Sodium hyaluronate gel compound for wound repair and preparation process of sodium hyaluronate gel compound

Abstract

The invention provides a sodium hyaluronate gel compound for wound repair and a preparation process of the sodium hyaluronate gel compound, and belongs to the technical field of gel compound preparation. The preparation process comprises the following steps: preparing a pachymaran-antibacterial peptide compound; preparation of catechol graft modified gelatin; preparing oxidized sodium alginate; preparing a sodium alginate compound; and preparing the sodium hyaluronate gel compound. The preparation method comprises the following steps: grafting a catechol group on the side chain of gelatin, and carrying out a coordination effect between ferric ions and the catechol group and a Schiff base reaction between an amino group on the gelatin and an aldehyde group on oxidized sodium alginate to prepare the double dynamic bond cross-linked sodium alginate compound. The pachymaran-antibacterial peptide compound is introduced through total doping of the sodium alginate compound, and then the sodium alginate compound is compounded with sulfhydrylated sodium hyaluronate to prepare the sodium hyaluronate gel compound which has good biocompatibility and efficient antibacterial property and can remarkably promote wound healing.

Description

Technical Field

[0001] The invention relates to the technical field of gel compound preparation, and in particular to a sodium hyaluronate gel compound for wound repair and a preparation process thereof. Background Art

[0002] In the field of trauma treatment, wound repair is a complex and critical process that affects the patient's recovery process, quality of life, and medical costs. Ideal wound repair materials must have good biocompatibility, moisture retention, antibacterial properties, promote cell proliferation and migration, and suitable mechanical properties to meet the treatment needs of different types of wounds. At present, although there are many types of wound repair materials on the market, they still face many challenges. The development of new and efficient wound repair materials has important clinical significance and broad market prospects.

[0003] Gauze is the most common traditional wound dressing, and its advantages are low cost and easy use. However, it has many disadvantages. On the one hand, the air permeability and moisture retention of gauze are poor. During the wound healing process, excessive water loss will cause the wound to dry out, hinder the migration and proliferation of epithelial cells, prolong the healing time, and may also cause pain and secondary injury. On the other hand, gauze is easy to adhere to the wound surface. When changing the dressing, it will tear the new tissue, aggravate the patient's pain, and increase the risk of infection.

[0004] Some natural polymer materials, such as collagen and chitosan, have been used in the field of wound repair due to their good biocompatibility. But they also have their own problems. Collagen sources are limited, the extraction and purification process is complex, the cost is high, and it is easy to induce immune response. Although chitosan has antibacterial properties, its degradation rate in the body is difficult to accurately control, and the degradation products may have adverse effects on tissues. In addition, its mechanical properties are relatively weak, and it is difficult to meet the repair needs of complex wounds when used alone.

[0005] Sodium hyaluronate is a biomaterial widely used in wound repair. It has excellent moisturizing properties, can maintain a moist environment on the wound surface, and promote cell migration and proliferation. However, pure sodium hyaluronate gel has problems such as poor mechanical properties and limited antibacterial ability. In order to improve these properties, researchers have tried to modify sodium hyaluronate or compound it with other materials, but the current compounding methods and modification technologies still have certain defects. For example, some simple physical mixing methods make it difficult for sodium hyaluronate to achieve good synergy with other materials, resulting in no obvious improvement in the overall performance of the complex; some chemical modification methods may destroy the original structure and biological activity of sodium hyaluronate, affecting its efficacy in wound repair.

[0006] With the development of society and the change of people's lifestyle, the incidence of trauma is on the rise. Various types of wounds, including burns, cuts, diabetic foot ulcers, etc., bring great pain to patients. Clinically, there is an urgent need for wound repair materials that can quickly promote wound healing, reduce infection, reduce scar formation and are easy to operate. Especially in special populations such as diabetic patients and the elderly, due to their poor healing ability, the demand for high-quality wound repair materials is more prominent. In addition, with the advancement of medical technology, patients have higher and higher requirements for the beauty and functional recovery of wounds after repair, which has prompted researchers to continuously explore and develop new wound repair materials and preparation processes.

[0007] Therefore, we proposed a sodium hyaluronate gel complex and its preparation process which has good biocompatibility, high antibacterial property, high mechanical strength and can significantly promote wound healing. Summary of the invention

[0008] In view of the shortcomings of the prior art, the object of the present invention is to provide a sodium hyaluronate gel complex for wound repair and a preparation process thereof.

[0009] A preparation process of a sodium hyaluronate gel complex for wound repair comprises the following steps:

[0010] S1: Preparation of Pachymaran-antimicrobial peptide complex

[0011] Microwave-ultrasound-assisted extraction of pachymaran was used, and then pachymaran was covalently bound to the antimicrobial peptide LL-37 through the cross-linking agent 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimidyl ester to obtain a pachymaran-antimicrobial peptide complex.

[0012] S2: Preparation of catechol-grafted modified gelatin

[0013] NHS and EDC are added to a 3-3-(3,4-dihydroxyphenyl) propionic acid solution, mixed, and then added to a gelatin solution, and reacted under an argon atmosphere and protected from light to prepare catechol-grafted modified gelatin;

[0014] S3: Preparation of oxidized sodium alginate

[0015] Sodium metaperiodate is reacted with sodium alginate to prepare oxidized sodium alginate;

[0016] S4: Preparation of sodium alginate complex

[0017] The catechol grafted modified gelatin solution is mixed with the FeCl3 solution to obtain a mixed solution, and then the pachymaran-antimicrobial peptide complex is added to the oxidized sodium alginate solution and then mixed with the mixed solution to prepare a sodium alginate complex;

[0018] S5: Preparation of Sodium Hyaluronate Gel Complex

[0019] The thiolated sodium hyaluronate is prepared by mixing N-hydroxysuccinimide, 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide, cysteamine hydrochloride and sodium hyaluronate freeze-dried powder, and then the thiolated sodium hyaluronate is compounded with a sodium alginate complex to prepare a sodium hyaluronate gel complex.

[0020] Furthermore, step S1 of preparing the pachymaran-antimicrobial peptide complex specifically comprises the following steps:

[0021] S1.1: Select the Poria cocos blocks with uniform color and no mold spots, dry them in an oven at 50-60℃, crush them with a high-speed grinder, and pass them through a 40-50 mesh sieve to obtain Poria cocos powder;

[0022] S1.2: 10-20 parts by weight of Poria powder are added to pure water at a solid-liquid ratio of 1:25-30, and then placed in a microwave-ultrasonic extraction instrument. After microwave-ultrasonic extraction, centrifuge at 3000-4000 r / min for 20-30 min, filter the supernatant, and freeze-dry to obtain Poria polysaccharide;

[0023] S1.3: adding tuckahoe polysaccharide to a PBS buffer at pH 7.4 to obtain a tuckahoe polysaccharide solution with a concentration of 2-3% w / v, and adding the antimicrobial peptide LL-37 to a PBS buffer at pH 7.4 to obtain a 1-2% w / v antimicrobial peptide LL-37 solution;

[0024] S1.4: Add 0.01-0.02 parts by weight of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimide ester to 20-30 parts by weight of the Poria polysaccharide solution, and then add 4-5 parts by weight of the antimicrobial peptide LL-37 solution. Oscillating reaction at 37-38°C for 4-5 hours. After the reaction is completed, dialyze and freeze-dry to obtain the Poria polysaccharide-antimicrobial peptide complex.

[0025] Furthermore, step S2 of preparing catechol-grafted modified gelatin specifically comprises the following steps:

[0026] S2.1: dissolving 5-8 parts by weight of gelatin in 50-60 parts by weight of a phosphate buffer having a pH value of 5.5 at 37-38° C. to obtain a gelatin solution, and dissolving 0.23-0.25 parts by weight of 3-(3,4-dihydroxyphenyl)propionic acid in 50-60 parts by weight of a phosphate buffer having a pH value of 5.5 at 37-38° C. to obtain a 3-(3,4-dihydroxyphenyl)propionic acid solution;

[0027] S2.2: Add 0.2-0.3 parts by weight of NHS and 0.4-0.5 parts by weight of EDC to the 3-3-(3,4-dihydroxyphenyl)propionic acid solution, stir and mix for 20-30 minutes under an argon atmosphere, then add to the gelatin solution, react for 20-24 hours under an argon atmosphere in the dark, then dialyze and freeze-dry to obtain catechol-grafted modified gelatin.

[0028] Furthermore, step S3 of preparing oxidized sodium alginate specifically comprises the following steps:

[0029] S3.1: 6-8 parts by weight of sodium alginate are dispersed in 30-40 parts by weight of ethanol, and 30-40 parts by weight of a 17-20% sodium metaperiodate solution is slowly added under vigorous stirring, and then reacted in a dark environment for 4-6 hours, and then 2-3 parts by weight of ethylene glycol are added, and stirred and mixed for 20-30 minutes to obtain a reaction solution;

[0030] S3.2: The reaction solution is dialyzed with distilled water for 3-4 days through a dialysis bag with a molecular weight cutoff of 3500Da, and then the purified solution is freeze-dried to obtain oxidized sodium alginate.

[0031] Furthermore, step S4 of preparing the sodium alginate complex specifically comprises the following steps:

[0032] S4.1: Prepare 250 mg / mL of catechol grafted modified gelatin solution and 150 mg / mL of oxidized sodium alginate solution with distilled water, and mix the catechol grafted modified gelatin solution with 5 mg / mL FeCl3 solution to obtain a mixed solution;

[0033] S4.2: Add 2-3 parts by weight of the Poria polysaccharide-antimicrobial peptide complex to 20-30 parts by weight of the oxidized sodium alginate solution, then add 2-3 mol / L of sodium hydroxide solution, adjust the pH to 8-10, then add 10-20 parts by weight of the mixed solution and pre-mix on a vortex oscillator for 2-3 hours to obtain the sodium alginate complex.

[0034] Furthermore, step S5 of preparing the sodium hyaluronate gel complex specifically comprises the following steps:

[0035] S5.1: 4-5 parts by weight of sodium hyaluronate freeze-dried powder is added to 80-100 parts by weight of deionized water, followed by adding 0.23-0.25 parts by weight of N-hydroxysuccinimide and 0.57-0.59 parts by weight of 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide, stirring and mixing, adjusting the pH to 4.57 with 1 mol / L hydrogen chloride solution, and then reacting at room temperature for 2-3 hours to obtain a mixed reaction solution;

[0036] S5.2: Add 0.34-0.36 parts by weight of cysteamine hydrochloride to the mixed reaction solution, stir and mix, adjust the pH to 4.57 with 1 mol / L sodium hydroxide solution, react at room temperature in the dark for 24-25 hours, then transfer to a dialysis bag, dialyze with deionized water at pH 3.5 at room temperature in the dark for 3-4 days, and finally freeze-dry to obtain thiolated sodium hyaluronate;

[0037] S5.3: Dissolve the thiolated sodium hyaluronate in a PBS solution with a pH of 7.4 to prepare a precursor solution with a concentration of 2% (w / v), then add the sodium alginate complex to the precursor solution, stir and mix for 2-3 hours to obtain the sodium hyaluronate gel complex.

[0038] Furthermore, the parameters of the microwave-ultrasonic extractor in step S1.2 are ultrasonic power 100-120w, ultrasonic interval time 2-3s, microwave power 100-120w, temperature 45-50°C, and time 30-40min.

[0039] Furthermore, in step S4.1, Fe 3+ The molar ratio to catechol group is 1:3-4.

[0040] Furthermore, in step S5.3, the amount of sodium alginate complex added is 2-3% of the precursor solution.

[0041] A sodium hyaluronate gel complex for wound repair is prepared by any of the preparation processes for a sodium hyaluronate gel complex for wound repair.

[0042] Compared with the prior art, the present invention has at least the following beneficial effects:

[0043] 1. In the present invention, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimide ester is a bifunctional crosslinking agent, which has two different active groups, one end of which can react with active groups such as amino groups on pachymaran, and the other end of which can react with active groups such as thiol groups on antimicrobial peptide LL-37, thereby connecting pachymaran and antimicrobial peptide LL-37 together through covalent bonds to form a pachymaran-antimicrobial peptide complex. Antimicrobial peptide LL-37 itself has good antibacterial activity. After being combined with pachymaran, it can be used to prepare sodium hyaluronate gel. When the complex is formed, the antimicrobial peptide can continue to play an antibacterial role, effectively inhibit the growth and reproduction of bacteria and other microorganisms in the gel system, reduce the risk of infection, and improve the safety and stability of the gel. Poria polysaccharide has multiple biological activities, such as immunomodulation and anti-tumor. Introducing the Poria polysaccharide-antimicrobial peptide complex into the sodium hyaluronate gel can make the gel complex not only have antibacterial and good biocompatibility, but also obtain other biological activities brought by Poria polysaccharide, thereby regulating the local immune microenvironment, promoting cell proliferation, differentiation and tissue repair, and thus significantly promoting wound healing.

[0044] 2. The present invention prepares a sodium alginate complex with double dynamic bond cross-linking by grafting catechol groups on gelatin side chains, through the coordination between trivalent iron ions and catechol groups and the Schiff base reaction between amino groups on gelatin and aldehyde groups on oxidized sodium alginate, and the sodium alginate complex is doped with a tuckahoe polysaccharide-antimicrobial peptide complex, and then compounded with thiolated sodium hyaluronate to prepare a sodium hyaluronate gel complex, the coordination between trivalent iron ions and catechol groups and the Schiff base reaction between amino groups on gelatin and aldehyde groups on oxidized sodium alginate form a double dynamic bond cross-linked porous structure, this cross-linking method gives the sodium alginate complex a unique network structure, can effectively improve the mechanical strength of the gel, making it tougher and less prone to rupture, the porous structure is conducive to cell proliferation and migration, and the dynamic bond The presence of gives the gel the property of self-repair. When the gel is damaged by external force, the coordination bonds between the trivalent iron ions and the catechol groups and the Schiff base bonds can automatically restore the damaged structure and performance to a certain extent through a dynamic dissociation and recombination process, thereby extending the service life of the gel. The gelatin in the gel complex can promote the migration and proliferation of wound cells such as fibroblasts and epithelial cells, accelerate the healing of the wound, and sodium hyaluronate can absorb a large amount of water and retain it in the gel, providing a moist environment for the skin or tissue, which is conducive to wound healing and tissue repair. The compounding of thiolated sodium hyaluronate with sodium alginate complex and Poria cocos polysaccharide-antimicrobial peptide complex can produce a synergistic effect, further enhance the biological activity and function of the gel, and can jointly regulate cell behavior to promote tissue regeneration and repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The accompanying drawings, which are incorporated herein and constitute a part of the specification, illustrate embodiments of the invention and, together with the description, further serve to explain the principles of the invention and to enable those skilled in the relevant art to make and use the invention.

[0046] Figure 1 The present invention is a flow chart of the preparation process of a sodium hyaluronate gel complex for wound repairing used in an embodiment of the present invention. DETAILED DESCRIPTION

[0047] The following is a detailed description of the preparation process of a sodium hyaluronate gel complex for wound repair provided by the present invention in conjunction with the accompanying drawings and specific embodiments. At the same time, it is explained here that in order to make the embodiments more detailed, the following embodiments are the best and preferred embodiments, and those skilled in the art may also adopt other alternative methods to implement some known technologies; and the accompanying drawings are only for a more specific description of the embodiments, and are not intended to specifically limit the present invention.

[0048] Example 1

[0049] A preparation process of a sodium hyaluronate gel complex for wound repair, such as Figure 1 As shown, the following steps are included:

[0050] S1: Preparation of Pachymaran-antimicrobial peptide complex

[0051] S1.1: Select the Poria cocos blocks with uniform color and no mold spots, dry them in an oven at 50°C, grind them with a high-speed grinder, and pass them through a 40-mesh sieve to obtain Poria cocos powder;

[0052] S1.2: 10 parts by weight of Poria powder were added to pure water at a solid-liquid ratio of 1:25, and then placed in a microwave-ultrasonic extractor with the following parameters: ultrasonic power of 100w, ultrasonic interval of 2s, microwave power of 100w, temperature of 45°C, time of 30min, and then centrifuged at 3000r / min for 20min, and the supernatant was filtered and freeze-dried to obtain Poria polysaccharide;

[0053] S1.3: adding tuckahoe polysaccharide to a PBS buffer at pH 7.4 to obtain a tuckahoe polysaccharide solution with a concentration of 2% w / v, and adding the antimicrobial peptide LL-37 to a PBS buffer at pH 7.4 to obtain a 1% w / v antimicrobial peptide LL-37 solution;

[0054] S1.4: Add 0.01 parts by weight of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimide ester to 20 parts by weight of the tuckahoe polysaccharide solution, and then add 4 parts by weight of the antimicrobial peptide LL-37 solution, shake and react at 37°C for 4 hours, dialyze after the reaction is completed, and freeze-dry to obtain a tuckahoe polysaccharide-antimicrobial peptide complex;

[0055] S2: Preparation of catechol-grafted modified gelatin

[0056] S2.1: dissolving 5 parts by weight of gelatin in 50 parts by weight of a phosphate buffer having a pH value of 5.5 at 37° C. to obtain a gelatin solution, and dissolving 0.23 parts by weight of 3-(3,4-dihydroxyphenyl)propionic acid in 50 parts by weight of a phosphate buffer having a pH value of 5.5 at 37° C. to obtain a 3-(3,4-dihydroxyphenyl)propionic acid solution;

[0057] S2.2: 0.2 parts by weight of NHS and 0.4 parts by weight of EDC were added to the 3-3-(3,4-dihydroxyphenyl)propionic acid solution, stirred and mixed for 20 minutes under an argon atmosphere, and then added to the gelatin solution, reacted for 20 hours under an argon atmosphere and protected from light, and then dialyzed and freeze-dried to obtain catechol-grafted modified gelatin;

[0058] S3: Preparation of oxidized sodium alginate

[0059] S3.1: 6 parts by weight of sodium alginate are dispersed in 30 parts by weight of ethanol, and 30 parts by weight of a 17% sodium metaperiodate solution are slowly added under vigorous stirring, and then reacted in a dark environment for 4 hours, and then 2 parts by weight of ethylene glycol are added, and stirred and mixed for 20 minutes to obtain a reaction solution;

[0060] S3.2: The reaction solution was dialyzed with distilled water for 3 days through a dialysis bag with a molecular weight cutoff of 3500 Da, and then the purified solution was freeze-dried to obtain oxidized sodium alginate;

[0061] S4: Preparation of sodium alginate complex

[0062] S4.1: Prepare 250 mg / mL of catechol-grafted gelatin solution and 150 mg / mL of oxidized sodium alginate solution with distilled water, mix the catechol-grafted gelatin solution with 5 mg / mL FeCl3 solution, and 3+ The molar ratio of the catechol group is 1:3 to obtain a mixed solution;

[0063] S4.2: Add 2 parts by weight of the pachymannan-antimicrobial peptide complex to 20 parts by weight of the oxidized sodium alginate solution, then add 2 mol / L sodium hydroxide solution to adjust the pH to 8, then add 10 parts by weight of the mixed solution and pre-mix on a vortex oscillator for 2 hours to obtain a sodium alginate complex;

[0064] S5: Preparation of Sodium Hyaluronate Gel Complex

[0065] S5.1: 4 parts by weight of sodium hyaluronate freeze-dried powder are added to 80 parts by weight of deionized water, followed by adding 0.23 parts by weight of N-hydroxysuccinimide and 0.57 parts by weight of 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide, stirring and mixing, adjusting the pH to 4.57 with 1 mol / L hydrogen chloride solution, and reacting at room temperature for 2 hours to obtain a mixed reaction solution;

[0066] S5.2: Add 0.34 parts by weight of cysteamine hydrochloride to the mixed reaction solution, stir and mix, adjust the pH to 4.57, react at room temperature in the dark for 24 hours, then transfer to a dialysis bag, dialyze with deionized water at pH 3.5 at room temperature in the dark for 3 days, and finally freeze-dry to obtain thiolated sodium hyaluronate;

[0067] S5.3: Dissolve thiolated sodium hyaluronate in a PBS solution with a pH of 7.4 to prepare a precursor solution with a concentration of 2% (w / v), then add sodium alginate complex to the precursor solution, the amount of sodium alginate complex added is 2% of the precursor solution, stir and mix for 2 hours to obtain a sodium hyaluronate gel complex.

[0068] Example 2

[0069] A preparation process of a sodium hyaluronate gel complex for wound repair, such as Figure 1 As shown, the following steps are included:

[0070] S1: Preparation of Pachymaran-antimicrobial peptide complex

[0071] S1.1: Select the Poria cocos blocks with uniform color and no mold spots, dry them in an oven at 50°C, grind them with a high-speed grinder, and pass them through a 40-mesh sieve to obtain Poria cocos powder;

[0072] S1.2: 20 parts by weight of Poria powder were added to pure water at a solid-liquid ratio of 1:30, and then placed in a microwave-ultrasonic extraction instrument, and the parameters were set as ultrasonic power of 100w, ultrasonic interval time of 2s, microwave power of -120w, temperature of 45°C, time of 30min, and then centrifuged at 3000r / min for 20min, and the supernatant was filtered and freeze-dried to obtain Poria polysaccharide;

[0073] S1.3: adding tuckahoe polysaccharide to a PBS buffer at pH 7.4 to obtain a tuckahoe polysaccharide solution with a concentration of 3% w / v, and adding the antimicrobial peptide LL-37 to a PBS buffer at pH 7.4 to obtain a 2% w / v antimicrobial peptide LL-37 solution;

[0074] S1.4: Add 0.02 parts by weight of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimide ester to 30 parts by weight of the tuckahoe polysaccharide solution, and then add 5 parts by weight of the antimicrobial peptide LL-37 solution, shake and react at 37°C for 4 hours, dialyze after the reaction is completed, and freeze-dry to obtain a tuckahoe polysaccharide-antimicrobial peptide complex;

[0075] S2: Preparation of catechol-grafted modified gelatin

[0076] S2.1: dissolving 8 parts by weight of gelatin in 60 parts by weight of a phosphate buffer having a pH value of 5.5 at 37° C. to obtain a gelatin solution, and dissolving 0.25 parts by weight of 3-(3,4-dihydroxyphenyl)propionic acid in 60 parts by weight of a phosphate buffer having a pH value of 5.5 at 37° C. to obtain a 3-(3,4-dihydroxyphenyl)propionic acid solution;

[0077] S2.2: 0.3 parts by weight of NHS and 0.5 parts by weight of EDC were added to the 3-3-(3,4-dihydroxyphenyl)propionic acid solution, stirred and mixed for 20 minutes under an argon atmosphere, and then added to the gelatin solution, reacted for 20 hours under an argon atmosphere and protected from light, and then dialyzed and freeze-dried to obtain catechol-grafted modified gelatin;

[0078] S3: Preparation of oxidized sodium alginate

[0079] S3.1: 8 parts by weight of sodium alginate are dispersed in 40 parts by weight of ethanol, and 40 parts by weight of a 20% sodium metaperiodate solution are slowly added under vigorous stirring, and then reacted in a dark environment for 4 hours, and then 3 parts by weight of ethylene glycol are added, and stirred and mixed for 20 minutes to obtain a reaction solution;

[0080] S3.2: The reaction solution was dialyzed with distilled water for 3 days through a dialysis bag with a molecular weight cutoff of 3500 Da, and then the purified solution was freeze-dried to obtain oxidized sodium alginate;

[0081] S4: Preparation of sodium alginate complex

[0082] S4.1: Prepare 250 mg / mL of catechol-grafted gelatin solution and 150 mg / mL of oxidized sodium alginate solution with distilled water, mix the catechol-grafted gelatin solution with 5 mg / mL FeCl3 solution, and 3+ The molar ratio of the catechol group is 1:4 to obtain a mixed solution;

[0083] S4.2: 3 parts by weight of the tuckahoe polysaccharide-antimicrobial peptide complex is added to 30 parts by weight of the oxidized sodium alginate solution, and then a 3 mol / L sodium hydroxide solution is added to adjust the pH to 10, and then 20 parts by weight of the mixed solution is added and pre-mixed on a vortex oscillator for 2 hours to obtain a sodium alginate complex;

[0084] S5: Preparation of Sodium Hyaluronate Gel Complex

[0085] S5.1: 5 parts by weight of sodium hyaluronate freeze-dried powder are added to 100 parts by weight of deionized water, followed by adding 0.25 parts by weight of N-hydroxysuccinimide and 0.59 parts by weight of 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide, stirring and mixing, adjusting the pH to 4.57 with 1 mol / L hydrogen chloride solution, and reacting at room temperature for 2 hours to obtain a mixed reaction solution;

[0086] S5.2: Add 0.36 parts by weight of cysteamine hydrochloride to the mixed reaction solution, stir and mix, adjust the pH to 4.57, react at room temperature in the dark for 24 hours, then transfer to a dialysis bag, dialyze with deionized water at pH 3.5 at room temperature in the dark for 3 days, and finally freeze-dry to obtain thiolated sodium hyaluronate;

[0087] S5.3: Dissolve the thiolated sodium hyaluronate in a PBS solution with a pH of 7.4 to prepare a precursor solution with a concentration of 2% (w / v), and then add sodium alginate complex to the precursor solution. The amount of sodium alginate complex added is 2-3% of the precursor solution. Stir and mix for 2 hours to obtain a sodium hyaluronate gel complex.

[0088] Example 3

[0089] A preparation process of a sodium hyaluronate gel complex for wound repair, such as Figure 1 As shown, the following steps are included:

[0090] S1: Preparation of Pachymaran-antimicrobial peptide complex

[0091] S1.1: Select the Poria cocos blocks with uniform color and no mold spots, dry them in an oven at 60°C, crush them with a high-speed grinder, and pass them through a 50-mesh sieve to obtain Poria cocos powder;

[0092] S1.2: 10 parts by weight of Poria powder were added to pure water at a solid-liquid ratio of 1:25, and then placed in a microwave-ultrasonic extractor, with the parameters set as ultrasonic power of 120w, ultrasonic interval time of 3s, microwave power of 120w, temperature of 50°C, time of 40min, and then centrifuged at 4000r / min for 30min, the supernatant was filtered, and freeze-dried to obtain Poria polysaccharide;

[0093] S1.3: adding tuckahoe polysaccharide to a PBS buffer at pH 7.4 to obtain a tuckahoe polysaccharide solution with a concentration of 2% w / v, and adding the antimicrobial peptide LL-37 to a PBS buffer at pH 7.4 to obtain a 1% w / v antimicrobial peptide LL-37 solution;

[0094] S1.4: Add 0.01 parts by weight of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimide ester to 20 parts by weight of the tuckahoe polysaccharide solution, and then add 4 parts by weight of the antimicrobial peptide LL-37 solution, shake and react at 38°C for 5 hours, dialyze after the reaction is completed, and freeze-dry to obtain a tuckahoe polysaccharide-antimicrobial peptide complex;

[0095] S2: Preparation of catechol-grafted modified gelatin

[0096] S2.1: dissolving 5 parts by weight of gelatin in 50 parts by weight of a phosphate buffer having a pH value of 5.5 at 38° C. to obtain a gelatin solution, and dissolving 0.23 parts by weight of 3-(3,4-dihydroxyphenyl)propionic acid in 50 parts by weight of a phosphate buffer having a pH value of 5.5 at 38° C. to obtain a 3-(3,4-dihydroxyphenyl)propionic acid solution;

[0097] S2.2: 0.2 parts by weight of NHS and 0.4 parts by weight of EDC were added to the 3-3-(3,4-dihydroxyphenyl)propionic acid solution, stirred and mixed for 30 minutes under an argon atmosphere, and then added to the gelatin solution, reacted for 24 hours under an argon atmosphere and protected from light, and then dialyzed and freeze-dried to obtain catechol-grafted modified gelatin;

[0098] S3: Preparation of oxidized sodium alginate

[0099] S3.1: 6 parts by weight of sodium alginate are dispersed in 30 parts by weight of ethanol, and 30 parts by weight of a 17% sodium metaperiodate solution are slowly added under vigorous stirring, and then reacted in a dark environment for 6 hours, and then 2 parts by weight of ethylene glycol are added, and stirred and mixed for 30 minutes to obtain a reaction solution;

[0100] S3.2: The reaction solution was dialyzed with distilled water for 4 days through a dialysis bag with a molecular weight cutoff of 3500Da, and then the purified solution was freeze-dried to obtain oxidized sodium alginate;

[0101] S4: Preparation of sodium alginate complex

[0102] S4.1: Prepare 250 mg / mL of catechol-grafted gelatin solution and 150 mg / mL of oxidized sodium alginate solution with distilled water, mix the catechol-grafted gelatin solution with 5 mg / mL FeCl3 solution, and 3+ The molar ratio of the catechol group is 1:3 to obtain a mixed solution;

[0103] S4.2: Add 2 parts by weight of the pachymannan-antimicrobial peptide complex to 20 parts by weight of the oxidized sodium alginate solution, then add 2 mol / L sodium hydroxide solution to adjust the pH to 8, then add 10 parts by weight of the mixed solution and pre-mix on a vortex oscillator for 3 hours to obtain a sodium alginate complex;

[0104] S5: Preparation of Sodium Hyaluronate Gel Complex

[0105] S5.1: 4 parts by weight of sodium hyaluronate freeze-dried powder are added to 80 parts by weight of deionized water, followed by adding 0.23 parts by weight of N-hydroxysuccinimide and 0.57 parts by weight of 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide, stirring and mixing, adjusting the pH to 4.57 with 1 mol / L hydrogen chloride solution, and reacting at room temperature for 3 hours to obtain a mixed reaction solution;

[0106] S5.2: Add 0.34 parts by weight of cysteamine hydrochloride to the mixed reaction solution, stir and mix, adjust the pH to 4.57, react at room temperature in the dark for 25 hours, then transfer to a dialysis bag, dialyze with deionized water at pH 3.5 at room temperature in the dark for 4 days, and finally freeze-dry to obtain thiolated sodium hyaluronate;

[0107] S5.3: Dissolve the thiolated sodium hyaluronate in a PBS solution with a pH of 7.4 to prepare a precursor solution with a concentration of 2% (w / v), and then add sodium alginate complex to the precursor solution. The amount of sodium alginate complex added is 2% of the precursor solution. Stir and mix for 3 hours to obtain a sodium hyaluronate gel complex.

[0108] Comparative Example 1

[0109] Compared with Example 1, the difference of Comparative Example 1 is that the Pachymaran-antimicrobial peptide complex in steps S1 and S4.2 is removed in Comparative Example 1, and the sodium hyaluronate gel complex is prepared without changing the other steps, which is recorded as Comparative Example 1.

[0110] Comparative Example 2

[0111] Compared with Example 1, the difference of Comparative Example 2 is that Comparative Example 2 removes steps S2-S4, and the sodium alginate complex in step S5.3 is replaced by a sodium alginate and a pachymaran-antimicrobial peptide complex in equal weight parts, and the other steps remain unchanged to prepare a sodium hyaluronate gel complex, which is recorded as Comparative Example 2.

[0112] Comparative Example 3

[0113] Compared with Example 1, the difference of Comparative Example 3 is that, in Comparative Example 3, steps S5.1-S5.2 are removed, the thiolated sodium hyaluronate in step S5.3 is replaced with sodium hyaluronate, and the remaining steps remain unchanged to prepare the sodium hyaluronate gel complex, which is recorded as Comparative Example 3.

[0114] Antimicrobial test results:

[0115] Table 1. Antibacterial performance test results of Example 1 and Comparative Example 1

[0116]

[0117] It can be seen from the data in Table 1 that the introduction of the pachymaran-antimicrobial peptide complex into the sodium hyaluronate gel in the present invention can effectively inhibit the growth and reproduction of microorganisms such as bacteria in the gel system and reduce the risk of infection.

[0118] Antimicrobial test results:

[0119] Table 2. Antibacterial performance test results of Example 1 and Comparative Example 1

[0120]

[0121] It can be seen from the data in Table 2 that the sodium alginate complex with double dynamic bond cross-linking introduced into the sodium hyaluronate gel complex prepared by the present invention can effectively improve the mechanical strength of the gel, making it tougher, less prone to rupture, and improving the mechanical strength.

[0122] Determination of healing rate of rat burn model:

[0123] Healthy adult rats were selected, and their body weight was usually between 200-250g. Adaptive feeding was performed for 1 week before the experiment to ensure that the rats were in good health. The rats were anesthetized with an anesthetic, and the hair was shaved on the back to ensure that the skin was exposed. 90°C hot water was used to contact the skin for a certain period of time to cause standardized deep II degree burns. The rats were randomly divided into seven groups, with 10 rats in each group. The first six groups were treated with the sodium hyaluronate gel complex prepared in Examples 1-3 and Comparative Examples 1-3, and the seventh group was a blank control group treated only with physiological saline to determine the healing rate.

[0124] Healing rate (%) = [(initial wound area - current wound area) / initial wound area] × 100%. Refer to Table 3 for the measurement results.

[0125] Table 3. Healing rate test results of Examples and Comparative Examples 1-3

[0126]

[0127] It can be seen from the data in Table 3 that the data in Comparative Example 1 indicate that the introduction of the pachymannan-antimicrobial peptide complex into the sodium hyaluronate gel in the present invention can promote cell proliferation, differentiation and tissue repair, thereby significantly promoting wound healing.

[0128] The data of Comparative Example 2 show that the prepared double dynamic bond cross-linked sodium alginate complex can improve the wound healing effect.

[0129] From the data of Comparative Example 3, it can be seen that the compounding of thiolated sodium hyaluronate and sodium alginate and the pachymannan-antimicrobial peptide complex can produce a synergistic effect and promote tissue regeneration and repair.

[0130] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A preparation process of a sodium hyaluronate gel complex for wound repair, characterized in that: The steps include: S1: Preparation of Pachymaran-antimicrobial peptide complex Microwave-ultrasound-assisted extraction of pachymaran was used, and then pachymaran was covalently bound to the antimicrobial peptide LL-37 through the cross-linking agent 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimidyl ester to obtain a pachymaran-antimicrobial peptide complex. S2: Preparation of catechol-grafted modified gelatin NHS and EDC are added to a 3-3-(3,4-dihydroxyphenyl) propionic acid solution, mixed, and then added to a gelatin solution, and reacted under an argon atmosphere and protected from light to prepare catechol-grafted modified gelatin; S3: Preparation of oxidized sodium alginate Sodium metaperiodate is reacted with sodium alginate to prepare oxidized sodium alginate; S4: Preparation of sodium alginate complex The catechol grafted modified gelatin solution is mixed with the FeCl3 solution to obtain a mixed solution, and then the pachymaran-antimicrobial peptide complex is added to the oxidized sodium alginate solution and then mixed with the mixed solution to prepare a sodium alginate complex; S5: Preparation of Sodium Hyaluronate Gel Complex The thiolated sodium hyaluronate is prepared by mixing N-hydroxysuccinimide, 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide, cysteamine hydrochloride and sodium hyaluronate freeze-dried powder, and then the thiolated sodium hyaluronate is compounded with a sodium alginate complex to prepare a sodium hyaluronate gel complex.

2. The preparation process of the sodium hyaluronate gel complex for wound repair according to claim 1, characterized in that: Step S1: Preparation of the pachymaran-antimicrobial peptide complex, specifically comprising the following steps: S1.1: Select the Poria cocos blocks with uniform color and no mold spots, dry them in an oven at 50-60℃, crush them with a high-speed grinder, and pass them through a 40-50 mesh sieve to obtain Poria cocos powder; S1.2: 10-20 parts by weight of Poria powder are added to pure water at a solid-liquid ratio of 1:25-30, and then placed in a microwave-ultrasonic extraction instrument. After microwave-ultrasonic extraction, centrifuge at 3000-4000 r / min for 20-30 min, filter the supernatant, and freeze-dry to obtain Poria polysaccharide; S1.3: adding tuckahoe polysaccharide to a PBS buffer at pH 7.4 to obtain a tuckahoe polysaccharide solution with a concentration of 2-3% w / v, and adding the antimicrobial peptide LL-37 to a PBS buffer at pH 7.4 to obtain a 1-2% w / v antimicrobial peptide LL-37 solution; S1.4: Add 0.01-0.02 parts by weight of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimide ester to 20-30 parts by weight of the Poria polysaccharide solution, and then add 4-5 parts by weight of the antimicrobial peptide LL-37 solution. Oscillating reaction at 37-38°C for 4-5 hours. After the reaction is completed, dialyze and freeze-dry to obtain the Poria polysaccharide-antimicrobial peptide complex.

3. The preparation process of a sodium hyaluronate gel complex for wound repair according to claim 2, characterized in that: Step S2: Preparation of catechol grafted modified gelatin, specifically comprising the following steps: S2.1: dissolving 5-8 parts by weight of gelatin in 50-60 parts by weight of a phosphate buffer having a pH value of 5.5 at 37-38° C. to obtain a gelatin solution, and dissolving 0.23-0.25 parts by weight of 3-(3,4-dihydroxyphenyl)propionic acid in 50-60 parts by weight of a phosphate buffer having a pH value of 5.5 at 37-38° C. to obtain a 3-(3,4-dihydroxyphenyl)propionic acid solution; S2.2: Add 0.2-0.3 parts by weight of NHS and 0.4-0.5 parts by weight of EDC to the 3-3-(3,4-dihydroxyphenyl)propionic acid solution, stir and mix for 20-30 minutes under an argon atmosphere, then add to the gelatin solution, react for 20-24 hours under an argon atmosphere in the dark, then dialyze and freeze-dry to obtain catechol-grafted modified gelatin.

4. The preparation process of the sodium hyaluronate gel complex for wound repair according to claim 3, characterized in that: Step S3 is the preparation of oxidized sodium alginate, which specifically comprises the following steps: S3.1: 6-8 parts by weight of sodium alginate are dispersed in 30-40 parts by weight of ethanol, and 30-40 parts by weight of a 17-20% sodium metaperiodate solution is slowly added under vigorous stirring, and then reacted in a dark environment for 4-6 hours, and then 2-3 parts by weight of ethylene glycol are added, and stirred and mixed for 20-30 minutes to obtain a reaction solution; S3.2: The reaction solution is dialyzed with distilled water for 3-4 days through a dialysis bag with a molecular weight cutoff of 3500Da, and then the purified solution is freeze-dried to obtain oxidized sodium alginate.

5. The preparation process of a sodium hyaluronate gel complex for wound repair according to claim 4, characterized in that: Step S4: Preparation of sodium alginate complex, specifically comprising the following steps: S4.1: Prepare 250 mg / mL of catechol grafted modified gelatin solution and 150 mg / mL of oxidized sodium alginate solution with distilled water, and mix the catechol grafted modified gelatin solution with 5 mg / mL FeCl3 solution to obtain a mixed solution; S4.2: Add 2-3 parts by weight of the Poria polysaccharide-antimicrobial peptide complex to 20-30 parts by weight of the oxidized sodium alginate solution, then add 2-3 mol / L of sodium hydroxide solution, adjust the pH to 8-10, then add 10-20 parts by weight of the mixed solution and pre-mix on a vortex oscillator for 2-3 hours to obtain the sodium alginate complex.

6. The preparation process of the sodium hyaluronate gel complex for wound repair according to claim 5, characterized in that: Step S5: Preparation of sodium hyaluronate gel complex, specifically comprising the following steps: S5.1: 4-5 parts by weight of sodium hyaluronate freeze-dried powder is added to 80-100 parts by weight of deionized water, followed by adding 0.23-0.25 parts by weight of N-hydroxysuccinimide and 0.57-0.59 parts by weight of 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide, stirring and mixing, adjusting the pH to 4.57 with 1 mol / L hydrogen chloride solution, and then reacting at room temperature for 2-3 hours to obtain a mixed reaction solution; S5.2: Add 0.34-0.36 parts by weight of cysteamine hydrochloride to the mixed reaction solution, stir and mix, adjust the pH to 4.57 with 1 mol / L sodium hydroxide solution, react at room temperature in the dark for 24-25 hours, then transfer to a dialysis bag, dialyze with deionized water at pH 3.5 at room temperature in the dark for 3-4 days, and finally freeze-dry to obtain thiolated sodium hyaluronate; S5.3: Dissolve the thiolated sodium hyaluronate in a PBS solution with a pH of 7.4 to prepare a precursor solution with a concentration of 2% (w / v), then add the sodium alginate complex to the precursor solution, stir and mix for 2-3 hours to obtain the sodium hyaluronate gel complex.

7. The preparation process of a sodium hyaluronate gel complex for wound repair according to claim 2, characterized in that: The parameters of the microwave-ultrasonic extractor in step S1.2 are ultrasonic power 100-120w, ultrasonic interval time 2-3s, microwave power 100-120w, temperature 45-50°C, and time 30-40min.

8. The preparation process of the sodium hyaluronate gel complex for wound repair according to claim 5, characterized in that: Step S4.1 Fe 3+ The molar ratio to catechol group is 1:3-4.

9. The process for preparing a sodium hyaluronate gel complex for wound repair according to claim 6, characterized in that: In step S5.3, the amount of sodium alginate complex added is 2-3% of the precursor solution.

10. A sodium hyaluronate gel complex for wound repair, characterized in that: It is prepared by the preparation process of a sodium hyaluronate gel complex for wound repairing as described in any one of claims 1 to 9.

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