Polylactic acid-polycitrate composite microspheres, and preparation method and application thereof

Abstract

The application provides a kind of polylactic acid-poly citrate composite microspheres and its preparation method and application, the preparation raw material of the polylactic acid-poly citrate composite microspheres includes poly citrate prepolymer and poly-l-lactic acid.The polylactic acid-poly citrate composite microspheres provided by the application can realize the mechanical-degradation "double period" matching of material by introducing poly citrate prepolymer, also can reduce the hydrophobicity of poly-l-lactic acid, realize the balance of the surface of composite microspheres, improve cell affinity and biological activity, and by using poly citrate prepolymer and poly-l-lactic acid, the polylactic acid-poly citrate composite microspheres as a whole have biomimetic viscoelasticity, so as to promote fibroblast spreading, secrete more integrin and collagen monomer, which is conducive to the behavior regulation of cells, and gently stimulates collagen regeneration.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a polylactic acid-polycitric acid composite microsphere, its preparation method, and its application. Background Technology

[0002] Poly-L-lactic acid (PLLA) microspheres have become one of the most widely studied polyester materials in the field of cosmetic fillers due to their excellent biodegradability, biocompatibility, and ability to stimulate collagen regeneration. Numerous studies and patents focus on particle size control, crystallinity optimization, cross-linking modification, and surface functionalization, aiming to prolong the duration of filler effects and reduce inflammatory responses. However, PLLA still has inherent drawbacks: its degradation cycle is lengthy (12 months), and the degradation rate does not match the collagen regeneration rate, easily leading to late-stage nodules or granulomas; the hydrophobicity resulting from high crystallinity weakens its interaction with cells / matrix, resulting in insufficient early mechanical support; furthermore, the acidic degradation product lactic acid accumulates locally, potentially causing delayed acidosis and chronic inflammation. These issues limit the further promotion of PLLA microspheres in refined, safe, and controllable cosmetic applications.

[0003] Polycitrate is a class of polyester elastomers constructed through melt polycondensation with aliphatic diols, with citric acid as the core. Its main chain is rich in hydroxyl and carboxyl groups, allowing it to degrade into non-toxic citric acid under physiological conditions. This citric acid directly enters the tricarboxylic acid cycle and is utilized by cells as an energy substrate, achieving simultaneous "degradation-energy supply-regeneration." In 2004, Yang Jian's team first reported that polycitrate glycol ester could be completely degraded in vitro over 26 weeks, exhibiting tensile strengths of 2.9-11.2 MPa and elongation at break of 117-502%. It interacts well with human arterial smooth muscle and endothelial cells, meeting the mechanical matching requirements of soft tissues. A review by Xu Peng and Lei Bo points out that polycitrate also possesses advantages such as simple synthetic routes, biomimetic mechanics, and the ability to further graft RGD or fluorescent probes onto its surface. It has been used for the repair of multiple tissues, including blood vessels, cartilage, skin, and nerves, and has achieved zero-order release in drug, gene, and growth factor delivery, demonstrating excellent drug loading stability and bioactivity. In summary, polycitrate, with its comprehensive advantages of being "metabolic-friendly, mechanically tunable, functionally scalable, and clinically validated," has become the most promising next-generation bioactive polyester platform to replace traditional PLA and PCL. However, there is little research on the application of polycitrate in medical aesthetic fillers. How to leverage the technological advantages of polycitrate and design a high-performance material that can be used as a filler in medical aesthetics is an urgent technical problem to be solved. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a polylactic acid-polycitric acid composite microsphere, its preparation method, and its application. By introducing a polycitric acid prepolymer, the mechanical-degradation "dual-phase" matching of the material is achieved, and the hydrophobicity of poly-L-lactic acid is reduced, achieving a hydrophilic-hydrophobic balance on the surface of the composite microsphere, enhancing cell affinity and bioactivity. Furthermore, by using the polycitric acid prepolymer in combination with poly-L-lactic acid, the polylactic acid-polycitric acid composite microsphere exhibits a viscoelasticity closer to that of the dermal matrix, promoting fibroblast spreading, secretion of more integrins and collagen monomers, and achieving a positive coupling between mechanics and biology.

[0005] To achieve this objective, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a polylactic acid-polycitric acid composite microsphere, wherein the raw materials for preparing the polylactic acid-polycitric acid composite microsphere include polycitric acid prepolymer and poly-L-lactic acid.

[0007] Poly-L-lactic acid (PLLA) has lactic acid units linked by ester bonds in its main chain, and its side chains contain hydrophobic methyl groups. Its low surface free energy results in a water contact angle often exceeding 80°, meaning it interacts almost no with the surrounding aquatic environment during the initial implantation stage. This "hydrophobic barrier" not only inhibits rapid protein adsorption but also weakens early fibroblast attachment and extension, delaying the initiation of collagen regeneration. Polycitrate prepolymer (POC) has a main chain rich in hydroxyl, carboxyl, and incompletely esterified citric acid residues, which can form a hydrogen-bonded / electrostatic double hydration layer at the interface, significantly reducing interfacial tension. When embedded in the PLLA framework as nanoislands or gradient shells, the contact angle of the composite microsphere surface can be lowered, thus endowing the material with a hydrophilic active interface that is "protein-adsorbable and cell-recognizable" without sacrificing mechanical support, laying the first "molecular springboard" for subsequent biological cascade reactions.

[0008] POCs are elastic cross-linkers that exist in a highly elastic state in physiological environments. When these elastic cross-linkers are uniformly distributed at the submicron scale within a rigid PLLA matrix, they can form "hard-soft" interpenetrating microregions within the same particle. Under stress, the PLLA crystalline regions provide instantaneous elastic recovery, while the POC network dissipates energy through segment extension and hydrogen bond dissociation, exhibiting an overall viscoelasticity closer to that of the dermal matrix: the storage modulus decreases, the loss factor increases, and the "softness" and "energy dissipation" signals perceived by cells are similar to those of the natural ECM, thereby promoting fibroblast spread, secretion of more integrins and collagen monomers, and achieving a positive coupling between mechanics and biology.

[0009] This invention, by combining POC with PLLA, not only achieves a "dual-phase" matching of the material's mechanical and degradation processes and realizes a hydrophilic-hydrophobic balance on the surface of the composite microspheres, thus enhancing cell affinity and bioactivity; the surface carboxyl / hydroxyl groups of POC can also be further grafted with RGD, fluorescent probes, or loaded with whitening, antioxidant, and fat-reducing active ingredients, achieving a three-in-one effect of "filling-rejuvenation-functionalization," providing a universal carrier for high-end personalized rejuvenation formulas, and has promising clinical and industrial applications.

[0010] The following are preferred technical solutions of the present invention, but are not intended to limit the technical solutions provided by the present invention. The purpose and beneficial effects of the present invention can be better achieved and realized through the following preferred technical solutions.

[0011] As a preferred technical solution, the D of the polylactic acid-polycitric acid composite microspheres 50 The particle size is 20-70μm, for example, it can be 25μm, 30μm, 35μm, 40μm, 45μm, 50μm, 55μm, 60μm, 65μm, etc.

[0012] Preferably, the D of the polylactic acid-polycitric acid composite microspheres is... 90 The particle size is 20-90μm, for example, it can be 25μm, 30μm, 35μm, 40μm, 45μm, 50μm, 55μm, 60μm, 65μm, 70μm, 75μm, 80μm, 85μm, etc.

[0013] Preferably, the mass ratio of the polycitric acid prepolymer to the polylactic acid is 1:(1.5-20), for example, it can be 1:1.5, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, etc., and more preferably 1:(6-15).

[0014] Preferably, the poly-L-lactic acid has a number average molecular weight of 40,000 to 200,000, for example, it can be 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 110,000, 120,000, 130,000, 140,000, 150,000, 160,000, 170,000, 180,000, 190,000, etc., and more preferably 80,000 to 120,000.

[0015] In this invention, the number-average molecular weight of the poly-L-lactic acid is obtained by gel permeation chromatography.

[0016] Preferably, the raw materials for preparing the polycitric acid ester prepolymer include citric acid and aliphatic diols.

[0017] Preferably, the aliphatic diol includes 1,8-octanediol and / or ethylene glycol.

[0018] Preferably, the molar ratio of citric acid to aliphatic diol is 1:(0.8-1.5), for example, it can be 1:0.85, 1:0.9, 1:0.95, 1:1, 1:1.05, 1:1.1, 1:1.15, 1:1.2, 1:1.25, 1:1.3, 1:1.35, 1:1.4, 1:1.45, etc., and more preferably 1:(1-1.2).

[0019] Preferably, the polycitrate prepolymer is prepared by the following method, the method comprising:

[0020] Citric acid and aliphatic diols are polycondensed at 140-160℃ (e.g., 142℃, 144℃, 146℃, 148℃, 150℃, 152℃, 154℃, 156℃, 158℃, etc.) for 0.1-2h (0.2h, 0.4h, 0.6h, 0.8h, 1h, 1.2h, 1.4h, 1.6h, 1.8h, etc.) to obtain the polycitric acid ester prepolymer.

[0021] Preferably, the polycondensation is carried out in the presence of a protic acid catalyst.

[0022] Preferably, the protic acid catalyst comprises p-toluenesulfonic acid.

[0023] Preferably, the mass of the protic acid catalyst is 0.01-1% based on the total mass of the citric acid and aliphatic diols as 100%, for example, it can be 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.7%, 0.8%, 0.9%, etc.

[0024] Preferably, the polycondensation is carried out under nitrogen protection.

[0025] In a second aspect, the present invention provides a method for preparing polylactic acid-polycitric acid ester composite microspheres as described in the first aspect, the method comprising the following steps:

[0026] (1) The polycitric acid prepolymer, poly-L-lactic acid and organic solvent are mixed to obtain the oil phase;

[0027] (2) The oil phase is added dropwise to the aqueous phase to obtain an O / W emulsion;

[0028] (3) Remove the organic solvent from the O / W emulsion to obtain microspheres;

[0029] (4) The microspheres are subjected to heat treatment to obtain the polylactic acid-polycitric acid composite microspheres.

[0030] Preferably, the organic solvent comprises a combination of dichloromethane and other organic solvents; the other organic solvents include any one or a combination of at least two of tetrahydrofuran, ethyl acetate, dodecanol, hexafluoroisopropanol, or dimethyl sulfoxide. The combination of dichloromethane with other organic solvents can improve the mixing uniformity and emulsion stability of POC and PLLA.

[0031] Preferably, the volume ratio of dichloromethane to other organic solvents is (1-12):1, for example, it can be 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, etc.

[0032] Preferably, the total solids content of the polycitric acid prepolymer and poly-L-lactic acid in the oil phase is 1-500 mg / mL, for example, it can be 10 mg / mL, 30 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 90 mg / mL, 100 mg / mL, 120 mg / mL, 150 mg / mL, 180 mg / mL, 200 mg / mL, 250 mg / mL, 300 mg / mL, 350 mg / mL, 400 mg / mL, 450 mg / mL, 500 mg / mL, etc.

[0033] Preferably, the aqueous phase comprises a first aqueous solution of polyvinyl alcohol.

[0034] Preferably, the mass percentage of polyvinyl alcohol in the first polyvinyl alcohol aqueous solution is 0.2-5%, for example, it can be 0.2%, 0.5%, 1%, 1.5%, 1.8%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, etc.

[0035] Preferably, the volume ratio of the oil phase to the water phase is 1:(5-15), for example, it can be 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, etc.

[0036] Preferably, the dripping rate is 1-5 mL / min, for example, it can be 1.5 mL / min, 2 mL / min, 2.5 mL / min, 3 mL / min, 3.5 mL / min, 4 mL / min, 4.5 mL / min, 4.8 mL / min, etc.

[0037] Preferably, the temperature of the aqueous phase is 14-16℃, for example, it can be 14.2℃, 14.4℃, 14.6℃, 14.8℃, 15℃, 15.2℃, 15.4℃, 15.6℃, 15.8℃, etc.

[0038] Preferably, the aqueous phase is stirred during the dropwise addition of the oil phase.

[0039] Preferably, the first stirring speed is 200-500 rpm, for example, it can be 220 rpm, 250 rpm, 280 rpm, 300 rpm, 320 rpm, 350 rpm, 380 rpm, 400 rpm, 420 rpm, 450 rpm, 480 rpm, etc.

[0040] Preferably, the process further includes a second stirring step after the addition is completed;

[0041] Preferably, the second stirring speed is 600-1000 rpm, for example, it can be 650 rpm, 700 rpm, 750 rpm, 800 rpm, 850 rpm, 900 rpm, 950 rpm, 980 rpm, etc.

[0042] Preferably, the second stirring time is 1-5 min, for example, it can be 1.5 min, 2 min, 2.5 min, 3 min, 3.5 min, 4 min, 4.5 min, etc.

[0043] Preferably, in step (3), the organic solvent in the O / W emulsion is removed by gradient decompression.

[0044] Preferably, the gradient decompression is 350-450 mbar (e.g., 355 mbar, 360 mbar, 365 mbar, 370 mbar, 375 mbar, 380 mbar, 385 mbar, 390 mbar, 395 mbar, 400 mbar, 405 mbar, 410 mbar, 415 mbar, 420 mbar, 425 mbar, 430 mbar, 435 mbar, 440 mbar, 445 mbar, etc.) maintained for 20-40 minutes (e.g., 22 min, 24 min, 26 min, 28 min, 30 min, 32 min, 34 min, 36 min, 38 min, etc.), or 90-110 mbar (92 mbar, 94 mbar, 96 mbar, 98 mbar, 100 mbar, 102 mbar, 104 mbar, 106 mbar, 108 mbar, etc.). Maintain at 50-80 min (52 min, 55 min, 58 min, 60 min, 62 min, 65 min, 68 min, 70 min, 72 min, 75 min, 78 min, etc.) at 20-30 mbar (e.g., 20.5 mbar, 21 mbar, 21.5 mbar, 22 mbar, 22.5 mbar, 23 mbar, 23.5 mbar, 24 mbar, 24.5 mbar, 25 mbar, 25.5 mbar, 26 mbar, 26.5 mbar, 27 mbar, 27.5 mbar, 28 mbar, 28.5 mbar, 29 mbar, 29.5 mbar, etc.) and maintain at 90-120 min (e.g., 92 min, 95 min, 98 min, 100 min, 102 min, 105 min, 108 min, 110 min, 112 min, 115 min, 118 min, etc.).

[0045] Preferably, during the gradient decompression process, the temperature of the O / W emulsion is maintained at 14-16°C, for example, 14.2°C, 14.4°C, 14.6°C, 14.8°C, 15°C, 15.2°C, 15.4°C, 15.6°C, 15.8°C, etc.; maintaining the temperature at 14-16°C can prevent the polycitrate prepolymer from deteriorating due to T... g Low and sticky.

[0046] POCs are amphiphilic polymer materials. In traditional emulsion methods, the rapid evaporation of solvents causes the hydrophobic layer to harden instantly, trapping the hydrophilic components with low glass transition temperatures into clusters. This results in particles of varying sizes and loose interfacial bonding. Furthermore, the simultaneous action of high shear and temperature rise accelerates molecular chain breakage, making it difficult for residual solvent to dissipate completely. During the drying stage, the particles stick together, requiring additional crushing and reshaping, which reduces the integrity of the morphology. This invention removes the organic solvent through a low-temperature gradient decompression process, first quickly and then slowly. The organic solvent, while forming a shell, simultaneously draws the molecules of the two phases to interpenetrate, forming a dense and flexible integrated structure. The resulting composite microspheres have a smooth surface and are freely dispersed, making them suitable for direct use in subsequent filling.

[0047] Preferably, the O / W emulsion is subjected to a third stirring while the gradient decompression is being performed.

[0048] Preferably, the speed of the third stirring is 100-500 rpm, for example, it can be 150 rpm, 200 rpm, 250 rpm, 300 rpm, 350 rpm, 400 rpm, 450 rpm, etc.

[0049] Preferably, after removing the organic solvent from the O / W emulsion, the process further includes sequentially passing the resulting product through a 200-500 mesh sieve and washing it.

[0050] Preferably, the washing process involves washing with a 0.01-0.2% aqueous solution of polyvinyl alcohol 2-5 times.

[0051] Preferably, the heat treatment is carried out at 40-80℃ (e.g., 42℃, 45℃, 48℃, 50℃, 52℃, 55℃, 58℃, 60℃, 62℃, 65℃, 68℃, 70℃, 72℃, 75℃, 78℃, etc.) and 30-50%RH (e.g., 32%RH, 34%RH, 36%RH, 38%RH, 40%RH, 42%RH, 44%RH, 46%RH, 48%RH, etc.) for 12-48h (e.g., 15h, 18h, 20h, 22h, 25h, 28h, 30h, 32h, 35h, 38h, 40h, 42h, 45h, etc.).

[0052] The heat treatment causes the hydroxyl groups of the polycitric acid ester prepolymer to undergo non-catalytic thermal crosslinking with the remaining carboxyl groups, forming a nanoscale elastic network.

[0053] Thirdly, the present invention provides an application of polylactic acid-polycitric acid composite microspheres as described in the first aspect, wherein the polylactic acid-polycitric acid composite microspheres are used as medical aesthetic filler materials.

[0054] The numerical range described in this invention includes not only the point values ​​listed above, but also any point values ​​within the numerical ranges not listed above. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific point values ​​included in the range.

[0055] Compared with the prior art, the present invention has the following beneficial effects:

[0056] The polylactic acid-polycitric acid composite microspheres provided by this invention, by introducing polycitric acid prepolymer, can achieve a "dual-phase" matching of the material's mechanical and degradation processes, and can also reduce the hydrophobicity of poly-L-lactic acid, achieving a hydrophilic-hydrophobic balance on the surface of the composite microspheres, enhancing cell affinity and bioactivity. Furthermore, by using polycitric acid prepolymer in combination with poly-L-lactic acid, the polylactic acid-polycitric acid composite microspheres as a whole can have biomimetic viscoelasticity, promoting fibroblast spreading, secreting more integrins and collagen monomers, which is beneficial for cell behavior regulation and gently stimulating collagen regeneration.

[0057] The polylactic acid-polycitric acid composite microspheres provided by this invention have a contact angle of 61-79°, a local tissue reaction score of 1.9-2.7 after implantation, and a collagen content of 25-35%. Attached Figure Description

[0058] Figure 1 These are cell viability and mortality staining images after treatment with the polylactic acid-polycitric acid composite microspheres provided in Example 1;

[0059] Figure 2 These are cell viability and mortality staining images after treatment with polylactic acid-polycitric acid composite microspheres provided in Example 2;

[0060] Figure 3 These are cell live / dead staining images after treatment with the polylactic acid-polycitric acid composite microspheres provided in Example 3;

[0061] Figure 4 The image shows cell viability and mortality staining after treatment with polylactic acid microspheres provided in Comparative Example 1.

[0062] Figure 5 This is a Sirius red staining image of site 1 after implantation of the polylactic acid-polycitric acid composite microspheres provided in Example 1;

[0063] Figure 6 This is a Sirius red staining image of site 1 after implantation of the polylactic acid-polycitric acid composite microspheres provided in Example 2;

[0064] Figure 7 This is a Sirius red staining image of site 1 after implantation of the polylactic acid-polycitric acid composite microspheres provided in Example 3;

[0065] Figure 8This is a Sirius red staining image of site 1 after implantation of polylactic acid microspheres provided in Comparative Example 1. Detailed Implementation

[0066] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be considered as specific limitations thereof.

[0067] The sources of some components in the following examples and comparative examples are as follows:

[0068] (1) Poly-L-lactic acid

[0069] Poly-L-lactic acid-1, S23075, Shanghai Yuanye Biotechnology Co., Ltd.;

[0070] Poly-L-lactic acid-2, S33261, Shanghai Yuanye Biotechnology Co., Ltd.;

[0071] (2) Polyvinyl alcohol: P139542, Aladdin Biochemical Technology Co., Ltd.

[0072] Example 1

[0073] A polylactic acid-polycitric acid composite microsphere, wherein the raw materials for preparing the polylactic acid-polycitric acid composite microsphere include polycitric acid prepolymer and poly-L-lactic acid-1;

[0074] The preparation method of the polylactic acid-polycitric acid composite microspheres includes the following steps:

[0075] (1) Under nitrogen protection, 0.5 mol of citric acid and 1,8-octanediol were fed in a molar ratio of 1:1.1, and 0.02 wt% of p-toluenesulfonic acid (total mass of citric acid and 1,8-octanediol) were added. Vacuum melt polycondensation was carried out at 160 °C for 1.5 h to obtain polycitric acid ester prepolymer POC.

[0076] 0.5g of the POC and poly-L-lactic acid-1 were co-dissolved in a dichloromethane-tetrahydrofuran (DCM / THF=8:1v / v) mixed solvent at a mass ratio of 1:9 to obtain an oil phase; the total solid content of POC and poly-L-lactic acid-1 in the oil phase was 200mg / mL;

[0077] (2) Under mechanical stirring at 400 rpm and water bath conditions at 15±1℃, 100 mL of the oil phase was uniformly dropped into 500 mL of 2% polyvinyl alcohol aqueous solution at a speed of 5 mL / min, and sheared at 1000 rpm for 5 min to obtain O / W emulsion;

[0078] (3) Maintain slow stirring at 300 rpm and remove organic solvent from the O / W emulsion by gradient pressure reduction, specifically: maintain 400 mbar for 30 min, 100 mbar for 60 min, and 20 mbar for 90 min; maintain temperature at 15 ± 1 °C throughout the process. Then, pass the microsphere suspension through a 325 mesh sieve and wash it three times with 0.1% PVA aqueous solution to remove free PVA and residual solvent, and obtain microspheres.

[0079] (4) The microspheres were heat-treated in a vacuum oven at 40°C and 30%RH for 12 hours, dried and stored to obtain the polylactic acid-polycitric acid composite microspheres.

[0080] Example 2

[0081] A polylactic acid-polycitric acid composite microsphere, wherein the raw materials for preparing the polylactic acid-polycitric acid composite microsphere include polycitric acid prepolymer and poly-L-lactic acid-1;

[0082] The preparation method of the polylactic acid-polycitric acid composite microspheres includes the following steps:

[0083] (1) Under nitrogen protection, 0.5 mol of citric acid and 1,8-octanediol were fed in a molar ratio of 1:1.5, and 0.3 wt% of p-toluenesulfonic acid (total mass of citric acid and 1,8-octanediol) were added. Vacuum melt polycondensation was carried out at 160 °C for 2 h to obtain polycitric acid ester prepolymer POC.

[0084] 1g of the POC and poly-L-lactic acid-1 were co-dissolved in a dichloromethane-hexafluoroisopropanol (DCM / HFIP=8:1v / v) mixed solvent at a mass ratio of 1:6 to obtain an oil phase; the total solid content of POC and poly-L-lactic acid-1 in the oil phase was 400mg / mL;

[0085] (2) Under the conditions of mechanical stirring at 300 rpm and water bath at 15±1℃, 100 mL of the oil phase was uniformly dropped into 500 mL of 2% polyvinyl alcohol aqueous solution at a speed of 3 mL / min, and sheared at 800 rpm for 5 min to obtain O / W emulsion;

[0086] (3) Maintain slow stirring at 100 rpm and remove organic solvent from the O / W emulsion by gradient pressure reduction, specifically: maintain 400 mbar for 35 min, 90 mbar for 60 min, and 20 mbar for 100 min; maintain temperature at 15 ± 1 °C throughout the process. Then, pass the microsphere suspension through a 200-mesh sieve and wash it three times with 0.1% PVA aqueous solution to remove free PVA and residual solvent, and obtain microspheres.

[0087] (4) The microspheres were heat-treated in a vacuum oven at 40°C and 35%RH for 12 hours, dried and stored to obtain the polylactic acid-polycitric acid composite microspheres.

[0088] Example 3

[0089] A polylactic acid-polycitric acid composite microsphere, wherein the raw materials for preparing the polylactic acid-polycitric acid composite microsphere include polycitric acid prepolymer and poly-L-lactic acid-2;

[0090] The preparation method of the polylactic acid-polycitric acid composite microspheres includes the following steps:

[0091] (1) Under nitrogen protection, 0.6 mol of citric acid and 1,8-octanediol were fed in a molar ratio of 1:0.8, and 0.01 wt% of p-toluenesulfonic acid (total mass of citric acid and 1,8-octanediol) were added. Vacuum melt polycondensation was carried out at 140 °C for 1 h to obtain polycitric acid ester prepolymer POC.

[0092] 0.5 g of the POC and poly-L-lactic acid-2 were co-dissolved in a dichloromethane-ethyl acetate (DCM / EA=8:1v / v) mixed solvent at a mass ratio of 1:12 to obtain an oil phase; the total solid content of POC and poly-L-lactic acid-2 in the oil phase was 80 mg / mL;

[0093] (2) Under the conditions of mechanical stirring at 500 rpm and water bath at 15±1℃, 100 mL of the oil phase was uniformly dropped into 600 mL of 2% polyvinyl alcohol aqueous solution at a speed of 5 mL / min, and sheared at 1000 rpm for 5 min to obtain O / W emulsion;

[0094] (3) Maintain slow stirring at 500 rpm and remove organic solvents from the O / W emulsion by gradient pressure reduction, specifically: maintain 400 mbar for 40 min, 100 mbar for 50 min, and 25 mbar for 90 min; maintain temperature at 15 ± 1 °C throughout the process. Then, pass the microsphere suspension through a 500-mesh sieve and wash it three times with 0.2% PVA aqueous solution to remove free PVA and residual solvents, and obtain microspheres.

[0095] (4) The microspheres were heat-treated in a vacuum oven at 50°C and 30%RH for 12 hours, dried and stored to obtain the polylactic acid-polycitric acid composite microspheres.

[0096] Example 4

[0097] A polylactic acid-polycitric acid composite microsphere and its preparation method are disclosed. The only difference between this method and Example 1 is that the total amount of POC and poly-L-lactic acid-1 remains unchanged in step (1), and the mass ratio of POC to poly-L-lactic acid-1 is adjusted to 1:20. The other raw materials, process parameters and steps are the same as in Example 1.

[0098] Example 5

[0099] A polylactic acid-polycitric acid composite microsphere and its preparation method are disclosed. The only difference between this method and Example 1 is that the total amount of POC and poly-L-lactic acid-1 remains unchanged in step (1), and the mass ratio of POC to poly-L-lactic acid-1 is adjusted to 1:15. The other raw materials, process parameters and steps are the same as in Example 1.

[0100] Example 6

[0101] A polylactic acid-polycitric acid composite microsphere and its preparation method are disclosed. The only difference between this method and Example 1 is that the total amount of POC and poly-L-lactic acid-1 remains unchanged in step (1), and the mass ratio of POC to poly-L-lactic acid-1 is adjusted to 1:2. The other raw materials, process parameters and steps are the same as in Example 1.

[0102] Example 7

[0103] A polylactic acid-polycitric acid composite microsphere and its preparation method are disclosed. The only difference between this method and Example 1 is that the equimolar amount of 1,8-octanediol in step (1) is replaced with ethylene glycol. All other raw materials, process parameters and steps are the same as in Example 1.

[0104] Example 8

[0105] A polylactic acid-polycitric acid composite microsphere and its preparation method are disclosed. The only difference between this method and Example 1 is that step (3) is performed as follows, while the other raw materials, process parameters and steps are performed in accordance with Example 1.

[0106] (3) Maintain slow stirring at 100 rpm and slowly heat the O / W emulsion to remove organic solvents, specifically: heat to 35°C and maintain for 35 min, 40°C and maintain for 60 min, and 66°C and maintain for 100 min; then wash the microsphere suspension three times with 0.1% PVA aqueous solution to remove free PVA and residual solvents to obtain microspheres.

[0107] Comparative Example 1

[0108] A polylactic acid microsphere and its preparation method are disclosed. The only difference between this method and Example 1 is that the operation of step (1) is as follows, while the other raw materials, process parameters and steps are carried out in accordance with Example 1.

[0109] (1) 5g of poly-L-lactic acid-1 was dissolved in a mixed solvent of dichloromethane-tetrahydrofuran (DCM / THF=8:1v / v) as the oil phase, with a solid content of 200mg / mL.

[0110] Product characterization and performance testing

[0111] (1) Appearance: The appearance of the sample was observed using an optical microscope (Zeiss Axio Lab.A1);

[0112] (2) Microscopic morphology: The roundness, presence of dents and cracks of the sample were observed by SEM (Zeiss Gemini 300);

[0113] (3) Particle size and distribution: determined by laser particle size analyzer (Malvin Mastersizer 2000 / 3000);

[0114] The polylactic acid-polycitric acid composite microspheres provided in Examples 1-8 and the polylactic acid microspheres provided in Comparative Example 1 were tested according to the above method. The test results are shown in Table 1 below:

[0115] Table 1

[0116]

[0117] As shown in Table 1, the polylactic acid-polycitric acid composite microspheres provided in Examples 1-7 are in the form of off-white powder solids with a fine texture. They have no obvious particle feel when observed with the naked eye, their surface is relatively smooth, and their particle size distribution range is narrow. In Example 8, the organic solvent was removed using a traditional method, but the microspheres adhered to each other, had poor sphericity, and were not of practical use.

[0118] (4) Cytotoxicity safety experiment:

[0119] Microspheres were incubated at 0.2 g / mL in DMEM complete medium at 37°C with constant shaking for 72 h before toxicity testing. The MTT assay was used. Mouse fibroblasts were seeded in 96-well plates at 2000 cells / well. After treatment with the microsphere extract for 72 hours, MTT reagent was added and incubated for 4 h. The supernatant was discarded, and DMSO was added to dissolve formazan crystals. Cell viability and mortality staining was performed. The test results are shown below. Figure 1-4 As shown; by Figure 1-4 It can be seen that the living cells (green) have normal morphology and adhere well to the wall, while the dead cells (red) are very few, indicating that the microspheres in each group have no obvious cytotoxicity and good biocompatibility.

[0120] (5) Contact angle: The microspheres were pressed into flat sheets (approximately 1-2 mm thick) at 8 MPa using a tablet press, with a holding time of 60 seconds to ensure that the sheet surface was dense, uniform, and free of cracks. The sheets were statically titrated with deionized water at room temperature, and the contact angle was measured using a contact angle meter (Dataphysics OCA20) after standing.

[0121] (6) Biocompatibility:

[0122] 150 mg of microspheres, 45 mg of CMC, and 145 mg of mannitol were mixed evenly to prepare a lyophilized powder, which was then mixed with 3 mL of physiological saline before use. A dorsal skin model was established using adult SD rats. Standardized injections were performed subcutaneously at the same anatomical level at four injection sites as parallel samples. The injection dose was 0.25 mL / site. Samples were collected after 13 weeks of feeding. Sections were stained using routine histopathological methods. The degree of local inflammatory infiltration and the extent of fibrosis were assessed based on HE staining. Scoring was performed according to the "Histological Analysis Scoring System" in Appendix E of IOS 10993-6:2016; a lower score indicated a smaller local tissue reaction. The average score of the four sites was recorded as the local tissue reaction score in Table 2.

[0123] The slide from injection site 1 was stained with Sirius red and examined using an upright microscope (NIKON Eclipse ci). The test results are as follows: Figure 5-8 As shown; by Figure 5-8 It can be seen that, compared with Comparative Example 1, the polylactic acid-polycitric acid composite microspheres provided by this invention have a denser and more uniform collagen fiber arrangement, exhibiting good collagen deposition promotion ability, and demonstrating both safety and effectiveness in inducing collagen regeneration in vivo; quantitative statistics Figure 5-8 The area of ​​fluorescence in the test area is used to calculate the collagen percentage. Collagen percentage = S1 / S × 100%, where S1 is the area of ​​the fluorescent region and S is the area of ​​the test region. The collagen percentage of the remaining three injection sites is calculated in the same way, and the average value is taken.

[0124] The polylactic acid-polycitric acid composite microspheres provided in Examples 1-7 and the polylactic acid microspheres provided in Comparative Example 1 were tested according to tests (5) and (6), and the test results are shown in Table 2 below:

[0125] Table 2

[0126]

[0127] As shown in Table 2, compared to Comparative Example 1, the introduction of polycitrate prepolymer significantly reduced the contact angle, indicating that the hydrophilicity of the polylactic acid-polycitrate composite microspheres provided by this invention is significantly improved. Experimental results show that local tissue reaction was minimal after implantation of the polylactic acid-polycitrate composite microspheres provided by this invention. The collagen content percentage indicates increased collagen fiber synthesis, suggesting that the polylactic acid-polycitrate composite microspheres provided by this invention possess good biocompatibility and promote repair.

[0128] The applicant declares that this invention illustrates the polylactic acid-polycitric acid composite microspheres, their preparation method, and applications through the above embodiments. However, this invention is not limited to the above embodiments, meaning that this invention does not necessarily rely on the above embodiments for implementation. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials, additions of auxiliary components, and selection of specific methods, etc., all fall within the protection and disclosure scope of this invention.

Claims

1. A polylactic acid-polycitrate composite microsphere, characterized by, The raw materials for preparing the polylactic acid-polycitric acid composite microspheres include polycitric acid prepolymer and poly-L-lactic acid; The D of the polylactic acid-polycitrate composite microspheres is 0.5-1.5 μm. 50 The particle size is 20-70 μm. The D of the polylactic acid-polycitric acid composite microspheres 90 Particle size is 20-90 μm; The mass ratio of the polycitric acid prepolymer to the poly-L-lactic acid is 1:(1.5-20). The number-average molecular weight of the poly-L-lactic acid is 40,000 to 200,000.

2. The polylactic acid-polycitrate composite microspheres according to claim 1, characterized in that, The raw materials for preparing the polycitric acid ester prepolymer include citric acid and aliphatic diols; The aliphatic diols include 1,8-octanediol and / or ethylene glycol; The molar ratio of citric acid to aliphatic diol is 1:(0.8-1.5).

3. The polylactic acid-polycitrate composite microspheres according to claim 2, characterized in that, The polycitric acid ester prepolymer is prepared by the following method, the method comprising: Citric acid and aliphatic diols are polycondensed at 140-160°C for 0.1-2 hours to obtain the polycitric acid ester prepolymer. The polycondensation is carried out in the presence of a protic acid catalyst.

4. A method for preparing the polylactic acid-polycitrate composite microspheres according to any one of claims 1 to 3, characterized by, The preparation method includes the following steps: (1) The polycitric acid prepolymer, poly-L-lactic acid and organic solvent are mixed to obtain the oil phase; (2) The oil phase is added dropwise to the aqueous phase to obtain an O / W emulsion; (3) Remove the organic solvent from the O / W emulsion to obtain microspheres; (4) The microspheres are subjected to heat treatment to obtain the polylactic acid-polycitric acid composite microspheres.

5. The preparation method according to claim 4, characterized in that, The organic solvent includes a combination of dichloromethane and other organic solvents; the other organic solvents include any one or a combination of at least two of tetrahydrofuran, ethyl acetate, dodecanol, hexafluoroisopropanol, or dimethyl sulfoxide. The volume ratio of the dichloromethane to the other organic solvent is (1-12):1; The total solids content of the polycitric acid prepolymer and poly-L-lactic acid in the oil phase is 1-500 mg / mL; The aqueous phase includes a first polyvinyl alcohol aqueous solution; The volume ratio of the oil phase to the water phase is 1:(5-15).

6. The preparation method according to claim 4, characterized in that, The dropping rate is 1-5 mL / min; The temperature of the aqueous phase is 14-16℃; The aqueous phase is first stirred during the dropwise addition of the oil phase; The first stirring speed is 200-500 rpm; After the dripping is completed, a second stirring step is also included; The second stirring speed is 600-1000 rpm; The second stirring time is 1-5 minutes.

7. The preparation method according to claim 4, characterized in that, In step (3), the organic solvent in the O / W emulsion is removed by gradient decompression; The gradient decompression is 350-450 mbar maintained for 20-40 min, 90-110 mbar maintained for 50-80 min, and 20-30 mbar maintained for 90-120 min; During the gradient decompression process, the temperature of the O / W emulsion is maintained at 14-16°C; While the gradient decompression is being performed, the O / W emulsion is being stirred for the third time. The third stirring speed is 100-500 rpm.

8. The preparation method according to claim 4, characterized in that, After removing the organic solvent from the O / W emulsion, the process further includes the steps of sequentially passing the resulting product through a 200-500 mesh sieve and washing it. The heat treatment is carried out at 40-80℃ and 30-50%RH for 12-48 hours.

9. Use of the polylactic acid-polycitrate composite microspheres according to any one of claims 1 to 3, characterized in that, The polylactic acid-polycitric acid composite microspheres are used as filler materials in medical aesthetics.

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