Poly citrate modified bg / plla composite microspheres, preparation method and application thereof

Abstract

This invention discloses a polycitrate-modified BG / PLLA composite microsphere, its preparation method, and its applications, belonging to the field of biomedical materials technology. The preparation method includes: reacting citric acid and 1,8-octanediol to obtain a polycitrate prepolymer; hydrolyzing a silicon source and a phosphorus source to obtain an intermediate product; reacting the intermediate product with a calcium source, gelling, aging, drying, and sintering to obtain bioactive glass; reacting the polycitrate prepolymer with the bioactive glass and thermosetting to obtain polycitrate-modified BG; mixing polycitrate-modified BG and PLLA in an organic solvent to obtain an oil phase; emulsifying the oil phase with an aqueous phase containing an emulsifier to obtain an emulsion; evaporating the organic solvent to obtain the polycitrate-modified BG / PLLA composite microsphere. The polycitrate-modified BG / PLLA composite microsphere obtained by this invention exhibits excellent encapsulation efficiency and mechanical properties, good sustained-release effect, and can significantly enhance collagen and elastin synthesis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a polycitrate-modified BG / PLLA composite microsphere, its preparation method, and its application. Background Technology

[0002] Skin aging, wrinkles, and soft tissue depressions have become the focus of the medical aesthetics industry. Injectable filler products are gradually being widely used due to their advantages such as minimal trauma, quick recovery, and significant effects. An ideal filler material should simultaneously possess good biocompatibility, suitable filling effect, controllable degradation rate, and the ability to promote collagen regeneration.

[0003] Polylactic acid (PLLA) microspheres possess excellent biocompatibility, biodegradability, and good mechanical properties, and have been widely used in the field of dermal fillers. However, the degradation products (lactic acid) of PLLA microspheres are acidic, which can lower the local pH value and trigger inflammatory responses (nodules, granulomas, etc.). Simultaneously, its inherent hydrophobicity affects cell-material interactions and cell adhesion. Furthermore, PLLA lacks biological activity, relying solely on physical stimulation to induce collagen regeneration, resulting in poor collagen regeneration efficiency.

[0004] Bioactive glass (BG) exhibits excellent bioactivity, releasing silicon and calcium ions in bodily fluids to activate fibroblasts and promote collagen and elastin synthesis. It also possesses antioxidant and anti-inflammatory properties. 2+ The release of BG can also regulate the local pH value and maintain the stability of the local microenvironment. Furthermore, a hydroxyapatite layer can form on the surface of BG. This hydroxyapatite layer is a perfect bridge connecting the material to the surrounding tissue, effectively adsorbing growth factors and guiding cell adhesion and proliferation. However, direct application of BG powder can cause a sharp increase in the local pH value, adversely affecting surrounding tissues and cells. It also presents problems such as easy particle diffusion, lack of carrier support, and difficulty in maintaining a sustained volume.

[0005] To address the aforementioned issues, the common approach is to combine PLLA with bioactive glass, hoping to balance the structural support of PLLA with the bioactivity of bioactive glass (BG) while resolving the inflammation caused by acidic byproducts of PLLA and the transient high alkalinity of BG. However, bioactive glass has a highly hydrophilic surface, while PLLA is hydrophobic, resulting in poor compatibility between the two. Furthermore, the interfacial bonding between them is extremely weak, making it prone to premature detachment during in vivo degradation, thus affecting the application results. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a polycitric acid ester modified BG / PLLA composite microsphere, its preparation method, and its application.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a method for preparing polycitrate-modified BG / PLLA composite microspheres, the preparation method comprising:

[0009] S1. Citric acid and 1,8-octanediol react to obtain a polycitric acid ester prepolymer;

[0010] S2. The silicon and phosphorus sources are hydrolyzed to obtain intermediate products. The intermediate products are reacted with calcium sources, gelled, aged, dried and sintered to obtain bioactive glass.

[0011] S3. Polycitrate prepolymer is reacted with bioactive glass and thermosetting to obtain polycitrate modified BG;

[0012] S4. Polycitric acid ester modified BG and PLLA are mixed in an organic solvent to obtain an oil phase. The oil phase is then mixed and emulsified with an aqueous phase containing an emulsifier to obtain an emulsion. The organic solvent in the emulsion evaporates to obtain the polycitric acid ester modified BG / PLLA composite microspheres.

[0013] Currently, the general method for preparing bioactive glass / PLLA composite microspheres is the emulsification method, including single emulsification and double emulsification. The single emulsification (O / W) process is simple, but due to the strong hydrophilicity of bioactive glass, it is easy to migrate to the oil-water interface during the emulsification process, resulting in uneven microsphere coating and severe exposure of bioactive materials in the microspheres. Although the double emulsification method can be used to encapsulate hydrophilic bioactive glass, the structure is unstable, and the migration of the internal aqueous phase can easily lead to microsphere breakage or the formation of low-strength sponge-like porous microspheres.

[0014] The preparation method and the obtained polycitrate-modified BG / PLLA composite microspheres provided by this invention have the following advantages:

[0015] i. By preparing polycitrate prepolymer and using the prepolymer to modify bioactive glass, the polycitrate molecular chain has abundant carboxyl and hydroxyl groups, which can form strong hydrogen bonds with the silanol groups on the surface of bioactive glass. At the same time, the carboxyl groups included in polycitrate can also coordinate / ion bond with calcium ions on the surface of bioactive glass. Furthermore, during thermosetting, some carboxyl groups can also undergo esterification reaction with silanol groups to generate covalent bonds (silanol ester bonds). The above triple action can firmly anchor polycitrate on the surface of bioactive glass and change the surface hydrophilicity of bioactive glass to hydrophobicity, which is beneficial to the preparation of composite microspheres.

[0016] ii. The degradation product of polycitrate is citric acid, which is an intermediate product of the tricarboxylic acid cycle. It has high safety and has antioxidant and cell metabolism-promoting effects. It can synergistically promote collagen regeneration with the bioactivity of bioactive glass and improve the filling effect in applications.

[0017] iii. In the preparation method, by introducing polycitrate-modified BG into the PLLA material, the segments of polycitrate and PLLA have good compatibility, which can improve the dispersibility of bioactive glass in the oil phase and avoid the problem of incomplete microsphere structure caused by the migration of bioactive glass to the oil-water interface during the preparation process. Therefore, the polycitrate-modified BG / PLLA composite microspheres provided by the present invention have excellent encapsulation efficiency for bioactive glass, and can realize the sustained release of bioactive glass in subsequent applications, ensuring the safety of subsequent applications and the stability of filling effect;

[0018] iv. Furthermore, by combining bioactive glass with PLLA, the bioactive glass can neutralize the acidic products generated by PLLA degradation, reduce the inflammatory response of subsequent applications, and the silicon and calcium ions released by the bioactive glass can activate fibroblasts, significantly enhance the synthesis of collagen and elastin, endow PLLA with bioactivity, and enable it to induce hydroxyapatite deposition.

[0019] v. At the same time, the rigid particles of bioactive glass reinforce and toughen the PLLA matrix, which can improve the mechanical properties and structural stability of the microspheres;

[0020] vi. The preparation method provided by this invention adopts an improved single emulsification method, which completes emulsification in one step. The process is simple, has few parameters, is easy to control, and has good batch-to-batch repeatability. Compared with the double emulsification method, it does not require the preparation of unstable double emulsion structures, shortens the preparation cycle, greatly improves the encapsulation rate of bioactive glass, and significantly improves production efficiency and product quality stability.

[0021] Therefore, the preparation method provided by the present invention can obtain polycitrate modified BG / PLLA composite microspheres with stable quality control. The bioactive glass in the obtained polycitrate modified BG / PLLA composite microspheres has excellent encapsulation efficiency, the composite microspheres have excellent mechanical properties, and good sustained-release effect. They can significantly enhance the synthesis of collagen and elastin and have excellent application effects.

[0022] Preferably, the molar ratio of citric acid to 1,8-octanediol is 1.1:(0.8-1.2), for example, 1.1:0.8, 1.1:0.9, 1.1:1.0, 1.1:1.1, 1.1:1.2 or any range of the above values.

[0023] Preferably, the reaction in step S1 is a melting reaction, the reaction temperature is 130-145℃, for example 130℃, 132℃, 135℃, 138℃, 140℃, 142℃, 145℃ or any range between the above values, and the time is 1-4 h, for example 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h or any range between the above values.

[0024] Preferably, step S1 further includes dialysis after the reaction to remove unreacted monomers and oligomers. If the molecular weight of the obtained polycitrate prepolymer is too low, it will affect the subsequent surface modification of the bioactive glass.

[0025] Preferably, the molecular weight of the polycitric acid ester prepolymer is ≥1000 Da, for example, 1000 Da, 2000 Da, 3000 Da, 4000 Da, 5000 Da, 6000 Da, 8000 Da, 10000 Da or any of the above values.

[0026] Preferably, the molar ratio of the silicon source to the phosphorus source is (4-12):1, for example, 4:1, 6:1, 8:1, 10:1, 12:1 or any of the above values.

[0027] Preferably, the silicon source includes tetraethyl silicate, and the phosphorus source includes tributyl phosphate.

[0028] Preferably, the hydrolysis is carried out under an acidic catalyst, and the hydrolysis time is 1-3 h, for example, 1 h, 1.5 h, 2 h, 2.5 h, 3 h or any range of the above values.

[0029] Preferably, the acidic catalyst includes dilute hydrochloric acid, and the concentration of the dilute hydrochloric acid is preferably 0.3-0.6 mol / L, for example 0.3 mol / L, 0.35 mol / L, 0.4 mol / L, 0.45 mol / L, 0.5 mol / L, 0.55 mol / L, 0.6 mol / L or any range between the above values.

[0030] Preferably, the molar ratio of the calcium source to the phosphorus source is (2-8):1, for example, 2:1, 4:1, 5:1, 6:1, 8:1 or any of the above values.

[0031] Preferably, the calcium source includes calcium nitrate.

[0032] Preferably, the reaction time with the calcium source is 1-3 h, for example, 1 h, 1.5 h, 2 h, 2.5 h, 3 h or any of the above values; the gel temperature is 50-80℃, for example, 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, 80℃ or any of the above values; the gelation time is 1-3 days, for example, 1 day, 1.5 days, 2 days, 2.5 days, 3 days or any of the above values; and the aging time is 4-6 days, for example, 4 days, 4.5 days, 5 days, 5.5 days, 6 days or any of the above values.

[0033] Preferably, the average particle size of the bioactive glass is 0.5-2 μm, for example, 0.5 μm, 1.0 μm, 1.5 μm, 2.0 μm or any of the above values.

[0034] The preparation method provided by this invention can obtain bioactive glass with an average particle size of micro-nano structure, and the particle size is relatively concentrated. It has the advantages of large specific surface area, abundant surface active sites, and good dispersibility.

[0035] Preferably, the mass ratio of the polycitric acid ester prepolymer to the bioactive glass is (0.2-5):1, for example, 0.2:1, 0.5:1, 1:1, 2:1, 3:1, 4:1, 5:1 or any range of the above values.

[0036] This invention, by limiting the mass ratio of polycitrate prepolymer to bioactive glass, has advantages such as high interfacial bonding strength, avoidance of particle agglomeration, and controllable bioactivity. If the amount of polycitrate added is too low, the modification effect of bioactive glass will be poor, and bioactive glass will easily precipitate during the subsequent preparation of composite microspheres, resulting in a decrease in encapsulation rate and a deterioration in application effect. If the amount of polycitrate added is too high, the polymer coating layer on the surface of BG will be too thick, hindering the effective contact between glass particles and the external environment and reducing the bioactivity of composite microspheres.

[0037] Preferably, the reaction in step S3 is carried out under a protective atmosphere for a time of 12-48 h, such as 12 h, 20 h, 25 h, 30 h, 35 h, 40 h, 48 h or any range between the above values.

[0038] Preferably, the temperature for heat curing in step S3 is 60-120℃, for example, 60℃, 70℃, 80℃, 90℃, 100℃, 110℃, 120℃ or any of the above values, and the time is 6-24 h, for example, 6 h, 10 h, 15 h, 20 h, 24 h or any of the above values.

[0039] Preferably, step S3 further includes: vacuum drying the bioactive glass at 80-120°C (e.g., 80°C, 85°C, 90°C, 95°C, 100°C, 105°C, 110°C, 115°C, 120°C or any of the above values) for 12-24 h (e.g., 12 h, 15 h, 18 h, 21 h, 24 h or any of the above values), and then dispersing it in anhydrous ethanol to obtain a dispersion.

[0040] Preferably, the concentration of the dispersion is 0.04-0.1 g / mL, for example, 0.004 g / mL, 0.025 g / mL, 0.050 g / mL, 0.075 g / mL, 0.100 g / mL or any range between the above values.

[0041] Preferably, the mass ratio of the polycitric acid ester modified BG to PLLA is (0.01-0.5):1, for example, 0.01:1, 0.1:1, 0.2:1, 0.25:1, 0.3:1, 0.4:1, 0.5:1 or any range of the above values.

[0042] In this invention, by controlling the mass ratio of polycitrate-modified BG and PLLA, the composite microspheres can achieve better overall performance. If the amount of polycitrate-modified BG added is too low, it may lead to insufficient bioactivity of the composite microspheres. If the amount of polycitrate-modified BG added is too high, it may easily lead to a decrease in the encapsulation rate of the bioactive glass.

[0043] Preferably, in the oil phase, the concentration of the polycitrate-modified BG is 2.5-12.5 mg / mL, for example, 2.5 mg / mL, 5 mg / mL, 8 mg / mL, 10 mg / mL, 12.5 mg / mL or any range between the above values.

[0044] Preferably, the volume ratio of the oil phase to the water phase is 1:(5-20), for example, 1:5, 1:8, 1:10, 1:12, 1:15, 1:18, 1:20 or any of the above values.

[0045] Preferably, the concentration of the emulsifier in the aqueous phase is 0.5-5 wt%, for example, 0.5 wt%, 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, or any of the above values.

[0046] Preferably, the organic solvent used in the oil phase includes, but is not limited to, any one or a combination of at least two of dichloromethane, chloroform, or tetrahydrofuran.

[0047] Preferably, the emulsifier includes, but is not limited to, any one or a combination of at least two of polyvinyl alcohol, gelatin, or Tween 80.

[0048] Preferably, the method for evaporating the organic solvent includes stirring at room temperature for 12-36 h (e.g., 12 h, 15 h, 20 h, 25 h, 30 h, 36 h or any range between the above values) to allow the organic solvent to evaporate.

[0049] During the evaporation of the organic solvent, PLLA solidifies into microspheres, which can then be centrifuged, washed, and freeze-dried.

[0050] As a preferred embodiment of the present invention, the preparation method includes:

[0051] S1. Citric acid and 1,8-octanediol were mixed and melted in a molar ratio of 1.1:(0.8-1.2) under a nitrogen atmosphere. After melting, the mixture was reacted at 130-145℃ for 1-4 h. After the reaction, the mixture was dialyzed to remove components with a molecular weight less than 1000 Da to obtain polycitric acid ester prepolymer.

[0052] S2. A silicon source and a phosphorus source are mixed in a solvent at a molar ratio of (4-12):1. The mixture is then acid-catalyzed and hydrolyzed at room temperature to obtain an intermediate product. A calcium source is added at a molar ratio of (2-8):1 to the phosphorus source, and the mixture is reacted for 1-3 hours. The mixture is then gelled at 50-80°C for 1-3 days, aged for 4-6 days, dried, sintered, ground, and sieved to obtain a bioactive glass with an average particle size of 0.5-2 μm. Preferably, the solvent is a mixture of ethanol and water.

[0053] S3. After vacuum drying the bioactive glass at 80-120℃ for 12-24 h, disperse it in ethanol to prepare a dispersion. Mix the polycitrate prepolymer with the bioactive glass dispersion at a mass ratio of (0.2-5):1, and react at room temperature under nitrogen protection for 12-48 h. After the reaction, separate and dry the mixture, and heat-cur it at 60-120℃ for 6-24 h to obtain polycitrate modified BG.

[0054] S4. Following a mass ratio of polycitric acid ester modified BG to PLLA of (0.01-0.5):1, mix the polycitric acid ester modified BG and PLLA in an organic solvent to obtain an oil phase. The concentration of the polycitric acid ester modified BG is 2.5-12.5 mg / mL. At a stirring rate of 2000-10000 rpm (e.g., 2000 rpm, 3000 rpm, 4000 rpm, 5000 rpm, 6000 rpm, 7000 rpm, 8000 rpm, 9000 rpm, 10000 rpm, or any range above), add the oil phase dropwise to an aqueous phase containing 0.5-5 wt% emulsifier at a volume ratio of 1:(5-20). After addition, continue emulsifying for 1-5 min (e.g., 1 min, 2 min, 3 min, 4 min, 5 min, or any range above) to obtain an emulsion. Then, continue stirring at room temperature for 12-36 minutes. h, allowing the organic solvent to evaporate, centrifuge, wash, and freeze-dry to obtain the polycitric acid ester modified BG / PLLA composite microspheres.

[0055] In this invention, when the reaction temperature is not specified, the reaction is at room temperature, which can also be understood as 10-30°C, such as 10°C, 15°C, 20°C, 25°C, 30°C or any of the above values, preferably a reaction temperature of 15-25°C, and more preferably 20-25°C.

[0056] Preferably, in step S2, the sintering temperature is 600-800℃, for example, 600℃, 650℃, 700℃, 750℃, 800℃ or any of the above values, and the time is 2-6 h, for example, 2 h, 3 h, 4 h, 5 h, 6 h or any of the above values.

[0057] In the preparation method provided by the present invention, polycitrate is used to modify the surface of bioactive glass powder, introducing active functional groups on the surface of bioactive glass and changing the surface of bioactive glass from hydrophilic to hydrophobic. Then, a PLLA coating layer is formed on the surface of polycitrate-modified BG by a single emulsification-solvent evaporation method, and finally a composite microsphere with a complete core-shell structure is obtained. The composite microsphere has excellent collagen regeneration ability and long-lasting filling effect.

[0058] In a second aspect, the present invention provides polycitric acid ester modified BG / PLLA composite microspheres prepared by the preparation method described in the first aspect.

[0059] Preferably, the average particle size of the polycitric acid ester modified BG / PLLA composite microspheres is 20-50 μm, for example, 20 μm, 25 μm, 30 μm, 40 μm, 50 μm or any of the above values.

[0060] The polycitric acid ester modified BG / PLLA composite microspheres prepared by the preparation method provided by the present invention have a regular spherical shape, good sphericity, and a smooth and dense surface; they also have good dispersibility and no obvious agglomeration. At the same time, the white polycitric acid ester modified BG powder is evenly distributed in the transparent PLLA matrix.

[0061] Thirdly, the present invention provides an application of polycitrate-modified BG / PLLA composite microspheres as described in the second aspect in injection-filled materials.

[0062] Compared with the prior art, the present invention has the following beneficial effects:

[0063] The preparation method provided by this invention can obtain polycitrate-modified BG / PLLA composite microspheres with stable quality control. The bioactive glass in the obtained polycitrate-modified BG / PLLA composite microspheres has excellent encapsulation efficiency, the composite microspheres have excellent mechanical properties, and good sustained-release effect. They can significantly enhance the synthesis of collagen and elastin and have excellent application effects. Attached Figure Description

[0064] Figure 1 The image shown is a microscope image of the polycitric acid ester modified BG / PLLA composite microspheres provided in Example 1 of this invention, with a magnification of 150x. Detailed Implementation

[0065] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0066] Unless otherwise specified, the raw materials involved in the following specific embodiments of the present invention are all conventional materials in the art, and can be purchased from commercially available products. Some raw material information is as follows:

[0067] PLLA-1: Powder, purchased from Shenzhen Jusheng Biotechnology Co., Ltd., brand name PLLA18;

[0068] PLLA-2: Powder form, purchased from Shenzhen Jusheng Biotechnology Co., Ltd., brand name PLLA24;

[0069] PLLA-3: Powder form, purchased from Shenzhen Jusheng Biotechnology Co., Ltd., brand name PLLA32;

[0070] Polyvinyl alcohol: purchased from Aladdin, 3.5-4.5 mPa·s;

[0071] Gelatin: Purchased from Aladdin, gel strength ~100 g Bloom;

[0072] Twain: Purchased from Aladdin, Twain-80 pharmaceutical grade.

[0073] Preparation Example 1-1

[0074] This preparation example provides a method for preparing a polycitric acid ester prepolymer, as follows:

[0075] Citric acid and 1,8-octanediol were added to a three-necked flask at a molar ratio of 1.1:0.8. Under nitrogen protection, the mixture was heated in an oil bath at 140°C and stirred vigorously until completely melted. After the reactants melted, the temperature was kept constant at 130°C and the reaction was continued with stirring for 4 hours. After the reaction was completed, the product was dialyzed in a dialysis bag (purchased from Sigma-Aldrich, 1000 Da) for 3 days to obtain purified POC prepolymer with an average molecular weight of 1800 Da.

[0076] Preparation Examples 1-2

[0077] This preparation example provides a method for preparing a polycitric acid ester prepolymer, as follows:

[0078] Citric acid and 1,8-octanediol were added to a three-necked flask at a molar ratio of 1.1:1.0. Under nitrogen protection, the mixture was heated in an oil bath at 150°C and stirred vigorously until completely melted. After the reactants melted, the temperature was kept constant at 135°C and the reaction was continued with stirring for 3 hours. After the reaction was completed, the product was dialyzed in a dialysis bag for 4 days to obtain purified POC prepolymer with an average molecular weight of 3000 Da.

[0079] Preparation Examples 1-3

[0080] This preparation example provides a method for preparing a polycitric acid ester prepolymer, as follows:

[0081] Citric acid and 1,8-octanediol were added to a three-necked flask at a molar ratio of 1.1:1.2. Under nitrogen protection, the mixture was heated in an oil bath at 160°C and stirred vigorously until completely melted. After the reactants melted, the temperature was kept constant at 140°C and the reaction was continued with stirring for 2 hours. After the reaction was completed, the product was dialyzed in a dialysis bag for 5 days to obtain purified POC prepolymer with an average molecular weight of 4200 Da.

[0082] Preparation Example 2-1

[0083] This preparation example provides a method for preparing bioactive glass, as follows:

[0084] 8 ml of tetraethyl silicate, 1.3 ml of triethyl phosphate, 7 ml of ethanol, and 7 ml of purified water were added sequentially to a three-necked flask. 0.3 mol / L dilute hydrochloric acid solution was added dropwise as a catalyst. After reacting for 1 h, 4 g of calcium nitrate was added, and the reaction was continued for 1 h to obtain a homogeneous solution. The solution was gelled at 60 °C for 1 day, aged at room temperature for 4 days, dried at 80 °C for 1 day, sintered at 600 °C for 2 h, ground, and sieved to obtain BG powder with an average particle size of 0.5 μm.

[0085] Preparation Example 2-2

[0086] This preparation example provides a method for preparing bioactive glass, as follows:

[0087] 9 ml of tetraethyl silicate, 1.2 ml of triethyl phosphate, 8 ml of ethanol, and 8 ml of purified water were sequentially added to a three-necked flask. 0.4 mol / L dilute hydrochloric acid solution was added dropwise as a catalyst. After reacting for 1.5 h, 5 g of calcium nitrate was added, and the reaction was allowed to proceed for another 1.5 h to obtain a homogeneous solution. The solution was then gelled at 70 °C for 1.5 days, aged at room temperature for 4.5 days, dried at 80 °C for 1.5 days, sintered at 650 °C for 3 h, ground, and sieved to obtain BG powder with an average particle size of 0.8 μm.

[0088] Preparation Examples 2-3

[0089] This preparation example provides a method for preparing bioactive glass, as follows:

[0090] 10 ml of tetraethyl silicate, 1.1 ml of triethyl phosphate, 9 ml of ethanol, and 9 ml of purified water were added sequentially to a three-necked flask. 0.5 mol / L dilute hydrochloric acid solution was added dropwise as a catalyst. After reacting for 2 h, 6 g of calcium nitrate was added, and the reaction was allowed to proceed for another 2 h to obtain a homogeneous solution. The solution was then gelled at 80 °C for 2 days, aged at room temperature for 5 days, dried at 80 °C for 2 days, sintered at 700 °C for 4 h, ground, and sieved to obtain BG powder with an average particle size of 1.2 μm.

[0091] Preparation Examples 2-4

[0092] This preparation example provides a method for preparing bioactive glass, as follows:

[0093] 12 ml of tetraethyl silicate, 0.8 ml of triethyl phosphate, 10 ml of ethanol, and 10 ml of purified water were added sequentially to a three-necked flask. 0.6 mol / L dilute hydrochloric acid solution was added dropwise as a catalyst. After reacting for 3 h, 8 g of calcium nitrate was added, and the reaction was continued for another 3 h to obtain a homogeneous solution. The solution was then gelled at 80 °C for 3 days, aged at room temperature for 6 days, dried at 80 °C for 3 days, sintered at 800 °C for 5 h, ground, and sieved to obtain BG powder with an average particle size of 2 μm.

[0094] Preparation Example 3-1

[0095] This preparation example provides a method for preparing polycitrate-modified BG, as follows:

[0096] BG powder (Preparation Example 2-1) was placed in a vacuum drying oven and dried at 80°C for 12 hours. Then, 0.1 g of the dried BG powder was dispersed in 5 ml of anhydrous ethanol and ultrasonically dispersed for 30 min to obtain a BG dispersion. The dispersion was mixed with 0.5 g of POC prepolymer (Preparation Example 1-1) and stirred at room temperature for 12 h under nitrogen protection. After the reaction was completed, the mixture was centrifuged, washed, dried, and thermocured at 80°C for 6 h to obtain polycitric acid ester modified BG (BG@POC).

[0097] Preparation Example 3-2

[0098] This preparation example provides a method for preparing polycitrate-modified BG, as follows:

[0099] BG powder (Preparation Example 2-2) was placed in a vacuum drying oven and dried at 90°C for 16 hours. Then, 0.2 g of the dried BG powder was dispersed in 10 ml of anhydrous ethanol and ultrasonically dispersed for 30 min to obtain a BG dispersion. The dispersion was mixed with 0.4 g of POC prepolymer (Preparation Example 1-2) and stirred at room temperature for 20 h under nitrogen protection. After the reaction was completed, the mixture was centrifuged, washed, dried, and thermocured at 90°C for 10 h to obtain polycitric acid ester modified BG (BG@POC).

[0100] Preparation Example 3-3

[0101] This preparation example provides a method for preparing polycitrate-modified BG, as follows:

[0102] BG powder (Preparation Examples 2-3) was placed in a vacuum drying oven and dried at 100°C for 20 hours. Then, 0.3 g of the dried BG powder was dispersed in 15 ml of anhydrous ethanol and ultrasonically dispersed for 30 min to obtain a BG dispersion. The dispersion was mixed with 0.3 g of POC prepolymer (Preparation Examples 1-3) and stirred at room temperature for 28 h under nitrogen protection. After the reaction was completed, the mixture was centrifuged, washed, dried, and thermocured at 100°C for 14 h to obtain polycitric acid ester modified BG (BG@POC).

[0103] Preparation Examples 3-4

[0104] This preparation example provides a method for preparing polycitrate-modified BG, as follows:

[0105] BG powder (Preparation Examples 2-4) was placed in a vacuum drying oven and dried at 120°C for 24 hours. Then, 0.5 g of the dried BG powder was dispersed in 20 ml of anhydrous ethanol and ultrasonically dispersed for 30 min to obtain a BG dispersion. The dispersion was mixed with 0.1 g of POC prepolymer (Preparation Examples 1-3) and stirred at room temperature for 48 h under nitrogen protection. After the reaction was completed, the mixture was centrifuged, washed, dried, and thermocured at 120°C for 18 h to obtain polycitric acid ester modified BG (BG@POC).

[0106] Preparation Examples 3-5

[0107] This preparation example provides a method for preparing polycitrate-modified BG.

[0108] The difference from Preparation Example 3-1 is that, in this preparation example, the bioactive glass was replaced with commercially available bioactive glass (purchased from Ipuri, model 2 µm).

[0109] Example 1

[0110] This embodiment provides a method for preparing polycitrate-modified BG / PLLA composite microspheres, as follows:

[0111] Polyvinyl alcohol was dissolved in deionized water to prepare an emulsifier aqueous solution with a concentration of 0.5 wt%, which served as the aqueous phase. 0.05 g of BG@POC powder (Preparation Example 3-1) was ultrasonically dispersed in 20 ml of dichloromethane, and then 3 g of PLLA-3 powder was added and fully dissolved to form an oil phase. The oil phase was slowly added dropwise to the aqueous phase at a volume ratio of 1:10, and emulsified at 5000 rpm for 3 min to form an oil-in-water emulsion. The emulsion was then stirred continuously at room temperature for 12 h to allow the organic solvent to evaporate completely and the PLLA to solidify into microspheres. The microspheres were collected by centrifugation, washed three times with deionized water, and freeze-dried to obtain polycitric acid ester modified BG / PLLA composite microspheres ((BG@POC) / PLLA composite microspheres).

[0112] Figure 1 The image shows a microscope photograph of the polycitric acid ester modified BG / PLLA composite microspheres prepared in this embodiment, magnified 150 times. As can be seen from the image, the composite microspheres prepared by the method provided by this invention are regular spheres with good sphericity, smooth and dense surfaces, and no obvious cracks or depressions. At the same time, the composite microspheres have good dispersibility and no obvious agglomeration. In addition, since the polycitric acid ester modified BG is a white powder and the PLLA matrix is ​​a transparent material, it can also be seen from the image that, at the macroscopic level, the white polycitric acid ester modified BG is uniformly distributed inside the PLLA matrix.

[0113] Example 2

[0114] This embodiment provides a method for preparing polycitrate-modified BG / PLLA composite microspheres, as follows:

[0115] Gelatin was dissolved in deionized water to prepare an emulsifier aqueous solution with a concentration of 1 wt%, which served as the aqueous phase. 0.10 g of BG@POC powder (Preparation Example 3-2) was ultrasonically dispersed in 20 ml of chloroform, and then 2 g of PLLA-2 powder was added and fully dissolved to form an oil phase. The oil phase was slowly added dropwise to the aqueous phase at a volume ratio of 1:10, and emulsified at 5000 rpm for 3 min to form an oil-in-water emulsion. The emulsion was then stirred continuously at room temperature for 20 h to allow the organic solvent to evaporate completely and the PLLA to solidify into microspheres. The microspheres were collected by centrifugation, washed three times with deionized water, and freeze-dried to obtain polycitric acid ester modified BG / PLLA composite microspheres ((BG@POC) / PLLA composite microspheres).

[0116] Example 3

[0117] This embodiment provides a method for preparing polycitrate-modified BG / PLLA composite microspheres, as follows:

[0118] Tween was dissolved in deionized water to prepare an emulsifier aqueous solution with a concentration of 2 wt%, which served as the aqueous phase. 0.2 g of BG@POC powder (Preparation Example 3-3) was ultrasonically dispersed in 20 ml of tetrahydrofuran, and then 1 g of PLLA-1 powder was added and fully dissolved to form an oil phase. The oil phase was slowly added dropwise to the aqueous phase at a volume ratio of 1:10, and emulsified at 5000 rpm for 3 min to form an oil-in-water emulsion. The emulsion was then stirred continuously at room temperature for 28 h to allow the organic solvent to evaporate completely and the PLLA to solidify into microspheres. The microspheres were collected by centrifugation, washed three times with deionized water, and freeze-dried to obtain polycitric acid ester modified BG / PLLA composite microspheres ((BG@POC) / PLLA composite microspheres).

[0119] Example 4

[0120] This embodiment provides a method for preparing polycitrate-modified BG / PLLA composite microspheres.

[0121] Polyvinyl alcohol was dissolved in deionized water to prepare an emulsifier aqueous solution with a concentration of 5 wt%, which served as the aqueous phase. 0.25 g of BG@POC powder (Preparation Example 3-4) was ultrasonically dispersed in 20 ml of dichloromethane, and then 0.6 g of PLLA-1 powder was added and fully dissolved to form an oil phase. The oil phase was slowly added dropwise to the aqueous phase at a volume ratio of 1:20, and emulsified at 5000 rpm for 3 min to form an oil-in-water emulsion. The emulsion was then stirred continuously at room temperature for 36 h to allow the organic solvent to evaporate completely and the PLLA to solidify into microspheres. The microspheres were collected by centrifugation, washed three times with deionized water, and freeze-dried to obtain polycitric acid ester modified BG / PLLA composite microspheres ((BG@POC) / PLLA composite microspheres).

[0122] Example 5

[0123] This embodiment provides a method for preparing polycitrate-modified BG / PLLA composite microspheres.

[0124] The difference from Example 1 is that, in this example, the BG@POC powder is replaced with the BG@POC powder provided in Preparation Example 3-2.

[0125] Example 6

[0126] This embodiment provides a method for preparing polycitrate-modified BG / PLLA composite microspheres.

[0127] The difference from Example 1 is that, in this example, the BG@POC powder is replaced with the BG@POC powder provided in Preparation Example 3-3.

[0128] Example 7

[0129] This embodiment provides a method for preparing polycitrate-modified BG / PLLA composite microspheres.

[0130] The difference from Example 1 is that, in this example, the BG@POC powder is replaced with the BG@POC powder provided in Preparation Examples 3-4.

[0131] Example 8

[0132] This embodiment provides a method for preparing polycitrate-modified BG / PLLA composite microspheres.

[0133] The difference from Example 1 is that, in this example, the BG@POC powder is replaced with the BG@POC powder provided in Preparation Examples 3-5.

[0134] Comparative Example 1

[0135] This comparative example provides a method for preparing PLLA microspheres.

[0136] The difference from Example 1 is that BG@POC powder is not added in this comparative example.

[0137] Comparative Example 2

[0138] This comparative example provides a method for preparing bioactive glass / PLLA microspheres.

[0139] The difference from Example 1 is that, in this comparative example, the BG@POC powder is replaced with the BG powder provided in Preparation Example 2-1.

[0140] Comparative Example 3

[0141] This comparative example provides a method for preparing bioactive glass / PLLA microspheres (double emulsion method), as follows:

[0142] Polyvinyl alcohol (PVA) was dissolved in deionized water to prepare a 2 wt% emulsifier aqueous solution as aqueous phase 1, and a 0.5 wt% emulsifier aqueous solution as aqueous phase 2. 0.1 g of BG powder (Preparation Example 2-1) was ultrasonically dispersed in 3 ml of aqueous phase 1, and 3 g of PLLA powder was fully dissolved in 20 ml of dichloromethane to form an oil phase. The two phases were then mixed and emulsified at 5000 rpm for 5 min to form an emulsion. The emulsion was rapidly added to aqueous phase 2 at a volume ratio of 1:10 to form a water-in-oil-in-water emulsion. The mixture was then continuously stirred at room temperature for 12 h to allow the organic solvent to completely evaporate and the PLLA to solidify into microspheres. The microspheres were collected by filtration, washed three times with deionized water, and freeze-dried to obtain BG / PLLA composite microspheres.

[0143] Performance Test 1

[0144] The performance of the composite microspheres provided in the examples and comparative examples was tested using the following methods:

[0145] (1) Bioactive glass encapsulation efficiency test: Accurately weigh a certain amount (m 总 The composite microspheres were dissolved in dichloromethane to dissolve PLLA, and BG was separated by centrifugation. After drying, the contents were weighed (m). BG The encapsulation ratio is calculated using the following formula:

[0146] BG Encapsulation Ratio, % = (m BG / m 理论 )×100%;

[0147] Where, m 理论 The mass of BG or BG@POC added during preparation;

[0148] (2) Surface BG exposure rate test: The surface morphology of the microspheres was observed using a scanning electron microscope (SEM). Five different fields of view were randomly selected at 200x magnification. The number of microspheres with obvious exposed BG particles on the surface was counted, and the exposure rate was calculated.

[0149] Surface BG exposure rate, % = (number of microspheres with exposed BG on the surface / total number of observed microspheres) × 100%;

[0150] (3) Strength test: The hardness and indentation modulus of the microspheres were determined by a nanoindenter. A flat-headed indenter (100 μm in diameter) was used with a maximum load of 100 mN, a loading rate of 2 mN / s, and a holding time of 10 s. The microspheres were uniformly dispersed on the silicon wafer surface. Individual microspheres were located using an optical microscope. After loading to the maximum load, the load was unloaded and the load-displacement curve was recorded. The hardness and modulus were calculated according to the Oliver-Pharr method. The average value of 10 microspheres in each group was taken.

[0151] The test results are as follows:

[0152] Table 1

[0153]

[0154] As can be seen from the examples and performance tests, the preparation method provided by the present invention can obtain composite microspheres with an encapsulation rate of over 95%, while the exposure rate of bioactive glass on the surface is less than 5%. Moreover, by introducing polycitrate-modified BG as rigid particles, it has a significant reinforcing and toughening effect on the PLLA matrix, which can significantly improve the mechanical properties of the composite microspheres, wherein the nanoindentation hardness is above 0.25 GPa and the indentation modulus is above 4 GPa.

[0155] In Comparative Example 2, the direct introduction of bioactive glass resulted in an extremely low encapsulation rate of only 42%, and a relatively high exposure rate of 46%. In Comparative Example 3, the encapsulation rate of bioactive glass was increased by using a double emulsification method, but the encapsulation rate was still low at only 58%, and the exposure rate was 22%. The comparison between the examples and Comparative Examples 2 and 3 shows that this invention modifies bioactive glass using polycitrate, changing the surface of the bioactive glass from hydrophilic to hydrophobic. Simultaneously, the improved single emulsification method effectively confines the polycitrate-modified BG within the oil phase during emulsification, avoiding the disadvantages of low encapsulation rate and high exposure rate caused by the migration of bioactive glass to the oil-water interface. This results in uniform dispersion of the bioactive glass in the oil phase and reduced leakage, achieving complete encapsulation of the bioactive glass by PLLA, significantly improving the encapsulation rate, and greatly reducing the exposure rate.

[0156] In Comparative Example 3, polymer microspheres were prepared using a double emulsion method. Due to the large number of pores left by the internal aqueous phase droplets, the microspheres had a sponge-like porous structure with low density and the worst mechanical properties.

[0157] Performance Test 2

[0158] The performance of the composite microspheres provided in the examples and comparative examples was tested using the following methods:

[0159] (1) Ion release concentration test: The composite microspheres were soaked in PBS solution at a solid-liquid ratio of 0.2 g / mL. The release concentrations of silicon ions and calcium ions released by the composite microspheres in the soaking solution were tested at 7 and 28 days using an ICP spectrometer.

[0160] (2) Cell proliferation rate (CCK-8 assay): Human skin fibroblasts were subjected to a 1×10⁻⁶ saturation assay. 4 Cells / well were seeded into 96-well plates. Microspheres were sterilized with ethylene oxide and added to the wells at a concentration of 1 mg / mL. After incubation at 37°C and 5% CO2 for 7 days, 10 μL of CCK-8 reagent was added to each well for 2 h. The absorbance at 450 nm was measured using a microplate reader.

[0161] Cell proliferation rate = (OD value of experimental group / OD value of blank control group) × 100%;

[0162] (3) Type I collagen secretion (ELISA method): Collect the supernatant after 7 days of cell culture, centrifuge to remove cell debris, and follow the instructions of the ELISA kit (purchased from Elite, brand number E-EL-H0869) to add the standard or test sample, biotinylated detection antibody, HRP-labeled secondary antibody, and TMB substrate in sequence. After terminating the reaction, use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance at 450 nm and calculate the type I collagen concentration (μg / mL) according to the standard curve.

[0163] The test results are as follows:

[0164] Table 2

[0165]

[0166] As demonstrated by the examples and performance tests, the composite microspheres provided by this invention achieve complete encapsulation of bioactive glass by the PLLA matrix, effectively avoiding ion burst release and inflammatory reactions caused by excessively high local concentrations. Furthermore, by controlling the degree of modification of the bioactive glass with polycitrate, the sustained-release effect of the bioactive glass can be precisely regulated. Simultaneously, cell proliferation rate and type I collagen secretion are significantly increased. That is, this invention avoids cytotoxicity caused by burst release by controlling the sustained release of silicon and calcium ions from the bioactive glass, and, in conjunction with the synergistic effect of PLLA stimulating collagen regeneration, significantly improves cell proliferation rate and type I collagen secretion.

[0167] The composite microspheres provided in Comparative Example 2 have a high exposure rate of bioactive glass, which leads to the problem of ion burst release. Although Comparative Example 3 alleviates the ion burst release, the microspheres are still porous, so the problem of ion burst release still exists. This can easily lead to inflammatory reactions due to excessively high local concentrations and cause cytotoxicity, resulting in a low cell proliferation rate.

[0168] The present invention has been illustrated through the above embodiments, but the present invention is not limited to the above process steps, that is, it does not mean that the present invention must rely on the above process steps to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials used in the present invention, additions of auxiliary components, and selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for preparing poly-citrate modified BG / PLLA composite microspheres, characterized in that, The preparation method includes: S1. Citric acid and 1,8-octanediol react to obtain a polycitric acid ester prepolymer; S2. The silicon and phosphorus sources are hydrolyzed to obtain intermediate products. The intermediate products are reacted with calcium sources, gelled, aged, dried and sintered to obtain bioactive glass. S3. Polycitrate prepolymer is reacted with bioactive glass and thermosetting to obtain polycitrate modified BG; S4. Polycitric acid ester modified BG and PLLA are mixed in an organic solvent to obtain an oil phase. The oil phase is then mixed and emulsified with an aqueous phase containing an emulsifier to obtain an emulsion. The organic solvent in the emulsion evaporates to obtain the polycitric acid ester modified BG / PLLA composite microspheres.

2. The production method according to claim 1, characterized by, The molar ratio of citric acid to 1,8-octanediol is 1.1:(0.8-1.2); The reaction described in step S1 is a melting reaction, and the reaction temperature is 130-145℃, with a time of 1-4 h; Step S1 also includes dialysis after the reaction; The molecular weight of the polycitric acid ester prepolymer is ≥1000 Da.

3. The preparation method according to claim 1, characterized in that, The molar ratio of the silicon source to the phosphorus source is (4-12):1; The hydrolysis is carried out under an acidic catalyst for a time of 1-3 hours. The molar ratio of the calcium source to the phosphorus source is (2-8):1; The reaction time with the calcium source is 1-3 hours, the gel temperature is 50-80°C, the gelation time is 1-3 days, and the aging time is 4-6 days.

4. The method of claim 1, wherein, The average particle size of the bioactive glass is 0.5-2 μm.

5. The preparation method according to claim 1, characterized in that, The mass ratio of the polycitric acid ester prepolymer to the bioactive glass is (0.2-5):1; The reaction described in step S3 is carried out under a protective atmosphere for 12-48 hours. The temperature for heat curing in step S3 is 60-120℃, and the time is 6-24 h.

6. The method of claim 1, wherein, The mass ratio of the polycitric acid ester modified BG to PLLA is (0.01-0.5):1; In the oil phase, the concentration of the polycitrate-modified BG is 2.5-12.5 mg / mL; The volume ratio of the oil phase to the water phase is 1:(5-20); The concentration of emulsifier in the aqueous phase is 0.5-5 wt%; The method for evaporating the organic solvent includes stirring at room temperature for 12-36 h to allow the organic solvent to evaporate.

7. The preparation method according to claim 1, characterized in that, The preparation method includes: S1. Citric acid and 1,8-octanediol were mixed and melted in a molar ratio of 1.1:(0.8-1.2) under a nitrogen atmosphere. After melting, the mixture was reacted at 130-145℃ for 1-4 h. After the reaction, the mixture was dialyzed to remove components with a molecular weight less than 1000 Da to obtain polycitric acid ester prepolymer. S2. Silicon source and phosphorus source are mixed in solvent at a molar ratio of (4-12):1 and acid-catalyzed hydrolysis is performed to obtain intermediate product. Calcium source is added at a molar ratio of (2-8):1 to react for 1-3 h. The mixture is gelled at 50-80℃ for 1-3 days, aged for 4-6 days, dried, sintered, ground, and sieved to obtain bioactive glass with an average particle size of 0.5-2 μm. S3. Mix the polycitric acid ester prepolymer with the bioactive glass dispersion at a mass ratio of (0.2-5):1, and react under nitrogen protection for 12-48 h. After reaction, separate and dry, and heat-cur at 60-120℃ for 6-24 h to obtain polycitric acid ester modified BG. S4. The polycitrate-modified BG and PLLA are mixed in an organic solvent at a mass ratio of (0.01-0.5):1 to obtain an oil phase. The concentration of the polycitrate-modified BG is 2.5-12.5 mg / mL. At a stirring speed of 2000-10000 rpm, the oil phase is added dropwise to an aqueous phase containing 0.5-5 wt% emulsifier at a volume ratio of 1:(5-20). After addition, emulsification continues for 1-5 min to obtain an emulsion. Then, stirring continues for 12-36 h to allow the organic solvent to evaporate, yielding the polycitrate-modified BG / PLLA composite microspheres.

8. A polycitric acid ester modified BG / PLLA composite microsphere prepared by the preparation method according to any one of claims 1-7.

9. The polycitrate-modified BG / PLLA composite microspheres according to claim 8, characterized in that, The average particle size of the polycitrate-modified BG / PLLA composite microspheres is 20-50 μm.

10. The application of polycitric acid ester modified BG / PLLA composite microspheres as described in claim 8 or 9 in injection-filled materials.

Images