A two-component pu / sis injectable hydrogel material and a preparation method thereof

Abstract

This invention discloses a two-component PU / SIS injectable hydrogel material and its preparation method. The preparation method includes the following steps: S1. Preparing an isocyanate-terminated aqueous polyurethane prepolymer solution; S2. Preparing an aminated SIS porous microsphere suspension; S3. Dispensing component A and component B into a dual-syringe or parallel syringe, and sterilizing them to obtain the two-component PU / SIS injectable hydrogel material. The two-component PU / SIS injectable hydrogel material prepared by this invention can be conveniently injected subcutaneously or deeply using a standard syringe, and can rapidly form a stable gel with excellent mechanical support and degradation resistance in situ. It can meet the requirements of an ideal soft tissue filler material and is expected to further broaden the application of SIS in medical aesthetic soft tissue filling and repair.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a two-component PU / SIS injectable hydrogel material and its preparation method. Background Technology

[0002] Injectable fillers play a crucial role in the field of soft tissue filling and repair in medical aesthetics. Ideal injectable fillers should possess good biocompatibility, suitable injectability, certain mechanical support properties, and a controllable degradation rate. Hyaluronic acid is currently the most widely used injectable filler, achieving its filling effect through physical occupancy and water-locking effects. However, hyaluronic acid lacks the bioactivity to actively promote tissue regeneration, and its persistence in the body is limited, requiring frequent injections to maintain the effect; furthermore, some individuals may experience allergic reactions to hyaluronic acid products. Animal-derived collagen (such as porcine collagen and bovine collagen) is also used in injectable fillers. They possess certain bioactivity, but pose a potential risk of immunogenicity, and their degradation rate in the body is relatively fast, with less than ideal support and durability.

[0003] Small intestinal submucosa (SIS), a natural decellularized extracellular matrix material, is mainly composed of type I and type III collagen, and retains various growth factors (such as VEGF and FGF-2) and glycoproteins. SIS possesses excellent biocompatibility and the ability to guide tissue regeneration, making it a theoretically promising injectable filler material. However, traditional SIS materials are usually in sheet, sponge, or powder form, which presents significant challenges for their application in injectable formulations. If SIS powder is dispersed in physiological saline to prepare a suspension using conventional methods, its rheological properties are poor, its injectability is low, and it is prone to loss and displacement after injection, making it difficult to accurately position and maintain its shape at the target site. Furthermore, pure SIS degrades too quickly and has weak mechanical strength, failing to provide sufficient physical support for tissue regeneration for a prolonged period, thus hindering long-term filling or repair effects.

[0004] Polyurethane (PU), as a synthetic polymer material, possesses excellent mechanical properties and controllable degradability. By combining it with SIS (solid-exposed silicone) gel, it is expected to compensate for the shortcomings of SIS in terms of mechanical strength and durability. Chinese patent application CN105999373B discloses a two-component injectable hydrogel product kit of polyurethane / small intestinal submucosa (PU / SIS). This kit achieves in-situ coverage and repair of luminal tissue wounds (such as the esophagus and gastric antrum) under endoscopic guidance by mixing an aqueous polyurethane emulsion (component A) with an acidic SIS sol (component B). This technology provides a minimally invasive solution for ulcer repair after ESD (electrode dissection). However, its application method involves applying it to the wound layer by layer using an endoscope. This is suitable for open ulcers, but it does not address the issues of uniform mixing, in-situ stability, and shape maintenance after injection in medical aesthetic soft tissue repair scenarios that require deep injection to achieve volume filling. Furthermore, it cannot be conveniently injected through a single needle hole using a standard syringe. On the other hand, the formation of its gel depends on an acidic environment that causes SIS collagen fibers to swell and interact electrostatically with negatively charged polyurethane emulsions. This mechanism is not suitable for pH-sensitive tissue environments (such as subcutaneous or facial soft tissues), and residual acidity may cause irritation.

[0005] Therefore, there is a need to develop a reliable solution for SIS-based injectable formulations to further broaden the application of SIS in soft tissue filling and repair in medical aesthetics. Summary of the Invention

[0006] To overcome the shortcomings of the existing technology, the present invention aims to provide a method for preparing a two-component PU / SIS injectable hydrogel material. The prepared hydrogel material is suitable for soft tissue filling and repair in medical aesthetics. It can be conveniently injected subcutaneously or deeply using a standard syringe and can quickly form a stable gel in situ with good mechanical support and degradation resistance.

[0007] This invention is achieved through the following technical solution:

[0008] In a first aspect, the present invention provides a method for preparing a two-component PU / SIS injectable hydrogel material, comprising the following steps:

[0009] S1. Preparation of isocyanate-terminated aqueous polyurethane prepolymer solution:

[0010] Under inert gas protection, polycaprolactone diol and isophorone diisocyanate are reacted at 75-85℃ for 2-4 hours to generate isocyanate-terminated prepolymer; then N-methyldiethanolamine is added, and the reaction is continued at 50-60℃ for 1-3 hours; after the reaction is completed, the pH is adjusted to 6.5-7.5, and then dispersed in water to obtain an isocyanate-terminated aqueous polyurethane prepolymer solution, which is component A; wherein the molar ratio of polycaprolactone diol, isophorone diisocyanate and N-methyldiethanolamine is 1 : (2.5-3.5) : (0.4-0.6);

[0011] S2. Preparation of aminated SIS porous microsphere suspension:

[0012] a. Disperse SIS powder in an acidic aqueous solution with a pH of 2-3, add pepsin, digest at 2-8℃ for 24-72h, adjust the pH to neutral, and obtain SIS sol with a mass-volume concentration of 2%-4%;

[0013] b. Mix SIS sol and gelatin aqueous solution at a dry weight ratio of (1-2):1 to obtain a mixed solution;

[0014] c. Add the emulsifier to the oil phase and stir until homogeneous. Then, add the mixed adhesive solution dropwise to the oil phase and stir to emulsify, forming an emulsion.

[0015] d. The emulsion was cooled and solidified in an ice bath, the microspheres were collected by centrifugation, washed, and freeze-dried to obtain SIS-gelatin composite porous microspheres;

[0016] e. Immerse SIS-gelatin composite porous microspheres in a 0.1%-0.5% (w / v) glutaraldehyde aqueous solution and crosslink them at 20-30°C for 1-4 hours;

[0017] f. After cross-linking, the microspheres are collected by centrifugation and washed repeatedly with buffer solution until no free glutaraldehyde residue remains. Finally, the microspheres are resuspended in buffer solution to obtain an aminated SIS porous microsphere suspension with a mass-volume concentration of 3%-8%, which is component B.

[0018] S3. Dispense component A and component B into a dual-syringe or parallel syringe, sterilize them, and prepare a two-component PU / SIS injectable hydrogel material.

[0019] In this invention, the mass-volume concentration refers to the mass / volume percentage, % (w / v), which represents the number of grams of solute contained in 100 ml of solution.

[0020] Further, in step S1, the number average molecular weight of the polycaprolactone diol is 1000-2000; the solid content of the isocyanate-terminated aqueous polyurethane prepolymer solution is 15%-25%, and the isocyanate group content is 2.0%-4.0%. The isocyanate group content is tested using the di-n-butylamine method in standard GB / T 12009.4-2016.

[0021] Further, in step S1, the step of dispersing in water is as follows: under high-speed shearing of 8000-10000 rpm, the reaction product is slowly added to ice water and sheared emulsified for 30-60 minutes.

[0022] The SIS powder of the present invention can be obtained commercially or prepared by existing methods. Preferably, the particle size of the SIS powder is ≤100μm.

[0023] Further, in step S2a, the acidic aqueous solution is a 0.1M-0.5M hydrochloric acid solution or acetic acid solution; the amount of pepsin added is 2%-5% of the mass of the SIS powder.

[0024] Furthermore, in step S2b, the mass-volume concentration of the gelatin aqueous solution is 4%-6%.

[0025] This invention provides a method for preparing a gelatin solution, comprising the following steps: accurately weighing gelatin powder, dispersing and swelling it in cold water at 4-10℃, stirring it in a water bath at 40-60℃ until completely dissolved, finally adjusting the volume with warm water at 40-60℃, and storing it in an environment at 37-50℃ for later use.

[0026] Preferably, the gelatin is selected from type A gelatin; the Bloom strength of the gelatin is 250-300 Bloom.

[0027] Further, in step S2c, the oil phase is liquid paraffin; the emulsifier is Span 80.

[0028] Further, in step S2c, the volume concentration of the emulsifier in the oil phase is 1.0%-2.0%.

[0029] Furthermore, in step S2c, the stirring and emulsification speed is 8000-12000 rpm, and the emulsification time is 5-15 minutes.

[0030] Further, in step S2d, the washing involves using acetone pre-cooled to 0-10°C to centrifuge and wash the collected microspheres multiple times until the washing liquid is clear and transparent, removing the residual oil phase.

[0031] Further, in step S2d, the freeze-drying conditions are: pre-freezing at -40°C to -80°C for 8-16 hours, and then drying at -40°C to -60°C and a pressure below 0.1 mbar for 20-28 hours.

[0032] Further, in step S2f, the buffer solution is a phosphate buffer solution.

[0033] Further, in step S3, the volume ratio of component A to component B is 1:(1-2).

[0034] Further, in step S3, the sterilization process is performed by irradiation sterilization using γ-rays generated by a Co-60 source, with an irradiation dose of 15-25 kGy.

[0035] Secondly, the present invention provides a two-component PU / SIS injectable hydrogel material prepared by the above method.

[0036] Thirdly, the present invention provides the application of the above-mentioned two-component PU / SIS injectable hydrogel material in the preparation of medical devices for subcutaneous or soft tissue filling and repair.

[0037] The present invention has the following beneficial effects:

[0038] The two-component PU / SIS injectable hydrogel material prepared by this invention has two components before injection: component A (isocyanate-terminated polyurethane prepolymer solution) and component B (aminated SIS porous microsphere suspension), both of which are easily transportable fluids that can be smoothly injected using a standard syringe. After the two components are mixed by a mixer and injected into the body, the amino groups enriched on the surface of the microspheres and the isocyanate groups at the ends of the polyurethane prepolymer undergo a rapid and efficient click chemical reaction, forming a three-dimensional stable network gel in situ within tens of seconds. The hybrid network structure gives the hydrogel good mechanical strength, provides effective physical support for soft tissue, and significantly enhances the material's resistance to enzymatic degradation, delaying its degradation rate in vivo, thereby achieving long-term filling effect.

[0039] The two-component PU / SIS injectable hydrogel material of this invention can be conveniently injected subcutaneously or deeply using a standard syringe, and can rapidly form a stable gel in situ with excellent mechanical support and degradation resistance. It meets the requirements of an ideal soft tissue filler material and is expected to further broaden the application of SIS in medical aesthetic soft tissue filling and repair. Detailed Implementation

[0040] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0041] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0042] Unless otherwise specified, room temperature or normal temperature in the embodiments of the present invention refers to 25±1℃.

[0043] Polycaprolactone diol: number average molecular weight 1250, Perstorp GmbH, Germany, brand name Capa 2125;

[0044] Isophorone diisocyanate: Sigma-Aldrich, USA, purity ≥99.0%;

[0045] N-Methyldiethanolamine: Sigma-Aldrich, USA, purity ≥99.0%;

[0046] Pepsin: Sigma-Aldrich, USA, derived from porcine gastric mucosa, activity ≥2500 U / mg;

[0047] Gelatin: Type A gelatin, Bloom strength 300, Sigma-Aldrich, USA, derived from pigskin.

[0048] SIS powder: self-made, preparation method is as follows:

[0049] (1) Pretreatment: Take fresh pig small intestine, wash it with physiological saline, remove the main muscle layer and serosa layer with a scraper, and cut it into 15-20cm long segments; wash it in physiological saline 3 times, 5 minutes each time.

[0050] (2) Degreasing: Soak the intestinal segments in a degreasing solution made of chloroform and methanol in a 1:1 volume ratio, place them in a fume hood for 12 hours, and change the degreasing solution every 6 hours; after degreasing, discard the degreasing solution and rinse the membrane with deionized water until there is no organic solvent odor.

[0051] (3) Trypsin digestion: Immerse the membrane in a 0.25% trypsin solution overnight at 4°C, and wash repeatedly with deionized water to remove trypsin.

[0052] (4) Decellularization: Prepare a 0.3% sodium dodecyl sulfate (SDS) solution, immerse the material in the SDS solution, let it stand at room temperature for 4 hours, and wash repeatedly with deionized water to remove SDS.

[0053] (5) Freeze-drying: Unfold and flatten the SIS membrane, pre-freeze at -20℃ for 3 hours, and freeze-dry in a vacuum freeze dryer.

[0054] (6) Grinding and sieving: Cut SIS into small fragments, place them in a ball mill, seal, and treat with liquid nitrogen at low temperature twice, 5 min each time. Assemble the ball mill onto the ball milling machine and grind at a vibration frequency of 25 times / min for 5 min. After the ball mill returns to room temperature, take out the powdered material, sieve it, and obtain SIS powder with a particle size range of 60-100 μm. Example

[0055] This embodiment provides a two-component PU / SIS injectable hydrogel material, the preparation method of which is as follows:

[0056] S1. Preparation of isocyanate-terminated aqueous polyurethane prepolymer solution:

[0057] In a dry four-necked flask, 0.10 mol of polycaprolactone diol and 0.30 mol of isophorone diisocyanate were added. Under nitrogen protection, the mixture was stirred at 300 rpm for 3 hours at 80°C to obtain an isocyanate-terminated prepolymer. The system was then cooled to 50°C, and 0.05 mol of N-methyldiethanolamine was added. The reaction was continued at 50°C for 2 hours. After the reaction was complete, the pH was neutralized to 7.0 with lactic acid. Subsequently, the reaction product was slowly added to 500 mL of ice water under high-speed shearing at 8000 rpm, and shear emulsification was continued for 30 minutes. Finally, water was added to bring the volume to a final concentration to obtain an aqueous polyurethane prepolymer solution with a solid content of 20% and an isocyanate group content of 3.2%, which was designated as component A and stored at 4°C for later use.

[0058] S2. Preparation of aminated SIS porous microsphere suspension:

[0059] a. Disperse 15.0 g of SIS powder in 500 mL of 0.1 M hydrochloric acid solution (pH≈2.0), add 0.45 g of pepsin, and digest magnetically at 4 °C for 48 hours. After digestion, adjust the pH to 7.0 with 1 M NaOH solution to obtain a SIS sol with a mass-volume concentration of approximately 3%.

[0060] b. Take 200 g of the above SIS sol and mix it with 100 g of gelatin solution with a mass-volume concentration of 5%, and stir magnetically for 2 hours to obtain a homogeneous mixed solution.

[0061] c. Add 4.5 mL of Span 80 to 300 mL of liquid paraffin and stir well. Then, while stirring at 10,000 rpm, add the mixed solution dropwise to the oil phase and emulsify for 10 minutes to form an emulsion.

[0062] d. The emulsion was quickly transferred to an ice-water bath to cool and stand for 1 hour to allow the gelatin to fully solidify. Then, the microspheres were collected by centrifugation at 4°C and 4000 rpm for 10 minutes and washed three times with acetone pre-cooled to 4°C. The washed microspheres were pre-frozen at -80°C for 12 hours and then transferred to a freeze dryer and freeze-dried at -50°C and 0.05 mbar for 24 hours to obtain SIS-gelatin composite porous microspheres.

[0063] e. Weigh 2.0 g of lyophilized microspheres and soak them in 200 mL of 0.25% (w / v) glutaraldehyde aqueous solution (prepared with 0.01 MPBS, pH 7.4). Place them in a shaker at 25°C and crosslink them at 100 rpm for 2 hours.

[0064] f. After cross-linking, collect the microspheres by centrifugation at 4000 rpm for 5 minutes at 4°C. Wash the microspheres repeatedly with PBS by centrifugation 6 times until the supernatant shows no obvious purple color when tested with Schiff's reagent. Finally, resuspend the microspheres in PBS and bring the volume to 40 mL to obtain a 5% (w / v) aminated SIS porous microsphere suspension, which is designated as component B and stored at 4°C.

[0065] S3. Dispensing and sterilization:

[0066] Component A and component B were filled into the two lumens of a dual syringe at a 1:1 (v / v) ratio and sealed. The syringe was then sterilized by irradiation with Co-60 gamma rays at a dose of 25 kGy to obtain the final product. Example

[0067] This embodiment provides a two-component PU / SIS injectable hydrogel material, the preparation method of which is as follows:

[0068] S1. Preparation of isocyanate-terminated aqueous polyurethane prepolymer solution:

[0069] In a dry four-necked flask, 0.10 mol of polycaprolactone diol and 0.28 mol of isophorone diisocyanate were added. Under nitrogen protection, the mixture was stirred at 300 rpm for 3 hours at 80°C to obtain an isocyanate-terminated prepolymer. The system was then cooled to 55°C, and 0.045 mol of N-methyldiethanolamine was added. The reaction was continued at 55°C for 1.5 hours. After the reaction was complete, the pH was neutralized to 7.0 with lactic acid. Subsequently, the reaction product was slowly added to 400 mL of ice water under high-speed shearing at 8000 rpm, and shear emulsification was continued for 30 minutes. Finally, water was added to bring the volume to a final concentration to obtain an aqueous polyurethane prepolymer solution with an isocyanate-terminated group content of 18% and an isocyanate group content of 3.8%, which was designated as component A and stored at 4°C for later use.

[0070] S2. Preparation of aminated SIS porous microsphere suspension:

[0071] a. Disperse 10.0 g of SIS powder in 500 mL of 0.1 M acetic acid solution (pH≈2.0), add 0.30 g of pepsin, and digest magnetically at 6 °C for 36 hours. After digestion, adjust the pH to 7.0 with 1 M NaOH solution to obtain a SIS sol with a mass-volume concentration of approximately 2%.

[0072] b. Take 300 g of the above SIS sol and mix it with 100 g of gelatin solution with a mass-volume concentration of 4%, and stir magnetically for 1.5 hours to obtain a homogeneous mixed solution.

[0073] c. Add 4.50 mL of Span 80 to 300 mL of liquid paraffin and stir until homogeneous. Then, while stirring at 10,000 rpm, add the mixture dropwise to the oil phase and emulsify for 10 minutes to form an emulsion.

[0074] d. The emulsion was quickly transferred to an ice-water bath to cool and stand for 1 hour to allow the gelatin to fully solidify. Then, the microspheres were collected by centrifugation at 4°C and 4000 rpm for 10 minutes and washed three times with acetone pre-cooled to 4°C. The washed microspheres were pre-frozen at -80°C for 12 hours and then transferred to a freeze dryer and freeze-dried at -50°C and 0.05 mbar for 24 hours to obtain SIS-gelatin composite porous microspheres.

[0075] e. Weigh 2.0 g of lyophilized microspheres and soak them in 200 mL of 0.1% (w / v) glutaraldehyde aqueous solution (prepared with 0.01 MPBS, pH 7.4). Place them in a shaker at 25°C and crosslink them at 100 rpm for 4 hours.

[0076] f. After cross-linking, collect the microspheres by centrifugation at 4000 rpm for 5 minutes at 4°C. Wash the microspheres repeatedly with PBS by centrifugation 6 times until the supernatant shows no obvious purple color when tested with Schiff's reagent. Finally, resuspend the microspheres in PBS and bring the volume to 40 mL to obtain a 5% (w / v) aminated SIS porous microsphere suspension, which is designated as component B and stored at 4°C.

[0077] S3. Dispensing and sterilization:

[0078] Component A and component B were filled into the two lumens of a dual-syllable syringe at a ratio of 1:1.5 (v / v) and sealed. The syringe was then sterilized by irradiation with a Co-60 source gamma rays at a dose of 25 kGy to obtain the final product. Example

[0079] This embodiment provides a two-component PU / SIS injectable hydrogel material, the preparation method of which is as follows:

[0080] S1. Preparation of isocyanate-terminated aqueous polyurethane prepolymer solution:

[0081] In a dry four-necked flask, 0.10 mol of polycaprolactone diol and 0.32 mol of isophorone diisocyanate were added. Under nitrogen protection, the mixture was stirred at 300 rpm for 3 hours at 80°C to obtain an isocyanate-terminated prepolymer. The system was then cooled to 50°C, and 0.055 mol of N-methyldiethanolamine was added. The reaction was continued at 50°C for 2 hours. After the reaction was complete, the pH was neutralized to 7.0 with lactic acid. Subsequently, the reaction product was slowly added to 500 mL of ice water under high-speed shearing at 8000 rpm, and shear emulsification was continued for 30 minutes. Finally, water was added to bring the volume to a final concentration to obtain an aqueous polyurethane prepolymer solution with a solid content of 22% and an isocyanate group content of 2.5%, which was designated as component A and stored at 4°C for later use.

[0082] S2. Preparation of aminated SIS porous microsphere suspension:

[0083] a. Disperse 20.0 g of SIS powder in 500 mL of 0.1 M hydrochloric acid solution (pH≈2.0), add 0.80 g of pepsin, and digest magnetically at 4 °C for 48 hours. After digestion, adjust the pH to 7.0 with 1 M NaOH solution to obtain a SIS sol with a mass-volume concentration of approximately 4%.

[0084] b. Take 150 g of the above SIS sol and mix it with 150 g of gelatin solution with a mass-volume concentration of 5%, and stir magnetically for 2 hours to obtain a homogeneous mixed solution.

[0085] c. Add 6.0 mL of Span 80 to 300 mL of liquid paraffin and stir until homogeneous. Then, while stirring at 10,000 rpm, add the mixed solution dropwise to the oil phase and emulsify for 10 minutes to form an emulsion.

[0086] d. The emulsion was quickly transferred to an ice-water bath to cool and stand for 1.5 hours to allow the gelatin to fully solidify. Then, the microspheres were collected by centrifugation at 4°C and 4000 rpm for 10 minutes and washed three times with acetone pre-cooled to 4°C. The washed microspheres were pre-frozen at -80°C for 12 hours and then transferred to a freeze dryer and freeze-dried at -50°C and 0.05 mbar for 24 hours to obtain SIS-gelatin composite porous microspheres.

[0087] e. Weigh 2.0 g of lyophilized microspheres and soak them in 200 mL of 0.4% (w / v) glutaraldehyde aqueous solution (prepared with 0.01 MPBS, pH 7.4). Place them in a shaker at 25°C and crosslink at 100 rpm for 1.5 hours.

[0088] f. After cross-linking, collect the microspheres by centrifugation at 4000 rpm for 5 minutes at 4°C. Wash the microspheres repeatedly with PBS by centrifugation 6 times until the supernatant shows no obvious purple color when tested with Schiff's reagent. Finally, resuspend the microspheres in PBS and bring the volume to 40 mL to obtain a 5% (w / v) aminated SIS porous microsphere suspension, which is designated as component B and stored at 4°C.

[0089] S3. Dispensing and sterilization:

[0090] Component A and component B were filled into the two lumens of a dual syringe at a ratio of 1:2 (v / v) and sealed. The product was then sterilized by irradiation with a Co-60 source gamma rays at a dose of 25 kGy to obtain the final product.

[0091] Comparative Example 1

[0092] This embodiment provides a two-component PU / SIS material. The difference between its preparation method and that of Example 1 is that the SIS-gelatin composite porous microspheres obtained in step S2d do not use glutaraldehyde for crosslinking. The remaining steps are the same as in Example 1. The specific preparation steps are as follows:

[0093] S1. Preparation of isocyanate-terminated aqueous polyurethane prepolymer solution:

[0094] In a dry four-necked flask, 0.10 mol of polycaprolactone diol and 0.30 mol of isophorone diisocyanate were added. Under nitrogen protection, the mixture was stirred at 300 rpm for 3 hours at 80°C to obtain an isocyanate-terminated prepolymer. The system was then cooled to 50°C, and 0.05 mol of N-methyldiethanolamine was added. The reaction was continued at 50°C for 2 hours. After the reaction was complete, the pH was neutralized to 7.0 with lactic acid. Subsequently, the reaction product was slowly added to 500 mL of ice water under high-speed shearing at 8000 rpm, and shear emulsification was continued for 30 minutes. Finally, water was added to bring the volume to a final concentration to obtain an aqueous polyurethane prepolymer solution with a solid content of 20% and an isocyanate group content of 3.2%, which was designated as component A and stored at 4°C for later use.

[0095] S2. Preparation of SIS porous microsphere suspension:

[0096] a. Disperse 15.0 g of SIS powder in 500 mL of 0.1 M hydrochloric acid solution (pH≈2.0), add 0.45 g of pepsin, and digest magnetically at 4 °C for 48 hours. After digestion, adjust the pH to 7.0 with 1 M NaOH solution to obtain a SIS sol with a mass-volume concentration of approximately 3%.

[0097] b. Take 200 g of the above SIS sol and mix it with 100 g of gelatin solution with a mass-volume concentration of 5%, and stir magnetically for 2 hours to obtain a homogeneous mixed solution.

[0098] c. Add 4.5 mL of Span 80 to 300 mL of liquid paraffin and stir well. Then, while stirring at 10,000 rpm, add the mixed solution dropwise to the oil phase and emulsify for 10 minutes to form an emulsion.

[0099] d. The emulsion was quickly transferred to an ice-water bath to cool and stand for 1 hour to allow the gelatin to fully solidify. Then, the microspheres were collected by centrifugation at 4°C and 4000 rpm for 10 minutes and washed three times with acetone pre-cooled to 4°C. The washed microspheres were pre-frozen at -80°C for 12 hours and then transferred to a freeze dryer and freeze-dried at -50°C and 0.05 mbar for 24 hours to obtain SIS-gelatin composite porous microspheres.

[0100] e. Weigh 2.0 g of lyophilized microspheres, suspend them in PBS, and bring the volume to 40 mL to obtain a 5% (w / v) SIS porous microsphere suspension, which is used as component B and stored at 4 °C.

[0101] S3. Dispensing and sterilization:

[0102] Component A and component B were filled into the two lumens of a dual syringe at a 1:1 (v / v) ratio and sealed. The syringe was then sterilized by irradiation with Co-60 gamma rays at a dose of 25 kGy to obtain the final product.

[0103] Comparative Example 2

[0104] This embodiment provides a two-component PU / SIS material. The difference between this material and Example 1 is that the concentration of the glutaraldehyde aqueous solution in step S2e is increased from 0.25% (w / v) to 2.0% (w / v), and the crosslinking time is extended from 2 hours to 12 hours. The remaining steps are the same as in Example 1. The specific preparation steps are as follows:

[0105] S1. Preparation of isocyanate-terminated aqueous polyurethane prepolymer solution:

[0106] In a dry four-necked flask, 0.10 mol of polycaprolactone diol and 0.30 mol of isophorone diisocyanate were added. Under nitrogen protection, the mixture was stirred at 300 rpm for 3 hours at 80°C to obtain an isocyanate-terminated prepolymer. The system was then cooled to 50°C, and 0.05 mol of N-methyldiethanolamine was added. The reaction was continued at 50°C for 2 hours. After the reaction was complete, the pH was neutralized to 7.0 with lactic acid. Subsequently, the reaction product was slowly added to 500 mL of ice water under high-speed shearing at 8000 rpm, and shear emulsification was continued for 30 minutes. Finally, water was added to bring the volume to a final concentration to obtain an aqueous polyurethane prepolymer solution with a solid content of 20% and an isocyanate group content of 3.2%, which was designated as component A and stored at 4°C for later use.

[0107] S2. Preparation of aminated SIS porous microsphere suspension:

[0108] a. Disperse 15.0 g of SIS powder in 500 mL of 0.1 M hydrochloric acid solution (pH≈2.0), add 0.45 g of pepsin, and digest magnetically at 4 °C for 48 hours. After digestion, adjust the pH to 7.0 with 1 M NaOH solution to obtain a SIS sol with a mass-volume concentration of approximately 3%.

[0109] b. Take 200 g of the above SIS sol and mix it with 100 g of gelatin solution with a mass-volume concentration of 5%, and stir magnetically for 2 hours to obtain a homogeneous mixed solution.

[0110] c. Add 4.5 mL of Span 80 to 300 mL of liquid paraffin and stir well. Then, while stirring at 10,000 rpm, add the mixed solution dropwise to the oil phase and emulsify for 10 minutes to form an emulsion.

[0111] d. The emulsion was quickly transferred to an ice-water bath to cool and stand for 1 hour to allow the gelatin to fully solidify. Then, the microspheres were collected by centrifugation at 4°C and 4000 rpm for 10 minutes and washed three times with acetone pre-cooled to 4°C. The washed microspheres were pre-frozen at -80°C for 12 hours and then transferred to a freeze dryer and freeze-dried at -50°C and 0.05 mbar for 24 hours to obtain SIS-gelatin composite porous microspheres.

[0112] e. Weigh 2.0 g of lyophilized microspheres and soak them in 200 mL of 2.0% (w / v) glutaraldehyde aqueous solution (prepared with 0.01 MPBS, pH 7.4). Place them in a shaker at 25°C and crosslink at 100 rpm for 12 hours.

[0113] f. After cross-linking, collect the microspheres by centrifugation at 4000 rpm for 5 minutes at 4°C. Wash the microspheres repeatedly with PBS by centrifugation 6 times until the supernatant shows no obvious purple color when tested with Schiff's reagent. Finally, resuspend the microspheres in PBS and bring the volume to 40 mL to obtain a 5% (w / v) aminated SIS porous microsphere suspension, which is designated as component B and stored at 4°C.

[0114] S3. Dispensing and sterilization:

[0115] Component A and component B were filled into the two lumens of a dual syringe at a 1:1 (v / v) ratio and sealed. The syringe was then sterilized by irradiation with Co-60 gamma rays at a dose of 25 kGy to obtain the final product.

[0116] Performance testing:

[0117] 1. Gel Time Determination: The inverted test tube method was used. 0.5 mL of component A and 0.5 mL of component B were placed in a 2 mL transparent glass vial, rapidly vortexed for 10 seconds, and placed in a 37°C water bath. Timing was started. The vial was tilted every 15 seconds to observe the flow. The gel time was recorded when the liquid level remained horizontal and stopped flowing at a 90° tilt. Each sample was tested independently three times, and the results are expressed as mean ± standard deviation.

[0118] 2. Compression Mechanical Properties Test: Equal volumes of components A and B were mixed and injected into a cylindrical mold (Φ8mm × H5mm). The mixture was reacted at 37℃ for 1 hour to form a gel. Using a universal testing machine, the gel cylinder was subjected to unconfined compression to 50% strain at a speed of 1 mm / min. The compressive modulus was calculated from the slope of the stress-strain curve in the 10%-20% strain zone. Each sample was tested independently 5 times, and the results are expressed as mean ± standard deviation.

[0119] 3. In vitro enzyme degradation test: Equal volumes of components A and B were mixed and injected into a cylindrical mold (Φ8mm × H5mm). The mixture was reacted at 37℃ for 1 hour to form a gel, and the initial wet weight (W0) was recorded. The sample was then immersed in 10 mL of PBS solution (pH=7.4) containing 100 U / mL esterase (from pig liver, Sigma E3019). The solution was placed in a 37℃ constant temperature shaker and shaken at 60 rpm. The enzyme digest was replaced with fresh solution every 2 days. The samples were removed on days 7 and 14, and the surface moisture was blotted dry with filter paper before weighing (W0). t ), calculate the quality retention rate (%) = (W t / W0)×100%. Five parallel tests were conducted for each sample group, and the results are expressed as mean ± standard deviation.

[0120] 4. Injection Force Test: The dual syringes containing components A and B from the examples and comparative examples were fitted with static mixers and 27G needles. Using a universal testing machine, the plunger was pushed at a constant speed of 100 mm / min. The maximum force exerted during the entire plunger stroke was recorded as the maximum injection force. Each sample was tested independently three times, and the results are expressed as mean ± standard deviation.

[0121] Table 1: Performance test results of Examples 1-3 and Comparative Examples 1-2

[0122]

[0123] The results above show that the two-component PU / SIS injectable hydrogel materials prepared in Examples 1-3 all exhibit good injectability, with a maximum injection force of less than 30N, allowing them to pass smoothly through a 27G fine needle. After mixing, they can rapidly gel in situ within a reasonable time (35-65 seconds), forming a stable gel with a significant compressive modulus (10.2-14.8 kPa). Furthermore, they demonstrate significant resistance to degradation in in vitro enzymatic hydrolysis experiments, with a mass retention rate still exceeding 50% after 14 days. This indicates that the two-component PU / SIS injectable hydrogel materials prepared by the method of this invention can be conveniently injected subcutaneously or deeply using a standard syringe, and can rapidly form a stable gel in situ with excellent mechanical support and degradation resistance. This meets the requirements of ideal soft tissue filling materials and is expected to further broaden the application of SIS in medical aesthetic soft tissue filling and repair.

[0124] Comparative Example 1, which did not undergo cross-linking treatment with glutaraldehyde, lacked sufficient active amino groups on its microsphere surface, making it unable to effectively cross-link with the polyurethane prepolymer to form a monolithic gel. In the in vitro enzymatic hydrolysis experiment, it severely disintegrated by day 7, and its structure could not be maintained.

[0125] Comparative Example 2 was cross-linked using a high concentration of glutaraldehyde aqueous solution, and the cross-linking time was extended, resulting in excessive cross-linking of the microspheres. This consumed a large amount of surface amino groups and caused the microsphere structure to become dense, hindering the reaction and resulting in an excessively long gelation time and poor effect. Furthermore, the fluidity of the suspension decreased, the injection force increased significantly, and the injectability deteriorated.

[0126] The above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing a two-component PU / SIS injectable hydrogel material, characterized in that, Includes the following steps: S1. Preparation of isocyanate-terminated aqueous polyurethane prepolymer solution: Under inert gas protection, polycaprolactone diol and isophorone diisocyanate are reacted at 75-85℃ for 2-4 hours to generate isocyanate-terminated prepolymer; then N-methyldiethanolamine is added, and the reaction is continued at 50-60℃ for 1-3 hours; after the reaction, the pH is adjusted to 6.5-7.5, and then dispersed in water to obtain an isocyanate-terminated aqueous polyurethane prepolymer solution, which is component A; wherein, the molar ratio of polycaprolactone diol, isophorone diisocyanate, and N-methyldiethanolamine is 1:(2.5-3.5):(0.4-0.6); the number average molecular weight of polycaprolactone diol is 1000-2000; the solid content of the isocyanate-terminated aqueous polyurethane prepolymer solution is 15%-25%, and the isocyanate group content is 2.0%-4.0%; S2. Preparation of aminated SIS porous microsphere suspension: a. Disperse SIS powder in an acidic aqueous solution with a pH of 2-3, add pepsin, digest at 2-8℃ for 24-72h, adjust the pH to neutral, and obtain SIS sol with a mass-volume concentration of 2%-4%; b. Mix SIS sol and gelatin aqueous solution at a dry weight ratio of (1-2):1 to obtain a mixed solution; c. Add the emulsifier to the oil phase and stir until homogeneous. Then, add the mixed adhesive solution dropwise to the oil phase and stir to emulsify, forming an emulsion. The oil phase is liquid paraffin. The emulsifier is Span 80. The volume concentration of the emulsifier in the oil phase is 1.0%-2.0%. The stirring speed for emulsification is 8000-12000 rpm, and the emulsification time is 5-15 minutes. d. The emulsion was cooled and solidified in an ice bath, the microspheres were collected by centrifugation, washed, and freeze-dried to obtain SIS-gelatin composite porous microspheres; e. Immerse SIS-gelatin composite porous microspheres in a 0.1%-0.5% (w / v) glutaraldehyde aqueous solution and crosslink them at 20-30°C for 1-4 hours; f. After cross-linking, the microspheres are collected by centrifugation and washed repeatedly with buffer solution until no free glutaraldehyde residue remains. Finally, the microspheres are resuspended in buffer solution to obtain an aminated SIS porous microsphere suspension with a mass-volume concentration of 3%-8%, which is component B. S3. Dispense component A and component B into a dual-syringe or parallel syringe, sterilize them, and prepare a two-component PU / SIS injectable hydrogel material.

2. The method for preparing the two-component PU / SIS injectable hydrogel material according to claim 1, characterized in that, In step S2a, the acidic aqueous solution is a 0.1M-0.5M hydrochloric acid solution or acetic acid solution; the amount of pepsin added is 2%-5% of the mass of the SIS powder.

3. The method for preparing the two-component PU / SIS injectable hydrogel material according to claim 1, characterized in that, In step S2b, the mass-volume concentration of the gelatin aqueous solution is 4%-6%.

4. The method for preparing the two-component PU / SIS injectable hydrogel material according to claim 1, characterized in that, In step S2d, the freeze-drying conditions are as follows: pre-freezing at -40°C to -80°C for 8-16 hours, and then drying at -40°C to -60°C and pressure below 0.1 mbar for 20-28 hours.

5. The method for preparing the two-component PU / SIS injectable hydrogel material according to claim 1, characterized in that, In step S2f, the buffer solution is a phosphate buffer solution.

6. The method for preparing the two-component PU / SIS injectable hydrogel material according to claim 1, characterized in that, In step S3, the volume ratio of component A to component B is 1:(1-2); the sterilization process is performed by irradiation sterilization using γ-rays generated by a Co-60 source, with an irradiation dose of 15-25 kGy.

7. A two-component PU / SIS injectable hydrogel material prepared by the method of any one of claims 1-6.

8. The use of the two-component PU / SIS injectable hydrogel material of claim 7 in the preparation of medical devices for subcutaneous or soft tissue filling and repair.

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