Emulsifying agent for microspheres, PLLA microspheres containing emulsifying agent and application of emulsifying agent in composite filling agent for medical beauty

Abstract

The invention belongs to the technical field of biomedical polymer materials, and discloses an emulsifier for microspheres, PLLA microspheres containing the emulsifier and application of the emulsifier in a composite filler for medical beauty. Sodium alginate with the molecular weight of 5-8 kDa is obtained through directional hydrolysis, and Alg-MP is prepared through octadecyl glycosylation and sulfation modification; alg-MP is used as an emulsifier, porous PLLA microspheres are prepared by adopting a pore-forming technology, the PLLA microspheres are mixed with a Ca < + >-containing sodium alginate solution with high guluronic acid (G) content and high G sodium alginate (G content is larger than or equal to 70%) to form a composite system, the storage modulus of the composite system at 37 DEG C reaches 850-1200 Pa, and the injection thrust is smaller than or equal to 12 N when the mass ratio of the microspheres to an excipient is 1: 0.3-1: 0.7. Animal experiments show that the six-month volume retention rate is 92.3 + / -3.5%, the density of new collagen is increased by 2.9 times compared with that of a control group, the composite system can be used as injectable gel for facial contour shaping or skin aging resistance, and a new solution is provided for development of medical beauty filling 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 applications of the emulsifier in composite fillers for medical aesthetics. Background Art

[0002] With the rapid development of the medical beauty industry, the demand for safe, effective and biocompatible filling materials is increasing. As a degradable polymer material, poly (L-lactic acid) (PLLA) microspheres have broad application prospects in the field of medical beauty. PLLA microspheres are widely used in facial contouring, skin anti-aging and other fields due to their good biocompatibility, controllable degradation rate and ability to promote collagen production.

[0003] However, the emulsifiers used in the traditional preparation of PLLA microspheres often have cytotoxicity problems. These small molecule emulsifiers are difficult to completely remove in the body and may cause inflammatory reactions or other adverse reactions. Therefore, the development of a new emulsifier that can reduce cytotoxicity and improve the hydrophilicity of microspheres has become the focus of current research. 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 purpose of the present invention is to provide an emulsifier for microspheres, PLLA microspheres containing the same, and their use in composite fillers for medical aesthetics, so as to solve at least one of the above technical problems. Specifically, the present invention provides a degradable PLLA microsphere based on a sulfated alginate emulsification system, and the microspheres are composited 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: 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 then reacting the sodium alginate with a C16-C18 alkyl bromide in the presence of a phase transfer catalyst to obtain a glycosylated product; S3. After dissolving the glycosylated 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).

[0006] The microsphere emulsifier of the present invention uses sodium alginate with good biocompatibility as a raw material. The sodium alginate is connected by β-D-mannuronic acid (M) and α-L-guluronic acid (G) through β-1,4-glycosidic bonds. The sodium alginate is directionally hydrolyzed in an acidic environment to obtain sodium alginate with a molecular weight of 5-8 kDa, and then glycosylated with alkyl bromide and modified with chlorosulfonic acid to obtain a sodium alginate-based emulsifier. The preparation method is simple. Chlorosulfonic acid is added to introduce sulfate groups into the glycosylated 5-8 kDa sodium alginate, which 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 finally obtained emulsifier has low cytotoxicity and high hydrophilicity, and further improves the safety of PLLA microspheres as an emulsifier.

[0007] In some embodiments, the molar ratio of C16-C18 alkyl bromide to sodium alginate structural unit is (1-1.5):1.

[0008] In some embodiments, the phase transfer catalyst is tetrabutylammonium bromide, and the amount of tetrabutylammonium bromide used is 5%-6% of the total molar number of sodium alginate, alkyl bromide and chlorosulfonic acid.

[0009] In some embodiments, in step S1, the pH of the aqueous solution of sodium alginate 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.

[0010] In some embodiments, the purification is performed using dialysis.

[0011] In some embodiments, in step S3, the molar ratio of chlorosulfonic acid to glycosylated product is 2.8:1-3.2:1.

[0012] 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%, and a hierarchical pore structure of 0.5-2 μm is formed on the surface, the residual dichloromethane content is ≤120 ppm, the average particle size is 35-55 μm, the span coefficient (D90-D10) / D50≤0.8; the BET specific surface area is ≥15 m² / g; and the Zeta potential is -35 mV to -45 mV.

[0013] The PLLA microspheres of the present invention use sulfated sodium alginate derivatives (Alg-MP) as emulsifiers, which significantly reduce the cytotoxicity problems caused by traditional small molecule emulsifiers and improve the hydrophilicity and biocompatibility of PLLA microspheres. Through pore-forming technology and porous structure design, the mechanical properties and elasticity of the microspheres are enhanced, while promoting the generation of new collagen.

[0014] According to a third aspect of the present invention, there is provided a method for preparing the above-mentioned PLLA microspheres, comprising the following steps: (1) dissolving L-polylactic acid in dichloromethane to form 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 as an aqueous phase; (3) slowly injecting the oil phase into the water phase at a homogenization speed of 8000-12000 rpm to obtain a W / O emulsion; (4) stirring the W / O emulsion at 30-55° C. for 6-8 h to evaporate the solvent; (5) The stirred W / O emulsion is washed with anhydrous ethanol and freeze-dried to obtain PLLA microspheres.

[0015] 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.

[0016] In some embodiments, in step (3), the stirring is magnetic stirring with a rotation speed of 300-400 rpm.

[0017] In some embodiments, in step (4), the stirring process includes the following heating program: Hours 0-3, temperature 35±1°C; 3-5 hours, raise the temperature to 42±1℃; During the last hour, the temperature was 50±1°C.

[0018] Specifically, when the stirring time is 6 hours, the stirring 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 control the porosity of the PLLA microspheres to 65%-80%.

[0019] According to a fourth aspect of the present invention, a composite filler for medical aesthetics is provided, which comprises, by weight percentage, 40-60wt% of the above-mentioned PLLA microspheres, 20-35wt% 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.

[0020] The composite filler for medical beauty of the present invention combines PLLA microspheres with high-G sodium alginate to form a new type of medical beauty filling system, which improves the safety and effectiveness of the material. Since sodium alginate with a high content of guluronic acid (G) has better gel-forming ability and mechanical strength, it can be used as an excipient to prepare fillers. This new medical beauty filling system not only has good mechanical properties and injection thrust, but also can significantly promote the generation of new collagen, providing a new idea for the development of medical beauty filling materials.

[0021] In some embodiments, the high G sodium alginate has a molecular weight of 80-120 kDa and a degree of esterification of ≤5%.

[0022] In some embodiments, the Ca 2+ Phosphate buffered saline 2+ The concentration is 0.5-1.2 mM.

[0023] 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 Ca-containing 2+ Phosphate buffered saline to obtain a high G alginate solution.

[0024] 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: (1) CaCl 2 Dissolve in PBS solution to obtain a solution containing 0.5-1.2 mM CaCl 2 phosphate buffer, and then dissolve high G sodium alginate in Ca 2+ Phosphate buffer to obtain high-G sodium alginate solution; (2) mixing the PLLA microspheres with a high-G sodium alginate solution and performing a vacuum degassing treatment; (3) Allow to stand for cross-linking at 25-30°C for 30-45 minutes to form an injectable gel.

[0025] 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, and the gel storage modulus (G') is 850-1200 Pa.

[0026] According to a sixth aspect of the present invention, there is provided an application of a composite filler for medical aesthetics as a facial contouring or skin anti-aging preparation, wherein the application is to inject the composite filler for medical aesthetics into the dermis, subcutaneous tissue layer or fascia layer of the face.

[0027] 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 for facial contour shaping or skin anti-aging.

[0028] 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.

[0029] The present invention obtains sodium alginate with a molecular weight of 5-8kDa by directional hydrolysis, and prepares sulfated sodium alginate derivative (Alg-MP) (HLB value 11.2-12.8) by octadecyl glycosylation and sulfation modification; then, Alg-MP is used as an emulsifier and pore-forming technology is used to prepare porous PLLA microspheres (porosity 65-80%), and then mixed with Ca² + The high guluronic acid (G) content sodium alginate and high G sodium alginate (G content ≥ 70%) solution (excipient) were mixed to form a composite system to prepare an injectable gel. The storage modulus of the composite system at 37°C reached 850-1200 Pa, and the injection thrust was ≤12 N when the microsphere / excipient mass ratio was 1:0.3-1:0.7. Animal experiments showed that the volume retention rate was 92.3±3.5% after 6 months, and the density of new collagen was 2.9 times higher than that of the control group. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The synthetic route of the emulsifier for microspheres in Example 1 of the present invention; Figure 2 This is a SEM panoramic image of the PLLA microspheres obtained in Example 2 of the present invention; 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 5 This is a SEM image of the PLLA microspheres obtained in Comparative Example 1 of the present invention; Figure 6 This is a SEM image of the PLLA microspheres obtained in Comparative Example 2 of the present invention; Figure 7 H&E staining results of the PLLA microsphere group in Experimental Example 2; Figure 8 This is the H&E staining result of the HA group in Experimental Example 2; Fig. 9 This is the H&E staining result of the experimental group in Experimental Example 2. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below in conjunction with 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.

[0032] Example 1 This example provides a synthetic route for the emulsifier (Alg-MP) for microspheres. Figure 1 , and its preparation method comprises the following steps: About 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 about 2.8, heated to about 75 ° C, and stirred for about 2.2 hours for hydrolysis reaction. After the reaction was completed, the unreacted small molecules and by-products were removed by dialysis to obtain sodium alginate with a molecular weight of 5-8 kDa; then 5 g of sodium alginate with a molecular weight of 5-8 kDa was weighed and added to 100 mL of dimethyl sulfoxide (DMSO), 10 g of octadecyl bromide and 0.3 g of tetrabutylammonium bromide were added, and the mixture was stirred at room temperature for 24 hours for glycosylation reaction. After the reaction was completed, the glycosylation product was separated and purified by column chromatography to obtain the glycosylation product. The glycosylation product was dissolved in an appropriate amount of dichloromethane, chlorosulfonic acid was added dropwise to dichloromethane under ice bath conditions, and the molar ratio of chlorosulfonic acid to glycosylation product was controlled to be 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.

[0033] The relevant substances were characterized by hydrogen nuclear magnetic resonance spectroscopy, and the data were recorded as follows: 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). Intermediate 1 1 H 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). Final product (emulsifier for microspheres) 1H 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). Compared with the H NMR spectrum of sodium alginate, the H NMR spectrum of intermediate 1 has several new characteristic peaks at 0.89-1.78 ppm, which are attributed to the methylene peaks of the long aliphatic chains (-CH 2 -) and the methyl peak (-CH 3 ), 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 at 4.48 and 4.00 ppm of the final product increases, and a new characteristic peak appears at 4.06 ppm, indicating the successful synthesis of the emulsifier for microspheres.

[0034] In this example, sodium alginate with a molecular weight of 5-8 kDa was obtained by directional hydrolysis, and Alg-MP was prepared by octadecyl glycosylation and sulfation modification. The HLB value was measured to be 11.2-12.8, indicating that Alg-MP has strong hydrophilicity and has the potential to be used as an emulsifier or surfactant.

[0035] Example 2 This embodiment provides a method for preparing poly (L-lactic acid) (PLLA) microspheres, comprising the following steps: Weigh 10 g of PLLA and dissolve it in 80 mL of dichloromethane to form an oil phase containing 10 wt% PLLA. Dissolve the Alg-MP prepared in Example 1 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; then slowly inject the oil phase into the aqueous phase at a homogenization speed of 10,000 rpm to form a W / O emulsion; then place the W / O emulsion at 25°C and stir it uniformly at a speed of 350 rpm for 7 hours. The stirring process is controlled by step-by-step temperature increase: maintain 35±1°C for the first 3 hours; then gradually increase the temperature to 42±1°C for the next 3 hours; and finally increase the temperature to 50±1°C for 1 hour to accelerate the removal of solvent from the W / O emulsion. Then wash the stirred W / O emulsion three times with anhydrous ethanol and freeze-dry it to obtain PLLA microspheres.

[0036] 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 It can be observed that the PLLA microspheres have a porous structure, with an average particle size of 35-55μm, a hierarchical pore structure of 0.5-2μm formed on the surface, a porosity of 65%-80% for the L-polylactic acid microspheres, and a residual dichloromethane content of ≤120 ppm (GC-MS detection). The span coefficient of the PLLA microspheres was determined to be (D90-D10) / D50≤0.8, indicating that the particle size distribution of the L-polylactic acid microspheres is relatively uniform.

[0037] Example 3 This embodiment provides a method for preparing a composite filler for medical aesthetics, comprising the following steps: Dissolve 10 g of high-G sodium alginate (G content ≥ 70%, molecular weight 100 kDa, esterification ≤ 5%) in 100 mL of 0.8 mM CaCl 2 PBS solution, 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, and after vacuum degassing, they were allowed to stand and cross-link at 28°C for 40 minutes to form an injectable gel.

[0038] Example 4 This embodiment provides a method for preparing a composite filler for medical aesthetics, comprising the following steps: Dissolve 10 g of high-G sodium alginate (G content 70%-90%, molecular weight 100 kDa, esterification degree 0.1%-5%) in 100 mL of 0.8 mM CaCl 2 PBS solution, 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, and after vacuum degassing, they were allowed to stand and cross-link at 28°C for 40 minutes to form an injectable gel.

[0039] Comparative Example 1 This comparative example provides a method for preparing PLLA microspheres, which uses sodium dodecylbenzene sulfonate as an emulsifier and comprises the following steps: 10 g of PLLA was weighed and dissolved in 80 mL of dichloromethane to form an oil phase containing 10 wt% PLLA. Sodium dodecylbenzene sulfonate was dissolved in 0.1 M phosphate buffer solution to prepare 100 mL of a mixed solution containing 2.5 wt% sodium dodecylbenzene sulfonate as the aqueous phase. Then, the oil phase was slowly injected into the aqueous phase at a homogenization 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 a speed of 350 rpm for 7 hours. The stirring process was controlled by stepwise temperature increase: 35±1°C was maintained for the first 3 hours; then the temperature was gradually increased to 42±1°C for 2 hours; and the temperature was increased to 50±1°C for the last hour to accelerate the removal of the solvent in the W / O emulsion. The stirred W / O emulsion was then washed three times with anhydrous ethanol and freeze-dried to obtain PLLA microspheres.

[0040] Comparative Example 2 This comparative example provides a method for preparing PLLA microspheres, which uses PVA as an emulsifier and comprises the following steps: 10 g of PLLA was weighed and 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 solution to prepare 100 mL of a mixed solution containing 2.5 wt% PVA as the aqueous phase. Then, the oil phase was slowly injected into the aqueous phase at a homogenization 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 a speed of 350 rpm for 7 hours. The stirring process was controlled by stepwise temperature increase: 35±1°C was maintained for the first 3 hours; then the temperature was gradually increased to 42±1°C for 2 hours; and the temperature was increased to 50±1°C for the last 1 hour to accelerate the removal of the solvent in the W / O emulsion. The stirred W / O emulsion was then washed three times with anhydrous ethanol and freeze-dried to obtain PLLA microspheres.

[0041] 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 selection of emulsifier will affect the morphology and pore structure of PLLA microspheres.

[0042] Experimental Example 1: Rheological Test The medical aesthetic composite filler of Example 3 is placed in the parallel plate fixture of a rheometer (such as Anton Paar MCR 302), the plate spacing is set to 1 mm, and the test temperature is controlled at 37°C to simulate human physiological conditions; then, the oscillatory shear mode is started, the frequency range is set to 0.1-10 Hz, and a constant strain of 1% is applied to ensure that the sample is in the linear viscoelastic region; then, the storage modulus (G') and loss modulus (G'') are recorded by the instrument software as a function of frequency. The results are shown in Table 1.

[0043] Table 1 Rheological test results

[0044] The storage modulus of the medical aesthetic composite filler prepared in Example 3 was measured to be 950 Pa and the loss modulus was 120 Pa at 37°C, indicating that the medical aesthetic composite filler is elastically dominant, has strong supporting force, low viscosity response and stable structure. At the same time, the viscosity of the medical aesthetic composite filler was measured to be about 4500 mPa·s, indicating that the medical aesthetic composite filler is moderately shear-thinning and easy to push, and the thixotropic recovery rate was measured to be 98.2% after 3 cycles, indicating that the medical aesthetic composite filler has a high thixotropic recovery rate, which enables it to quickly restore its shape and remain stable after injection. The yield stress of the medical aesthetic composite filler is 85Pa, indicating that the medical aesthetic composite filler has good resistance to deformation.

[0045] According to the test, 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.

[0046] Experimental Example 2: Animal Experiment 2.1 Experimental methods Thirty 8-week-old SD rats were selected and 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, and the acute inflammatory response was observed in the short term (1 week). The volume retention rate was quantified by Micro-CT in the medium term (1, 3 months) and long term (6 months), and the tissues in the implanted area were taken for H&E staining (inflammatory score) and Masson staining (determination of collagen density). Among them, the implanted sample in the experimental group was the medical aesthetic composite filler prepared in Example 3, the implanted sample in the PLLA microsphere group was the PLLA microsphere prepared in Example 2, and the implanted sample in the HA group was a commercial hyaluronic acid gel.

[0047] 2.2 Experimental Results Figure 7 This is the staining result of PLLA microsphere group. Figure 8This is the staining result of HA group. Fig. 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 new collagen is higher than that in the PLLA microsphere group and the HA group.

[0048] The results of inflammation score, volume retention rate and new collagen density are shown in Table 2. As can be seen from Table 2, in terms of inflammation score, the score of the experimental group at week 1 was 1.2±0.3, which was lower than 2.5±0.6 of the PLLA microsphere group and 1.8±0.4 of the HA group. Among them, the composite filler for medical aesthetics neutralizes the acidic products produced during the degradation of PLLA microspheres through the antioxidant effect of sodium alginate, thereby avoiding the acidic microenvironment caused by the sudden drop in pH after the implantation of the filler, and reducing the excessive activation of macrophages. The low inflammatory response after the implantation of the composite filler for medical aesthetics verifies the biocompatibility advantage of the material and provides a basis for safe clinical application.

[0049] 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 implant in the experimental group was significantly higher than that in the PLLA microsphere group and the HA group, P < 0.01, which was statistically significant.

[0050] 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 3 The density of new collagen in the experimental group was about 2.9 times higher than that in the PLLA group. The experimental results show that compared with the implantation of PLLA microspheres or hyaluronic acid gel, the implantation of medical aesthetic composite fillers can significantly promote the generation of new collagen under the skin of rats, which is suitable for facial contouring and skin anti-aging treatment.

[0051] Table 2 Animal experiment results

[0052] 2.3 Results Analysis High volume retention due to sodium alginate-Ca 2+ The durability of the cross-linked network and the synergistic effect of the degradation and sustained release of PLLA microspheres, sodium alginate and Ca 2+The formed cross-linked network provides initial mechanical support for the subcutaneous tissue in the early stage after implantation. PLLA microspheres release lactic acid during degradation, stimulating fibroblasts to secrete collagen and gradually replacing composite fillers for medical aesthetics. Collagen regeneration enhancement is related to sodium alginate oligosaccharide fragments (M fragments), which reduce collagen degradation by inhibiting MMP-1 activity and activate TGF-β / Smad pathway to promote ECM synthesis. The low inflammatory response in the experimental group verifies the biocompatibility advantage of the material and provides a basis for safe clinical application.

[0053] 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 creative concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. An emulsifier for microspheres, characterized in that: 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 then reacting with C16-C18 alkyl bromide in the presence of a phase transfer catalyst to obtain a glycosylated product; S3. After dissolving the glycosylation product in dichloromethane, chlorosulfonic acid is added dropwise to react to obtain a sulfated sodium alginate derivative.

2. The emulsifier for microspheres according to claim 1, characterized in that The phase transfer catalyst is tetrabutylammonium bromide, and its usage is 5%-6% of the total molar number of the sodium alginate, C16-C18 alkyl bromide and chlorosulfonic acid.

3. The emulsifier for microspheres according to claim 2, characterized in that The molar ratio of the chlorosulfonic acid to the glycosylation product is 2.8:1-3.2:

1. 4.PLLA microspheres, characterized in that The microsphere emulsifier is prepared from raw materials containing any one of claims 1 to 3, has a porosity of 65-80%, a graded pore structure of 0.5-2 μm is formed on the surface, an average particle size of 35-55 μm, and a Zeta potential of -35 mV to -45 mV.

5. The method for preparing PLLA microspheres according to claim 4, 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 a 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) stirring the W / O emulsion at 30-55°C for 6-8h; (5) Wash the stirred W / O emulsion and freeze-dry it to obtain the product.

6. The method for preparing PLLA microspheres according to claim 5, characterized in that: In step (4), the stirring process includes the following heating procedure: Hours 0-3, temperature 35±1°C; 3-5 hours, raise the temperature to 42±1℃; During the last hour, the temperature was 50±1°C.

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

8. The composite filler for medical aesthetics according to claim 7, 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+ Phosphate buffered saline 2+ The concentration is 0.5-1.2 mM.

9. The method for preparing the composite filler for medical aesthetics according to claim 7 or 8, characterized in that: The following steps are involved: (1) Dissolve CaCl2 in PBS to obtain a phosphate buffer solution containing 0.5-1.2 mM CaCl2. Dissolve high-G sodium alginate in Ca 2+ Phosphate buffer to obtain high-G sodium alginate solution; (2) mixing the PLLA microspheres with a high-G sodium alginate solution and performing a vacuum degassing treatment; (3) Allow to crosslink at 25-30°C for 30-45 minutes.

10. Use of the medical aesthetic composite filler according to claim 7 or 8 as a facial contour shaping or skin anti-aging preparation.

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