Preparation method and application of injectable high-oxidation-resistance chitosan-based hydrogel capable of promoting wound healing
The hydrogel prepared by crosslinking chitosan, polyvinylpyrrolidone and gallic acid solves the problem of insufficient antibacterial and antioxidant properties of traditional dressings, achieving efficient wound healing and biocompatibility, suitable for various wound shapes and possessing self-healing ability.
Patent Information
- Application Number
- CN202410451030.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-04-15
- Publication Date
- 2025-10-21
AI Technical Summary
Traditional medical wound dressings have poor antibacterial and antioxidant properties, and conventional polymer antibacterial hydrogels pose potential environmental and biosafety risks.
A hydrogel with high antioxidant properties and promoting wound healing was prepared by crosslinking chitosan, polyvinylpyrrolidone, and gallic acid in an acetic acid solution through stirring and static crosslinking.
The prepared hydrogel has significant antibacterial, antioxidant and biocompatibility properties, and is non-toxic and harmless. It is suitable for various wound shapes and exhibits self-healing ability.
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Figure CN120815212A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of medical polymer materials, and in particular to a preparation method and application of an injectable chitosan-based hydrogel with high oxidation resistance and the ability to promote wound healing. Background Art
[0002] The skin is the largest organ in the human body, protecting it from external damage and infection by pathogens. Every year, tens of thousands of people are affected by acute and chronic skin injuries. The inflammatory state following skin injury can lead to slow local tissue healing and even necrosis. Various metabolites that appear in the early stages of wound healing, such as tissue fluid, proteins, and dead cells, increase the probability of microbial infection of the wound. An ideal wound dressing can serve as a protective barrier to prevent further damage to the wound and prevent microorganisms from invading the wound. It can also absorb wound exudate, remove metabolites, and serve as a medium for gas exchange between the wound surface and the outside world. In addition to flexibility and mechanical stability, an ideal wound dressing also needs to exhibit appropriate anti-inflammatory, antioxidant, and antibacterial properties.
[0003] Traditional wound dressings such as absorbent cotton and gauze do not have good antibacterial properties and are not suitable for all wound types. Antimicrobial drugs used in wound dressings can be divided into three categories: antibiotics, nanoparticles, and natural biomaterials. Nanoparticles such as silver and zinc oxide nanoparticles are considered to be effective alternatives to traditional antibiotics. Natural biomaterials such as sericin, alginate, and chitosan have good antibacterial properties and good biocompatibility. Studies have shown that under moist conditions, the wound closure rate is almost twice that under dry conditions. Therefore, dry absorbent cotton and gauze dressings are gradually being replaced by wet dressings. Hydrogels are considered to be an ideal alternative to traditional dry wound dressings in wet dressings.
[0004] Chitosan is a natural polysaccharide obtained by deacetylation of chitin. It is non-toxic, biodegradable, and has broad-spectrum antibacterial properties. Polyvinylpyrrolidone is a synthetic water-soluble polymer compound with excellent film-forming, adhesive, and biocompatibility properties. Gallic acid, chemically known as 3,4,5-trihydroxybenzoic acid, is a polyphenolic organic compound that is widely found in plants such as Rheum palmatum, Eucalyptus globulus, and Cornus officinalis. It has the effects of strengthening the stomach and promoting digestion, promoting the production of body fluids and quenching thirst, stopping bleeding and cooling blood, and inhibiting pathogenic bacteria. The hydrogel formed by cross-linking chitosan, polyvinylpyrrolidone, and gallic acid has good injectability, self-healing ability, antioxidant properties, antibacterial properties, and biocompatibility. Summary of the Invention
[0005] The present invention addresses the shortcomings of traditional medical wound dressings, such as poor antibacterial and antioxidant properties, and potential environmental and biosafety risks of conventional polymer antibacterial hydrogels. The invention provides an injectable chitosan-based hydrogel with high antioxidant properties that can promote wound healing, as well as its preparation method and application.
[0006] To solve the above problems, the first aspect of the present invention provides an injectable chitosan-based hydrogel with high antioxidant properties that can promote wound healing, which mainly includes the following components: chitosan, polyvinyl pyrrolidone, and gallic acid.
[0007] Preferably, by weight, the content of the chitosan is 4-8 wt %, the content of the polyvinyl pyrrolidone is 1-3 wt %, and the content of the gallic acid is 0.2-0.5 wt %.
[0008] Preferably, the chitosan (C) is analytically pure and has a deacetylation degree of ≥95%.
[0009] Preferably, the polyvinylpyrrolidone (P) is analytically pure.
[0010] Preferably, the gallic acid (G) is analytically pure.
[0011] Compared with the prior art, the injectable chitosan-based hydrogel with high antioxidant properties that can promote wound healing provided by the present invention has the following beneficial effects:
[0012] 1) The chitosan and gallic acid used are biomass and its derivative materials, which are non-toxic and harmless and have obvious antibacterial effects
[0013] 2) Compared with chitosan / polyvinyl pyrrolidone hydrogel, chitosan / polyvinyl pyrrolidone / gallic acid hydrogel has better antibacterial, antioxidant and biocompatibility properties.
[0014] The second aspect of the present invention provides a method for preparing the chitosan / polyvinyl pyrrolidone hydrogel (CP) or chitosan / polyvinyl pyrrolidone / gallic acid (CPG) having the above-mentioned effect 2, comprising the following steps:
[0015] S1. Add appropriate amount of chitosan, polyvinyl pyrrolidone and gallic acid to 1% acetic acid solution at room temperature and stir thoroughly for half an hour.
[0016] S2: Pour the chitosan, polyvinyl pyrrolidone and gallic acid prepared in step S1 into the mold after thorough stirring and let it stand for 30 minutes until the hydrogel is fully cross-linked.
[0017] Compared with the prior art, the preparation method of the injectable chitosan-based hydrogel with high antioxidant properties that can promote wound healing provided by the present invention has the following beneficial effects:
[0018] 1) Chitosan contains cations, which can effectively inhibit bacteria without causing drug resistance
[0019] 2) The hydrogel formed by polyvinyl pyrrolidone has certain adhesion
[0020] 3) Gallic acid has excellent antioxidant properties and can enhance the adhesion and antibacterial effects of hydrogels BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 The effect diagram of the injectability and self-healing performance of CPG hydrogel in Example 2
[0022] Figure 2 IR spectra of CP, CPG-1, CPG-2, and CPG-3 hydrogels in Example 3;
[0023] Figure 3 These are SEM scanning electron micrographs of CP, CPG-1, CPG-2, and CPG-3 hydrogels of Example 4.
[0024] Figure 4 The water absorption capacity test chart of CP, CPG-1, CPG-2 and CPG-3 hydrogels in Example 5
[0025] Figure 5 Adhesion test diagram of CP, CPG-1, CPG-2, and CPG-3 hydrogels in Example 6
[0026] Figure 6 DPPH free radical removal effect diagram of CP, CPG-1, CPG-2, and CPG-3 hydrogels in Example 7
[0027] Figure 7 The inhibitory effect of CP, CPG-1, CPG-2, and CPG-3 hydrogels in Example 8 on Gram-negative and Gram-positive bacteria is shown in FIG.
[0028] Figure 8 This is the experimental effect diagram of the hemolysis rate of CP, CPG-1, CPG-2, and CPG-3 hydrogels in Example 9
[0029] Figure 9 The results of the cell scratch test of CP, CPG-1, CPG-2, and CPG-3 hydrogels in Example 10 are shown in FIG.
[0030] Figure 10 The cell activity graph of CP, CPG-1, CPG-2, and CPG-3 hydrogels of Example 11 co-cultured with mouse L929 fibroblasts for 24 hours DETAILED DESCRIPTION
[0031] To make the above-mentioned objects, features, and advantages of the present invention more clearly understood, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings. It should be noted that the following embodiments are only intended to illustrate the implementation methods and typical parameters of the present invention, and are not intended to limit the parameter ranges described in the present invention. Reasonable variations derived therefrom are still within the scope of protection of the claims of the present invention.
[0032] It should be noted that the endpoints of the ranges and any values disclosed herein are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be considered to be specifically disclosed herein.
[0033] An embodiment of the present invention provides an antibacterial and antioxidant hydrogel that can be used as a medical dressing. The hydrogel comprises the following components: chitosan, polyvinyl pyrrolidone, and gallic acid. The chitosan content is 4-8 wt%, the polyvinyl pyrrolidone content is 1-3 wt%, and the gallic acid content is 0.2-0.5 wt%.
[0034] Example 1
[0035] Preparation of CP, CPG-1, CPG-2, and CPG-3 hydrogels
[0036] S1. Add appropriate amount of chitosan and polyvinyl pyrrolidone to 1% acetic acid solution at room temperature and stir thoroughly for half an hour.
[0037] S2, after fully stirring the chitosan and polyvinyl pyrrolidone in step S1, pour them into a mold and let them stand for 30 minutes to wait for the hydrogel to be fully cross-linked to obtain chitosan / polyvinyl pyrrolidone hydrogel (CP)
[0038] S3. Add appropriate amount of chitosan, polyvinyl pyrrolidone and different amounts of gallic acid to 1% acetic acid solution at room temperature and stir thoroughly for half an hour.
[0039] S4, the chitosan, polyvinyl pyrrolidone and gallic acid in step S1 were poured into a mold after being fully stirred and allowed to stand for 30 minutes to wait for the hydrogel to be fully cross-linked to obtain chitosan / polyvinyl pyrrolidone / gallic acid hydrogels (CPG-1, CPG-2, CPG-3)
[0040] In a specific embodiment, the preparation method is simple, gentle, and controllable. The resulting hydrogel is non-toxic and harmless, exhibits significant antibacterial effects, and the hydrogel's main components, chitosan, polyvinyl pyrrolidone, and gallic acid, exhibit excellent gelling properties, making it an ideal material for medical dressings. The present invention will be described in detail below through specific examples.
[0041] Example 2
[0042] Injectability and self-healing properties of CPG hydrogel
[0043] The CPG hydrogel can be continuously squeezed through a needle, creating the letters "NFU," demonstrating its injectability and adaptability to various wound shapes. Two rectangular CPG hydrogels were prepared, one dyed blue. In the absence of external force, the CPG hydrogel's self-healing behavior was observed, and even when pulled, it remained intact.
[0044] Example 3
[0045] FTIR spectra of CP, CPG-1, CPG-2, and CPG-3 hydrogels
[0046] The groups of CP, CPG-1, CPG-2 and CPG-3 hydrogels were characterized by FTIR spectrometer. First, CP, CPG-1, CPG-2 and CPG-3 hydrogels were freeze-dried using a vacuum freeze dryer. Then, the freeze-dried CP, CPG-1, CPG-2 and CPG-3 hydrogels were uniformly mixed with potassium bromide and prepared into ultrafine particles. After tableting with a tablet press, FTIR testing was performed. Figure 2 As shown, the CP hydrogel has a peak at 439 cm -1 Chuhe 1657cm -1 The peaks at 3432 cm-1 are the stretching vibrations of chitosan hydroxyl and amino groups and the overlapping peaks formed by the carbonyl group in PVP and the amide I group of chitosan. After the addition of gallic acid, the peaks moved to 3432 cm-1. -1 Chuhe 1646cm -1 It was verified that hydrogen bonds were formed between gallic acid and chitosan and polyvinyl pyrrolidone.
[0047] Example 4
[0048] SEM scanning electron microscope test of CP, CPG-1, CPG-2, and CPG-3
[0049] The prepared CP, CPG-1, CPG-2, and CPG-3 hydrogels were frozen and placed in a freeze dryer for freeze drying, and then subjected to SEM scanning electron microscopy testing. Figure 3 As shown in the figure, it can be seen that the pore size of the CP hydrogel is large and irregular. After adding gallic acid, the pore size of the hydrogel becomes smaller and the structure of the hydrogel becomes compact.
[0050] Example 5
[0051] Bioadhesion test of CP, CPG-1, CPG-2, and CPG-3 hydrogels First, after removing the hair and excess pig skin fat, fresh pig skin was cut into several rectangular blocks. The pig skin was dehydrated in PBS buffer for 2 hours. Subsequently, CP, CPG-1, CPG-2, and CPG-3 were adhered to the overlapping position of the two pieces of pig skin. They were fixed with a clamp and stretched at a speed of 0.5 mm / min. Figure 4 As can be seen in the figure, the CPG hydrogel with added gallic acid has a higher elastic strain than the CP hydrogel, but the adhesion strength is reduced. With the increase of gallic acid addition, the adhesion strength and strain of the CPG hydrogel are enhanced. This is because the newly added gallic acid contains a large number of phenolic hydroxyl groups that can enhance adhesion properties. The phenolic hydroxyl groups also increase the degree of cross-linking between molecules, so the adhesion strength and strain of the hydrogel are improved.
[0052] Example 6
[0053] CP, CPG-1, CPG-2, and CPG-3 hydrogel water absorption test: Freshly prepared hydrogels were freeze-dried and weighed (recorded as W0) and immersed in 10 mL of PBS (pH 5.0) at 37°C. The swollen hydrogels were removed from the buffer at predetermined time intervals. The surface water was gently removed with filter paper, and then the hydrogels were weighed (recorded as Wt). After the experiment was completed, the water absorption rate of the hydrogel was calculated using the formula. Figure 5 It can be seen that with the increase of the amount of gallic acid added, the water absorption of CPG hydrogel is significantly lower than that of CP hydrogel, and CPG-1>CPG-2>CPG-3. This is because the increase in the amount of gallic acid added increases the internal cross-linking degree of the hydrogel, the internal pores of the hydrogel become smaller, and the water absorption rate decreases.
[0054] Example 7
[0055] Antioxidant test of CP, CPG-1, CPG-2 and CPG-3 hydrogels
[0056] First, weigh 0.016g of 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) and dissolve it in 40ml of anhydrous ethanol for later use. Weigh 500mg of CP, CPG-1, CPG-2, and CPG-3 hydrogels respectively and put them into test tubes. Add 5ml of DPPH solution to each test tube, keep it away from light for 5 minutes, and then centrifuge it. Then test its ultraviolet absorption light value. The results are as follows: Figure 5 As shown. Take the absorbance value corresponding to 517nm wavelength according to the formula The free radical removal rates of each were calculated and their antioxidant effects were compared. The results are shown in the following table:
[0057] sample CP CPG-1 CPG-2 CPG-3 Removal rate 15.58％ 93.27％ 93.21％ 93.14％
[0058] Example 8
[0059] Antibacterial effect test of CP, CPG-1, CPG-2 and CPG-3 hydrogels
[0060] The purchased Escherichia coli freeze-dried powder and Staphylococcus aureus freeze-dried powder were mixed with the matching broth for bacterial recovery. 2.5g LB powder was dissolved in 100ml deionized water, and then 1.3g agar powder was added. After dissolving evenly, it was placed in an autoclave and sterilized at 121 degrees Celsius for 20 minutes to prepare a sterile solid culture medium for use. The sterilized solid culture medium was cooled to 40-50 degrees Celsius and poured into a bacterial culture dish. After solidification, the two bacteria were streaked on the plate and then placed at 37 degrees Celsius for constant temperature culture for 24 hours. The cultured bacteria were taken out, and the better strains were placed in a liquid culture medium for constant temperature shaking culture for 10-14 hours to prepare a bacterial suspension. Finally, the prepared bacterial suspension was taken out, 20 microliters of bacterial suspension were taken and mixed evenly with the liquid culture medium, and co-cultured with the hydrogel in a 12-well plate for 24 hours. The OD value was measured with ultraviolet light and the antibacterial rate was calculated using the formula. The results are shown in the following table:
[0061]
[0062] Example 9
[0063] Hemolytic test of CP, CPG-1, CPG-2, and CPG-3 hydrogels
[0064] Fresh sheep blood was collected using an anticoagulant tube and centrifuged at 2000 rpm for 10 minutes. The obtained particles were resuspended in PBS to obtain a red blood cell suspension (2wt%). CP, CPG-1, CPG-2, and CPG-3 hydrogel solutions were prepared in 1.5 mL centrifuge tubes at a concentration of 100 mg / μL. 200 μL of red blood cell suspension was added to each sample and incubated at 37°C for 4 hours. After removing the hydrogel, the supernatant was centrifuged at 2000 rpm for 10 minutes. Then, 100 μL of supernatant was added to each sample on a 96-well plate, and the absorbance value was measured at 540 nm. The same volume of 1% Triton X-100 and PBS were used as positive controls, respectively. The results showed that the hemoglobin levels of the CP hydrogel and CPG hydrogel groups remained within the normal range (2%), and no hemolysis occurred.
[0065] Example 10
[0066] Cell scratch test of CP and CPG-3 hydrogels
[0067] The effect of hydrogel on L929 fibroblast migration was evaluated by scratch test. Briefly, L929 cells were seeded at 1×10 5Cells were cultured in 24-well plates at a cell density of 100 μL and cultured until fully confluent. A new 200 μL pipette tip was used to gently and slowly scratch the cell monolayer through the center, and then washed twice with PBS. The cells were then treated with 1 mL of CP and CPG-3 hydrogel extracts and fresh DMEM. After incubation for 24 h, the morphology of the cells was observed using a fluorescence microscope, and the migration behavior was quantitatively measured using Image J software. The migration percentage was calculated as: migration percentage = (1-S t / S i )×100%, where S t is the unhealed scratch area, S i is the initial scratch area, and the results are shown in the following table:
[0068] sample CP CPG-3 Mobility (%) 25.95％ 38.87％
[0069] Example 11
[0070] Biocompatibility testing of CP, CPG-1, CPG-2, and CPG-3 hydrogels
[0071] The CP, CPG-1, CPG-2, and CPG-3 hydrogels prepared in Example 1 were prepared into 10% aqueous solutions, and 10 μL of the hydrogel aqueous solution was added to 90 μL of the cell suspension. 100 μL of the hydrogel and cell suspension were then added to a 96-well plate and co-cultured in a 37°C constant temperature carbon dioxide incubator (containing 5% CO2) for 24 hours. After 24 hours, the well plate was taken out, washed twice with PBS solution, and 10 μL of CCK-8 working solution and 90 μL of cell culture medium were added to each well. After incubation in a 37°C constant temperature carbon dioxide incubator (containing 5% CO2) for 1 to 2 hours, the absorbance (OD) was measured at a wavelength of 450 nm using a microplate reader, and the cell viability was calculated according to the formula: Cell viability (%) = OD 实验 Group / OD 对照组 ×100%. The result is Figure 7 As shown in the table below, the number of cells in each group, as determined by the CCK-8 assay, increased over time without any inter-group differences. This indicates that the hydrogel has good biocompatibility and can be used for in vivo applications. The cell survival rates are shown in the table below:
[0072] sample CP CPG-1 CPG-2 CPG-3 Survival rate (%) 104.30％ 110.62％ 157.40％ 142.48％
Claims
1. An injectable chitosan-based hydrogel with high antioxidant properties that can promote wound healing, characterized in that: The invention mainly comprises the following components: chitosan, polyvinyl pyrrolidone and gallic acid.
2. The injectable chitosan hydrogel with high antioxidant properties that can promote wound healing according to claim 1, characterized in that: By weight, the content of the chitosan is 4-8wt%, the content of the polyvinyl pyrrolidone is 1-3wt%, and the content of the gallic acid is 0.2-0.5wt%.
3. The injectable chitosan hydrogel with high antioxidant properties that can promote wound healing according to claim 2, characterized in that: The chitosan deacetylation degree is ≥95%.
4. The injectable high antioxidant hydrogel capable of promoting wound healing according to claim 1 or 2, characterized in that: Reversible hydrogen bonds can be formed among the chitosan, polyvinyl pyrrolidone and gallic acid.
5. The injectable chitosan hydrogel with high antioxidant properties and the ability to promote wound healing according to claim 1 or 2, characterized in that: The chitosan, polyvinyl pyrrolidone and gallic acid are all analytically pure reagents.
6. A method for preparing the injectable chitosan hydrogel with high antioxidant properties and the ability to promote wound healing according to any one of claims 1 to 5, characterized in that: The following steps are involved: S1. Add appropriate amount of chitosan, polyvinyl pyrrolidone and gallic acid to 1% acetic acid solution at room temperature and stir thoroughly for half an hour. S2. Pour the chitosan, polyvinyl pyrrolidone and gallic acid prepared in step S1 into a mold after thorough stirring and let it stand for 30 minutes to allow the hydrogel to be fully cross-linked.
7. The use of the injectable chitosan-based hydrogel with high antioxidant properties that can promote wound healing as claimed in any one of claims 1 to 6, characterized in that: The biodegradable multifunctional chitosan hydrogel is used as a medical dressing for wound treatment.