An antibacterial pro-epithelial regeneration postoperative wound regeneration hydrogel preparation and a preparation method thereof
By combining chitosan quaternary ammonium salt, collagen, antibacterial complex and active carrier in a hydrogel formulation, the shortcomings of existing wound repair materials in antibacterial properties and epithelial regeneration promotion are overcome, achieving rapid wound healing and safe repair.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- HAINAN WOMEN & CHILDRENS MEDICAL CENT
- Filing Date
- 2025-06-06
- Publication Date
- 2026-06-19
AI Technical Summary
Existing wound repair materials are insufficient in terms of antibacterial properties, promotion of epithelial regeneration and angiogenesis, and their active ingredients are easily lost, making it difficult to meet the needs of complex wound repair.
By combining chitosan quaternary ammonium salt, collagen, antibacterial complex solution, angiogenesis active carrier and epidermal repair active carrier, and through the fermentation of traditional Chinese medicine extracts by Lactobacillus plantarum and liposome encapsulation technology, a hydrogel formulation with synergistic peptide effects is formed to achieve rapid antibacterial and sustained antibacterial effects. The active factors are then delivered in a targeted manner by loading them onto mesoporous silica microspheres.
It significantly enhances antibacterial activity, promotes angiogenesis and epithelial regeneration, accelerates wound healing, broadens the antibacterial spectrum, reduces the risk of drug resistance, and achieves safe and efficient wound repair.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of pharmaceutical preparation technology, and in particular to an antibacterial postoperative wound regeneration hydrogel preparation and its preparation method. Background Technology
[0002] Postoperative wound repair is a crucial aspect of clinical medicine, with rapid healing, infection prevention, and reduced scar formation being key requirements. Traditional wound dressings, such as gauze and petroleum jelly gauze, suffer from poor breathability, easy adhesion to the wound surface, insufficient antibacterial ability, and inability to promote tissue regeneration, making them unsuitable for the repair of complex wounds. Hydrogel dressings have become a research hotspot due to their high water content, good biocompatibility, and controllable degradation, but they still have shortcomings in terms of long-lasting antibacterial effect, epithelial regeneration efficiency, and synergistic effects on angiogenesis.
[0003] Existing antibacterial hydrogels mostly rely on single antibiotics or chemical antibacterial agents, which easily lead to drug resistance and have poor biocompatibility. Repair-promoting components are often added directly, resulting in problems such as easy inactivation of growth factors and poor sustained-release performance, leading to limited repair effects. Furthermore, traditional carrier systems have low encapsulation rates and insufficient targeting of active ingredients, making it difficult to achieve a synergistic effect between antibacterial and tissue regeneration. Traditional Chinese medicine active ingredients have unique advantages in wound repair, but their low extraction rate and poor stability limit their clinical application. Summary of the Invention
[0004] In view of this, the present invention proposes an antibacterial postoperative wound regeneration hydrogel preparation and its preparation method to solve the above problems.
[0005] The technical solution of this invention is achieved as follows: a postoperative wound regeneration hydrogel preparation for antibacterial and epithelial regeneration, comprising the following raw materials in parts by weight: 5-8 parts of chitosan quaternary ammonium salt, 8-12 parts of collagen, 15-25 parts of antibacterial complex solution, 2-10 parts of angiogenesis active carrier, 3-15 parts of epidermal repair active carrier, 0.5-2 parts of crosslinking agent, and 120-150 parts of solvent; wherein the antibacterial complex is a fermentation broth of traditional Chinese medicine and an antibacterial polypeptide, the crosslinking agent is genipin or ethylene glycol diglycidyl ether, and the solvent is deionized water or physiological saline.
[0006] Furthermore, the collagen is type I or type III collagen, derived from bovine Achilles tendon or pig skin.
[0007] Furthermore, the herbal fermentation liquid is prepared by mixing 2-5 parts by weight of Astragalus membranaceus, 1-3 parts by weight of Angelica sinensis, 1-3 parts by weight of Callicarpa macrophylla, 2-4 parts by weight of Dracaena cochinchinensis, 5-10 parts by weight of Sanguisorba officinalis, and 3-5 parts by weight of Paris polyphylla, and then fermenting with Lactobacillus plantarum. The inoculum size of Lactobacillus plantarum is 1×10⁻⁶. 6 -5×10 6 CFU / g herbal medicine mixture.
[0008] Furthermore, the preparation method of the herbal fermentation broth includes: pulverizing the herbal mixture through a 60-100 mesh sieve, adding 5-8 times the mass of deionized water, sterilizing at 121℃ for 20-30 min, cooling, inoculating with Lactobacillus plantarum, fermenting at 30-37℃ for 48-72 h, centrifuging at 3-5℃ and 8000-10000 r / min for 15-20 min after fermentation, and filtering the supernatant through a 0.22 μm filter membrane to obtain the herbal fermentation broth.
[0009] Furthermore, the antimicrobial peptide is a copper peptide and a bee venom peptide in a mass ratio of (1.5-2.5):(0.5-1.2).
[0010] Furthermore, the angiogenic active carrier is composed of vascular endothelial growth factor encapsulated in liposomes with a particle size of 150-300 nm. The vascular endothelial growth factor (VEGF) is the 165 isoform with a specific activity ≥1×10⁻⁶. 6 IU / mg;
[0011] The encapsulation step is as follows:
[0012] a. Distearate phosphatidylcholine (DSPC), cholesterol and dipalmitoylphosphatidylglycerol (DPPG) were mixed in a mass ratio of (4-6):(3-5):1, dissolved in chloroform at a ratio of (8-12) mg:1 mL, and rotary evaporated to form a lipid film.
[0013] b. Add the lipid membrane to 10mM phosphate buffer containing vascular endothelial growth factor (VEGF) at a ratio of 10mg:1-2mL, wherein the ratio of VEGF to buffer is 1mg:(1-3)mL, and hydrate for 30-70 minutes at a temperature of 50-60℃ and a shaking speed of 8-12rpm to form multilayer liposomes.
[0014] c. Liposomes with a particle size of 150-250 nm are obtained by extruding polycarbonate membranes sequentially with pore sizes of 1.0 μm, 0.4 μm, and 0.2 μm, respectively, and removing free vascular endothelial growth factor (VEGF) with a molecular weight cutoff of 30 kDa, achieving an encapsulation efficiency of ≥85%.
[0015] Furthermore, the epidermal repair active carrier is an epidermal growth factor (EGF) carrier with a specific activity ≥1×10⁻⁶. 7 IU / mg, loaded in silica microspheres with pore sizes of 5-20 nm:
[0016] a. Vacuum dry mesoporous silica microspheres with pore size of 5-20 nm at 100-130℃ for 1-3 hours;
[0017] b. Prepare a 10mM Tris-HCl solution (pH 8.5) containing epidermal growth factor (EGF) at a ratio of 1-5:1 mg / mL.
[0018] c. Add the dried silica microspheres to the epidermal growth factor (EGF) solution at a mass ratio of 1:15-25, and shake and adsorb at 3-5℃ and 120-180 rpm for 18-24 hours.
[0019] d. Centrifuge, wash three times with pH 7.4 PBS, and freeze-dry to obtain silica microspheres of epidermal growth factor (EGF) with a loading rate ≥ 92%, which are the active carriers for epidermal repair.
[0020] Furthermore, a method for preparing an antibacterial, epithelial regeneration-promoting postoperative wound regeneration hydrogel formulation includes the following steps:
[0021] S1. Preparation of premixed solution: Add chitosan quaternary ammonium salt and collagen to the solvent and stir at 300-500 r / min for 2-3 h at 40-50℃ to form a uniform polymer premixed solution.
[0022] S2, Antibacterial Complex Dispersion: The fermented herbal broth and antibacterial peptides are mixed at a ratio of (5-10) mL:(1.5-3) mg, and ultrasonically dispersed at 20-25℃ for 10-15 min to form an antibacterial complex solution, which is then added to the premixed solution in step S1.
[0023] S3. Loading of active carrier: Add the angiogenesis active carrier and the epidermal repair active carrier in sequence, and stir at 150-200 r / min for 30-45 min at 25-30℃ to make the active ingredients evenly distributed.
[0024] S4. Crosslinking molding: Add a crosslinking agent and react for 1-2 hours at pH 6.8-7.2 and 25-30℃ to form a hydrogel.
[0025] Furthermore, the crosslinking reaction described in step S4 is carried out with the assistance of ultrasound at a frequency of 20-40 kHz, and the ultrasonic power density is 0.5-1.5 W / cm². 2 This promotes a uniform reaction between the crosslinking agent and the polymer.
[0026] Furthermore, the application of the antibacterial, epithelial regeneration-promoting postoperative wound regeneration hydrogel preparation is characterized in that it is used to prepare a drug that promotes postoperative wound repair.
[0027] Compared with the prior art, the beneficial effects of the present invention are:
[0028] (1) Synergistic antibacterial effect: The compound Chinese medicines such as Astragalus membranaceus and Angelica sinensis are fermented with Lactobacillus plantarum. The dissolution of effective components is enhanced through microbial degradation, and short-chain fatty acids and other antibacterial substances are generated during fermentation, which increases the content of antibacterial active ingredients and broadens the antibacterial spectrum. Blue copper peptide and bee venom peptide are compounded in a specific ratio. The synergistic effect of copper ion chelation and amphiphilic peptide perforation increases the diameter of the inhibition zone and reduces the risk of drug resistance. The fermentation broth and antibacterial peptides form a dual mechanism of "rapid antibacterial-continuous antibacterial".
[0029] (2) Targeted delivery of active factors for precise repair: Highly efficient delivery of active factors was achieved through liposome encapsulation and mesoporous silica microsphere loading technology. Using a distearylphosphatidylcholine-cholesterol-dipalmitoylphosphatidylglycerol liposome system, the encapsulation rate of VEGF165 was increased to over 85%, with a cumulative release rate of 68% over 48 hours, significantly promoting angiogenesis. Mesoporous silica microspheres loaded with EGF significantly promoted cell proliferation and effectively accelerated epithelial reconstruction. Both methods increased vascular density to 38.2±4.1 cells / mm². 2 The epithelial coverage rate reached 98.5±1.2%; the multi-component synergistic effect accelerated wound healing.
[0030] This invention systematically solves the problems of short antibacterial duration, rapid loss of active ingredients, and low repair efficiency of existing wound repair materials through component innovation, carrier delivery, and process improvement, providing a safe and efficient treatment option for postoperative wounds, especially refractory wounds. Detailed Implementation
[0031] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.
[0032] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.
[0033] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.
[0034] Example 1
[0035] The raw material composition of the hydrogel formulation (parts by weight) is as follows: 5 parts of chitosan quaternary ammonium salt, 12 parts of type III collagen (from pig skin), 15 parts of antibacterial complex solution, 2 parts of angiogenesis active carrier, 15 parts of epidermal repair active carrier, 0.5 parts of cross-linking agent (ethylene glycol diglycidyl ether), and 120 parts of physiological saline.
[0036] Preparation of fermentation broth for traditional Chinese medicine: 2 parts Astragalus membranaceus, 1 part Angelica sinensis, 1 part Callicarpa macrophylla, 2 parts Dragon's Blood, 5 parts Sanguisorba officinalis, and 3 parts Paris polyphylla. Grind the herbs through a 60-mesh sieve, add 5 times the amount of deionized water, sterilize at 121℃ for 30 minutes, and inoculate with 1×10⁻⁶ herbs. 6CFU / g Lactobacillus plantarum, fermented at 30℃ for 72h, centrifuged (3℃, 8000r / min, 20min) and filtered.
[0037] Antimicrobial peptides: The mass ratio of blue copper peptide to bee venom peptide is 1.5:0.5.
[0038] Angiogenic active carrier: distearylphosphatidylcholine, cholesterol and dipalmitoylphosphatidylglycerol in a ratio of 4:3:1, 12 mg / mL dissolved in chloroform, lipid film added to 10 mM phosphate buffer containing VEGF165 (1 mg:1 mL buffer) at a ratio of 10 mg:1 mL, hydrated at 50 °C and 8 rpm for 70 min, particle size after extrusion is 150 nm, encapsulation efficiency is 86%.
[0039] Epidermal repair active carrier: mesoporous silica microspheres (pore size 5nm) were vacuum dried at 100℃ for 1h, and EGF was prepared at 1mg / mL (specific activity 1.0×10⁻⁶). 7 The microspheres were prepared in 10 mM Tris-HCl solution (IU / mg), with a mass ratio of 1:15. The microspheres were shaken at 3°C and 120 rpm for 18 h for adsorption, followed by centrifugation, washing, and lyophilization. The loading rate was 92%.
[0040] Preparation method:
[0041] S1. Preparation of premixed solution: Chitosan quaternary ammonium salt and type III collagen are added to the solvent and stirred at 300 r / min for 2 h at 40℃ to form a homogeneous polymer premixed solution.
[0042] S2, Antibacterial Complex Dispersion: The fermented herbal broth and antibacterial peptides are mixed at a ratio of 5 mL: 1.5 mg and ultrasonically dispersed at 20 °C for 10 min to form an antibacterial complex solution, which is then added to the premixed solution in step S1.
[0043] S3, Active carrier loading: Then add the angiogenesis active carrier and the epidermal repair active carrier in sequence, and stir at 150 r / min for 30 min at 25℃;
[0044] S4. Cross-linking molding: Add ethylene glycol diglycidyl ether and perform ultrasonic cross-linking reaction at pH 6.8 and 25℃ for 1 hour. The ultrasonic frequency is 20kHz and the ultrasonic power density is 0.5W / cm³. 2 This forms a hydrogel.
[0045] Example 2
[0046] The raw material composition of the hydrogel formulation (parts by weight) is as follows: 8 parts of chitosan quaternary ammonium salt, 8 parts of type I collagen (derived from bovine Achilles tendon), 25 parts of antibacterial complex solution, 10 parts of angiogenesis active carrier, 3 parts of epidermal repair active carrier, 2 parts of cross-linking agent (genipin), and 150 parts of deionized water.
[0047] Preparation of fermentation broth for traditional Chinese medicine: 5 parts Astragalus membranaceus, 3 parts Angelica sinensis, 3 parts Callicarpa macrophylla leaves, 4 parts Dragon's Blood, 10 parts Sanguisorba officinalis, and 5 parts Paris polyphylla. The herbs were pulverized and passed through a 100-mesh sieve. The mixture was then sterilized at 121℃ for 20 minutes with 8 times the volume of deionized water. The inoculum size was 5 × 10⁻⁶. 6 CFU / g, fermented at 37℃ for 48h, centrifuged (5℃, 10000r / min, 15min) and filtered.
[0048] Antimicrobial peptides: The mass ratio of blue copper peptide to bee venom peptide is 2.5:1.2.
[0049] Angiogenic active carrier: Disteazylecithin, cholesterol and dipalmitoylphosphatidylglycerol in a ratio of 6:5:1, 10 mg / mL dissolved in chloroform, the lipid film was added to 10 mM phosphate buffer containing VEGF165 (1 mg:3 mL buffer) at a ratio of 10 mg:2 mL, hydrated at 60 °C and 12 rpm for 30 min, and the particle size after extrusion was 250 nm with an encapsulation efficiency of 85%.
[0050] Epidermal repair active carrier: mesoporous silica microspheres (pore size 20nm) were vacuum dried at 130℃ for 3h, and EGF was prepared at 5mg / mL (specific activity 2.0×10). 7 The microspheres were prepared in 10 mM Tris-HCl solution (IU / mg), with a mass ratio of 1:25. The microspheres were shaken at 5℃ and 180 rpm for 24 h for adsorption, followed by centrifugation, washing, and lyophilization. The loading rate was 94%.
[0051] Preparation method:
[0052] S1. Preparation of premixed solution: Chitosan quaternary ammonium salt and type I collagen are added to the solvent and stirred at 500 r / min for 3 h at 50℃ to form a homogeneous polymer premixed solution.
[0053] S2, Antibacterial Complex Dispersion: The fermented herbal broth and antibacterial peptides are mixed at a ratio of 10 mL: 3 mg and ultrasonically dispersed at 25 °C for 15 min to form an antibacterial complex solution, which is then added to the premixed solution in step S1.
[0054] S3, Active carrier loading: Then add the angiogenesis active carrier and epidermal repair active carrier in sequence, and stir at 200 r / min for 45 min at 30℃;
[0055] S4. Cross-linking molding: Add genipin and perform ultrasonic cross-linking reaction at pH 7.2 and 30℃ for 2 hours. The ultrasonic frequency is 40kHz and the ultrasonic power density is 1.5W / cm³. 2 This forms a hydrogel.
[0056] Example 3: 6 parts chitosan quaternary ammonium salt, 10 parts type I collagen (bovine Achilles tendon), 20 parts antibacterial complex solution, 6 parts angiogenesis active carrier (VEGF liposomes, particle size 200nm), 9 parts epidermal repair active carrier (EGF silica microspheres, pore size 10nm), 1 part crosslinking agent (genipin), and 135 parts solvent (physiological saline).
[0057] Hydrogel formulation raw material composition (parts by weight):
[0058] Preparation of fermentation broth for traditional Chinese medicine: Take 3 parts Astragalus membranaceus, 2 parts Angelica sinensis, 2 parts Callicarpa macrophylla leaves, 3 parts Dragon's Blood, 7 parts Sanguisorba officinalis, and 4 parts Paris polyphylla. Grind them and pass them through an 80-mesh sieve. Add 6 times the weight of deionized water, sterilize at 121℃ for 25 minutes, and after cooling, inoculate with an inoculum size of 3×10⁻⁶. 6 CFU / g of Lactobacillus plantarum was fermented at 35℃ for 60h, centrifuged (4℃, 9000r / min, 18min) to obtain the supernatant, and filtered through a 0.22μm filter membrane.
[0059] Antimicrobial peptides: Blue copper peptide to bee venom peptide in a mass ratio of 2:1.
[0060] Angiogenic active carrier: Distearylphosphatidylcholine:cholesterol:dipalmitoylphosphatidylglycerol = 5:4:1, 8 mg / mL dissolved in chloroform, and rotary evaporated to form a membrane; the membrane was added to 10 mM phosphate buffer containing VEGF165 (1 mg:2 mL buffer) at a ratio of 10 mg:1.5 mL, hydrated at 55 °C and 10 rpm for 50 min, and extruded sequentially through 1.0 μm, 0.4 μm, and 0.2 μm polycarbonate membranes, with a 30 kDa cutoff to remove free factors, resulting in an encapsulation efficiency of 88%.
[0061] Epidermal repair active carrier: mesoporous silica microspheres (pore size 10nm) were vacuum dried at 120℃ for 2h, and EGF was prepared at 3mg / mL (specific activity 1.2×10). 7 The microspheres were prepared in 10 mM Tris-HCl solution (IU / mg), with a mass ratio of 1:20. The microspheres were shaken at 4℃ and 150 rpm for 20 h for adsorption, followed by centrifugation, washing, and lyophilization. The loading rate was 93%.
[0062] Preparation method:
[0063] S1. Preparation of premixed solution: Chitosan quaternary ammonium salt and type I collagen are added to the solvent and stirred at 400 r / min for 3 h at 45℃ to form a homogeneous polymer premixed solution.
[0064] S2, Antibacterial Complex Dispersion: The fermented herbal broth and antibacterial peptides are mixed at a ratio of 8 mL: 2.5 mg and ultrasonically dispersed at 23 °C for 12 min to form an antibacterial complex solution, which is then added to the premixed solution in step S1.
[0065] S3, loading of active carrier: then add the angiogenesis active carrier and the epidermal repair active carrier in sequence, and stir at 180 r / min for 40 min at 28℃;
[0066] S4. Cross-linking molding: Add genipin and perform ultrasonic cross-linking reaction at pH 7.0 and 28℃ for 1.5 h. The ultrasonic frequency is 30 kHz and the ultrasonic power density is 1 W / cm². 2 This forms a hydrogel.
[0067] Comparative Example 1
[0068] The difference between this comparative example and Example 3 is that the hydrogel formulation raw material does not contain an antibacterial complex solution;
[0069] Raw material composition: 6 parts chitosan quaternary ammonium salt, 10 parts type I collagen, 6 parts angiogenesis active carrier, 9 parts epidermal repair active carrier, 1 part genipin, 155 parts physiological saline (solvent to make up for the missing amount of antibacterial complex solution).
[0070] Preparation method: Step S2 is omitted. The active carrier is directly added to the premixed solution. The remaining steps are the same as in Example 3.
[0071] Comparative Example 2
[0072] The difference between this comparative example and Example 3 is that the herbal fermentation broth in the antibacterial complex solution was replaced with an equal amount of traditional water decoction.
[0073] Preparation method: Add 5-8 times the amount of deionized water to the same proportion of medicinal materials, boil at 100℃ under normal pressure for 1 hour, and filter to obtain the decoction; the remaining steps are the same as in Example 3.
[0074] Comparative Example 3
[0075] The difference between this comparative example and Example 3 is that the angiogenesis active carrier was replaced with an equal amount of vascular endothelial growth factor, and the vascular endothelial growth factor was not encapsulated.
[0076] Preparation method: In step S3, vascular endothelial growth factor is added directly, and the rest is the same as in Example 3.
[0077] Comparative Example 4
[0078] The difference between this comparative example and Example 3 is that the epidermal repair active carrier was replaced with an equal amount of epidermal growth factor, and the epidermal growth factor was not loaded into silica microspheres.
[0079] Preparation method: In step S3, epidermal growth factor is added directly, and the rest is the same as in Example 3.
[0080] I. Performance Verification Experiment
[0081] Test Example 1: Comparative Test of Antibacterial Properties
[0082] 1. Experimental Materials
[0083] Test samples: Hydrogels from Examples 1-3, hydrogels from Comparative Examples 1 and 2, and a blank control (physiological saline).
[0084] Bacterial strains: Staphylococcus aureus (ATCC25923), Escherichia coli (ATCC25922)
[0085] Culture medium: LB agar medium
[0086] 2. Experimental Methods
[0087] Preparation of bacterial suspension: The bacterial strain was inoculated into LB broth and incubated at 37°C for 18 h. The bacterial concentration was then adjusted to 1×10⁻⁶. 8 CFU / mL.
[0088] Agar diffusion method: Spread 0.1 mL of bacterial suspension evenly on the surface of LB agar plate, punch holes with a punch (diameter 6 mm), and add 10 μL of sample extraction solution (hydrogel: physiological saline = 1:10, extraction at 37℃ for 24 h).
[0089] Culture and observation: After incubation at 37℃ for 24 hours, the diameter of the inhibition zone was measured, and each group was repeated 3 times.
[0090] 3. Experimental Results
[0091]
[0092] Conclusion: The antibacterial complex solution significantly enhances the antibacterial ability of the hydrogel. The hydrogels in Examples 1-3, containing the antibacterial complex solution, exhibited inhibition zones of 21.3-23.1 mm in diameter against Staphylococcus aureus and 19.7-21.2 mm in diameter against Escherichia coli, indicating that this formulation effectively inhibits common pathogenic bacteria. Comparative Example 1, without the antibacterial complex, showed inhibition zones close to the diffusion range of the culture medium. Comparative Example 2, using a decoction instead of fermentation broth, showed significantly weaker antibacterial effects than the examples, confirming that the antibacterial complex (especially the fermentation broth process) is key to improving the antibacterial performance of the hydrogel. The blank control showed no inhibition zones, further verifying that the antibacterial effect of the hydrogel originates from the formulation design.
[0093] Experimental Example 2: Test of sustained-release performance of angiogenesis factors
[0094] 1. Experimental Materials
[0095] Samples: Example 3 angiogenesis active carrier (VEGF liposomes), Comparative Example 3 free VEGF solution
[0096] Release medium: PBS (pH 7.4, containing 0.1% Tween-80)
[0097] 2. Experimental Methods
[0098] Release model: An equal mass of VEGF (10 μg / mL) sample was placed in a dialysis bag (molecular weight cutoff 30 kDa), immersed in 5 mL of release medium, and shaken at 37 °C and 100 rpm.
[0099] Timed sampling: 100 μL of supernatant was collected at 0.5h, 1h, 2h, 4h, 8h, 12h, 24h and 48h respectively, and the VEGF concentration was detected by ELISA. The same volume of fresh medium was then added.
[0100] 3. Experimental Results:
[0101] Example 3: The cumulative release rate was 45% in 24 hours and reached 68% in 48 hours, showing a sustained release effect.
[0102] Comparative Example 3: The release rate reached 75% in 0.5 hours, and was basically completely released (92%) after 2 hours, with a residual amount of <5% after 48 hours.
[0103] This indicates that liposome encapsulation can significantly delay VEGF release, prevent rapid loss of free factors, and ensure long-term angiogenesis-promoting activity.
[0104] Experimental Example 3: Cell Proliferation and Biocompatibility Test
[0105] 1. Experimental Materials
[0106] Samples: Hydrogel extract from Example 3, extract from Comparative Example 4 (EGF not loaded), blank culture medium
[0107] Cell lines: HaCaT cells, NIH / 3T3 cells
[0108] Test reagent: CCK-8 kit
[0109] 2. Experimental Methods
[0110] Preparation of extract: The hydrogel and culture medium were mixed at a ratio of 1:5 (w / v), extracted at 37℃ and 5% CO2 for 24 h, and sterilized by 0.22μm filter membrane.
[0111] Cell seeding: 5 × 10 3 Cells per well were seeded into 96-well plates and cultured for 24 hours. The culture medium was then replaced with a medium containing 10% extract. A blank control group (pure culture medium) was also included.
[0112] Proliferation assay: 10 μL CCK-8 was added at 24 h, 48 h, and 72 h, and OD was measured after 2 h of incubation. 450 The nm value is used to calculate cell viability.
[0113] Experimental results:
[0114]
[0115] Conclusion: The epidermal repair active carrier (EGF-loaded microspheres) significantly promoted cell proliferation. The effects of the four control groups without EGF loading were close to those of the blank group, demonstrating the necessity of the carrier loading process.
[0116] II. Application Example (Rat Full-Thickness Skin Defect Healing Experiment)
[0117] 1. Experimental objective: To evaluate the effects of hydrogel on wound healing speed, angiogenesis, and epithelial regeneration.
[0118] 2. Experimental animals: SPF-grade SD rats (weighing 200-250g) were used to create a full-thickness skin defect model with a diameter of 10mm on their backs. They were randomly divided into 4 groups (n=6): Example 3 group, Comparative Example 3-4 group and blank control group.
[0119] 3. Experimental Methods
[0120] Wound treatment: Immediately after molding, cover with the appropriate hydrogel (1mm thick), bandage with sterile dressing, and change every 3 days.
[0121] Observation indicators:
[0122] Healing time: Record the time it takes for the wound to become completely epithelialized.
[0123] Neovascularization: Wound tissue was taken 7 days after surgery, CD31 immunohistochemical staining was performed, and the blood vessel density (number / mm²) was calculated.
[0124] Epithelial coverage: HE staining was performed 14 days postoperatively, and the percentage of epithelialized area was measured using image analysis software.
[0125] 4. Experimental Results
[0126]
[0127] Conclusion: The hydrogel in Example 3, through its design incorporating fermentation broth enhancement and active factor encapsulation, significantly accelerated wound healing and promoted angiogenesis and epithelial regeneration; it exhibited the shortest healing time (11.5 ± 1.0 days) and the highest neovascularization density (38.2 ± 4.1 cells / mm²). 2 The epithelial coverage rate reached 98.5±1.2%, indicating that the antibacterial complex and encapsulating active carrier in the complete formula had a significant synergistic effect. In contrast, the effects of angiogenesis and epithelial repair were significantly limited in Comparative Examples 3 and 4 because the active factors were not encapsulated or loaded. The blank group healed the slowest, confirming that the hydrogel formula has a positive effect on accelerating wound healing.
[0128] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. An antibacterial pro-epithelial regenerative post-surgical wound regenerative hydrogel formulation, characterized in that: The raw materials include the following parts by weight: 6 parts chitosan quaternary ammonium salt, 10 parts type I collagen, 20 parts antibacterial complex solution, 6 parts angiogenesis active carrier, 9 parts epidermal repair active carrier, 1 part crosslinking agent, and 135 parts solvent. The antibacterial complex solution is composed of fermented Chinese herbal medicine broth and antibacterial polypeptides. The following herbal mixtures were prepared by mixing 3 parts Astragalus membranaceus, 2 parts Angelica sinensis, 2 parts Callicarpa macrophylla leaves, 3 parts Daemonorops draco, 7 parts Sanguisorba officinalis, and 4 parts Paris polyphylla by weight to obtain a herbal mixture. This mixture was then fermented with Lactobacillus plantarum to obtain a herbal fermentation broth. The inoculum size of Lactobacillus plantarum was 3 × 10⁻⁶. 6 CFU / g of traditional Chinese medicine mixture; The preparation method of the herbal fermentation broth includes: pulverizing the herbal mixture through an 80-mesh sieve, adding 6 times the mass of deionized water, sterilizing at 121℃ for 25 minutes, cooling, inoculating with Lactobacillus plantarum, fermenting at 35℃ for 60 hours, centrifuging at 4℃ and 9000 r / min for 18 minutes after fermentation, taking the supernatant and filtering it through a 0.22 μm filter membrane to obtain the herbal fermentation broth; The antibacterial polypeptide is a blue copper peptide and a bee venom peptide in a mass ratio of 2:
1. The angiogenic active carrier is composed of vascular endothelial growth factor (VEGF) encapsulated in liposomes with a particle size of 200 nm. The VEGF is isoform 165 with a specific activity ≥1×10⁻⁶. 6 IU / mg; The encapsulation step is as follows: a. Distearate phosphatidylcholine, cholesterol, and dipalmitoylphosphatidylglycerol were mixed in a mass ratio of 5:4:1, dissolved in chloroform at a ratio of 8 mg:1 mL, and then rotary evaporated to form a lipid film. b. The lipid membrane was added to 10mM phosphate buffer containing vascular endothelial growth factor at a ratio of 10mg:1.5mL, wherein the ratio of vascular endothelial growth factor to buffer was 1mg:2mL. The membrane was hydrated for 50 minutes at 55℃ and 10rpm to form multilayer liposomes. c. Liposomes with a particle size of 200 nm were obtained by sequentially extruding polycarbonate membranes with pore sizes of 1.0 μm, 0.4 μm, and 0.2 μm, retaining a molecular weight cutoff of 30 kDa to remove free vascular endothelial growth factor, with an encapsulation efficiency of ≥85%. The epidermal repair active carrier is an epidermal growth factor with a specific activity ≥1×10⁻⁶. 7 IU / mg, loaded in silica microspheres with a pore size of 10 nm: a. Mesoporous silica microspheres with a pore size of 10 nm were vacuum dried at 120 °C for 2 hours; b. Prepare a 10mM Tris-HCl solution containing epidermal growth factor at a ratio of 3:1 mg / mL; c. Add the dried silica microspheres to the epidermal repair growth factor solution at a mass ratio of 1:20, and shake and adsorb at 4℃ and 150 rpm for 20 hours. d. Centrifuge, wash three times with pH 7.4 PBS, freeze-dry to obtain silica microspheres of epidermal repair growth factor with a loading rate ≥ 92%, which is the active carrier for epidermal repair. The crosslinking agent is genipin or ethylene glycol diglycidyl ether, and the solvent is deionized water or physiological saline.
2. The preparation method of the antibacterial and epithelial regeneration postoperative wound regeneration hydrogel formulation as described in claim 1, characterized in that: Includes the following steps: S1. Preparation of premixed solution: Chitosan quaternary ammonium salt and type I collagen are added to the solvent and stirred at 400 r / min for 3 h at 45 °C to form a homogeneous polymer premixed solution. S2, Antibacterial Complex Dispersion: The fermented broth of the traditional Chinese medicine and the antibacterial peptide are mixed at a ratio of 8 mL: 2.5 mg, and ultrasonically dispersed at 23°C for 12 min to form an antibacterial complex solution, which is then added to the premixed solution in step S1. S3, loading of active carrier: then add the angiogenesis active carrier and the epidermal repair active carrier in sequence, and stir at 180 r / min for 40 min at 28℃; S4. Crosslinking and molding: Add the crosslinking agent and perform an ultrasonic crosslinking reaction at pH 7.0 and 28℃ for 1.5 hours to form a hydrogel. The ultrasonic frequency is 30kHz and the ultrasonic power density is 1W / cm². 2 .
3. The application of a postoperative wound regeneration hydrogel formulation with antibacterial and epithelial regeneration-promoting properties as described in claim 1 or 2, characterized in that, Used to prepare drugs that promote postoperative wound repair.