determined by nuclear magnetic resonance relaxation experiments, which monitor the self-diffusion behavior of solvent molecules in polymers, typically within a range of... ~ ; By measuring the diffusion coefficient at different temperatures, a plot was drawn. right The curve, the slope of the curve is For polylactic acid systems, the value is generally 15–25 kJ / mol. The diffusion coefficient is determined by measuring the diffusion coefficient at different polymer concentrations, and is generally between 1.0 and 3.0. It can be calculated based on the formula; Measured using a laser particle size analyzer.

[0072] This equation is based on molecular diffusion theory and uses spherical coordinates because the microspheres have a spherical structure. The exponential term reflects the strong temperature dependence (Arrhenius relation) and nonlinear dependence on polymer concentration of the diffusion coefficient. The diffusion coefficient increases with increasing temperature and decreases with increasing polymer concentration, which is consistent with the physical picture of how polymer chains impede the motion of solvent molecules. In the equation... The second derivative term represents the rate of change of the concentration gradient, while the first derivative term considers geometric effects in spherical coordinates. Compared to the traditional flat plate diffusion model, this equation more accurately describes the solvent diffusion behavior in spherical microspheres and can more precisely predict the hardening time and internal pore structure of the microspheres.

[0073] The phase separation kinetics model mentioned in step S07 is used to predict the polymer phase separation and microsphere solidification behavior during the evaporation of the emulsion solvent, as specifically expressed below:

[0074] ;

[0075] In the formula, This is the polymer volume fraction, dimensionless. Time, in seconds; Mobility is expressed in square meters per (joule-second). The free energy functional is expressed in joules per cubic meter. The gradient energy coefficient is expressed in joules per meter.

[0076] Free energy functional It can be represented as:

[0077] ;

[0078] In the formula, The value represents the molar volume of solvent molecules, expressed in cubic meters per mole. The parameters for the Flory-Huggins interaction are dimensionless.

[0079] migration rate Related to polymer viscosity:

[0080] ;

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

[0082] ;

[0083] In the formula, The coupling coefficient is dimensionless. This represents the corresponding force tensor for the polymer, in Pascals.

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

[0085] ;

[0086] In the formula, Let be the probability density function of particle size distribution; The diameter of the microsphere is in meters. The geometric mean diameter of the microsphere is in meters. denoted as the standard deviation of the distribution, which is dimensionless.

[0087] The parameter acquisition method is as follows:

[0088] The critical phase separation point can be determined by measuring the phase diagram of the polymer in the solvent; the value corresponding to this point is... It can also be estimated through solubility parameters: ,in and These are the solubility parameters for the polymer and the solvent, respectively. The characteristic length of the polymer phase separation structure was determined by measuring small-angle X-ray scattering. Determined through a combination of rheological measurements and phase separation kinetics experiments; and It was obtained by fitting the measured particle size distribution data.

[0089] This model is based on the Cahn-Hilliard equations and Flory-Huggins theory. The Cahn-Hilliard equations describe the phase separation dynamics of a conservative field system; the second-order operator in the first term on the right-hand side represents diffusion, and the derivative of the free energy functional represents the driving force of phase separation. The free energy functional, in logarithmic form, reflects the contribution of the mixing entropy. The term represents the interaction between polymer and solvent molecules. The introduction of a viscoelastic correction term is an innovation of this model, considering the influence of polymer chain relaxation behavior on phase separation kinetics, which 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, providing theoretical guidance for optimizing preparation parameters.

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

[0091] ;

[0092] In the formula, The viscosity is a dimensionless relative viscosity. The volume fraction of the microspheres is dimensionless. The maximum stacked fraction is dimensionless. Intrinsic viscosity, expressed in cubic centimeters per gram.

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

[0094] ;

[0095] In the formula, Let be the probability of microsphere aggregation, which is dimensionless; Frequency factor, in seconds ; Shear rate, in seconds ; This is a mixed time, expressed in seconds. The energy barrier for the interaction between microspheres is expressed in joules. Boltzmann's constant, Joules / Kelvin.

[0096] Equation for determining the optimal mixing parameters:

[0097] ;

[0098] In the formula, The optimal shear rate, in seconds. ; The optimal mixing time is expressed in seconds.

[0099] Energy Barrier Related to the surface charge and size of the microspheres:

[0100] ;

[0101] In the formula, The vacuum permittivity, Farads per meter; is the relative permittivity of the medium, which is dimensionless; The diameter of the microsphere is in meters. ν is the surface potential of the microspheres, measured in volts. It is the reciprocal of the length of Debye, in meters. .

[0102] The parameter acquisition method is as follows:

[0103] Calculate directly from the formula; The microsphere sedimentation experiment was conducted by measuring the maximum random packing density, which is typically 0.6 to 0.7. The viscosity of a dilute solution is measured using a capillary viscometer and then extrapolated to zero concentration. The aggregation kinetics of microspheres under different shear conditions were determined; Directly converted from the speed of the mixing equipment. ,in The shear coefficient of the equipment; Measured using a Zeta potentiometer; It can be calculated based on ionic strength. ,in Let Avogadro's constant be 1. For elementary charge, This represents the ionic strength.

[0104] The optimization function is constructed based on rheology and colloidal stability theory. The equation for relative viscosity adopts the Krieger-Dougherty model, describing the nonlinear relationship between the viscosity of the suspension system and particle concentration. Intrinsic viscosity is introduced into the exponent to account for the influence of particle shape and surface properties on rheological behavior. The aggregation probability function references Smoluchowski aggregation kinetics and DLVO theory, and the energy barrier term in the exponent indicates the influence of temperature and surface charge on microsphere stability. The optimal mixing parameters are determined by minimizing the aggregation probability. This method can automatically adjust mixing conditions in actual production, avoiding microsphere breakage under high shear or aggregation under low shear, thus improving the uniformity and stability of the injection filler.

[0105] Specifically, the principle of this invention is as follows: The core principle of this invention is based on the theory of phase separation and structure formation enhanced by a hypergravity field, combined with a multimodal deep learning prediction model to achieve precise control of process parameters. Firstly, its working principle stems from the stable shear environment provided by a hypergravity field. Compared to conventional stirring, hypergravity equipment can generate a centrifugal force field with 80-300 times the acceleration of gravity, forming a uniform shear force distribution throughout the reaction area. This eliminates dead zones and localized high-shear zones present in traditional stirring equipment, making the emulsification process spatially more uniform. Under this uniform shear environment, the droplet formation kinetics strictly follow the shear energy balance equation, significantly shortening the time for droplet size to reach equilibrium and significantly reducing batch-to-batch differences.

[0106] Secondly, the evaporation process of organic solvents is a key factor affecting the stability of microsphere formation. This invention employs the Fick 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 ambient temperature and stirring rate, the solvent evaporation rate can be precisely controlled, avoiding differences in the internal structure of the microspheres caused by uneven evaporation rates. Simultaneously, a phase separation kinetic model guides the phase transition behavior of the polymer during solvent evaporation, ensuring that different batches of microspheres maintain a similar internal structural evolution path during formation.

[0107] Third, the composite mechanism of amphiphilic polylactic acid (PLA) materials and inorganic particles is based on the balance of interfacial forces. Under hypergravity, 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, while the lipophilic groups maintain affinity with the organic phase, achieving uniform and stable coating of the inorganic particles. This precisely controlled interfacial interaction ensures the consistency of the inorganic component distribution in different batches of composite microspheres.

[0108] Finally, the polylactic acid (PLA) microsphere biological effect prediction model, based on a multimodal deep learning network architecture, integrates information on the physicochemical properties, surface morphology, and component ratios of the microspheres. It monitors the impact of fluctuations in process parameters on the final product performance in real time and adjusts parameters to maintain stable product quality. Trained on a large amount of historical data, this model can predict performance fluctuations caused by minute changes in process parameters, providing closed-loop feedback control for the preparation process and fundamentally ensuring preparation stability.

[0109] The following provides a specific embodiment 1 of the present invention, and the specific implementation of each step in this embodiment 1 is described in detail below.

[0110] The specific implementation of step S01 involves selecting a suitable polymer material and preparing an oil phase solution. First, weigh out the polylactic acid-glycolic acid copolymer or poly(L-lactic acid-polyethylene glycol) block copolymer, accurately measuring the required mass. Then, add the polymer to pre-prepared dichloromethane or ethyl acetate and stir until dissolved and transparent. The concentration of the oil phase solution is controlled within the range of 20 to 200 mg / mL; lower concentrations are beneficial for forming small-diameter microspheres, while higher concentrations increase the mechanical strength of the microspheres. Simultaneously, zinc oxide or magnesium oxide nanoscale inorganic particles are added to the oil phase solution, with the inorganic particle to polymer mass ratio controlled within the range of 1:200 to 1:5. The purpose of introducing inorganic particles is to enhance the mechanical properties of the microspheres and regulate the degradation rate, while simultaneously promoting extracellular matrix synthesis. In this process, polymer solubility parameter theory is used to predict the polymer's dissolution behavior in organic solvents, ensuring the formation of a stable and homogeneous oil phase solution. The polymer solubility parameter can be calculated using the group contribution method. In the formula, This is a polymer solubility parameter, in units of... ; Let be the molar attraction constant of the i-th group in the molecular structure, in units of . ; The molar volume of the i-th group in the molecular structure, in units of The solubility parameter of polylactic acid is generally 19–21. The solubility parameter for dichloromethane is 20.2. The solubility parameter for ethyl acetate is 18.1. When the difference between the solubility parameters of the polymer and the solvent is less than 2 When the polymer is in contact with the target material, it is easily dissolved.

[0111] The specific implementation of step S02 involves preparing an aqueous solution. First, prepare water for injection, filtering it through a 0.22-micron filter membrane for sterilization. Then, weigh either polyvinyl alcohol or Tween 80 surfactant and add it to the water for injection. Polyvinyl alcohol requires heating to 80°C and continuous stirring during preparation, while Tween 80 can dissolve directly 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 solution is controlled between 1:1 and 20:1. The surfactant is selected based on its hydrophilic-lipophilic balance value. Polyvinyl alcohol has a hydrophilic-lipophilic balance value of 18, suitable for preparing microspheres with high hydrophilicity; Tween 80 has a hydrophilic-lipophilic balance value of 15, suitable for preparing moderately hydrophilic microspheres. During the preparation of the aqueous solution, attention should be paid to the critical micelle concentration theory of surfactants, ensuring that the surfactant concentration is higher than the critical micelle concentration to provide sufficient interfacial stability. The critical micelle concentration can be determined by the surface tension method. In the formula, Surface tension, unit: ; The surface tension of pure water is expressed in units of 1. ; is the gas constant, 8.314. ; Absolute temperature, unit: ; The maximum adsorption capacity on the surface, in units of ; This represents the surfactant concentration, in units of... ; It is a constant, dimensionless. The critical micelle concentration is the concentration value corresponding to the abrupt change point on the surface tension-concentration curve; the critical micelle concentration of polyvinyl alcohol is approximately 0.03–0.05%. The critical micelle concentration for Tween 80 is approximately 0.01–0.02. .

[0112] The specific implementation of step S03 involves using a hypergravity apparatus for shear emulsification. First, the hypergravity apparatus is started and its rotation speed is adjusted to 300 to 3000 revolutions per minute, generating a centrifugal force field with an acceleration of 80 to 300 times gravity. The oil phase solution and the aqueous phase solution are simultaneously injected into the hypergravity apparatus at a constant flow rate using a precision metering pump. Under strong shear force, the oil phase is dispersed into tiny droplets that encapsulate inorganic particles, forming an oil-in-water emulsion. During emulsification, droplet formation follows the shear energy balance equation, which describes the relationship between the maximum stable droplet diameter at equilibrium and interfacial tension, continuous phase density, dispersed phase viscosity, continuous phase viscosity, angular velocity, and the characteristic radius of the hypergravity apparatus. The shear energy balance equation is specifically expressed as: In the formula, The maximum stable droplet diameter under equilibrium conditions, expressed in meters; The oil-water interfacial tension is expressed in Newtons per meter (N / m). This is the density of the continuous phase (aqueous phase), expressed in kilograms per cubic meter. Angular velocity for hypergravity equipment, measured in radians per second; The characteristic radius of the hypergravity equipment is expressed in meters. The viscosity of the dispersed phase (oil phase) is expressed in Pascals per second. Viscosity of the continuous phase (aqueous phase), measured in Pascals per second; This is a dimensionless constant related to the equipment geometry, typically ranging from 0.5 to 2.0. Microscopic turbulent fluctuations in a hypergravity field enhance the interface renewal rate, promoting the formation of microsphere precursor emulsions with uniform particle size distribution. In practice, microspheres with a narrower particle size distribution can be obtained when the ratio of the aqueous phase flow rate to the oil phase flow rate is between 5:1 and 15:1.

[0113] The specific implementation of step S04 involves collecting the microsphere suspension emulsion and promoting the evaporation of the organic solvent. First, a collection container is installed at the bottom outlet of the hypergravity equipment to collect the outflowing microsphere suspension emulsion. The collected emulsion is transferred to a glass reactor equipped with a mechanical stirrer, and the stirring speed is set to 200 to 1000 rpm to avoid excessive speed causing microsphere aggregation. Stirring is continued at room temperature for 6 to 12 hours to promote the diffusion of the organic solvent within the microspheres into the aqueous phase and its eventual evaporation. The solvent evaporation process follows the Fick diffusion equation, describing the relationship between solvent concentration and time and space. The Fick diffusion equation in spherical coordinates is expressed as: In the formula, This is the solvent concentration, expressed in grams per cubic meter. Time, in seconds; This represents the radial distance of the microspheres, in meters. This represents the diffusion coefficient of the solvent in the polymer, expressed in square meters per second. Related to temperature and polymer concentration: In the formula, This is a reference diffusion coefficient, measured in square meters per second, typically ranging from [value missing]. ; The activation energy is for diffusion, expressed in joules per mole, and is typically 15–25 for polylactic acid systems. ; The gas constant is 8.314 joules / (molar Kelvin); This is absolute temperature, measured in Kelvin. This is a coefficient, dimensionless, typically ranging from 1.0 to 3.0; The value represents the polymer volume fraction, which is dimensionless. During solvent evaporation, 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 pores formed, but the more uneven their distribution. The time required for solvent diffusion can be estimated as follows: In the formula, The time required for solvent diffusion, in seconds; The radius of the microsphere is in meters.

[0114] The specific implementation of step S05 involves centrifuging and washing the microsphere suspension. First, the microsphere suspension is transferred to a centrifuge tube and centrifuged at 3000 to 5000 rpm for 5 to 10 minutes. The precipitate formed after centrifugation is the microspheres, and the supernatant contains residual surfactants and organic solvents. The supernatant is carefully aspirated and discarded. Fresh water for injection is added to the precipitate, and the microspheres are resuspended using a vortex mixer. This centrifugation and washing process is repeated 3 to 5 times until the concentration of residual organic solvents in the supernatant is below 10 μg / mL and the concentration of residual surfactants is below 5 μg / mL. The optimal centrifugation time is calculated using Stokes sedimentation theory during the washing process. In the formula, Centrifugation time, in seconds; The viscosity is for continuous phases, measured in Pascals per second. This refers to the inner diameter of the centrifuge rotor, in meters. This refers to the outer diameter of the centrifuge rotor, in meters. Angular velocity, in radians per second; This refers to the density of the microspheres, expressed in kilograms per cubic meter. This is the density of the continuous phase, expressed in kilograms per cubic meter. The value is the diameter of the microspheres, in meters. For polylactic acid microspheres with a particle size of 10 to 50 micrometers, the optimal centrifugation time is 5 to 8 minutes at 3000 rpm. The number of washes is determined based on the solute dilution theory; after each wash, the concentration of impurities in the supernatant decreases by approximately 90%. The residual solvent concentration can be detected by gas chromatography, and the residual surfactant concentration can be detected by ultraviolet spectrophotometry.

[0115] The specific implementation of step S06 involves freeze-drying. First, the washed microsphere suspension is transferred to a freeze-drying flask and pre-frozen in an environment of -40 to -60°C for at least 4 hours to ensure complete freezing of water into ice crystals. Then, the freeze-drying flask is connected to a freeze dryer, with a vacuum of 10 to 50 Pa and a condenser temperature of -80°C. Freeze-drying is divided into three stages: the first stage maintains a temperature of -40°C and a pressure of 20 Pa for 12 hours, primarily removing free water; the second stage gradually increases the temperature to -20°C and decreases the pressure to 15 Pa for 8 hours, removing some bound water; the third stage increases the temperature to 0°C and further decreases the pressure to 10 Pa for 4 hours, removing residual bound water. During freeze-drying, the microsphere porosity is determined by the ice crystal sublimation rate, and this process follows a heat-mass transfer coupling model. In the formula, The sublimation rate is expressed in kilograms per second. The area of ​​the sublimated interface is expressed in square meters. is the diffusion coefficient of water vapor in the porous layer, expressed in square meters per second; The molar mass of water is 0.018 kg / mol. This is the water vapor pressure at the ice interface, measured in Pascals. The vapor pressure of water on the condenser surface is expressed in Pascals. The gas constant is 8.314 joules / (molar Kelvin); This is absolute temperature, measured in Kelvin. The thickness of the porous layer is measured in meters. A faster ice crystal sublimation rate results in higher porosity of the formed microspheres, but relatively lower mechanical strength. After drying, the microspheres are in a loose powder form; avoid contact with indoor air and seal immediately for storage.

[0116] The specific implementation of step S07 involves determining the microsphere size distribution. First, a suitable amount of dried microsphere sample is taken and dispersed evenly in a 0.1% polysorbate 80 solution. Measurement is performed using a laser particle size analyzer with a laser wavelength of 633 nm and a detection angle range of 0.02 to 165 degrees. Each sample is measured five times, and the average value is used to calculate the 10th percentile, 50th percentile, and 90th percentile of the microspheres, which are recorded as follows: , , The uniformity of microsphere size was evaluated using the Span value, calculated using the following formula: A Span value less than 1 indicates uniform particle size distribution. Simultaneously, a phase separation kinetic model was used for theoretical particle size prediction. This model, based on the Cahn-Hilliard equation, describes the polymer phase separation process. In the formula, This is the polymer volume fraction, dimensionless. Time, in seconds; Mobility is expressed in square meters per (joule-second). The free energy functional is expressed in joules per cubic meter. This represents the gradient energy coefficient, expressed in joules per meter (J / m). Free energy functional. Based on the Flory-Huggins theory, it can be expressed as follows: In the formula, The value represents the molar volume of solvent molecules, expressed in cubic meters per mole. Let be the Flory-Huggins interaction parameter, which is dimensionless. Considering the influence of polymer rheological properties on phase separation, a viscoelastic correction term is introduced: In the formula, The coupling coefficient is dimensionless. Let be the corresponding force tensor of the polymer, in Pascals. The final particle size distribution of the microspheres can be described as a log-normal distribution: In the formula, Let be the probability density function of particle size distribution; The diameter of the microsphere is in meters. The geometric mean diameter of the microsphere is in meters. The standard deviation is dimensionless. The deviation between theoretical predictions and measured values ​​should be controlled within 10%; otherwise, the preparation parameters need to be readjusted.

[0117] The specific implementation of step S08 is the preparation of an injectable filler. First, a certain amount of dried composite microspheres are taken and added to cross-linked sodium hyaluronate gel, with the mass ratio of microspheres to gel controlled within the range of 1:5 to 1:20. The sodium hyaluronate gel is pre-treated with 1,4-butanedialdehyde, diene disiloxane, or polyethylene glycol diglycidyl ether cross-linking agent, with a cross-linking degree of 1% to 5%. The mixing process uses a twin-screw extruder or a high-shear homogenizer at a speed of 500 to 2000 rpm for a mixing time of 5 to 30 minutes. The mixing parameters are optimized based on a microsphere dispersion optimization function, which treats the microspheres as suspended particles and establishes the relationship between relative viscosity, microsphere volume fraction, maximum packing fraction, and intrinsic viscosity. In the formula, The viscosity is a dimensionless relative viscosity. The volume fraction of the microspheres is dimensionless. The maximum stacking fraction is dimensionless and typically ranges from 0.6 to 0.7. Here, represents the intrinsic viscosity, expressed in cubic centimeters per gram. The probability function for microsphere aggregation can be expressed as: In the formula, Let be the probability of microsphere aggregation, which is dimensionless; Frequency factor, in seconds ; Shear rate, in seconds ; This is a mixed time, expressed in seconds. The energy barrier for the interaction between microspheres is expressed in joules. Boltzmann's constant, Joules / Kelvin. Equation for determining the optimal mixing parameters: In the formula, The optimal shear rate, in seconds. ; The optimal mixing time is specified in seconds. The mixed filler is sterilized by filtration through a 0.45-micron filter membrane, filled into sterile syringes, sealed, and then sterilized by gamma irradiation at a dose of 15 to 25 kGrey to ensure sterility without affecting the microsphere structure.

[0118] The specific implementation of step S09 involves conducting biocompatibility evaluation and collagen regeneration efficiency testing. First, an in vitro cell proliferation experiment is performed. An appropriate amount of filler is injected and co-cultured with fibroblasts for 72 hours. Cell viability is determined using the thiazolyl blue colorimetric method, and cytokine secretion levels are detected using enzyme-linked immunosorbent assay (ELISA). Then, an animal subcutaneous injection experiment is conducted, using rabbits or mice as experimental subjects. Histological changes are observed at 1, 7, 14, and 28 days after subcutaneous injection of the filler. Inflammatory response is assessed using hematoxylin-eosin staining, and collagen content is determined using Sirius red staining. The experimental results are analyzed using a polylactic acid microsphere biological effect prediction model. This model is a multimodal deep learning network containing three parallel feature extraction branches and a fusion prediction module. The evaluation indicators for the model prediction include the cell growth curve fitting results. In the formula, Let t be the number of cells at time t; This represents the initial number of cells; This is the cell growth rate constant, expressed in hours. ; This represents the maximum cell volume. Collagen content is assessed using quantitative indicators: In the formula, The collagen growth index is dimensionless. The collagen content of the sample group is expressed in micrograms per milligram of tissue. The collagen content in the control group is expressed in micrograms per milligram of tissue. If biocompatibility evaluation shows a cell viability rate below 80%, the inorganic particle content is adjusted; if the collagen regeneration efficiency is less than 40% of the control group, the molecular weight of polylactic acid is increased or the proportion of polymer components is adjusted. When both biocompatibility and collagen regeneration efficiency reach the preset thresholds, the preparation of the composite microsphere injection filler is complete.

[0119] To better understand and implement this invention, Example 2, a specific application scenario of this invention, is provided below: Researchers used a supergravity emulsification process to prepare microspheres composed of poly(L-lactic acid) polyethylene glycol block copolymer and zinc oxide for use as a filler in medical aesthetic injections. Amphiphilic polylactic acid organic materials and inorganic particles were emulsified using surfactants and supergravity equipment to form an S / O / W coated microsphere suspension emulsion, which was then separated and freeze-dried to obtain organic-inorganic composite microspheres.

[0120] The specific preparation method is as follows: First, prepare the aqueous phase solution by adding 1.8g of polyvinyl alcohol to 180ml of deionized water and heating and stirring at 80℃ for 1 hour, then naturally cooling to room temperature. Next, prepare the oil phase solution by dissolving 0.72g of PEG-PLLA with a molecular weight of 180,000 and 0.018g of zinc oxide particles with a particle size of 15nm in 36ml of dichloromethane. Set the rotation speed of the hypergravity equipment to 1200r / min using a frequency converter. The water and oil phase solutions are then introduced into the liquid inlet via pumps, with the aqueous phase feed rate set to 1500ml / min and the oil phase feed rate to 300ml / min. The motor and pump are then turned on to achieve feeding and hypergravity emulsification of the water and oil phase solutions. The S / O / W emulsion is collected from the liquid outlet at the bottom of the hypergravity equipment.

[0121] Under room temperature conditions, the emulsion emulsified by gravity was mechanically stirred at 450 r / min for 8 h to remove dichloromethane solvent. The solvent-removed and solidified microsphere suspension was centrifuged and washed at 5000 r / min, with 40 ml of deionized water used for each wash, for a total of 4 centrifugation washes. Finally, it was freeze-dried to obtain PEG-PLLA@ZnO composite microspheres.

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

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

[0124]

[0125] The prepared PEG-PLLA@ZnO composite microspheres were mixed with cross-linked sodium hyaluronate gel at a mass ratio of 1:15 and sterilized before being used as a filler for cosmetic injection. In vitro cell experiments and subcutaneous injection experiments in mice were conducted to evaluate its biological properties. The results showed that the composite microsphere filler had good biocompatibility, with a cell survival rate of 95.3% in in vitro cell proliferation experiments, higher than the 87.2% of pure sodium hyaluronate gel. Sixteen weeks after subcutaneous injection in mice, the collagen content at the injection site increased by 68.7%, significantly higher than the 23.4% in the sodium hyaluronate injection group and the 42.1% in the PEG-PLLA microsphere group without zinc oxide.

[0126] Analysis of collagen biosynthesis-related gene expression showed that the COL1A1 expression level in the PEG-PLLA@ZnO / HA group was upregulated by 3.6 times compared to the control group and by 1.8 times compared to the PEG-PLLA / HA group. Simultaneously, the expression of inflammation-related genes was reduced by 42% compared to 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 produced during polylactic acid degradation and alleviated the inflammatory response.

[0127] The following is a specific example, Example 3: Researchers used hypergravity emulsification technology to develop PLLA-magnesium oxide composite microspheres for use as a filler in medical injections. Example 3 investigated the effect of the rotational speed of the hypergravity device on the morphology and particle size distribution of the microspheres.

[0128] The specific preparation process is as follows: Prepare the aqueous phase solution by adding 1.5g of polyvinyl alcohol to 150ml of deionized water, heating and stirring at 80℃ for 1 hour, then naturally cooling to room temperature, and adding 25ml of glycerol solution and stirring until homogeneous. Prepare the oil phase solution by dissolving 0.06g of magnesium oxide particles with a particle size of 40nm and 0.6g of PLLA with a molecular weight of 120,000 in 15ml of dichloromethane.

[0129] Comparative experiments were conducted using a hypergravity apparatus with rotation speeds of 800 r / min, 1000 r / min, and 1200 r / min. The feed rate for the aqueous phase solution was uniformly set at 1000 ml / min, and the feed rate for the oil phase solution was set at 100 ml / min. The motor and pump were activated to feed the two-phase solution (water and oil) and perform hypergravity emulsification. The primary emulsion was collected from the liquid outlet at the bottom of the hypergravity apparatus. The primary emulsion was mechanically stirred at room temperature and 400 r / min for 12 hours to remove the dichloromethane solvent. The microsphere suspension was then centrifuged and washed at 5000 r / min, using 30 ml of deionized water for each wash, for a total of three centrifugation washes. Finally, the microspheres were freeze-dried to obtain PLLA@MgO composite microspheres.

[0130] The particle size distribution of PLLA@MgO composite microspheres prepared under different rotation speeds is shown in Table 2:

[0131] Table 2. Particle size distribution of PLLA@MgO composite microspheres at different rotation speeds of hypergravity equipment.

[0132]

[0133] As shown in Table 2, with the increase of the rotational speed of the hypergravity equipment, the particle size of the prepared PLLA@MgO composite microspheres gradually decreased, and the particle size distribution became more concentrated. This is because the increased rotational speed leads to enhanced 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 With the maximum stable droplet diameter It is inversely proportional, which is consistent with the experimental results.

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

[0135] The following is a specific example, Example 4: Researchers investigated the effects of different inorganic particles on the properties of polylactic acid (PLA)-based microspheres and designed a method for preparing composite microspheres using combined inorganic particles. PLGA was used as the organic material, and the combination of multiple inorganic particles improved the biological properties of the microspheres.

[0136] The specific preparation method is as follows: Polyvinyl alcohol (PVA) was weighed at a concentration of 40 mg / ml and added to deionized water. The mixture was heated and stirred at 80°C for 1 hour, then naturally cooled to room temperature to obtain an aqueous solution. Polyvinyl alcohol (PLGA) with a molecular weight of 150,000 and an LA / GA ratio of 80:20 was weighed at a concentration of 12 mg / ml, and three oil-phase solutions were prepared by adding different proportions of inorganic particles: ① zinc oxide particles at a concentration of 1.5 mg / ml; ② magnesium oxide particles at a concentration of 1.5 mg / ml; ③ a mixture of zinc oxide / magnesium oxide / magnesium carbonate particles with a total concentration of 1.5 mg / ml and a mass ratio of 2:2:1. Ethyl acetate was used as the solvent for all oil phases, and the water-oil volume ratio was 5:1.

[0137] The temperature of the circulating water in the jacket of the hypergravity equipment was adjusted to 8℃, and the rotation speed of the hypergravity equipment was set to 2000 r / min. The water-oil two-phase solution was introduced into the liquid inlet via separate pumps, with the water phase feed rate at 500 ml / min and the oil phase feed rate at 100 ml / min. The motor and pumps were turned on to achieve feeding of the water-oil two-phase solution and hypergravity emulsification. The S / O / W emulsion was collected from the liquid outlet at the bottom of the hypergravity equipment. The hypergravity emulsion was mechanically stirred at 600 r / min for 6 hours at room temperature to remove the ethyl acetate solvent. The solvent-removed and solidified microsphere suspension was centrifuged and washed at 5000 r / min, using 40 ml of deionized water for each wash, for a total of 4 centrifugal washes. The microspheres were then freeze-dried to obtain three types of PLGA-based composite microspheres.

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

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

[0140]

[0141] Three types of microspheres were mixed with cross-linked 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 PLGA@ZnO / MgO / MgCO3 / HA group showed the highest cell viability at 97.1%, while PLGA@ZnO / HA and PLGA@MgO / HA showed 94.3% and 95.2%, respectively. Inflammation-related gene expression assays showed that the PLGA@ZnO / MgO / MgCO3 / HA group had the lowest expression levels of inflammatory factors, decreasing by 58.6% compared to the control group. This is attributed to the synergistic effect of multiple inorganic particles, which not only neutralize the acidic substances produced by polylactic acid degradation but also slowly release metal ions to regulate the cellular microenvironment.

[0142] Sixteen weeks after subcutaneous injection in mice, collagen content at the injection sites of all three groups significantly increased, with the PLGA@ZnO / MgO / MgCO3 / HA group showing the most significant increase at 82.5%, while the PLGA@ZnO / HA and PLGA@MgO / HA groups showed increases of 73.6% and 76.2%, respectively. Expression level detection of the collagen biosynthesis-related gene COL1A1 showed a 4.2-fold upregulation in the PLGA@ZnO / MgO / MgCO3 / HA group compared to the control group, significantly higher than the other two groups.

[0143] Traditional methods for preparing polylactic acid (PLA) microspheres typically employ mechanical stirring emulsification or membrane emulsification. Mechanical stirring produces microspheres with a wide particle size distribution, typically with a span value between 1.5 and 3.0, requiring sieving before use in medical injections. Membrane emulsification, while producing a narrower particle size distribution, involves complex equipment and low production efficiency. This invention utilizes hypergravity emulsification technology, leveraging the strong shear force generated by a hypergravity field to disperse the oil phase into tiny droplets. The resulting microspheres have a narrow particle size distribution (span values ​​generally less than 1.2), allowing for direct use in medical injections without sieving, significantly improving production efficiency and reducing production costs.

[0144] Furthermore, traditional polylactic acid (PLA)-based fillers tend to create a localized acidic environment during in vivo degradation, inducing inflammatory responses and resulting in low patient satisfaction. This invention introduces inorganic particles such as zinc oxide, magnesium oxide, and magnesium carbonate into PLA-based microspheres, effectively neutralizing the acidic substances produced during PLA degradation and significantly reducing inflammatory responses. Simultaneously, these metal ions promote collagen synthesis, enhancing the filling effect. In particular, the combined use of multiple inorganic particles exhibits a synergistic effect, significantly improving the biocompatibility and efficacy of the filler. Experimental results show that the composite microsphere filler containing inorganic particles outperforms traditional products in terms of biocompatibility and collagen regeneration capacity, providing a new direction for the development of injectable fillers in medical aesthetics.

[0145] Figure 2 The comparison results of the percentage increase in collagen content and cell survival rate of different composite microsphere fillers in Examples 2-4 are presented. The charts show the percentage increase in collagen content in bar chart form (blue bars) and the cell survival rate in line chart form (red line). It is clear from the figures that the increase in collagen content shows a significant upward trend with the introduction and optimization of inorganic particles in the composite microspheres. The collagen increase was lowest in the sodium hyaluronate injection group (23.4%), while the PLGA@ZnO / MgO / MgCO3 / HA group (Example 4) showed the best effect (82.5%). Meanwhile, the cell survival rate data shows that the composite microsphere filler containing inorganic particles has better biocompatibility, and the cell survival rate of all examples is higher than that of the sodium hyaluronate group. Among them, the PLGA@ZnO / MgO / MgCO3 / HA group has the highest cell survival rate, reaching 97.1%. This chart intuitively demonstrates the superior performance of different embodiments of the present invention, especially the synergistic effect exhibited by the combination of multiple inorganic particles.

[0146] To better understand and implement this invention, Example 5, a specific application scenario of this invention, is provided below: Researchers prepared a composite microsphere injection filler using poly(L-lactic acid)-polyethylene glycol (PLLA-PEG) block copolymer and zinc oxide nanoparticles. First, 8.0 g of 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 mL of ethyl acetate to prepare an oil phase solution with a concentration of 100 mg / mL. The solution was stirred at 25°C for 4 hours until completely transparent. Subsequently, 0.4 g of zinc oxide nanoparticles (average particle size 52 nm) were added to the oil phase solution, making the mass ratio of zinc oxide nanoparticles to polymer 1:20. The solution was ultrasonically dispersed for 30 minutes to ensure that the inorganic particles were uniformly dispersed in the oil phase.

[0147] The aqueous solution was prepared by dissolving 12 g of polyvinyl alcohol (molecular weight 23000, degree of hydrolysis 88%) in 400 mL of water for injection. The solution was stirred in an 80 °C water bath 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 performed using a rotating disc hypergravity apparatus at a speed of 1500 rpm, equivalent to approximately 150 times the acceleration due to gravity. The oil phase solution was simultaneously injected into the hypergravity apparatus at a flow rate of 12 mL / min and the aqueous phase solution at a flow rate of 60 mL / min using a metering pump. Under shear force, an oil-in-water emulsion was formed, and the microsphere suspension emulsion flowing out from the bottom outlet of the apparatus was collected. The collected emulsion was transferred to a 500 mL reaction vessel and mechanically stirred at 500 rpm for 8 hours to promote the evaporation of organic solvents.

[0149] The microsphere suspension was washed by centrifugation at 4000 rpm for 8 minutes. The supernatant was discarded, and the suspension was resuspended in 200 mL of fresh water for injection. The centrifugation and washing were repeated 4 times until the residual concentration of ethyl acetate in the supernatant was less than 8 μg / mL (detected by gas chromatography) and the residual concentration of polyvinyl alcohol was less than 3 μg / mL (detected by ultraviolet spectrophotometry).

[0150] The washed microsphere suspension was transferred to a freeze-drying flask. After pre-freezing at ℃ for 6 hours, connect to a freeze dryer and set the condenser temperature to [temperature value missing]. ℃, vacuum degree 15 Pa. Three-stage drying is performed according to the procedure: first stage drying... ℃, 12 hours; secondary drying Drying at ℃ for 8 hours; then three-stage drying at 0℃ for 6 hours. After drying, 6.8 g of white, loose powdery composite microspheres were obtained, with a yield of 85%.

[0151] The particle size distribution of microspheres was determined 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 calculated Span value for the microsphere particle size uniformity was 0.97, indicating a relatively uniform particle size distribution. Scanning electron microscopy (SEM) observation of the microsphere morphology revealed that the microspheres were spherical with slight surface pores, and the zinc oxide nanoparticles were uniformly distributed within the microspheres. Thermal analysis determined that the composite microspheres had a glass transition temperature of 56℃, a melting point of 172℃, and a thermal degradation temperature of 235℃. The thermal analysis results are shown in Table 5.

[0155] Table 5. Thermal analysis results of composite microspheres

[0156]

[0157] 5 grams of dried composite microspheres were cross-linked with 75 grams of sodium hyaluronate gel (molecular weight 2.0×). The crosslinking degree is 3%, and the gel viscosity is 3500 mPa·s. It is a mixture of 1,4-butyraldehyde (crosslinked) at a mass ratio of 1:15. The physicochemical properties of sodium hyaluronate gel are shown in Table 6.

[0158] Table 6 Physicochemical properties of cross-linked sodium hyaluronate gel

[0159]

[0160] The mixture was mixed using a twin-screw extruder at a speed of 1200 rpm for 15 minutes. Based on the microsphere dispersion optimization function, the optimal mixing parameter was a shear rate of 1050. The mixing time was 14.8 minutes, and the theoretical microsphere aggregation probability was 0.032. The mixed filler was filtered through a 0.45-micron filter membrane, filled into 2 ml sterile syringes, and sterilized by gamma irradiation at a dose of 20 kGry.

[0161] The biocompatibility of the prepared injectable filler was evaluated. Human skin fibroblasts (HSF) were co-cultured with the filler for 72 hours. The cell viability was determined by the thiazolyl blue colorimetric method, and the cell viability was 92.5%, indicating that the filler has good biocompatibility. The cytokine detection results are shown in Table 7.

[0162] Table 7. Cytokine levels in fibroblast culture supernatant

[0163]

[0164] Histological changes were observed after subcutaneous injection of filler in rabbits, and collagen content was detected using Sirius red staining. Table 8 shows the changes in collagen content at different time points after injection.

[0165] Table 8. Changes in collagen content at different time points after injection.

[0166]

[0167] Research data showed that 28 days after injection, collagen content increased by 63.8% compared to the control group, far exceeding the preset threshold of 40%, indicating that the composite microsphere injection filler has a good collagen regeneration effect. Histological analysis did not observe obvious inflammatory response, and cell proliferation and angiogenesis were active in the injection area. Figure 3This paper presents the trend of collagen content changes at different time points (1 day, 7 days, 14 days, and 28 days) after filler injection in Example 5. The graphs use scatter plots and fitted curves to illustrate the increase in collagen content over time, and indicate the percentage increase relative to the control group at each time point. As can be seen from the graphs, collagen content increases significantly with prolonged injection time, exhibiting a non-linear growth trend. Initially (1 day after injection), the collagen content was 45.2 μg / mg tissue, with a growth rate of only 5.4%; by 28 days after injection, the collagen content reached 92.1 μg / mg tissue, with a growth rate as high as 63.8%. The red dashed line in the graph marks the critical collagen level (65 μg / mg), indicating that this level was reached approximately 8-9 days after injection, demonstrating that the filler has a rapid effect on promoting collagen regeneration.

[0168] The experimental data were analyzed using a polylactic acid microsphere biological effect prediction model. The predicted in vivo degradation cycle of the filler was 10–12 months, and the collagen regeneration duration was 12–16 months. The volume retention rate curves after injection are shown in Table 9.

[0169] Table 9. Prediction of Volume Retention Rate of Injectable Fillers

[0170]

[0171] Figure 4A predictive model for the volume retention rate of the injectable filler developed in Example 5 is presented. The graph uses scatter plots and exponential fitting curves to illustrate the volume retention of the filler in vivo for up to 18 months, comparing it with that of a conventional filler (blue dashed line). The time axis is divided into three phases: initial filling period (0-6 months, green area), maintenance period (6-12 months, yellow area), and degradation period (12-20 months, red area). The data shows that the filler of this invention retains 95.2% of its volume after 1 month, 72.3% after 6 months, and 42.8% after 12 months, significantly outperforming conventional fillers at the same time points (92.1%, 60.2%, and 25.8%, respectively). The graph indicates that the filler's half-life is approximately 10.5 months, at which point the volume retention rate is 50%. This graph visually demonstrates the long-lasting performance advantages of the filler of this invention, providing important predictive evidence for clinical applications. Traditional methods for preparing injectable fillers in cosmetic medicine mainly employ conventional emulsification-solvent evaporation methods, such as stirring and homogenization. While these methods offer advantages like simple processes and low equipment requirements, they also suffer from drawbacks such as wide microsphere size distribution (Span value typically between 1.5 and 3.0), low preparation efficiency, and poor batch-to-batch consistency. Furthermore, polylactic acid microspheres prepared using traditional methods often induce strong inflammatory responses after injection, exhibit low collagen regeneration efficiency (usually less than 30% of the control group), and poor volume stability, affecting the durability and naturalness of the cosmetic effect.

[0172] Compared to traditional methods, this invention employs hypergravity equipment for shear emulsification, significantly improving microsphere particle size uniformity (Span value reduced to below 1.0) and increasing preparation efficiency (yield increased by 15%–25%). By introducing inorganic particles to form organic-inorganic composite microspheres, the degradation rate of microspheres is effectively regulated, inflammatory responses are reduced, and collagen regeneration efficiency is improved (reaching 63.8% of the control group, approximately 30% higher than traditional methods). Simultaneously, this invention uses multiple theoretical models to guide process parameter optimization, including shear energy balance equations, Fick diffusion equations, phase separation kinetic models, and microsphere dispersion optimization functions, establishing a quantitative relationship between process, structure, and performance, enabling precise control and prediction of product performance. Furthermore, this invention establishes a predictive model for the biological effects of polylactic acid microspheres, accurately assessing and predicting the biological properties of the filler, providing a scientific basis for the clinical application of the product. Overall, the composite microsphere injection filler preparation method provided by this invention has significant technological innovation and practical value, offering new ideas and methods for the development of medical aesthetic fillers.

[0173] It should be noted that the variables involved in this invention are explained in detail in Tables 10 and 11 below.

[0174] Table 10 Variable Explanation Table (Part 1)

[0175]

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

[0177]

[0178] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing composite microspheres for medical aesthetic injectable fillers, comprising preparing a polymer oil phase solution, preparing a surfactant aqueous phase solution, performing shear emulsification in a high-gravity apparatus to form an oil-in-water emulsion, mechanically stirring at room temperature to promote the evaporation of organic solvents, centrifuging and washing to remove residues, freeze-drying to obtain composite microspheres, measuring the microsphere particle size distribution, and mixing with cross-linked sodium hyaluronate gel to prepare an injectable filler and evaluating it, characterized in that, A high-gravity apparatus was used to generate a strong shear force field to precisely control the emulsification process. The rotation speed of the high-gravity apparatus was set to 300 to 3000 rpm. The aqueous phase solution and the oil phase solution were simultaneously introduced into the high-gravity apparatus through a metering pump. Shear emulsification was carried out at room temperature to form a water-in-oil-in-solids emulsion. During the emulsification process, the droplet formation kinetics followed the shear energy balance equation, the solvent evaporation process followed the Fick diffusion equation, the microsphere particle size was theoretically estimated using a phase separation kinetic model, the optimal operating parameters for the mixing process were determined using a microsphere dispersion optimization function, and the analysis and optimization were carried out using a polylactic acid microsphere biological effect prediction model to achieve the preparation of highly stable composite microspheres. The shear energy balance equation is specifically expressed as follows: ； In the formula, This represents the maximum stable droplet diameter under equilibrium conditions. For oil-water interfacial tension, The density of the continuous phase, i.e., the water phase. To equip angular velocity in supergravity, The characteristic radius of the hypergravity equipment. The viscosity of the dispersed phase, i.e., the oil phase, The viscosity is for the continuous phase, i.e., the aqueous phase. These are constants related to the geometry of the equipment.

2. The method as described in claim 1, characterized in that, The polymer oil phase solution comprises dissolving polylactic acid glycolate copolymer or poly-L-lactic acid polyethylene glycol block copolymer in dichloromethane or ethyl acetate at a concentration of 20 to 200 mg / mL, and adding zinc oxide or magnesium oxide inorganic particles to the oil phase solution, wherein the mass ratio of inorganic particles to polymer is controlled in the range of 1:200 to 1:

5.

3. The method as described in claim 2, characterized in that, The aqueous surfactant solution comprises polyvinyl alcohol or Tween 80 surfactant dissolved in water for injection at a concentration of 5 to 100 mg / mL, with the ratio of aqueous solution to oil solution controlled at 1:1 to 20:

1.

4. The method as described in claim 3, characterized in that, Centrifugal washing to remove residues involves centrifuging the microsphere suspension 3 to 5 times, discarding the supernatant after each centrifugation, and resuspending it in fresh water for injection to remove residual organic solvents and surfactants.

5. The method as described in claim 4, characterized in that, The process of obtaining composite microspheres by freeze-drying involves freeze-drying a washed microsphere suspension at a temperature controlled between -40 and -60 degrees Celsius, maintaining a vacuum level between 10 and 50 Pa, and drying for at least 24 hours.

6. The method as described in claim 5, characterized in that, The determination of microsphere size distribution includes using a laser particle size analyzer to evaluate the uniformity of microsphere size and calculating the 10th percentile, 50th percentile, and 90th percentile of the microsphere size distribution.

7. The method as described in claim 6, characterized in that, The preparation of injectable fillers by mixing with cross-linked sodium hyaluronate gel involves mixing dried organic-inorganic composite microspheres with cross-linked sodium hyaluronate gel at a mass ratio of 1:5 to 1:20, and preparing injectable fillers through an aseptic filling process.

8. The method as described in claim 7, characterized in that, The oil-in-water emulsion refers to a three-phase system in which inorganic particles are coated with amphiphilic polylactic acid (PLA) material to form oil droplets dispersed in an aqueous phase. The solid phase is the inorganic particles, the oil phase is a polymer solution, and the aqueous phase is an aqueous surfactant solution. The amphiphilic PLA material refers to a PLA derivative containing both hydrophilic and lipophilic groups, including PLA copolymers with a lactic acid to glycolic acid ratio of 90:10, PLA copolymers with glycolic acid, or PLA block copolymers with poly(L-lactic acid) and polyethylene glycol, wherein the PLA molecular weight is 80,000 to 240,000 and the polyethylene glycol molecular weight is 700 to 6,000.

9. The method as described in claim 8, characterized in that, Microsphere particle size uniformity is assessed using the Span value, which is calculated as follows: Span value equals 90th percentile particle size minus 10th percentile particle size, divided by 50th percentile particle size. The smaller the Span value, the more uniform and concentrated the microsphere particle size distribution.

10. The method as described in 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 optimal duration for which the microspheres and sodium hyaluronate gel need to be mixed, and the optimal mixing speed is the optimal stirring rate during the mixing process.

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