A preparation method of a carboxymethylated curdlan composite hydrogel capable of promoting healing of infectious skin wounds

Abstract

The present application relates to a kind of carboxymethylated gellan gum composite hydrogel that can promote the preparation method of infectious skin wound healing, the present application will be a certain concentration of carboxymethylated gellan gum with the solution of active ion (Ca 2+ 、Cu 2+ 、Zn 2+ ) of proper proportion, using ion crosslinking method to prepare carboxymethylated gellan gum hydrogel.The prepared hydrogel has fast gelation, good injectability, self-healing, tissue adhesion performance and excellent bacteriostatic effect, and can effectively promote the healing of infected wound, has potential application prospect in the treatment of wound infection.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and in particular relates to a method for preparing a carboxymethylated curdlan composite hydrogel capable of promoting the healing of infectious skin wounds. Background Art

[0002] Infected wounds caused by large-scale trauma or surgery are a major clinical problem. The weak defense barrier of the wound and the plasma nutrients exposed to the air make it extremely easy for pathogenic microorganisms to adhere, aggregate and grow on the wound surface, resulting in delayed wound healing and even infectious complications. In addition, the accumulated bacteria and their metabolites are also embedded in the extracellular matrix, forming a biofilm that is difficult to eliminate and has antibiotic resistance. An explosive bacterial infection can turn an acute infection into a chronic infection, forming a chronic, difficult-to-heal wound. Therefore, it is crucial to develop a multifunctional wound dressing that can eliminate infection in a timely manner in the early stages and promote wound healing and tissue regeneration in the middle and late stages.

[0003] Curdlan is a linear glucan composed of β-1,3-D glycosidic bonds. However, due to its water insolubility and lack of antibacterial activity, curdlan itself is difficult to deal with infection problems in wound repair.

[0004] Inorganic active ions are important components of transcription factors, enzymes, and proteins, influencing a range of cellular activities during wound healing. However, a single ion cannot meet the diverse needs of wound healing. Furthermore, unstable ion release can be toxic to tissues. Therefore, it is crucial to develop new dressings that effectively regulate the release of active ions to achieve timed delivery and meet the diverse needs of wound healing stages. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for preparing a carboxymethylated curdlan composite hydrogel that can promote the healing of infected skin wounds, and in particular to a method for preparing a carboxymethylated curdlan hydrogel with a "fast and slow release of copper and zinc ions" effect and its application. 2+ 、Zn 2+ A novel polyol based on carboxymethylated curdlan and Ca 2+ As a substrate, Cu can be delivered in time 2+ 、Zn 2+ The composite hydrogel realizes Cu 2+ Early and rapid clearance of infection, Zn 2+ The later stage promotes the therapeutic effect of wound healing and tissue regeneration.

[0006] The present invention provides a method for preparing a carboxymethylated curdlan hydrogel, comprising:

[0007] (1) Preparation of carboxymethylated curdlan:

[0008] The curdlan gum and isopropyl alcohol are mixed, and then a sodium hydroxide solution is added and stirred, and then a chloroacetic acid solution is added and reacted. After the reaction is completed, the sample is filtered, washed, dialyzed, and freeze-dried to obtain a carboxymethylated curdlan gum sample;

[0009] (2) Preparation of carboxymethylated curdlan hydrogel:

[0010] Carboxymethylated curdlan gum and water are mixed to obtain a carboxymethylated curdlan gum aqueous solution, and then an active ion solution is added and stirred to obtain a carboxymethylated curdlan gum hydrogel.

[0011] Preferably, in step (1), the curdlan and isopropyl alcohol are mixed, and then sodium hydroxide solution is added and stirred to mix, and then heated in an oil bath, and then chloroacetic acid solution is added. After the reaction is completed, the carboxymethylated curdlan is obtained by filtration, washing, dialyzing, and freeze-drying.

[0012] Preferably, in step (1), the mass volume ratio of curdlan to isopropyl alcohol is 2-5 wt.%, and the degree of substitution of the carboxymethylated curdlan is 0.3-0.5. When the degree of substitution is less than 0.3, the curdlan does not dissolve well in water; when the degree of substitution is greater than 0.5, the curdlan cannot form a hydrogel with inorganic active ions.

[0013] Preferably, the sodium hydroxide solution is added in step (1) by slowly adding the sodium hydroxide solution dropwise to the reaction system in 5-10 portions, with an interval of 5-10 minutes between each addition, and stirring at a constant speed for 1-2 hours at room temperature.

[0014] The concentration of sodium hydroxide in step (1) is 25-35 wt.%.

[0015] Preferably, in step (1), the oil bath is heated at 50-60° C., and the chloroacetic acid solution is added dropwise to the reaction system in 5-10 times, with an interval of 5-10 minutes between each addition, and the reaction system is stirred at a constant temperature and uniform speed for 2-5 hours.

[0016] Preferably, the ratio of the curdlan, sodium hydroxide solution, and chloroacetic acid solution is 1-5 g: 8-12 mL: 8-12 mL;

[0017] Furthermore, the ratio of the curdlan, sodium hydroxide solution, and chloroacetic acid solution is 3 g: 10 mL: 10 mL;

[0018] The sodium hydroxide concentration is 25-35 wt.%, preferably 30 wt.%.

[0019] The chloroacetic acid solution is an isopropanol solution of chloroacetic acid, wherein the concentration of the isopropanol solution of chloroacetic acid is 30-40% g / mL, preferably 36%.

[0020] Preferably, the reaction in step (1) is carried out in an oil bath at 50-60° C. for 2-5 hours.

[0021] Preferably, the concentration of the aqueous solution of carboxymethylated curdlan in step (2) is 8-15 wt.%; and the concentration of the active ion solution is 0.1-1 mol / L.

[0022] Furthermore, in step (2), the concentration of the aqueous solution of carboxymethylated curdlan is 10-15 wt.%; and the concentration of the active ion solution is 1 mol / L.

[0023] The active ion solution is an active ion aqueous solution.

[0024] Preferably, the active ion mixture in step (2) is calcium ions and copper ions, calcium ions and zinc ions, or calcium ions, zinc ions and copper ions.

[0025] The calcium ion source includes calcium chloride; the copper ion source includes at least one of copper chloride and copper sulfate; and the zinc ion source includes zinc chloride.

[0026] Preferably, the molar ratio of calcium ions to copper ions, or calcium ions to zinc ions, or calcium ions to copper and zinc ions in the final carboxymethylated curdlan hydrogel in step (2) is 8:1-18:1.

[0027] More preferably, the molar ratio of calcium ions, copper ions and zinc ions in the active ions is 18:1:1.

[0028] Preferably, the volume ratio of the carboxymethylated curdlan aqueous solution to the active ion aqueous solution in step (2) is 2:1-8:1.

[0029] Further preferably, in step (2), the volume ratio of the carboxymethylated curdlan aqueous solution to the active ion aqueous solution is 5:1.

[0030] Preferably, the stirring time in step (2) is 2-5 min.

[0031] The invention provides a carboxymethylated curdlan hydrogel prepared by the method.

[0032] The present invention provides an application of the carboxymethylated curdlan hydrogel in preparing skin wound healing dressings, wound tissue repair, and regeneration dressings, such as a dressing for promoting healing of infectious skin wounds, such as injecting the hydrogel into the wound surface for wound tissue repair.

[0033] The present invention provides a novel injectable, self-healing, and rapidly releasing Cu 2+ , slowly release Zn 2+ CRc-Cu-Zn composite hydrogel and its application in rapid repair of bacterial-infected wounds.

[0034] The method firstly modifies curdlan by carboxymethylation through a solvent method, and then adds active ion solution to prepare a CRc composite hydrogel through ion coordination cross-linking.

[0035] The hydrogel exhibits excellent tissue adhesion, injectability, rapid self-healing properties, and broad-spectrum antimicrobial properties. Animal experiments have demonstrated that the hydrogel can promote the rapid repair of infected wounds. Combined with these characteristics, this novel injectable, self-healing, multifunctional hydrogel with excellent antimicrobial activity is expected to find application as a new wound dressing for the rapid repair of chronic wounds, including infected wounds, and as an anti-infective tissue engineering implant.

[0036] Beneficial effects

[0037] (1) The carboxymethylated curdlan hydrogel prepared by the present invention is an unbranched β-1,3 glucan;

[0038] (2) The carboxymethylated curdlan hydrogel prepared by the present invention is safe and simple in composition, has injectability and self-healing properties, has strong adhesion to skin tissue, can be used as a wound dressing to fill irregularly shaped deep wounds, and can effectively synergize the advantages of various components to provide a suitable microenvironment for tissue growth and function;

[0039] (3) The carboxymethylated curdlan hydrogel prepared by the present invention can realize the time-sharing delivery of active ions, wherein Cu 2+ In the early stage, Zn 2+ The drug is slowly and continuously released, thus achieving the effect of anti-infection in the early stage and promoting tissue repair in the later stage;

[0040] (4) The present invention is applicable to dressings for acute / chronic wounds in different environments and tissue regeneration materials, and has broad application prospects.

[0041] (5) The present invention combines a certain concentration of carboxymethylated curdlan with an appropriate proportion of active ions (Ca 2+ 、Cu 2+ 、Zn 2 +) solution and ionic crosslinking to produce a carboxymethylated curdlan hydrogel. This hydrogel exhibits rapid gelation, good injectability, self-healing properties, tissue adhesion, and excellent antibacterial properties. It can effectively promote the repair and regeneration of skin tissue defects and has potential application in treating wound infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Graphs showing the rheological behavior of the hydrogel prepared in Example 4; wherein A: viscosity variation curve of the CRc-Cu-Zn hydrogel with shear rate; B: storage modulus G′ and loss modulus G″ variation curve of the CRc-Cu-Zn hydrogel in a strain sweep test; C: storage modulus G′ and loss modulus G″ variation curve under 1-700% alternating step strain cycles;

[0043] Figure 2 Quantitative graph of tissue adhesion strength of the hydrogels prepared in Examples 1-4;

[0044] Figure 3 Graphs showing ion release from the hydrogels prepared in Examples 1-4; A: Cumulative ion release from the CRc-Cu hydrogel at different time points in PBS buffer; B: Cumulative ion release from the CRc-Zn hydrogel at different time points in PBS buffer; C: Cumulative ion release from the CRc-Cu-Zn hydrogel at different time points in PBS buffer;

[0045] Figure 4 This is a photo of the colonies after the hydrogel prepared in Example 1-4 was co-cultured with Escherichia coli and Staphylococcus aureus for 24 hours;

[0046] Figure 5 The proliferation status of L929 cells in the hydrogels prepared in Examples 1-4;

[0047] Figure 6 This is a quantitative analysis of the wound healing area in Example 3 and Example 4 during wound repair in SD rats. DETAILED DESCRIPTION

[0048] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.

[0049] Example 1

[0050] The invention discloses a carboxymethylated curdlan hydrogel capable of promoting the healing of infected skin wounds. The hydrogel comprises the following raw materials: carboxymethylated curdlan and active ions.

[0051] In this embodiment, the active ion is calcium chloride.

[0052] A method for preparing a carboxymethylated curdlan hydrogel capable of promoting healing of infected skin wounds comprises the following steps:

[0053] (1) Preparation of carboxymethylated curdlan: 3 g of curdlan was mixed with 80 mL of isopropanol and stirred at room temperature for 1 h to uniformly disperse the mixture. 10 mL of 30 wt.% sodium hydroxide solution was slowly added dropwise to the reaction system in 10 portions, with an interval of 10 min between each addition, and the mixture was stirred at a constant speed for 1.5 h at room temperature. The reaction system was then heated to 55°C, 3.6 g of chloroacetic acid was dissolved in 10 mL of isopropanol and added dropwise to the reaction system in 10 portions, with an interval of 10 min between each addition. The reaction system was stirred at a constant temperature for 4 h, the reaction was stopped, and the crude product was filtered to obtain the product. The crude product was washed with anhydrous ethanol and acetone, dissolved in water, dialyzed, and freeze-dried to obtain a carboxymethylated curdlan with a degree of substitution of 0.37.

[0054] (2) The carboxymethylated curdlan obtained in 1) was dissolved in water to prepare a 15 wt.% carboxymethylated curdlan solution. 400 μL of a 1 mol / L calcium chloride solution was slowly added dropwise to 2 mL of the carboxymethylated curdlan solution and stirred at room temperature to obtain a hydrogel CRc.

[0055] Example 2

[0056] The preparation method of carboxymethylated curdlan hydrogel (CRc-Cu) in this example is different from that in Example 1 in that 360 μL of 1 mol / L calcium chloride and 40 μL of 1 mol / L copper chloride are mixed to prepare the active ion solution. The rest is the same as in Example 1.

[0057] Example 3

[0058] The preparation method of carboxymethylated curdlan hydrogel (CRc-Zn) in this example is different from that in Example 1 in that 360 μL of 1 mol / L calcium chloride and 40 μL of 1 mol / L zinc chloride are mixed to prepare the active ion solution. The rest is the same as in Example 1.

[0059] Example 4

[0060] The preparation method of carboxymethylated curdlan hydrogel (CRc-Cu-Zn) in this example differs from that in Example 1 in that 360 μL of 1 mol / L calcium chloride, 20 μL of 1 mol / L copper chloride, and 20 μL of zinc chloride are mixed to prepare the active ion solution. The remaining steps are the same as in Example 1.

[0061] The final carboxymethylated curdlan hydrogel was subjected to performance tests, as follows:

[0062] Test 1: The injectability and self-healing properties of the hydrogel (CRc-Cu-Zn) prepared in Example 4 were tested, specifically: (1) Macroscopic injectability: The hydrogel was added to a syringe and then injected onto a flat plate.

[0063] (2) Micro-injectability: The test was conducted using a rheometer (RS150L, ​​Haake Rheometer, Germany). 0.5 mL of hydrogel was added to the vertebral plate with a distance of 1.0 mm between the rotor and the vertebral plate to ensure contact between the rotor and the hydrogel. The viscosity change was tested at different shear rates, ranging from 0.01 to 1000 s. -1 .

[0064] (3) Microscopic self-healing properties: First, 0.5 mL of hydrogel was added to the vertebral plate, and the shear strain was 0-10 3 %, shear frequency is 1rad·s -1 The experiment was conducted in a strain sweep mode to determine the critical strain point of the hydrogel. The self-healing behavior of the hydrogel was then tested using an oscillatory time sweep mode. The amplitude oscillation strain was switched from 1% (3 cycles, 50 seconds per cycle) to 700% (2 cycles, 50 seconds per cycle). The strains of 1% and 700% were alternating.

[0065] like Figure 1 As shown in Figure A, the hydrogel prepared in Example 4 was injected onto a flat plate using a syringe to form the word "DHU", indicating that the hydrogel has good injectability and can be injected into the wound in situ.

[0066] like Figure 1 As shown in Figure B, as the shear strain gradually increases, the G' and G" of the CRc-Cu-Zn hydrogel gradually decrease, and the G' and G" curves intersect at a strain of 643.7%. The intersection of G' and G" indicates the collapse of the hydrogel's internal structure, indicating that the hydrogel transitions from a solid with gel properties to a liquid-like structure with fluid properties. A larger critical strain means that the hydrogel can withstand greater deformation to resist shear stress. Compared with self-healing hydrogels previously reported in the literature, the CRc-Cu-Zn hydrogel has a higher critical strain, indicating that the CRc-Cu-Zn hydrogel can withstand greater strain.

[0067] The strains of 700% and 1% were selected to scan the hydrogel time. Figure 1 It can be seen from Figure 3 that after alternating strains of 1% and 700%, when the strain recovers from 700% to 1%, the G' and G" values ​​of the CRc-Cu-Zn hydrogel almost return to their original values, indicating that CRc-Cu-Zn has rebuilt its own structure and basically achieved self-healing even with partial loss.

[0068] Test 2: Tissue adhesion test was performed on the hydrogels (CRc, CRc-Cu, CRc-Zn, CRc-Cu-Zn) prepared in Examples 1-4, specifically:

[0069] Step 1: Take fresh pig skin, dehair, defat, and disinfect it. Then cut it into 30mm×10mm×1mm pieces and wash it in PBS for 20 minutes.

[0070] Step 2: The freshly prepared hydrogel (10 mm × 10 mm × 1 mm) was injected onto pigskin and then overlapped with another piece of pigskin of the same size. After curing for 30 minutes, a lap shear test was performed using a computerized tensile (compression) testing machine (HY-940FS) at a tensile speed of 5 mm / min. All groups were tested three times to obtain the average adhesion strength.

[0071] The average adhesion strength of the hydrogel is Figure 2 As shown in the figure, all CRc hydrogels have a certain adhesion strength to pig skin, among which CRc-Cu-Zn hydrogel shows the highest adhesion strength (24.50±3.503kPa). This shows that when used as a wound dressing, CRc hydrogel is conducive to adhering to the wound surface, sealing the wound, and providing protection for wound healing.

[0072] Test 3: Ion release test was performed on the hydrogels (CRc, CRc-Cu, CRc-Zn, CRc-Cu-Zn) prepared in Examples 1-4, specifically:

[0073] Step 1: Soak 600 μL of hydrogel sample in 10 mL of PBS;

[0074] Step 2: Collect supernatant at preset time points (1 day, 2 days, 4 days, 6 days) for ICP testing

[0075] exist Figure 3 The release of Cu from CRc-Cu, CRc-Zn and CRc-Cu-Zn hydrogels in PBS was studied. 2+ and Zn 2+ The amount of Figure 3 As shown, it can be observed that CRc-Cu hydrogel only releases Cu 2+Similarly, CRc-Zn hydrogels only released Zn 2+ , and the release rate gradually decreases with time. CRc-Cu-Zn hydrogel can release Cu 2+ and Zn 2+ , Cu 2+ The concentration of Zn 2+ .

[0076] Test 4: The hydrogels (CRc, CRc-Cu, CRc-Zn, CRc-Cu-Zn) prepared in Examples 1-4 were subjected to in vitro antibacterial tests, specifically:

[0077] Step 1: 600 μL of different hydrogel samples were sterilized by UV irradiation for 4 h and then placed in a 24-well plate;

[0078] Step 2: Add 100 μL of bacterial suspension (10 5 CFU / mL), and cultured in a 37°C incubator for 24 h;

[0079] Step 3: Use PBS to increase the concentration of the bacterial suspension co-cultured with the material in step 2 to 10 2 CFU / mL. Pipette 100 μL of the diluted bacterial solution onto the solid culture medium and evenly spread the solution across the entire plate using a sterile spreading rod. Place the plate in a 37°C incubator and incubate for 24 hours. Then, take a picture and count the colonies.

[0080] like Figure 4 As shown in the figure, compared with the control group, the CRc material had no antibacterial properties against Escherichia coli and Staphylococcus aureus, and showed a trend of bacterial growth; CRc-Zn hydrogel had no antibacterial properties against Escherichia coli and Staphylococcus aureus, and showed a trend of bacterial growth; 2+ The release amount in 24 hours (0.96ppm) is less than that of Zn 2+ The effective antibacterial concentration (6.5ppm) of CRc-Cu and CRc-Cu-Zn hydrogels could not effectively inhibit the growth of bacteria. Escherichia coli and Staphylococcus aureus were almost invisible in the plates of CRc-Cu and CRc-Cu-Zn hydrogels, indicating that the presence of Cu 2+ The CRc hydrogel has excellent antibacterial properties, which can resist bacterial infection in the wound and accelerate the wound healing process.

[0081] Test 5: The hydrogels (CRc, CRc-Cu, CRc-Zn, CRc-Cu-Zn) prepared in Examples 1-4 were tested for fibroblast activity and proliferation. Specifically:

[0082] Step 1: UV sterilize the material for 2.0 h each, prepare the extract, and then filter it using a 0.22 μm microporous filter membrane and a 10 mL syringe.

[0083] Step 2: 5 x 10 3 The recovered cells were inoculated into the well plates at a cell number of 5 x 10

[0084] Step 3: The CCK-8 method was used to determine the cell proliferation at the preset time points of 24 h, 48 h and 72 h. The cell proliferation is shown in Figure 5 The results show that all the CRc hydrogels promote the growth of fibroblasts and can be applied in clinics.

[0085] Test six: the infection wound healing effect evaluation was performed on the hydrogels (CRc-Cu, CRc-Cu-Zn) prepared in Examples 2 and 4, specifically:

[0086] Step 1: Male SD rats (160-200 g) were selected for animal experiments. First, the rats were anesthetized by intraperitoneal injection of sufatol. After the skin of the rats was disinfected, three full-thickness circular wounds with a diameter of 1 cm were made on the back of the rats with a surgical knife, and a 1 cm rubber ring was attached to the wound. 20 μL (10 7 CFU / mL) of Staphylococcus aureus suspension was dropped on the wound site using a sterile syringe, and a 3M film was used to fix it to prevent the bacteria solution from seeping out.

[0087] Step 2: After 24 h of infection, the wounds were treated with PBS, CRc-Cu and CRc-Cu-Zn, respectively, and the wound photos were taken at 5, 7 and 10 days of treatment. The wound area was calculated using ImageJ, and the wound closure rate was calculated according to the following formula:

[0088] Wound closure rate = (initial wound area - current wound area) / initial wound area x 100%

[0089] The percentage of different wound healing is shown in Figure 6 At day 10, the CRc-Cu group can make the wound basically closed, and the wound closure rate is 92.40 ± 0.81%, and the CRc-Cu-Zn group can make the wound completely closed, and the wound closure rate is as high as 99.01 ± 1.18%, which are higher than that of the PBS group (79.07 ± 1.02%). It is shown that during the wound recovery period, the CRc-Cu-Zn hydrogel dressing has obvious promotion advantage for the healing of infected wounds.

Claims

1. A method for preparing a carboxymethylated curdlan hydrogel, comprising: Step (1) mixing curdlan and isopropyl alcohol, then adding sodium hydroxide solution and stirring, then adding chloroacetic acid solution and reacting, and after the reaction is completed, filtering, washing, dialyzing, and freeze-drying to obtain carboxymethylated curdlan; Step (2) dissolving carboxymethylated curdlan in water to obtain an aqueous solution of carboxymethylated curdlan, then adding an active ion mixed solution and stirring to obtain a carboxymethylated curdlan hydrogel; wherein the concentration of the carboxymethylated curdlan aqueous solution is 8-15 wt.%; the concentration of the active ion mixed solution is 1.0-0.3 mol / L; the active ions in the active ion mixed solution are composed of calcium ions, zinc ions and copper ions; the ratio of the molar amount of calcium ions to the total molar amount of copper and zinc ions in the carboxymethylated curdlan hydrogel is 8:1-18:1; and the volume ratio of the carboxymethylated curdlan aqueous solution to the active ion mixed solution is 2:1-8:

1.

2. The preparation method according to claim 1, characterized in that In the step (1), the mass volume ratio of the curdlan to the isopropyl alcohol is 2-5 wt.%; and the degree of substitution of the carboxymethylated curdlan is 0.3-0.

5.

3. The preparation method according to claim 1, characterized in that: The reaction in step (1) is carried out in an oil bath at 50-60° C. for 2-5 h.

4. The preparation method according to claim 1, characterized in that In step (2), the calcium ion source includes calcium chloride; the copper ion source includes at least one of copper chloride and copper sulfate; and the zinc ion source includes zinc chloride.

5. The preparation method according to claim 1, characterized in that: The stirring time in step (2) is 2-5 min.

6. Use of the carboxymethylated curdlan hydrogel prepared by the method of claim 1 in preparing skin wound healing dressings and wound tissue repair and regeneration dressings.

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