Emulsifier for microspheres, PLLA microspheres containing the same, and their application in composite fillers for medical aesthetics

Abstract

The present invention belongs to the technical field of biomedical polymer materials, and discloses an emulsifier for microspheres, PLLA microspheres containing the same, and their application in composite fillers for medical aesthetics. The present invention obtains sodium alginate with a molecular weight of 5-8 kDa by directional hydrolysis, and prepares Alg-MP through octadecyl nucleophilic substitution and sulfate modification; then uses Alg-MP as an emulsifier and adopts pore-forming technology to prepare porous PLLA microspheres to prepare PLLA microspheres, and then mixes with Ca-containing 2+ A composite system was formed by mixing a high-guluronic acid (G)-content sodium alginate with a high-G sodium alginate (G content ≥70%) solution. This composite system exhibited a storage modulus of 850-1200 Pa at 37°C, and an injection force of ≤12 N at a microsphere / excipient mass ratio of 1:0.3-1:0.7. Animal experiments demonstrated a volume retention rate of 92.3±3.5% after 6 months, and a 2.9-fold increase in new collagen density compared to the control group. This composite system can be used as an injectable gel for facial contouring or skin anti-aging, providing a new solution for the development of medical aesthetic filler materials.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical polymer materials, and specifically relates to an emulsifier for microspheres, PLLA microspheres containing the emulsifier, and their application in composite fillers for medical aesthetics. Background Art

[0002] With the rapid development of the medical aesthetics industry, the demand for safe, effective, and biocompatible filler materials is increasing. As a biodegradable polymer, poly (L-lactic acid) (PLLA) microspheres hold broad application prospects in the medical aesthetics field. Due to their excellent biocompatibility, controlled degradation rate, and ability to promote collagen production, PLLA microspheres are widely used in facial contouring and anti-aging applications.

[0003] However, the emulsifiers used in the traditional preparation of PLLA microspheres often have cytotoxicity issues. These small molecule emulsifiers are difficult to completely remove from the body and may cause inflammatory reactions or other adverse reactions. Therefore, the development of a new emulsifier that can both reduce cytotoxicity and improve the hydrophilicity of the microspheres has become a current research focus. In addition, the hydrophilicity and biocompatibility of PLLA microspheres need to be improved to better adapt to the human tissue environment. Summary of the Invention

[0004] The present invention aims to provide an emulsifier for microspheres, PLLA microspheres containing the same, and their use in composite fillers for medical aesthetics, thereby addressing at least one of the aforementioned technical problems. Specifically, the present invention provides a biodegradable PLLA microsphere based on a sulfated alginate emulsifier system, which is then combined with high-G sodium alginate to form a medical aesthetic filling system, providing a new solution for the development of medical aesthetic filling materials.

[0005] According to a first aspect of the present invention, there is provided an emulsifier for microspheres, which is prepared by the following method:

[0006] S1, hydrolyzing sodium alginate at pH 2.5-3.2 to obtain sodium alginate with a molecular weight of 5-8 kDa;

[0007] S2, dissolving sodium alginate with a molecular weight of 5-8 kDa in dimethyl sulfoxide, and reacting the sodium alginate with a C16-C18 alkyl bromide in the presence of a phase transfer catalyst to obtain a nucleophilic substitution product;

[0008] S3. After dissolving the nucleophilic substitution product in dichloromethane, chlorosulfonic acid is added dropwise, and the product is modified by sulfation with chlorosulfonic acid to obtain a sulfated sodium alginate derivative (Alg-MP).

[0009] The microsphere emulsifier of the present invention uses sodium alginate with good biocompatibility as a raw material. The sodium alginate is composed of β-D-mannuronic acid (M) and α-L-guluronic acid (G) connected by a β-1,4-glycosidic bond. The sodium alginate is directionally hydrolyzed in an acidic environment to obtain sodium alginate with a molecular weight of 5-8 kDa. The sodium alginate is then subjected to nucleophilic substitution with an alkyl bromide and modified with chlorosulfonic acid to obtain a sodium alginate-based emulsifier. The preparation method is simple. By adding chlorosulfonic acid, sulfate groups are introduced into the 5-8 kDa sodium alginate after nucleophilic substitution. The sodium alginate not only has certain antibacterial and anti-inflammatory effects, but also improves its surface activity and hydrophilicity, thereby enhancing the emulsification ability of the emulsifier. The resulting emulsifier has low cytotoxicity and high hydrophilicity, and further improves the safety of PLLA microspheres as an emulsifier.

[0010] In some embodiments, the molar ratio of C16-C18 alkyl bromide to sodium alginate structural unit is (1-1.5):1.

[0011] In some embodiments, the phase transfer catalyst is tetrabutylammonium bromide, and the amount thereof is 5% to 6% of the total molar number of sodium alginate, alkyl bromide and chlorosulfonic acid.

[0012] In some embodiments, in step S1, the pH of the sodium alginate aqueous solution is adjusted to 2.8, the temperature is raised to 70-80° C., and the hydrolysis reaction is carried out for 2-2.5 hours.

[0013] In some embodiments, purification is performed using dialysis.

[0014] In some embodiments, in step S3, the molar ratio of chlorosulfonic acid to the nucleophilic substitution product is 2.8:1-3.2:1.

[0015] According to a second aspect of the present invention, a PLLA microsphere is provided, which is prepared from a raw material containing the above-mentioned emulsifier for microspheres, has a porosity of 65-80%, a hierarchical pore structure of 0.5-2 μm formed on the surface, a residual dichloromethane content of ≤120 ppm, an average particle size of 35-55 μm, a span coefficient (D90-D10) / D50 ≤0.8, and a BET specific surface area ≥15 m 2 / g; Zeta potential -35 mV to -45 mV.

[0016] The PLLA microspheres of this invention use a sulfated sodium alginate derivative (Alg-MP) as an emulsifier, significantly reducing the cytotoxicity associated with traditional small-molecule emulsifiers and improving the hydrophilicity and biocompatibility of the PLLA microspheres. Through pore-forming technology and porous structure design, the mechanical properties and elasticity of the microspheres are enhanced, while also promoting the production of new collagen.

[0017] According to a third aspect of the present invention, a method for preparing the above-mentioned PLLA microspheres is provided, comprising the following steps:

[0018] (1) dissolving L-polylactic acid in dichloromethane to form an oil phase containing 8-12 wt% of the polymer;

[0019] (2) preparing a 0.1 M phosphate buffer solution containing 1.5-3.0 wt% of an emulsifier for microspheres as the aqueous phase;

[0020] (3) slowly injecting the oil phase into the water phase at a homogenization speed of 8000-12000 rpm to obtain a W / O emulsion;

[0021] (4) stirring the W / O emulsion at 30-55°C for 6-8 hours to evaporate the solvent;

[0022] (5) The stirred W / O emulsion is washed with anhydrous ethanol and freeze-dried to obtain PLLA microspheres.

[0023] The present invention uses pore-forming technology and porous structure design in the preparation method to prepare PLLA microspheres with an average particle size of 35-55 μm and a graded pore structure of 0.5-2 μm on the surface, thereby enhancing the mechanical properties and elasticity of the PLLA microspheres and promoting the generation of new collagen.

[0024] In some embodiments, in step (3), the stirring is magnetic stirring at a rotation speed of 300-400 rpm.

[0025] In some embodiments, in step (4), the stirring process includes the following heating program:

[0026] During the first 3 hours, the temperature was 35 ± 1 °C;

[0027] During the 3rd to 5th hour, the temperature was raised to 42±1°C;

[0028] During the last hour, the temperature was 50±1°C.

[0029] Specifically, when the stirring time is 6 hours, the temperature is maintained at 35±1°C for the first 3 hours to evaporate the solvent in the W / O emulsion, then the temperature is raised to 42±1°C for 2 hours, and finally the temperature is raised to 50±1°C for 1 hour to accelerate the removal of the solvent. The solvent in the W / O emulsion is evaporated by gradient temperature program to achieve the porosity of the PLLA microspheres controlled to 65%-80%.

[0030] According to the fourth aspect of the present invention, a composite filler for medical aesthetics is provided, which comprises, by weight percentage, 40-60 wt% of the above-mentioned PLLA microspheres, 20-35 wt% of high G sodium alginate and a Ca-containing 2+Phosphate buffer solution 15-25 wt%; wherein, high-G sodium alginate having a G content of ≥70% is used as an excipient.

[0031] The present invention's composite filler for medical aesthetics combines PLLA microspheres with high-G sodium alginate to form a novel medical aesthetic filling system, enhancing the material's safety and effectiveness. Because sodium alginate, with its high guluronic acid (G) content, exhibits enhanced gel-forming ability and mechanical strength, it can be used as an excipient in filler preparations. This novel medical aesthetic filling system not only exhibits excellent mechanical properties and injection thrust, but also significantly promotes the production of new collagen, providing new insights into the development of medical aesthetic filling materials.

[0032] In some embodiments, the high-G sodium alginate has a molecular weight of 80-120 kDa and a degree of esterification of ≤5%.

[0033] In some embodiments, the Ca 2+ Ca in phosphate buffer 2+ The concentration is 0.5-1.2 mM.

[0034] In some embodiments, the mass ratio of the excipient to the PLLA microspheres is 0.4:1-0.6:1. The excipient is high-G sodium alginate dissolved in a solution containing Ca 2+ Phosphate buffer solution to obtain high G sodium alginate solution.

[0035] According to a fifth aspect of the present invention, a method for preparing a composite filler for medical aesthetics is provided, comprising the following steps:

[0036] (1) Dissolve CaCl2 in PBS solution to obtain phosphate buffer solution containing 0.5-1.2 mM CaCl2, and then dissolve high-G sodium alginate in CaCl2 solution. 2+ Phosphate buffer to obtain high-G sodium alginate solution;

[0037] (2) mixing PLLA microspheres with high-G sodium alginate solution and performing vacuum degassing treatment;

[0038] (3) Allow to stand for cross-linking at 25-30°C for 30-45 minutes to form an injectable gel.

[0039] The preparation method of the composite filler for medical aesthetics of the present invention is simple. High-G sodium alginate is used as an excipient to cross-link with PLLA microspheres to form an injectable gel. The gel storage modulus (G') is 850-1200 Pa.

[0040] According to a sixth aspect of the present invention, there is provided an application of a medical aesthetic composite filler as a facial contouring or skin anti-aging preparation, wherein the application is to inject the medical aesthetic composite filler into the dermis, subcutaneous tissue layer or fascia layer of the face.

[0041] The composite filler for medical aesthetics of the present invention significantly promotes the generation of new collagen, has good injection thrust, and can be injected into the dermis, subcutaneous tissue layer or fascia layer of the face to perform facial contour shaping or anti-aging of the skin.

[0042] In some embodiments, the injection dose of the composite filler for medical aesthetics is 0.8-1.2 mL / cm 2 , injection interval ≥ 6 months.

[0043] The present invention obtains sodium alginate with a molecular weight of 5-8 kDa by directional hydrolysis, and prepares sulfated sodium alginate derivative (Alg-MP) (HLB value 11.2-12.8) by octadecyl nucleophilic substitution and sulfation modification; then uses Alg-MP as an emulsifier and adopts pore-forming technology to prepare porous PLLA microspheres (porosity 65-80%), and then reacts with Ca-containing 2+ An injectable gel was prepared by mixing a high-guluronic acid (G)-content sodium alginate with a high-G sodium alginate solution (G content ≥70%) (as an excipient) to form a composite system. This composite system exhibited a storage modulus of 850-1200 Pa at 37°C, and an injection force of ≤12 N at a microsphere / excipient mass ratio of 1:0.3-1:0.7. Animal experiments demonstrated a volume retention rate of 92.3±3.5% after 6 months, and a 2.9-fold increase in the density of newly formed collagen compared to the control group. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is a synthetic route for the emulsifier for microspheres according to Example 1 of the present invention;

[0045] Figure 2 This is a SEM panoramic image of the PLLA microspheres obtained in Example 2 of the present invention;

[0046] Figure 3 This is the SEM magnified image of PLLA microspheres;

[0047] Figure 4 This is the SEM magnified image of a single PLLA microsphere;

[0048] Figure 5 This is a SEM image of the PLLA microspheres obtained in Comparative Example 1 of the present invention;

[0049] Figure 6 This is a SEM image of the PLLA microspheres obtained in Comparative Example 2 of the present invention;

[0050] Figure 7 H&E staining results of the PLLA microsphere group in Experimental Example 2;

[0051] Figure 8 This is the H&E staining result of the HA group in Experimental Example 2;

[0052] Figure 9 This is the H&E staining result of the experimental group in Experimental Example 2. DETAILED DESCRIPTION

[0053] The present invention will be further described in detail below with reference to the accompanying drawings, but the embodiments of the present invention are not limited thereto. The raw materials and reagents involved in the following examples can all be obtained from commercial channels.

[0054] Example 1

[0055] This example provides a synthetic route for the microsphere emulsifier (Alg-MP). Figure 1 , and its preparation method comprises the following steps:

[0056] Approximately 10 g of sodium alginate was dissolved in 1 L of deionized water to obtain a sodium alginate aqueous solution. The pH of the sodium alginate aqueous solution was adjusted to approximately 2.8, heated to approximately 75°C, and stirred continuously for approximately 2.2 hours to allow for hydrolysis. After the reaction, unreacted small molecules and byproducts were removed by dialysis to obtain sodium alginate with a molecular weight of 5-8 kDa. Next, 5 g of sodium alginate with a molecular weight of 5-8 kDa was weighed and added to 100 mL of dimethyl sulfoxide (DMSO), along with 10 g of octadecyl bromide and 0.3 g of tetrabutylammonium bromide. The solution was stirred at room temperature for 24 hours to allow for nucleophilic substitution. After the reaction, the product was separated and purified by column chromatography to obtain the nucleophilic substitution product. The nucleophilic substitution product was dissolved in an appropriate amount of dichloromethane. Chlorosulfonic acid was added dropwise to the dichloromethane under an ice bath, maintaining a molar ratio of chlorosulfonic acid to the nucleophilic substitution product of 3:1, and the reaction was continued for 1 hour. After the reaction is completed, an appropriate amount of ice water is added to terminate the reaction, and the unreacted chlorosulfonic acid is removed by dialysis to obtain a sulfated sodium alginate derivative (Alg-MP), which is also an emulsifier for microspheres.

[0057] The relevant substances were characterized by hydrogen nuclear magnetic resonance spectroscopy, and the data were recorded as follows:

[0058] Sodium alginate 1 H NMR: δ5.37 (5H), 5.07 (2H), 5.00 (2H), 4.95 (1H), 4.49(1H), 4.48 (4H), 3.50 (2H), 3.41 (3H), 3.39 (3H), 3.36 (2H), 2.34 (2H).

[0059] Intermediate 1 1H NMR: δ5.37 (5H), 5.07 (2H), 5.00 (2H), 4.95 (1H), 4.49(1H), 4.48 (1H), 4.00 (6H), 3.98 (1H), 3.95 (2H), 3.50 (2H), 3.41 (3H), 3.39(3H), 3.36 (2H), 2.34 (2H), 1.78 (6H), 1.45 (4H), 1.31 (2H), 1.29 (6H), 1.27(6H), 1.26 (72H), 0.89 (9H).

[0060] Final product (emulsifier for microspheres) 1 H NMR: δ5.37 (3H), 5.07 (2H), 5.00 (2H), 4.95(1H), 4.49 (1H), 4.48 (2H), 4.06 (1H), 4.00 (8H), 3.98 (1H), 3.95 (2H), 3.50(1H), 3.41 (3H), 3.39 (3H), 3.36 (1H), 2.34 (2H), 1.78 (6H), 1.45 (4H), 1.31(2H), 1.29 (6H), 1.27 (6H), 1.26 (72H), 0.89 (9H).

[0061] Compared with the H NMR spectrum of sodium alginate, the H NMR spectrum of intermediate 1 added multiple characteristic peaks at 0.89-1.78 ppm, attributed to the methylene peak (-CH2-) and methyl peak (-CH3) of the long aliphatic chain, indicating that the molecular chain of octadecyl bromide has been successfully introduced into intermediate 1. Comparing the H NMR spectrum of the final product with that of intermediate 1, it can be seen that the number of hydrogen atoms in the final product increased at 4.48 and 4.00 ppm, and a new characteristic peak appeared at 4.06 ppm, indicating the successful synthesis of the emulsifier for microspheres.

[0062] In this example, sodium alginate with a molecular weight of 5-8 kDa was obtained by directional hydrolysis. Alg-MP was prepared through octadecyl nucleophilic substitution and sulfation modification. The HLB value of Alg-MP was determined to be 11.2-12.8, indicating that Alg-MP has strong hydrophilic properties and has potential as an emulsifier or surfactant.

[0063] Example 2

[0064] This embodiment provides a method for preparing poly (L-lactic acid) (PLLA) microspheres, comprising the following steps:

[0065] 10 g of PLLA was weighed and dissolved in 80 mL of dichloromethane to form an oil phase containing 10 wt% PLLA. The Alg-MP prepared in Example 1 was dissolved in 0.1 mol / L phosphate buffer solution to prepare 100 mL of a mixed solution containing 2.5 wt% Alg-MP as the aqueous phase. The oil phase was then slowly injected into the aqueous phase at a homogenizer speed of 10,000 rpm to form a W / O emulsion. The W / O emulsion was then placed at 25°C and magnetically stirred at 350 rpm for 7 hours. The stirring process was controlled by a stepwise temperature increase: maintaining 35±1°C for the first 3 hours, then gradually increasing the temperature to 42±1°C for the next 3 hours, and finally increasing the temperature to 50±1°C for the final hour to accelerate solvent removal from the W / O emulsion. The stirred W / O emulsion was then washed three times with anhydrous ethanol and freeze-dried to obtain PLLA microspheres.

[0066] The PLLA microspheres were subjected to scanning electron microscopy (SEM) experiments. Figure 2-4 shown. Figure 2 This is the SEM panoramic image of PLLA microspheres. Figure 3 This is the SEM magnified image of PLLA microspheres. Figure 4 This is a magnified SEM image of a single PLLA microsphere. Figure 2-4 The PLLA microspheres exhibit a porous structure with an average particle size of 35-55 μm, a hierarchical pore structure of 0.5-2 μm, and a porosity of 65%-80%. The residual dichloromethane content is ≤120 ppm (GC-MS analysis). The span coefficient of the PLLA microspheres is (D90-D10) / D50 ≤0.8, indicating a relatively uniform particle size distribution.

[0067] Example 3

[0068] This embodiment provides a method for preparing a composite filler for medical aesthetics, comprising the following steps:

[0069] 10 g of high-G sodium alginate (G content ≥70%, molecular weight 100 kDa, esterification ≤5%) was dissolved in 100 mL of PBS solution containing 0.8 mM CaCl2 and stirred evenly to obtain a high-G sodium alginate solution. Then, 50 g of the PLLA microspheres prepared in Example 2 were mixed with 30 g of the high-G sodium alginate solution. After vacuum degassing, the mixture was allowed to stand and cross-link at 28°C for 40 minutes to form an injectable gel.

[0070] Example 4

[0071] This embodiment provides a method for preparing a composite filler for medical aesthetics, comprising the following steps:

[0072] 10 g of high-G sodium alginate (G content 70%-90%, molecular weight 100 kDa, degree of esterification 0.1%-5%) was dissolved in 100 mL of PBS solution containing 0.8 mM CaCl2 and stirred evenly to obtain a high-G sodium alginate solution. Then, 50 g of the PLLA microspheres prepared in Example 2 were mixed with 20 g of the high-G sodium alginate solution. After vacuum degassing, the mixture was allowed to stand and cross-link at 28°C for 40 minutes to form an injectable gel.

[0073] Comparative Example 1

[0074] This comparative example provides a method for preparing PLLA microspheres, which uses sodium dodecylbenzenesulfonate as an emulsifier and includes the following steps:

[0075] 10 g of PLLA was dissolved in 80 mL of dichloromethane to form an oil phase containing 10 wt% PLLA. Sodium dodecylbenzenesulfonate was dissolved in 0.1 M phosphate buffer to prepare 100 mL of a mixed solution containing 2.5 wt% sodium dodecylbenzenesulfonate as the aqueous phase. The oil phase was then slowly injected into the aqueous phase at a homogenizer speed of 10,000 rpm to form a water-in-water (W / O) emulsion. The W / O emulsion was then magnetically stirred at 350 rpm at 25°C for 7 hours. The stirring process was controlled by a stepwise temperature increase: 35 ± 1°C for the first 3 hours, then gradually increased to 42 ± 1°C for 2 hours, and finally to 50 ± 1°C for 1 hour to accelerate solvent removal from the W / O emulsion. The stirred W / O emulsion was then washed three times with anhydrous ethanol and freeze-dried to obtain PLLA microspheres.

[0076] Comparative Example 2

[0077] This comparative example provides a method for preparing PLLA microspheres, which uses PVA as an emulsifier and includes the following steps:

[0078] 10 g of PLLA was dissolved in 80 mL of dichloromethane to form an oil phase containing 10 wt% PLLA. Polyvinyl alcohol (PVA) was dissolved in 0.1 M phosphate buffer to prepare a 100 mL mixed solution containing 2.5 wt% PVA as the aqueous phase. The oil phase was then slowly injected into the aqueous phase at a homogenizer speed of 10,000 rpm to form a W / O emulsion. The W / O emulsion was then magnetically stirred at 350 rpm at 25°C for 7 hours. The stirring process was controlled by a stepwise temperature increase: maintaining 35 ± 1°C for the first 3 hours, then gradually increasing the temperature to 42 ± 1°C for 2 hours, and finally increasing the temperature to 50 ± 1°C for 1 hour to accelerate solvent removal from the W / O emulsion. The stirred W / O emulsion was then washed three times with anhydrous ethanol and freeze-dried to obtain PLLA microspheres.

[0079] The PLLA microspheres obtained in Comparative Example 1 and the PLLA microspheres obtained in Comparative Example 2 were subjected to scanning electron microscopy (SEM) experiments. The results are as follows: Figure 5 and Figure 6 shown. Figure 5 This is the SEM image of the PLLA microspheres obtained in Comparative Example 1. Figure 6 The SEM image of the PLLA microspheres obtained in Comparative Example 2. Figure 5 and Figure 6 It can be seen that the PLLA microspheres obtained in Comparative Example 1 and the PLLA microspheres obtained in Comparative Example 2 are both solid microspheres. The above results show that the choice of emulsifier will affect the morphology and pore structure of PLLA microspheres.

[0080] Experimental Example 1: Rheological Test

[0081] The medical aesthetic composite filler of Example 3 was placed in the parallel plate fixture of a rheometer (e.g., Anton Paar MCR 302), the plate spacing was set to 1 mm, and the test temperature was controlled at 37°C to simulate human physiological conditions. Then, the oscillatory shear mode was started, the frequency range was set to 0.1-10 Hz, and a constant strain of 1% was applied to ensure that the sample was in the linear viscoelastic region. Next, the storage modulus (G') and loss modulus (G'') were recorded as a function of frequency using the instrument software. The results are shown in Table 1.

[0082] Table 1 Rheological test results

[0083]

[0084] The storage modulus of the medical aesthetic composite filler prepared in Example 3 was measured at 37°C to be 950 Pa, and the loss modulus was 120 Pa, indicating that the medical aesthetic composite filler is elastically dominant, has strong supporting force, low viscous response, and stable structure. At the same time, the viscosity of the medical aesthetic composite filler was measured to be around 4500 mPa·s, indicating that the medical aesthetic composite filler is moderately shear-thinning and easy to inject. After 3 cycles, the thixotropic recovery rate was measured to be 98.2%, indicating that the medical aesthetic composite filler has a high thixotropic recovery rate, enabling it to quickly recover its shape and remain stable after injection. The yield stress of the medical aesthetic composite filler is 85 Pa, indicating that the medical aesthetic composite filler has good resistance to deformation.

[0085] According to tests, when the mass ratio of high-G sodium alginate to PLLA microspheres is 0.4:1-0.6:1, the gel storage modulus (G') is 850-1200 Pa, indicating that the composite filler for medical aesthetics of the present invention has good mechanical properties.

[0086] Experimental Example 2: Animal Experiment

[0087] 2.1 Experimental methods

[0088] Thirty 8-week-old SD rats were randomly divided into an experimental group, a control group 1 (PLLA microsphere group), and a control group 2 (HA group), with 10 rats in each group. 0.5 mL of the sample was symmetrically implanted subcutaneously on the back. Acute inflammatory responses were observed in the short term (1 week), and volume retention was quantified by Micro-CT in the medium term (1 and 3 months) and long term (6 months). Tissue from the implanted area was stained with H&E (inflammatory score) and Masson's stain (collagen density). The experimental group received the aesthetic composite filler prepared in Example 3, the PLLA microsphere group received the PLLA microspheres prepared in Example 2, and the HA group received a commercial hyaluronic acid gel.

[0089] 2.2 Experimental Results

[0090] Figure 7 This is the staining result of PLLA microsphere group. Figure 8 This is the staining result of HA group. Figure 9 The staining results of the experimental group show that the collagen fibers in the experimental group are arranged in an orderly manner, and the density of newly formed collagen is higher than that in the PLLA microsphere group and the HA group.

[0091] The results of inflammation scores, volume retention, and new collagen density are shown in Table 2. As can be seen from Table 2, the inflammatory score in the experimental group at week 1 was 1.2±0.3, significantly lower than the 2.5±0.6 in the PLLA microsphere group and the 1.8±0.4 in the HA group. The aesthetic composite filler, through the antioxidant effect of sodium alginate, neutralizes the acidic products produced during the degradation of the PLLA microspheres, thereby preventing the pH drop after implantation that triggers an acidic microenvironment and reduces macrophage overactivation. The low inflammatory response after implantation of the aesthetic composite filler demonstrates the material's biocompatibility and provides a basis for safe clinical application.

[0092] In terms of volume retention rate, 6 months after implantation, the volume retention rate of the experimental group was (92.3±3.5)%, the volume retention rate of the PLLA microsphere group was (78.2±4.1)%, and the volume retention rate of the HA group was (65.4±5.2)%. The results showed that the volume retention rate of the experimental group was significantly higher than that of the PLLA microsphere group and the HA group, P < 0.01, which was statistically significant.

[0093] In terms of the density of new collagen, according to the results of Masson staining, the density of new collagen in the experimental group was (1.82±0.21) g / cm 3 The density of new collagen in the PLLA group was (0.63±0.15) g / cm 3 The density of new collagen in the HA group was (0.48±0.12) g / cm 3The experimental group showed a 2.9-fold increase in new collagen density compared to the PLLA group. These results suggest that, compared to implanting PLLA microspheres or hyaluronic acid gel, implanting composite fillers for medical aesthetics significantly promotes new collagen production in rats' subcutaneous tissues, making them suitable for facial contouring and anti-aging treatments.

[0094] Table 2 Animal experiment results

[0095]

[0096] 2.3 Results Analysis

[0097] High volume retention due to sodium alginate-Ca 2+ The durability of the cross-linked network and the degradation and sustained release of PLLA microspheres synergistically, sodium alginate and Ca 2+ The resulting cross-linked network provides initial mechanical support for the subcutaneous tissue in the early stages after implantation. During degradation, the PLLA microspheres release lactic acid, stimulating fibroblasts to secrete collagen and gradually replacing composite fillers used in medical aesthetics. The enhanced collagen regeneration is associated with the alginate oligosaccharide fragment (M fragment), which inhibits MMP-1 activity, reduces collagen degradation, and simultaneously activates the TGF-β / Smad pathway to promote ECM synthesis. The low inflammatory response in the experimental group confirms the material's superior biocompatibility and provides evidence for its safe clinical application.

[0098] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the inventive concept of the present invention, which all fall within the scope of protection of the present invention.

Claims

1. A method for preparing PLLA microspheres, characterized in that: The following steps are involved: (1) dissolving L-polylactic acid in dichloromethane to obtain an oil phase containing 8-12 wt% of the polymer; (2) preparing a 0.1 M phosphate buffer solution containing 1.5-3.0 wt % of an emulsifier for microspheres to obtain an aqueous phase; (3) injecting the oil phase into the water phase at 8000-12000 rpm to obtain a W / O emulsion; (4) Stir the W / O emulsion at 30-55°C for 6-8 hours; (5) washing the stirred W / O emulsion and freeze-drying it to obtain; Wherein, the microsphere emulsifier is prepared by the following method: S1, hydrolyzing sodium alginate at pH 2.5-3.2 to obtain sodium alginate with a molecular weight of 5-8 kDa; S2, dissolving sodium alginate with a molecular weight of 5-8 kDa in dimethyl sulfoxide, and reacting the alginate with a C16-C18 alkyl bromide in the presence of a phase transfer catalyst to obtain a nucleophilic substitution product; S3, dissolving the nucleophilic substitution product in dichloromethane, and adding chlorosulfonic acid dropwise to react to obtain a sulfated sodium alginate derivative; The phase transfer catalyst is tetrabutylammonium bromide, and its amount is 5%-6% of the total molar number of the sodium alginate, C16-C18 alkyl bromide and chlorosulfonic acid; The molar ratio of the chlorosulfonic acid to the nucleophilic substitution product is 2.8:1-3.2:

1.

2. The method for preparing PLLA microspheres according to claim 1, wherein In step (4), the stirring process includes the following heating program: During the first 3 hours, the temperature was 35 ± 1 °C; During the 3rd to 5th hour, the temperature was raised to 42±1°C; During the last hour, the temperature was 50±1°C. 3.PLLA microspheres, characterized in that The PLLA microspheres are prepared by the preparation method of claim 1 or 2, with a porosity of 65-80%, a hierarchical pore structure of 0.5-2 μm formed on the surface, an average particle size of 35-55 μm, and a Zeta potential of -35 mV to -45 mV.

4. A composite filler for medical aesthetics, characterized in that: In terms of weight percentage, it comprises 40-60wt% of the PLLA microspheres according to claim 3, 20-35wt% of high G sodium alginate and Ca-containing 2+ 15-25 wt% phosphate buffer; wherein the G content of the high-G sodium alginate is ≥70%.

5. The composite filler for medical aesthetics according to claim 4, characterized in that: The molecular weight of the high G sodium alginate is 80-120 kDa, the degree of esterification is 0.1%-5%, and the content of Ca 2+ Ca in phosphate buffer 2+ The concentration is 0.5-1.2 mM.

6. The method for preparing the composite filler for medical aesthetics according to claim 4 or 5, characterized in that: The following steps are involved: (1) Dissolve CaCl2 in PBS solution to obtain phosphate buffer solution containing 0.5-1.2 mM CaCl2, and dissolve high-G sodium alginate in Ca 2+ Phosphate buffer to obtain high-G sodium alginate solution; (2) mixing PLLA microspheres with high-G sodium alginate solution and performing vacuum degassing treatment; (3) Allow to stand for cross-linking at 25-30°C for 30-45 minutes to obtain the product.

7. Use of the composite filler for medical aesthetics according to claim 4 or 5 in the preparation of an anti-aging preparation for skin.

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