Multifunctional lignin hydrogel for skin dressing and preparation method thereof

Abstract

The invention discloses a multifunctional lignin hydrogel for skin dressing and a preparation method thereof. The preparation method of the multifunctional lignin hydrogel comprises the following steps: dissolving curcumin powder and alkali lignin powder in a NaOH solution according to a certain ratio, slowly dropwise adding the curcumin powder and the alkali lignin powder into an isometric silver ammonia solution, reacting for 30-60 minutes at room temperature, adding a certain amount of nudianin, 2.7 g of acrylamide, 0.3 g of acrylic acid and 3 mg of N, N-dimethyl formamide, and reacting for 30-60 minutes at room temperature to obtain the multifunctional lignin hydrogel. The lignin-curcumin silver nanoparticle-poly (acrylamide-acrylic acid) hydrogel loaded with the nudianin is obtained by jointly dissolving the lignin, the N, N-methylene bisacrylamide and 45 mg of ammonium persulfate in a dispersion liquid of the nanoparticles, stirring to prepare a uniform solution, and standing for 20-30 minutes at room temperature to obtain the lignin-curcumin silver nanoparticle-poly (acrylamide-acrylic acid) hydrogel loaded with the nudianin. The multifunctional lignin hydrogel prepared by the invention has multiple functions of adhesion, antibiosis, antioxidation, hemostasis and the like, can be used for accelerating skin wound healing, and has a better application prospect in skin dressings.

Description

Technical Field

[0001] The present invention relates to a multifunctional lignin hydrogel for skin dressings and a preparation method thereof, belonging to the field of biomedical materials. Background Art

[0002] As the largest organ of the human body, the skin is the first line of defense against the invasion of bacteria and viruses. Once the skin is severely damaged, bacteria can easily multiply at the wound site, leading to serious infections. Traditional dressings, such as gauze and bandages, have certain limitations in effectively controlling bleeding and absorbing wound exudates, and their moisture retention and breathability are also very poor. Advanced materials such as nanofibers and porous foams can be used for wound healing. However, if the wound exudate is very little, it will cause wound dehydration, thus hindering the wound healing process; if the exudate is too much, the limited absorption capacity is likely to lead to the leakage of exudates and the risk of infection. Hydrogels, as a kind of polymer material with a three-dimensional network structure, have a high water content, good moisture retention, and their porous structure and swelling properties can effectively absorb wound exudates. However, a single-functional hydrogel can only rely on moisturizing and protective effects and cannot cope with problems such as infection, oxidative stress, persistent bleeding, and insufficient tissue regeneration ability brought about by chronic wounds. Therefore, it is of great significance to develop a multifunctional hydrogel dressing with adhesiveness, antibacterial property, antioxidant property, hemostasis, etc. for rapid wound healing.

[0003] To obtain multifunctional hydrogels, combining natural polymers and synthetic polymers to give full play to the advantages of both to obtain multifunctional composite hydrogels is a development trend. Among them, materials such as acrylic acid and acrylamide are favored due to their biocompatibility and high mechanical strength, but they lack inherent biological properties such as antibacterial, anti-inflammatory, and hemostatic abilities. Traditionally, antibiotics are the cornerstone of wound infection treatment. However, the overuse of antibiotics has led to the emergence of drug-resistant strains, seriously affecting their efficacy. Silver nanoparticles (AgNPs) have become a promising alternative due to their broad-spectrum antibacterial activity. However, their reduction and encapsulation in hydrogels are still challenging. Lignin has attracted extensive attention in biomedical applications due to its excellent biocompatibility and biodegradability. In particular, lignin has phenolic hydroxyl groups and methoxy groups, which can be used as reducing agents and capping agents for metal nanoparticles, and can solve the problems of silver nanoparticle reduction and encapsulation. In addition, lignin contains various reducing functional groups such as phenolic hydroxyl groups and methoxy groups, which endow antioxidant properties and can scavenge excessive reactive oxygen species (ROS) in wounds. However, even with the addition of silver-lignin nanoparticles, the antibacterial and antioxidant effects of lignin-based systems still need to be improved. Studies have shown that combining lignin with other polyphenolic compounds can improve its biocompatibility, antibacterial property, and antioxidant property. Curcumin also contains reducing groups such as phenolic hydroxyl groups and methoxy groups in its molecular structure, and can also be used as a reducing agent and capping agent for metal nanoparticles, and it has anti-tumor, antibacterial, anti-inflammatory, and antioxidant properties. Therefore, if lignin and curcumin are used simultaneously in the synthesis of silver nanoparticles, it is expected to promote the self-gelation of poly(acrylamide-acrylic acid) hydrogel [P(AM-AA) hydrogel] and enhance the antibacterial and antioxidant properties of the hydrogel.

[0004] In addition, the initial stage of wound healing includes hemostasis. Therefore, it is particularly important to develop a hydrogel dressing with hemostatic function. Current research mainly focuses on combining polymers or organic small molecules to achieve hemostasis. And integrating some traditional Chinese medicines with hemostatic effects into hydrogels to endow the hydrogels with hemostatic function is a new approach. Callicarpa nudiflora Hook. et Arn. is a traditional Chinese medicine with hemostatic effects and has been widely used in postpartum hemorrhage, traumatic hemorrhage, and gastrointestinal hemorrhage. It contains a large number of bioactive compounds such as phenethyl isothiocyanate, glycosides, flavonoids, and terpenoids, and also has anti-inflammatory, antithrombotic, and antioxidant effects. However, there has been no report on introducing Callicarpa nudiflora Hook. et Arn. and curcumin-lignin-reduced silver nanoparticles into the AM / AA hydrogel system at the same time, and it is expected to explore its potential efficacy in skin wound dressings. Summary of the Invention

[0005] The present invention provides a multifunctional lignin hydrogel for skin dressings and a preparation method thereof. The method adopts a one-pot preparation process under mild conditions, which can avoid damaging the activities of lignin / curcumin and achieve the efficient reduction of silver nanoparticles and the stable cross-linking of the hydrogel network. At the same time, silver nanoparticles reduced by curcumin-lignin and Callicarpa nudiflora are introduced, enabling the functional integration of antibacterial, antioxidant, hemostatic, and ultra-high stretchability in a single hydrogel system. A new type of high-performance hydrogel dressing can be developed, which provides new ideas for the high-value utilization of natural components, the construction of dynamic hydrogel networks, and chronic wound treatment strategies, and has good application prospects in the field of biomedical materials.

[0006] The preparation method of the multifunctional lignin hydrogel includes the following steps: Dissolve curcumin powder and alkali lignin powder in a certain proportion in 5 - 10 mL of sodium hydroxide solution (pH = 11), slowly add the above solution dropwise to an equal volume of silver ammonia solution with a concentration of 7.5 - 15 mg / mL, and react at room temperature for 30 - 60 minutes to obtain a lignin-curcumin-Ag nanoparticle dispersion; then dissolve an appropriate amount of Callicarpa nudiflora, 2.7 g of acrylamide (AM), 0.3 g of acrylic acid (AA), 3 mg of N, N-methylenebisacrylamide (MBA), and 45 mg of ammonium persulfate (APS) in the above nanoparticle dispersion and stir into a uniform solution, and let it stand at room temperature for 20 - 30 minutes to obtain a Callicarpa nudiflora-loaded lignin-curcumin silver nanoparticle-P(AM-AA) hydrogel.

[0007] The superiority of the multifunctional lignin hydrogel for skin dressings and the preparation method thereof provided by the present invention lies in: (1) In terms of hydrogel performance: For the first time, four functional modules of antibacterial (Ag NPs), antioxidant (lignin / curcumin), hemostatic (Callicarpa nudiflora), and tissue adhesion (quinone groups) are integrated into a single hydrogel system. Through the molecular-level synergistic effect between components (such as curcumin enhancing the antibacterial activity of silver nanoparticles, and Callicarpa nudiflora and lignin jointly scavenging free radicals), the functions are superimposed rather than simply mixed. Therefore, it has multiple functions such as high adhesiveness, antibacterial property, antioxidant property, hemostatic performance, and promoting wound healing, and can significantly improve the regulation efficiency of the chronic wound microenvironment.

[0008] (2) In terms of the preparation method: Breaking through the limitations of traditional multi-step preparation of hydrogels, the "one-pot method" is adopted to simultaneously complete the synthesis of silver nanoparticles. The quinone free radicals generated during this process will promote the self-gelation of the P(AM-AA) hydrogel. By adding different proportions of reactant concentrations, the dynamic balance between covalent cross-linking (polymerization initiated by quinone free radicals) and non-covalent interactions (hydrogen bonds, ionic bonds, π-π stacking) in the P(AM-AA) hydrogel network of Ag@Lig-Cur NPs and Callicarpa nudiflora is regulated, enhancing the tensile strain ability of the hydrogel, breaking through the bottleneck of the easy brittleness of traditional hydrogels, and meeting the stringent requirements for the flexibility of dressings during the dynamic healing process of chronic wounds.

[0009] (3) The raw materials for preparing this novel hydrogel are widely sourced. Among them, lignin, curcumin, and Callicarpa nudiflora are all natural components, so the production cost is low.

[0010] In summary, the present invention reflects a complete chain breakthrough in material design concepts, preparation processes, functional integration mechanisms, and verification methods. It can not only develop a multifunctional hydrogel dressing with adhesiveness, antibacterial properties, antioxidant properties, hemostatic properties, etc. for rapid wound healing, but also provide new ideas for the high-value utilization of natural products, the construction of dynamic hydrogel networks, and chronic wound treatment strategies, showing good application prospects and clinical transformation value in wound dressings. Description of the Drawings

[0011] Figure 1 Photographs of the hydrogels in Example 1 and Comparative Examples 1-4.

[0012] Figure 2 Adhesion results of the hydrogels in Example 1 and Comparative Examples 1-4.

[0013] Figure 3 Antibacterial results of the hydrogels in Example 1 and Comparative Examples 1-4.

[0014] Figure 4 Antioxidant results of the hydrogels in Example 1 and Comparative Examples 1-4.

[0015] Figure 5 Hemostatic results of the hydrogels in Example 1 and Comparative Examples 1-4.

[0016] Figure 6 Wound healing results of the hydrogels in Example 1 and Comparative Examples 1-4.

[0017] Among them, the hydrogel sample corresponding to Example 1 is abbreviated as Ag@Lig-Cur NPs / C. nudiflora, the hydrogel sample of Comparative Example 1 is abbreviated as Ag@Lig-Cur NPs, the hydrogel sample of Comparative Example 2 is abbreviated as Ag@Lig NPs, the hydrogel sample of Comparative Example 3 is abbreviated as Ag@Cur NPs, and the hydrogel sample of Comparative Example 4 is abbreviated as P(AM-AA). Detailed implementation mode

[0018] The following examples are hydrogels with a mass ratio of curcumin to alkali lignin of 1:300 to 4:300 and a content of Callicarpa nudiflora of 10 to 40 mg. For mass ratios outside this range, it is difficult to form the hydrogel, and various properties are poor. Therefore, only the experimental results obtained from the examples that meet the above range are given in the present invention.

[0019] Example 1: Dissolve 2 mg of curcumin powder and 300 mg of alkali lignin powder in 8 mL of NaOH solution (pH = 11). Slowly add the above solution dropwise to an equal volume of silver ammonia solution with a concentration of 7.5 mg / mL at room temperature and react for 50 minutes. Then dissolve 30 mg of Callicarpa nudiflora, 2.7 g of AM, 0.3 g of AA, 3 mg of MBA, and 45 mg of APS in the dispersion of silver-lignin-curcumin nanoparticles and stir into a uniform solution. Let it stand at room temperature for 30 minutes to obtain the Ag@Lig-Cur NPs-P(AM-AA) hydrogel loaded with Callicarpa nudiflora. Its adhesion strength to pigskin is 130 KPa, the tensile strain is 2100%, the antibacterial rate is greater than 99%, the free radical scavenging rate is 96.47%, the hemostatic amount and hemostatic time are 37 mg and 39 s respectively, and the wound healing rate at 12 days is 98.9%.

[0020] Example 2: Dissolve 1 mg of curcumin powder and 300 mg of alkali lignin powder in 5 mL of NaOH solution (pH = 11). Slowly add the above solution dropwise to an equal volume of silver ammonia solution with a concentration of 7.5 mg / mL at room temperature and react for 30 minutes. Then dissolve 20 mg of Callicarpa nudiflora, 2.7 g of AM, 0.3 g of AA, 3 mg of MBA, and 45 mg of APS in the dispersion of silver-lignin-curcumin nanoparticles and stir into a uniform solution. Let it stand at room temperature for 20 minutes to obtain the Ag@Lig-Cur NPs-P(AM-AA) hydrogel loaded with Callicarpa nudiflora. Its adhesion strength to pigskin is 120 KPa, the tensile strain is 1900%, the antibacterial rate is greater than 99%, the free radical scavenging rate is 90.47%, the hemostatic amount and hemostatic time are 48 mg and 42 s respectively, and the wound healing rate at 12 days is 94.8%.

[0021] Example 3: Dissolve 4 mg of curcumin powder and 300 mg of alkali lignin powder in 10 mL of NaOH solution (pH = 11). Slowly add the above solution dropwise to an equal volume of silver ammonia solution with a concentration of 15.0 mg / mL. React at room temperature for 60 minutes. Then dissolve 40 mg of Callicarpa nudiflora, 2.7 g of AM, 0.3 g of AA, 3 mg of MBA, and 45 mg of APS in the dispersion of silver-lignin-curcumin nanoparticles, and stir into a homogeneous solution. Let it stand at room temperature for 30 minutes to obtain the Callicarpa nudiflora-loaded Ag@Lig-Cur NPs-P(AM-AA) hydrogel. Its adhesion strength to pigskin is 140 KPa, the tensile strain is 2000%, the antibacterial rate is greater than 99%, the free radical scavenging rate is 97.39%, the hemostatic volume and hemostatic time are 33 mg and 35 s respectively, and the wound healing rate at 12 days is 98.6%.

[0022] Comparative Example 1: Dissolve 2 mg of curcumin powder and 300 mg of alkali lignin powder in 8 mL of NaOH solution (pH = 11). Slowly add the above solution dropwise to an equal volume of silver ammonia solution with a concentration of 7.5 mg / mL. React at room temperature for 40 minutes. Then dissolve 2.7 g of AM, 0.3 g of AA, 3 mg of MBA, and 45 mg of APS in the dispersion of silver-lignin-curcumin nanoparticles, and stir into a homogeneous solution. Let it stand at room temperature for 30 minutes to obtain the Ag@Lig-Cur NPs-P(AM-AA) hydrogel. Its adhesion strength to pigskin is 125 KPa, the tensile strain is 1694%, the antibacterial rate is greater than 99%, the free radical scavenging rate is 82.65%, the hemostatic volume and hemostatic time are 58 mg and 49 s respectively, and the wound healing rate at 12 days is 93.1%.

[0023] Comparative Example 2: Dissolve 300 mg of alkali lignin powder in 10 mL of NaOH solution (pH = 11). Slowly add the above solution dropwise to an equal volume of silver ammonia solution with a concentration of 7.5 mg / mL. React at room temperature for 60 minutes. Then dissolve 2.7 g of AM, 0.3 g of AA, 3 mg of MBA, and 45 mg of APS in the dispersion of silver-lignin nanoparticles, and stir into a homogeneous solution. Let it stand at room temperature for 25 minutes to obtain the Ag@Lig NPs-P(AM-AA) hydrogel. Its adhesion strength to pigskin is 108.1 KPa, the tensile strain is 1494%, the antibacterial rate is greater than 99%, the free radical scavenging rate is 52.94%, the hemostatic volume and hemostatic time are 64 mg and 61 s respectively, and the wound healing rate at 12 days is 87.4%.

[0024] Comparative Example 3: 2 mg of curcumin powder was dissolved in 5 mL of NaOH solution (pH = 11). The above solution was slowly added dropwise to an equal volume of silver ammonia solution with a concentration of 7.5 mg / mL. The reaction was carried out at room temperature for 50 minutes. Then, 2.7 g of AM, 0.3 g of AA, 3 mg of MBA, and 45 mg of APS were dissolved in the dispersion of silver-curcumin nanoparticles and stirred into a homogeneous solution. After standing at room temperature for 20 minutes, Ag@Cur NPs-P(AM-AA) hydrogel was obtained. Its adhesion strength to pig skin was 62.5 KPa, the tensile strain was 709%, the antibacterial rate was greater than 99%, the free radical scavenging rate was 78.24%, the hemostatic volume and hemostatic time were 86 mg and 78 s respectively, and the wound healing rate at 12 days was 89.4%.

[0025] Comparative Example 4: 2.7 g of AM, 0.3 g of AA, 3 mg of MBA, and 45 mg of APS were dissolved in 10 ml of deionized water. The reaction was carried out at room temperature for 30 minutes, and then left standing at room temperature for 2 days to obtain P(AM-AA) hydrogel. Its adhesion strength to pig skin was 37 KPa, the tensile strain was 1070%, it had no antibacterial property, the free radical scavenging rate was 8.82%, the hemostatic volume and hemostatic time were 99 mg and 87 s respectively, and the wound healing rate at 12 days was 81%.

[0026] Adhesion performance: The lap shear experiment of pig skin was carried out on a universal material testing machine (ANSCMT4503, SANS, China) to measure the adhesion strength of the hydrogel to tissues. Two pieces of fresh pig skin purchased from a local store were cut into samples with a length of 50 mm, a width of 30 mm, and a thickness of 2 mm, and were washed to remove the fat layer. The hydrogel (2 mm high, 20 mm in diameter) was placed between the two pig skin samples. After adhesion, the sample was pulled apart at a crosshead speed of 5 mm / min until failure. The adhesion strength was calculated as the measured maximum load divided by the adhesion area.

[0027] Determination of antibacterial property: 100 μL of Staphylococcus aureus or Escherichia coli suspension (1×10 6 CFU / mL) was added to the sample. After 12 h, 1900 μL of lysogenic broth was added. Then, the sample was placed in an incubator at 37 °C and continuously shaken. After 12 h, 1000 μL of the bacterial suspension was collected in a centrifuge tube. The optical density of the suspension at 600 nm was measured (denoted as OD Sample ), and the absorbance without the sample was denoted as OD Control . The antibacterial rate was calculated according to the following formula.

[0028]

[0029] Antioxidant performance: Weigh 50 mg of the hydrogel at the bottom of a centrifuge tube, add 4 mL of deionized water, and soak it for 12 h at ambient temperature. Take 1 mL of the supernatant extract to evaluate the scavenging activity of DPPH: Mix the supernatant extract (1 mL) with 1 mL of methanol solution of DPPH (50 mg / L) to obtain a 25 mg / L DPPH solution. Keep the mixture in the dark at room temperature for 60 minutes. Measure the absorbance at 517 nm using a UV-visible spectrophotometer (UV-2600i, Japan). Use the mixture solution of deionized water and DPPH methanol solution as a control. Calculate the DPPH radical scavenging activity using the following formula, where OD1 is the absorbance of the sample and OD0 is the absorbance of the control.

[0030]

[0031] Hemostatic performance: A liver bleeding model of 7-week-old male SD mice (36 - 40 g) was used to detect the hemostatic performance of the hydrogel. First, anesthetize the mice with 1 wt% sodium pentobarbital. Shave the abdominal hair of the anesthetized mice and fix them on a plastic platform. Wipe the abdomen with 75% alcohol, then cut the abdominal skin with surgical scissors, fix the abdominal wound with a needle, expose the liver, and remove the serous fluid around the liver with filter paper. Then, place the liver on a pre-weighed filter paper. Induce liver bleeding with a needle and immediately apply the hydrogel sample (18 mm in diameter × 3 mm in thickness) to the bleeding site. Determine the blood loss by weighing the hydrogel and the filter paper, and record the hemostatic time.

[0032] Wound healing: Anesthetize 4 7-week-old male SD mice (36 - 40 g) with 1 wt% sodium pentobarbital. Shave the dorsal hair of the anesthetized mice and create a full-thickness circular wound (wound diameter 8 mm) on the back of each mouse using an 8 mm skin biopsy punch. Implant the sterilized hydrogel (8 mm in diameter, 2 mm thick) into the wound site of the mice. The wound area is denoted as S 初始 . Observe the wound surface condition every day and record the wound healing condition of the mice on the 6th and 12th days. The wound area is denoted as S 愈合 . Calculate the skin wound healing rate according to the following formula.

[0033]

Claims

1. A method for preparing a multifunctional lignin hydrogel for skin dressing, characterized in that Curcumin powder and alkaline lignin powder were dissolved in sodium hydroxide solution to obtain solution A, and solution A was slowly added dropwise to an equal volume of silver ammonia solution with a concentration of 7.5-15 mg / ml, and reacted at room temperature for 30-60 minutes to obtain a lignin-curcumin-Ag nanoparticle dispersion; Callicarpa nudiflora, acrylamide, acrylic acid, N, N-methylenebisacrylamide, and ammonium persulfate were co-dissolved in the nanoparticle dispersion, and stirred to form a uniform solution B, which was allowed to stand at room temperature for 20-30 minutes to obtain a lignin-curcumin silver nanoparticle-poly (acrylamide-acrylic acid) hydrogel loaded with Callicarpa nudiflora.

2. According to the preparation method of the multifunctional lignin hydrogel for skin dressing according to claim 1, the mass ratio of curcumin to alkali lignin is 1:300~4:300, the content of Callicarpa nudiflora is 10~40 mg, the volume of the sodium hydroxide solution is 5~10 ml, the pH is 11, the acrylamide is 2.7 g, the acrylic acid is 0.3 g, the N, N-methylenebisacrylamide is 3 mg, and the ammonium persulfate is 45 mg.

3. The hydrogel prepared according to the method for preparing the multifunctional lignin hydrogel for skin dressing according to claim 1.

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