Preparation method of photosensitive induced cross-linked HA / PHBV composite filling material

Abstract

The invention relates to the field of biological materials, and discloses a preparation method of a photosensitive induced cross-linked HA / PHBV composite filling material. The preparation method comprises the following steps: S1, constructing photosensitive induced cross-linked hyaluronic acid gel in the presence of photosensitive polyphenol and a photocatalyst; s2, preparing uniform poly (hydroxybutyric acid)-pentanoic acid copolyester microspheres subjected to plasma activation; and S3, mixing the microspheres with the gel through a partial compounding process to construct a uniform suspension system. According to the invention, the defects of the traditional filling agent are effectively overcome; a photosensitive induction system is used for replacing a chemical cross-linking agent, so that the risk of residual toxicity is fundamentally avoided; the degradation curve of the material is more stable, and the action time is obviously prolonged; through microsphere activation and a specific mixing process, uniform and stable suspension of polyhydroxybutyrate-pentanoic acid copolyester microspheres in a gel carrier is ensured, and the stability and injection uniformity of a product are improved.

Description

Technical Field

[0001] This invention relates to the field of filler materials technology, specifically to a method for preparing a photosensitive induced crosslinked HA / PHBV composite filler material. Background Technology

[0002] Existing filler materials, especially hyaluronic acid (HA)-based fillers, still face challenges in terms of chemical safety, degradation behavior, and physical stability. Regarding chemical safety, traditional processes commonly use chemical cross-linking agents such as 1,4-butanediol diglycidyl ether (BDDE), posing a risk of residual cross-linking agents and potentially triggering sensitization. In terms of degradation performance, these fillers exhibit insufficiently smooth degradation curves in vivo, often being rapidly absorbed within 2 to 6 months, resulting in a short duration of action and difficulty in achieving long-lasting shaping. Regarding physical stability, composite systems introduced to extend support time (such as those containing PHBV, PLLA, or PCL polymer microspheres) often cause problems due to poor density matching. When the microsphere density is significantly higher than that of the HA gel carrier, this density mismatch leads to rapid microsphere sedimentation (24-hour sedimentation rate >30%). This phase separation phenomenon not only causes inhomogeneity within product batches and during injection but also creates localized microsphere-rich areas under the skin, causing uneven physical stimulation to surrounding tissues. Such differential stimulation may disrupt normal cellular responses, affect the uniformity of tissue repair and collagen regeneration, ultimately leading to unpredictable clinical outcomes and increasing the risk of adverse reactions such as inflammation and nodules. Summary of the Invention

[0003] This invention aims to provide a method for preparing a photosensitive induced crosslinked HA (crosslinked hyaluronic acid) / PHBV (polyhydroxybutyrate-valerate copolyester) composite filler, thereby solving several defects of traditional fillers: First, this invention uses a photosensitive induced crosslinking system to replace chemical crosslinking agents, fundamentally avoiding the risk of residual toxicity; second, this crosslinking system makes the degradation curve more stable and prolongs the reaction time; through uniform plasma-activated microspheres and a fractional composite mixing process, the uniform and stable suspension of microspheres in the gel carrier is ensured, improving the stability and injection uniformity of the product.

[0004] The technical solution of the present invention: A method for preparing a photosensitive induced crosslinked HA / PHBV composite filler material includes the following steps: S1. Construction of photosensitive induced cross-linked HA gel: Sodium hyaluronate solution was subjected to photoinduced cross-linking reaction in the presence of polyphenols and photocatalyst to form cross-linked HA gel. The gel was then crushed, sieved to a particle size of 10-100 μm and purified. S2. Preparation of PHBV microspheres: PHBV microspheres were prepared and their surface was activated by plasma. S3. Preparation of composite filler material: The PHBV microspheres prepared in step S2 are uniformly mixed with the cross-linked HA gel obtained by sieving and purification in step S1 to construct a suspension stable system and obtain the photosensitive induced cross-linked HA / PHBV composite filler material.

[0005] Preferably, step S1 includes the following steps: S11. Dissolve sodium hyaluronate in deionized water to prepare a 1–10% w / v solution; S12. Add polyphenols and photocatalysts to the sodium hyaluronate solution; S13, pretreatment at 2–30℃ for 1.5–3 hours; S14, photo-induced crosslinking reaction was carried out at 2–30℃. The crosslinking conditions were: power of 30–500W microwave and / or wavelength of 100–700nm and intensity of 5–50mW / cm. 2 Under light; S15. After cross-linking is completed, let it stand at 2–30℃ for 2–48h. After standing, crush the gel, sieve it uniformly, and obtain cross-linked HA gel with a particle size of 10–100μm. Then, use purified water to repeatedly vacuum wash and remove impurities present in the preparation process to obtain purified cross-linked HA gel.

[0006] Preferably, in step S11, the molecular weight of sodium hyaluronate is ≥ 1.5 MDa; The polyphenolic substances mentioned in step S12 include gallic acid, anthocyanins, tea polyphenols, curcumin, resveratrol, rutin and their derivatives or one or more thereof, and the amount of polyphenolic substances added is 0.1–5% w / v of the mass of sodium hyaluronate solution; In step S12, the photocatalyst is TiO2 or ZnO, added at a concentration of 0.001–0.05 mg / mL in sodium hyaluronate solution. TiO2 or ZnO are medical or pharmaceutical grade raw materials; TiO2 is used as a drug coating material, and ZnO is used as a drug raw material. Both have high biosafety, and the dosage used is far below their daily exposure limits (maximum oral dose 1387 mg; maximum oral dose 7.54 mg; maximum ophthalmic dose 0.4 mg; maximum topical dose 5%). The catalyst used in this step will be further removed during the subsequent vacuum cleaning process with purified water to ensure no residue remains.

[0007] In step S14, the crosslinking time under microwave is 0.5–24 h; the crosslinking time under light is 2–48 h; and the crosslinking time under both microwave and light is 1–48 h. The crosslinking reaction is carried out in stages, with each stage spaced 0.5–3 h apart.

[0008] Preferably, step S2 includes the following specific steps: S21. Dissolve PHBV in an organic solvent to form a PHBV organic solution; S22. Using microfluidic electrospray technology, the PHBV organic solution is made into droplets and solidified to obtain PHBV microspheres with a particle size of 20–150 μm and a particle size variation coefficient CV < 8%. S23. Perform plasma surface activation treatment on the PHBV microspheres.

[0009] Preferably, the specific preparation method of step S21 is as follows: PHBV and organic solvent are mixed at a mass-volume ratio of 5%-47.8% w / v, PHBV is added to the organic solvent, and the mixture is magnetically stirred at room temperature for 2-4 hours until completely dissolved to form an organic solution of PHBV; the organic solvent in step S21 is one or a mixture of dichloromethane, acetone, methanol, ethanol, chloroform, and ethyl acetate.

[0010] Preferably, the specific preparation method of step S22 is as follows: the microfluidic chip channel is ultrasonically cleaned sequentially with deionized water and anhydrous ethanol for 5-20 min at an ultrasonic power of 50-300 W, and then dried with nitrogen for 2-10 min before use; a micro-injection pump is connected to the microfluidic chip inlet, ensuring the tubing is free of air bubbles; the microfluidic chip nozzle is aligned with the collection device, which contains a solidification solution; the positive electrode is connected to the metal electrode of the microfluidic chip nozzle, and the negative electrode is connected to the collection device, which is grounded; fluid delivery is started: the flow rate of the micro-injection pump is set to 0.01–0.5 mL / h, the applied voltage is 3–12 kV, and the gas-liquid flow rate ratio is controlled to be 3:1, so that the particle size variation coefficient CV < 8%; the PHBV organic solution is pumped into the microfluidic chip channel, and after a stable Taylor cone is formed at the nozzle, the power is turned on for electrospraying, and the droplets are solidified in the solidification solution to obtain PHBV microspheres with a particle size of 20–150 μm.

[0011] Preferably, step S23 is performed as follows: the solidified PHBV microspheres are placed in the plasma chamber, the chamber door is closed, the vacuum pump is started, and the chamber pressure is evacuated to a vacuum of 1.0-5.0 × 10⁻⁶. -3 Pa; Open the gas valve and introduce process gas into the chamber, controlling the gas flow rate at 0.01-10 mL / min; the gas introduced into the chamber is nitrogen, oxygen, argon-nitrogen mixture, or argon-oxygen mixture; turn on the radio frequency power supply, set the radio frequency voltage to 100-500V and the bias voltage to 50-500V, and clean the solidified PHBV microspheres; the cleaning time for the solidified PHBV microspheres is 10-60 min. After the cleaning time is reached, turn off the radio frequency power supply to stop plasma generation, then close the gas valve to stop the gas supply, and open the vent valve to restore the chamber pressure to atmospheric pressure.

[0012] Preferably, step S3 includes the following steps: S31. Mix the cross-linked hyaluronic acid gel with phosphate buffer containing lidocaine and glycerol to obtain the initial cross-linked hyaluronic acid gel mixture. S32. Spray polyhydroxybutyrate-valerate copolyester microspheres with phosphate buffer containing hyaluronic acid to obtain wetted microspheres; S33. The wetted microspheres and the cross-linked hyaluronic acid gel initial mixture are mixed under revolution kneading and segmented ultrasonic conditions to obtain a pre-made gel. S34. The pre-prepared gel is dispersed and degassed at high speed under vacuum to obtain a composite suspension gel, which is then sterilized.

[0013] Preferably, step S3 includes the following steps: S31. Initial mixing: Under low-speed anchor stirring at 5–100 r / min (1–3 shafts, 5–25 kW), add the cross-linked hyaluronic acid gel finally prepared in step S1 to the mixing container, stir uniformly for 5–20 min, add phosphate buffer containing lidocaine solution and glycerol at pH 7.0–7.4 to obtain the initial mixture of cross-linked hyaluronic acid gel. S32, Microsphere wetting: Add 20–150 μm PHBV microspheres prepared in step S2 to the mixing container and spray with phosphate buffer containing sodium hyaluronate solution prepared in step S11 at pH=7.0–7.4 for 2–8 min to obtain pre-wetted microsphere powder. S33, Ultrasonic dispersion: Add the cross-linked hyaluronic acid gel initial mixture prepared in S31 to the pre-wetted microsphere powder, knead by revolution at 5–100 r / min, and sonicate in segments 1–10 times, each time for 1–120 min, with an ultrasonic power of 0.1–100 W to obtain the pre-mixed gel. S34. High-speed dispersion and degassing: The pre-mixed gel is placed in a vacuum environment and dispersed in a high-speed disperser at 0–2500 r / min. The high-speed disperser is a 1–3 axis high-speed disperser with a power of 2–200 kW. The gel is ultrasonicated in segments 1–10 times, each time for 1–120 min, to finally obtain a uniform, bubble-free suspension gel containing PHBV microspheres. The composite gel is then sterilized.

[0014] Preferably, in step S31, the mass concentration of lidocaine in the initial cross-linked hyaluronic acid gel mixture is 0.01%-10% (w / v), and the mass concentration of glycerol is 0.01%-15% (w / v). The mass ratio of the 20–150 μm PHBV microspheres used in step S32 to the phosphate buffer containing the sodium hyaluronate solution prepared in step S11 is 1:0.01–100. In step S33, the mass ratio of the initially wetted microsphere powder to the initial cross-linked hyaluronic acid gel mixture is 1:1-1000. In step S34, the obtained composite microsphere suspension gel is terminally sterilized: the terminal sterilization is performed by low-energy electron beam irradiation, 5-10 kGy, at a temperature below 40℃.

[0015] The beneficial effects of this invention are: 1. Safety: The cross-linked network is uniform with a linear density distribution, contains no chemical cross-linking agents, and has high biocompatibility with no acidic burst release; it improves the spatial cross-linking network by replacing traditional chemical cross-linking agents (BDDE, etc.) with photosensitized induction, eliminating residual toxicity; it retains the natural biological signals and hydrophilicity of HA, improving tissue integration; the degradation products are neutral and non-irritating, avoiding inflammatory reactions; the microspheres have uniform density and particle size, reducing injection resistance and the risk of needle blockage; it retains the natural hydrophilicity and biological signaling function of HA, improving tissue integration.

[0016] Durability: It exhibits a stable degradation curve with no abrupt volume changes, and the component retention rate is ≥60% after 6 months; From 0 to 3 months, HA plays a role in hydration and shaping, while from 3 to 12 months, PHBV microspheres provide mechanical support. The effect lasts longer than 12 months. The combined effect of HA and PHBV microspheres induces collagen regeneration and reconstructs tissue structure, resulting in a significant increase in collagen deposition compared to pure HA.

[0017] Excellent suspension stability and smooth operation: By precisely controlling the density of PHBV microspheres (difference from the carrier gel density <5%), and combining microfluidic electrospray technology to ensure uniform particle size (CV <8%), and with the assistance of optimized wetting, ultrasonic and high-speed dispersion composite processes, long-term uniform and stable suspension of microspheres in gel is achieved (sedimentation height <5% after 24 hours of standing).

[0018] This effectively avoids phase separation during injection, reduces injection resistance and the risk of needle blockage, and ensures a uniform distribution of mechanical properties, thereby improving the smoothness, safety, and reproducibility of clinical procedures.

[0019] 2. Wide range of indications: It can be widely used in medical aesthetics and regenerative medicine fields, including facial contouring and soft tissue defect repair. Detailed Implementation

[0020] Example 1. Preparation of cross-linked hyaluronic acid gel Cross-linked hyaluronic acid gel Example 1-1 1. Raw materials: High molecular weight sodium hyaluronate (molecular weight ≥ 1.5 MDa, medical grade); dissolve sodium hyaluronate in deionized water to prepare a 1% w / v sodium hyaluronate solution.

[0021] 2. Add gallic acid and TiO2 photocatalyst to the sodium hyaluronate solution. The amount of gallic acid added is 0.1% of the mass of the sodium hyaluronate solution; the amount of TiO2 added is 0.001 mg / mL. 3. Stir well and pre-treat at 2℃ for 1.5 hours.

[0022] 4. Photo-induced crosslinking was carried out at 30℃. The crosslinking conditions were: microwave power of 400W, microwave time of 12h, carried out in segments with an interval of 1h between each segment.

[0023] 5. After cross-linking, allow the gel to stand at 30℃ for 48 hours; then crush, sieve, and purify: Crush and sieve the gel after standing to obtain gel particles with a particle size of 100 μm. Subsequently, wash the gel particles repeatedly with purified water in a vacuum filtration device at least 3 times until the conductivity of the filtrate is stable and close to the background value of purified water, so as to completely remove unreacted gallic acid, TiO2 catalyst, and soluble impurities, and finally obtain purified cross-linked hyaluronic acid gel.

[0024] In vitro degradation and mechanical properties of cross-linked HA: In PBS buffer containing HA enzyme, the enzyme concentration was 20 U / ml, PBS buffer (pH 7.4), and the temperature was 37°C to simulate in vivo degradation; Results: 0-3 months: cross-linked hyaluronic acid gel degradation ≤30%; 6 months: gel degradation ≤40%; 9 months: overall degradation rate 65.3%; curve was stable with no sharp peaks.

[0025] Polyphenol modification gradient test of cross-linked hyaluronic acid gel: Results: The modification amount of 1.2 wt% showed the best stability of injection shear stress and the best bonding force between the continuous phase and the microspheres.

[0026] Cross-linked hyaluronic acid gel Examples 1-2 1. Raw materials: High molecular weight sodium hyaluronate (molecular weight ≥ 1.5 MDa, medical grade); Dissolve sodium hyaluronate in deionized water to prepare a 10% (w / v) sodium hyaluronate solution.

[0027] 2. Add anthocyanins and ZnO photocatalyst to the sodium hyaluronate solution; the amount of anthocyanins added is 5% of the mass of the sodium hyaluronate solution; the amount of ZnO added is 0.05 mg / mL; 3. Stir well and pretreat at 30℃ for 3 hours; 4. Photo-induced crosslinking was performed at 2℃, with the following conditions: crosslinking strength 50 mW / cm². 2 Irradiation was carried out; the illumination time was 48 hours, divided into segments with an interval of 0.5 hours between each segment.

[0028] 5. After cross-linking, allow the gel to stand at 2°C for 2 hours; then crush, sieve, and purify: crush and sieve the gel after standing to obtain gel particles with a particle size of 10 μm. Subsequently, wash and purify the gel with purified water (the specific operation is the same as in Example 1-1) to finally obtain the purified cross-linked hyaluronic acid gel.

[0029] In vitro degradation and mechanical properties of cross-linked HA: In PBS buffer containing HA enzyme, the enzyme concentration was 20 U / ml, PBS buffer (pH 7.4), and the temperature was 37°C to simulate in vivo degradation; Results: 0-3 months: cross-linked hyaluronic acid gel degradation ≤30%; 6 months: gel degradation ≤40%; 9 months: overall degradation rate 62.4%; curve was stable with no sharp peaks.

[0030] Polyphenol modification gradient test of cross-linked hyaluronic acid gel: Results: The modification amount of 1.24 wt% showed the best stability of injection shear stress and the best bonding force between the continuous phase and the microspheres.

[0031] Cross-linked hyaluronic acid gel Examples 1-3 1. Raw materials: High molecular weight sodium hyaluronate (molecular weight ≥ 1.5 MDa, medical grade); dissolve sodium hyaluronate in deionized water to prepare a 5% (w / v) sodium hyaluronate solution.

[0032] 2. Add tea polyphenols and TiO2 photocatalyst to the sodium hyaluronate solution; the amount of tea polyphenols added is 2.4% of the mass of the sodium hyaluronate solution; the amount of TiO2 added is 0.015 mg / mL; 3. Stir well and pretreat at 17℃ for 2.4 hours; 4. Photo-induced crosslinking was performed at 12℃. The crosslinking conditions were: microwave power of 100W, wavelength of 320nm, and intensity of 24mW / cm². 2 Irradiation was carried out; the illumination time was 6 hours, divided into segments with an interval of 0.5 hours between each segment.

[0033] 5. After cross-linking, the gel was allowed to stand at 14°C for 24 hours; pulverization, sieving, and purification: The gel after standing was pulverized and sieved to obtain gel particles with a particle size of 50 μm. Subsequently, it was washed and purified with purified water (the specific operation is the same as in Example 1-1), and finally purified cross-linked hyaluronic acid gel was obtained.

[0034] In vitro degradation and mechanical properties of cross-linked HA: In PBS buffer containing HA enzyme, the enzyme concentration was 20 U / ml, PBS buffer (pH 7.4), and the temperature was 37°C to simulate in vivo degradation; Results: 0-3 months: cross-linked hyaluronic acid gel degradation ≤30%; 6 months: gel degradation ≤40%; 9 months: overall degradation rate 65%; curve was stable with no sharp peaks.

[0035] Polyphenol modification gradient test of cross-linked hyaluronic acid gel: Results: The modification amount of 1.18 wt% showed the best stability of injection shear stress and the best bonding force between the continuous phase and the microspheres.

[0036] Preparation Example 2-1 of Polyhydroxybutyrate-valerate copolyester (PHBV) Microspheres 1. Preparation of PHBV organic solution: Weigh the raw materials according to the mass-volume ratio of PHBV and methanol-dichloromethane mixed solvent of 5% (w / v), dissolve PHBV in methanol and dichloromethane (volume ratio 1:1) mixed solvent, and stir magnetically at room temperature for 2 hours until completely dissolved to form PHBV methanol and dichloromethane solution. 2. Microfluidic preparation and curing of microspheres: The microfluidic chip channels are ultrasonically cleaned sequentially with deionized water and anhydrous ethanol for 5 minutes at a power of 50W. Nitrogen gas is then introduced to dry the microspheres for 2 minutes before use. A microinjection pump is connected to the microfluidic chip inlet, ensuring the tubing is free of air bubbles. The microfluidic chip nozzle is aligned with the collection device, which contains the curing solution. The positive electrode is connected to the metal electrode of the microfluidic chip nozzle, and the negative electrode is connected to the collection device, which is grounded. Initiating fluid delivery: Set the micro-injection pump flow rate, voltage to 12kV, flow rate to 0.01mL / h, gas-liquid ratio to 3:1, and particle size CV < 8%; inject the PHBV methanol and dichloromethane solution into the microfluidic chip channel; after a stable liquid cone forms at the injection nozzle, turn on the power; inject the curing solution; PHBV microspheres solidify and form, with a particle size of 150μm.

[0037] 3. Plasma activation of the surface: Place the cured PHBV microspheres on the carrier in the plasma chamber, close the equipment door, start the vacuum pump, and evacuate the chamber pressure to a vacuum of 1.0 × 10⁻⁶. -3 Pa; Open the gas valve and introduce the appropriate gas into the chamber, controlling the gas flow rate at 10 mL / min; Introduce nitrogen; Turn on the radio frequency power supply, set the radio frequency voltage to 100V and the bias voltage to 500V, and clean the cured PHBV microspheres. The cleaning time for the cured PHBV microspheres is 10 minutes. After the cleaning time is up, turn off the radio frequency power supply to stop plasma generation, then close the gas valve to stop the gas supply, and open the vent valve to restore the pressure inside the chamber to atmospheric pressure.

[0038] PHBV microsphere density verification: Density determination: The density of the PHBV microspheres prepared in this example was determined using the ASTM D792 standard method to be 1.12 g / cm³. 3 .

[0039] Density matching analysis: The density of the microspheres matches that of the cross-linked hyaluronic acid gel carrier prepared in Example 1-1. Calculations show that the density difference between the two is only 3.21%, meeting the design requirement of less than 5% for this invention.

[0040] Suspension stability verification: To verify the actual effect of the density matching described above, microspheres were uniformly dispersed in the cross-linked hyaluronic acid gel of Example 1-1. After standing for 30 min, the sedimentation height of the microspheres was measured to be only 3.20%. As a control, in a traditional pure HA gel carrier, the sedimentation height of particles under the same conditions is usually greater than 15%. This result directly proves that long-term stable suspension of microspheres in a gel carrier can be achieved by precisely controlling the density of the microspheres.

[0041] Preparation of polyhydroxybutyrate-valerate copolyester (PHBV) microspheres Example 2-2 1. Preparation of PHBV organic solution: Weigh the raw materials according to the mass-volume ratio of PHBV to acetone of 47.8% (w / v), add PHBV to acetone, and stir magnetically at room temperature for 4 hours until completely dissolved to form PHBV acetone solution; 2. Microfluidic preparation and curing of microspheres: The microfluidic chip channels are ultrasonically cleaned sequentially with deionized water and anhydrous ethanol for 20 minutes at 300W. Nitrogen gas is then introduced to dry the microspheres for 10 minutes before use. A microinjection pump is connected to the microfluidic chip inlet, ensuring the tubing is free of air bubbles. The microfluidic chip nozzle is aligned with the collection device, which contains the curing solution. The positive electrode is connected to the metal electrode of the microfluidic chip nozzle, and the negative electrode is connected to the collection device, which is grounded. Initiating fluid delivery: Set the micro-injection pump flow rate, voltage to 3kV, flow rate to 0.01mL / h, gas-liquid flow rate ratio to 3:1, and particle size CV < 8%; inject the PHBV acetone solution into the microfluidic chip channel, and turn on the power after a stable liquid cone forms at the injection nozzle; inject the curing solution; PHBV microspheres are cured and formed, with a particle size of 20μm.

[0042] 3. Plasma activation of the surface: Place the cured PHBV microspheres on the carrier in the plasma chamber, close the equipment door, start the vacuum pump, and evacuate the chamber pressure to a vacuum of 5.0 × 10⁻⁶. -3 Pa; Open the gas valve and introduce the corresponding gas into the chamber, controlling the gas flow rate at 0.01 mL / min; Introduce an argon-nitrogen mixture; Turn on the radio frequency power supply, set the radio frequency voltage to 500V and the bias voltage to 50V, and clean the cured PHBV microspheres. The cleaning time for the cured PHBV microspheres is 60 minutes. After the cleaning time is up, turn off the radio frequency power supply to stop plasma generation, then close the gas valve to stop the gas supply, and open the vent valve to restore the pressure inside the chamber to atmospheric pressure.

[0043] PHBV microsphere density verification: Density determination: The density of the PHBV microspheres prepared in this example was determined using the ASTM D792 standard method to be 1.09 g / cm³. 3 .

[0044] Density matching analysis: The density of the microspheres matches that of the cross-linked hyaluronic acid gel carriers prepared in Examples 1-2. Calculations show that the density difference between the two is 4.02%, which meets the design requirement of less than 5% for this invention.

[0045] Suspension stability verification: To verify the actual effect of the density matching described above, microspheres were uniformly dispersed in the cross-linked hyaluronic acid gels of Examples 1-2. After standing for 30 min, the sedimentation height of the microspheres was measured to be 3.23%. As a control, in a traditional pure HA gel carrier, the sedimentation height of particles is usually greater than 15% under the same conditions. This result directly proves that long-term stable suspension of microspheres in a gel carrier can be achieved by precisely controlling the density of the microspheres.

[0046] Preparation Examples of Polyhydroxybutyrate-valeric acid copolyester (PHBV) Microspheres 2-3 1. Preparation of PHBV organic solution: Weigh the raw materials according to the mass-volume ratio of PHBV and dichloromethane of 30% (w / v), add PHBV to dichloromethane, and stir magnetically at room temperature for 3 hours until completely dissolved to form PHBV dichloromethane solution; 2. Microfluidic preparation and curing of microspheres: The microfluidic chip channels are ultrasonically cleaned sequentially with deionized water and anhydrous ethanol for 10 minutes at 150W. Nitrogen gas is then introduced to dry the microspheres for 5 minutes before use. A microinjection pump is connected to the microfluidic chip inlet, ensuring the tubing is free of air bubbles. The microfluidic chip nozzle is aligned with the collection device, which contains the curing solution. The positive electrode is connected to the metal electrode of the microfluidic chip nozzle, and the negative electrode is connected to the collection device, which is grounded. Initiating fluid delivery: Set the micro-injection pump flow rate, voltage to 8kV, flow rate to 0.3mL / h, gas-liquid flow rate ratio to 3:1, and particle size CV < 8%; inject PHBV dichloromethane solution into the microfluidic chip channel, and turn on the power after a stable liquid cone forms at the injection nozzle; inject the curing solution; PHBV microspheres are cured and formed, with a particle size of 100μm.

[0047] 3. Plasma activation of the surface: Place the cured PHBV microspheres on the carrier in the plasma chamber, close the equipment door, start the vacuum pump, and evacuate the chamber pressure to a vacuum of 3.0 × 10⁻⁶. -3 Pa; Open the gas valve and introduce the corresponding gas into the chamber, controlling the gas flow rate at 1 mL / min; Introduce an argon-oxygen mixture; Turn on the radio frequency power supply, set the radio frequency voltage to 300V and the bias voltage to 300V, and clean the cured PHBV microspheres. The cleaning time for the cured PHBV microspheres is 30 minutes. After the cleaning time is up, turn off the radio frequency power supply to stop plasma generation, then close the gas valve to stop the gas supply, and open the vent valve to restore the pressure inside the chamber to atmospheric pressure.

[0048] PHBV microsphere density verification: Density determination: The density of the PHBV microspheres prepared in this example was determined to be 1.13 g / cm³ using the ASTM D792 standard method.

[0049] Density matching analysis: The density of the microspheres matches that of the cross-linked hyaluronic acid gel carriers prepared in Examples 1-3. Calculations show that the density difference between the two is 3.83%, which meets the design requirement of less than 5% for this invention.

[0050] Suspension stability verification: To verify the actual effect of the density matching described above, microspheres were uniformly dispersed in the cross-linked hyaluronic acid gels of Examples 1-3. After standing for 30 min, the sedimentation height of the microspheres was measured to be 3.11%. As a control, in a traditional pure HA gel carrier, the particle sedimentation height is usually greater than 15% under the same conditions. This result directly proves that long-term stable suspension of microspheres in a gel carrier can be achieved by precisely controlling the microsphere density.

[0051] Example 3-1: Preparation of Photosensitive Induced Crosslinked HA / PHBV Composite Soft Tissue Filler Injection 1. Initial mixing: Under low-speed anchor stirring at 100 r / min (single shaft, 5 kW power), the cross-linked hyaluronic acid gel finally prepared in Example 1-1 was added to the mixing container and stirred uniformly for 20 min. Then, a phosphate buffer solution containing lidocaine and glycerol at pH 7.0 was added to obtain the initial mixture of cross-linked hyaluronic acid gel. The mass concentration of lidocaine in the initial mixture of cross-linked hyaluronic acid gel was 10% (w / v), the mass concentration of glycerol was 15% (w / v), and the mass concentration of the phosphate buffer solution containing sodium hyaluronate solution was 0.01% (w / v). 2. Microsphere wetting: Add the PHBV microspheres prepared in Example 2-1 to the mixing container and spray with phosphate buffer containing sodium hyaluronate solution at pH=7.0 for 8 min to obtain preliminarily wetted microsphere powder; the mass ratio of PHBV microspheres to phosphate buffer containing sodium hyaluronate solution is 1:100. 3. Ultrasonic dispersion: Add cross-linked hyaluronic acid gel initial mixture to the pre-wetted microsphere powder, knead at 50 r / min, and sonicate in segments 5 times, 5 min each time, with an ultrasonic power of 100 W to obtain a pre-mixed gel; the mass ratio of pre-wetted microsphere powder to cross-linked hyaluronic acid gel initial mixture is 1:1000. 4. High-speed dispersion and degassing: The pre-mixed gel is fed into a vacuum environment high-speed disperser at 2500 r / min. The high-speed disperser is a 3-axis high-speed disperser with a power of 10 kW. It is ultrasonicated in segments for 10 times, each time for 1 min, to finally obtain a uniform, bubble-free suspension gel containing PHBV microspheres. The obtained cross-linked HA / PHBV gel was terminally sterilized by low-energy electron beam irradiation at 5 kGy and 35 °C; the terminally sterilized cross-linked HA / PHBV gel was used as a filler material.

[0052] Suspension stability verification: The composite gel prepared in this embodiment was stirred and mixed at a speed of 50 r / min and allowed to stand for 24 h. The sedimentation interface height was 1.86%. After a simulated transport test (1 g acceleration, 5 min cycle), it was allowed to stand for another 24 h. The sedimentation height was 1.48%, while the sedimentation height of the HA control was >15%.

[0053] Functional validation results of terminally sterilized composite microsphere suspension gel: biocompatibility and collagen deposition. In the co-culture experiment of human dermal fibroblasts, compared with the comparative sample (pure cross-linked hyaluronic acid gel carrier), the composite gel of this embodiment showed a 32% increase in cell adhesion rate and no significant difference in proliferation index. The amount of collagen deposition at week 4 was 48% higher than that of the comparative sample.

[0054] Example 3-2: Preparation of Photosensitive Induced Crosslinked HA / PHBV Composite Soft Tissue Filler Injection 1. Initial mixing: Under low-speed anchor stirring at 50 r / min (two-shaft anchor stirring, power 15 kW), the cross-linked hyaluronic acid gel finally prepared in Examples 1-2 was added to the mixing container and stirred uniformly for 20 min. Then, a phosphate buffer solution containing lidocaine and glycerol at pH 7.4 was added to obtain the initial mixture of cross-linked hyaluronic acid gel. The mass concentration of lidocaine in the initial mixture of cross-linked hyaluronic acid gel was 0.01% (w / v), the mass concentration of glycerol was 0.01% (w / v), and the mass concentration of the phosphate buffer solution containing the sodium hyaluronate solution prepared in step S11 was 0.1% (w / v). 2. Microsphere wetting: 20 μm PHBV microspheres were prepared in Example 2-2 and added to the mixing container. The microspheres were then sprayed with a phosphate buffer solution containing sodium hyaluronate at pH 7.4 for 2 min to obtain pre-wetted microsphere powder. The mass ratio of PHBV microspheres to phosphate buffer solution containing sodium hyaluronate was 1:0.01. 3. Ultrasonic dispersion: Add cross-linked hyaluronic acid gel initial mixture to the pre-wetted microsphere powder, knead at 10 r / min, and sonicate in segments 10 times, 1 min each time, with an ultrasonic power of 0.1 W to obtain a pre-mixed gel; the mass ratio of pre-wetted microsphere powder to cross-linked hyaluronic acid gel initial mixture is 1:1. 4. High-speed dispersion and degassing: The pre-mixed gel was fed into a high-speed disperser with a vacuum environment of 1500 r / min. The high-speed disperser was a single-axis high-speed disperser with a power of 200 kW. The mixture was ultrasonicated once in segments for 120 min each time, and finally a uniform, bubble-free suspension gel containing PHBV microspheres was obtained. The obtained cross-linked HA / PHBV gel was terminally sterilized by low-energy electron beam irradiation at 7 kGy and 30 °C; the terminally sterilized cross-linked HA / PHBV gel was used as a filler material.

[0055] Suspension stability verification: The composite gel prepared in this embodiment was stirred and mixed at a speed of 50 r / min and allowed to stand for 24 h. The sedimentation interface height was 1.92%. After a simulated transport test (1 g acceleration, 5 min cycle), it was allowed to stand for another 24 h. The sedimentation height was 1.34%, while the sedimentation height of the HA control was >15%.

[0056] Functional validation results of terminally sterilized composite microsphere suspension gel: Biocompatibility and collagen deposition: In the co-culture experiment of human dermal fibroblasts, the cell adhesion rate was increased by 24% compared with the control group (a composite filler composed of ordinary cross-linked hyaluronic acid and PLLA microspheres prepared in the laboratory according to the formula of similar commercially available composite filler materials), while the proliferation index was not significantly different. At week 4, the amount of collagen deposition was approximately 18.7% higher than that of the control group (a composite filler composed of ordinary cross-linked hyaluronic acid and PLLA microspheres prepared in the laboratory according to the formula of similar commercially available composite filler materials).

[0057] Preparation of Photosensitive Induced Crosslinked HA / PHBV Composite Soft Tissue Filler Injection - Example 3-3 1. Initial mixing: Under low-speed anchor stirring at 10 r / min (three-axis anchor stirring, power 25 kW), the cross-linked hyaluronic acid gel finally prepared in Examples 1-3 was added to the mixing container and stirred uniformly for 10 min. Then, a phosphate buffer solution containing lidocaine and glycerol at pH 7.3 was added to obtain the initial mixture of cross-linked hyaluronic acid gel. The mass concentration of lidocaine in the initial mixture of cross-linked hyaluronic acid gel was 5% (w / v), the mass concentration of glycerol was 5% (w / v), and the mass concentration of the phosphate buffer solution containing the sodium hyaluronate solution prepared in step S11 was 0.05% (w / v). 2. Microsphere wetting: 100 μm PHBV microspheres were prepared in Example 2-3 and added to the mixing container. The microspheres were then sprayed with a phosphate buffer solution containing sodium hyaluronate at pH 7.3 for 6 min to obtain pre-wetted microsphere powder. The mass ratio of PHBV microspheres to phosphate buffer solution containing sodium hyaluronate was 1:1. 3. Ultrasonic dispersion: Add cross-linked hyaluronic acid gel initial mixture to the pre-wetted microsphere powder, knead at 100 r / min, and sonicate in segments once for 120 min each time. The ultrasonic power is 10 W to obtain the pre-mixed gel. The mass ratio of pre-wetted microsphere powder to cross-linked hyaluronic acid gel initial mixture is 1:100. 4. High-speed dispersion and degassing: The pre-mixed gel was fed into a high-speed disperser with a vacuum environment of 2500 r / min. The high-speed disperser was a 2-axis high-speed disperser with a power of 100 kW. It was ultrasonicated in segments for 5 minutes each time, and finally a uniform, bubble-free suspension gel containing PHBV microspheres was obtained. The obtained cross-linked HA / PHBV gel was terminally sterilized by low-energy electron beam irradiation at 10 kGy and 25 °C; the terminally sterilized cross-linked HA / PHBV gel was used as a filler material.

[0058] Suspension stability verification: The composite gel prepared in this embodiment was stirred and mixed at a speed of 50 r / min and allowed to stand for 24 h. The sedimentation interface height was 1.77%. After a simulated transport test (1 g acceleration, 5 min cycle), it was allowed to stand for another 24 h. The sedimentation height was 1.36%, while the sedimentation height of the HA control was >15%.

[0059] Functional validation results of terminally sterilized composite microsphere suspension gel: Biocompatibility and collagen deposition: In the co-culture experiment of human dermal fibroblasts, the cell adhesion rate was increased by 28% compared with the control group (a composite filler composed of ordinary cross-linked hyaluronic acid and PCL microspheres prepared in the laboratory according to the formula of similar commercially available composite filler materials, with no significant difference in proliferation index, and the amount of collagen deposition increased by about 33.7% at week 4.

[0060] The technical solutions disclosed and proposed in this invention can be implemented by those skilled in the art by appropriately modifying the conditions and routes, etc. Although the methods and preparation techniques of this invention have been described through preferred embodiments, those skilled in the art can obviously modify or recombine the methods and technical routes described herein without departing from the content, spirit, and scope of this invention to achieve the final preparation technique. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the spirit, scope, and content of this invention.

Claims

1. A method for preparing a photosensitive induced crosslinked HA / PHBV composite filler, characterized in that, Includes the following steps: S1. Construction of photosensitive induced cross-linked hyaluronic acid gel: Sodium hyaluronate solution was subjected to photoinduced cross-linking reaction in the presence of polyphenols and photocatalyst to form cross-linked hyaluronic acid gel. The gel was then crushed, sieved and purified. S2. Prepare polyhydroxybutyrate-valerate copolyester microspheres and activate their surface with plasma; S3. Preparation of composite filler material: The polyhydroxybutyrate-valerate copolyester microspheres prepared in step S2 are uniformly mixed with the cross-linked hyaluronic acid gel obtained by sieving and purification in step S1 to construct a suspension stable system and obtain the photosensitive induced cross-linked hyaluronic acid / polyhydroxybutyrate-valerate copolyester composite filler material.

2. The preparation method according to claim 1, characterized in that, Step S1 includes the following steps: S11. Dissolve sodium hyaluronate in deionized water to prepare a 1–10% w / v sodium hyaluronate solution; S12. Add polyphenols and photocatalysts to the sodium hyaluronate solution; S13. Pretreatment at 2–30℃ for 1.5–3 hours; S14. Microwaves at 2–30°C, power 30–500W, and / or wavelengths of 100–700nm, intensity 5–50mW / cm². 2 Under illumination, the photo-induced crosslinking reaction was carried out for 0.5–48 h; S15. The cross-linked system is allowed to stand at 2–30℃ for 2–48h. The gel after standing is crushed, uniformly sieved to a particle size of 10–100μm, and then washed and purified with purified water to obtain cross-linked hyaluronic acid gel.

3. The preparation method according to claim 2, characterized in that, In step S11, the molecular weight of the sodium hyaluronate is ≥1.5 MDa; In step S12, the polyphenolic substance is selected from one or more of gallic acid, anthocyanins, tea polyphenols, curcumin, resveratrol, rutin and their derivatives, and the amount added is 0.1–5% w / v of the mass of sodium hyaluronate solution. In step S12, the photocatalyst is TiO2 or ZnO, and its addition amount is 0.001–0.05 mg / mL; In step S14, the crosslinking time under microwave is 0.5–24 h; the crosslinking time under light is 2–48 h; and the crosslinking time under both microwave and light is 1–48 h. The crosslinking reaction is carried out in stages, with each stage spaced 0.5–3 h apart.

4. The preparation method according to claim 1, characterized in that, Step S2 includes the following steps: S21. Dissolve polyhydroxybutyrate-valerate copolyester in an organic solvent to form a polyhydroxybutyrate-valerate copolyester organic solution; S22. Using microfluidic electrospray technology, the polyhydroxybutyrate-valerate copolyester organic solution is formed into droplets and solidified to obtain polyhydroxybutyrate-valerate copolyester microspheres with a particle size of 20-150μm and a particle size variation coefficient CV<8%. S23. The polyhydroxybutyrate-valerate copolyester microspheres are subjected to plasma surface activation treatment.

5. The preparation method according to claim 4, characterized in that, The specific method of step S21 is as follows: mix polyhydroxybutyrate-valerate copolyester and organic solvent according to the mass-volume ratio, stir at room temperature for 2-4 hours to dissolve, and form polyhydroxybutyrate-valerate copolyester organic solution; The specific method of step S22 is as follows: ultrasonically clean the microfluidic chip channel with deionized water and anhydrous ethanol, and start fluid delivery; pump the polyhydroxybutyrate-valerate copolyester organic solution into the microfluidic chip channel, and the droplets enter the curing liquid to solidify and form polyhydroxybutyrate-valerate copolyester microspheres with a particle size of 20–150 μm. The specific method of step S23 is as follows: place the cured polyhydroxybutyrate-valerate copolyester microspheres in a plasma chamber and evacuate the chamber; introduce process gas into the chamber to clean the polyhydroxybutyrate-valerate copolyester microspheres.

6. The preparation method according to claim 5, characterized in that, In step S21, the mass-to-volume ratio of the polyhydroxybutyrate-valerate copolyester to the organic solvent is 5%-47.8% w / v; the organic solvent is selected from one or more of dichloromethane, acetone, methanol, ethanol, chloroform, and ethyl acetate. In step S22, the ultrasonic cleaning process involves an ultrasonic time of 5-20 min and an ultrasonic power of 50-300 W; nitrogen gas is introduced for drying for 2-10 min; and the process parameters for microfluidic electrospray are: micro-injection pump flow rate of 0.01–0.5 mL / h, applied voltage of 3–12 kV, and gas-liquid flow rate ratio of 3:

1. The process parameters for plasma treatment in step S23 are: vacuum degree of 1.0–5.0 × 10⁻⁶. -3 Pa, the process gas is nitrogen, oxygen, argon-nitrogen mixture or argon-oxygen mixture, the gas flow rate is 0.01–10 mL / min, the radio frequency voltage is 100–500 V, the bias voltage is 50–500 V, and the cleaning time is 10–60 min.

7. The preparation method according to claim 1, characterized in that: Step S3 includes the following steps: S31. Mix the cross-linked hyaluronic acid gel with phosphate buffer containing lidocaine and glycerol to obtain the initial cross-linked hyaluronic acid gel mixture. S32. Spray polyhydroxybutyrate-valerate copolyester microspheres with phosphate buffer containing hyaluronic acid to obtain wetted microspheres; S33. The wetted microspheres and the cross-linked hyaluronic acid gel initial mixture are mixed under revolution kneading and segmented ultrasonic conditions to obtain a pre-made gel. S34. The pre-prepared gel is dispersed and degassed at high speed under vacuum to obtain a composite suspension gel, which is then sterilized.

8. The preparation method according to claim 7, characterized in that: S31. Initial mixing: Under low-speed anchor stirring at 5–100 r / min, with stirring shaft 1–3 and power 5–25 kW, add the cross-linked hyaluronic acid finally prepared in step S1 to the mixing container, stir uniformly for 5–20 min, add phosphate buffer containing lidocaine solution and glycerol at pH=7.0-7.4 to obtain the initial mixture of cross-linked hyaluronic acid gel; S32, Microsphere wetting: Add the 20–150 μm polyhydroxybutyrate-valerate copolyester microspheres prepared in step S2 to the mixing container, and spray with phosphate buffer solution containing sodium hyaluronate solution prepared in step S11 at pH=7.0-7.4 for 2–8 min to obtain pre-wetted microsphere powder. S33, Ultrasonic dispersion: Add the cross-linked hyaluronic acid gel initial mixture prepared in S31 to the pre-wetted microsphere powder, knead by revolution at 5–100 r / min, and sonicate in segments 1–10 times, each time for 1–120 min, with an ultrasonic power of 0.1–100 W to obtain the pre-mixed gel. S34. High-speed dispersion and degassing: The pre-mixed gel is placed in a vacuum environment and dispersed in a high-speed disperser at 0–2500 r / min. The high-speed disperser is a 1–3 axis high-speed disperser with a power of 2–200 kW. The gel is ultrasonicated in segments 1–10 times, each time for 1–120 min, to obtain a uniform, bubble-free polyhydroxybutyrate-valerate copolyester microsphere suspension gel. Finally, the composite gel is sterilized.

9. The preparation method according to claim 8, characterized in that: In step S31, the mass concentration of lidocaine in the initial mixture of hyaluronic acid gel is 0.01%–10% w / v, and the mass concentration of glycerin is 0.01%–15% w / v. In step S32, the mass ratio of the polyhydroxybutyrate-valerate copolyester microspheres to the phosphate buffer containing hyaluronic acid is 1:0.01–100; In step S33, the mass ratio of the wetted microspheres to the initial mixture of hyaluronic acid gel is 1:1–1000; In step S34, the terminal sterilization is performed using low-energy electron beam irradiation with an irradiation dose of 5–10 kGy and a temperature below 40°C.