A multifunctional lignin hydrogel for skin dressing and preparation method thereof

Abstract

The present invention discloses a multifunctional lignin hydrogel for skin dressings and a preparation method thereof. The multifunctional lignin hydrogel is prepared by first dissolving curcumin powder and alkali lignin powder in a NaOH solution in a certain proportion, slowly adding the solution dropwise to an equal volume of silver ammonia solution, and reacting at room temperature for 30 to 60 minutes. A certain amount of nudiflorin, 2.7 g acrylamide, 0.3 g acrylic acid, 3 mg N,N-methylenebisacrylamide, and 45 mg ammonium persulfate are then dissolved in a dispersion of the nanoparticles described above, stirred to form a uniform solution, and allowed to stand at room temperature for 20 to 30 minutes to obtain a nudiflorin-loaded lignin-curcumin silver nanoparticle-poly(acrylamide-acrylic acid) hydrogel. The multifunctional lignin hydrogel prepared by the present invention has multiple functions, including adhesion, antibacterial, antioxidant, and hemostatic properties. It can be used to accelerate skin wound healing and has good application prospects in skin dressings.

Description

Technical Field

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

[0002] As the largest organ in the human body, the skin is the first line of defense against bacterial and viral invasion. Once the skin is severely injured, bacteria can easily multiply at the wound site, leading to serious infection. Traditional dressings, such as gauze and bandages, have limitations in effectively controlling bleeding and absorbing wound exudate. They also have poor moisture retention and breathability. Advanced materials such as nanofibers and porous foams can be used for wound healing. However, low wound exudate levels can lead to dehydration, hindering the healing process. Excessive exudate levels and limited absorption capacity can lead to leakage and the risk of infection. Hydrogels, as polymer materials with a three-dimensional network structure, have high water content and excellent moisture retention. Their porous structure and swelling properties allow them to effectively absorb wound exudate. However, single-function hydrogels, relying solely on moisture retention and protection, cannot address the problems of infection, oxidative stress, persistent bleeding, and insufficient tissue regeneration associated with chronic wounds. Therefore, the development of multifunctional hydrogel dressings with adhesive, antibacterial, antioxidant, and hemostatic properties for rapid wound healing is of great significance.

[0003] To achieve multifunctional hydrogels, combining natural and synthetic polymers to leverage the advantages of both is a growing trend. Acrylic acid and acrylamide are highly favored for their biocompatibility and high mechanical strength, but they lack inherent biological properties such as antibacterial, anti-inflammatory, and hemostatic abilities. Traditionally, antibiotics have been the cornerstone of wound infection treatment. However, overuse of antibiotics has led to the emergence of drug-resistant strains, severely impacting their efficacy. Silver nanoparticles (AgNPs) have emerged as a promising alternative due to their broad-spectrum antimicrobial activity. However, their reduction and encapsulation in hydrogels remain challenging. Lignin has attracted considerable attention in biomedical applications due to its excellent biocompatibility and biodegradability. In particular, lignin possesses phenolic and methoxyl groups, making it a reducing and capping agent for metal nanoparticles, potentially addressing the challenges of silver nanoparticle reduction and encapsulation. Furthermore, lignin contains a variety of reducing functional groups, such as phenolic and methoxyl groups, which impart antioxidant properties and can scavenge excess reactive oxygen species (ROS) in wounds. However, even with the addition of silver-lignin nanoparticles, the antimicrobial and antioxidant effects of lignin-based systems still need to be improved. Studies have shown that combining lignin with other polyphenolic compounds can enhance its biocompatibility, antimicrobial, and antioxidant properties. Curcumin, which also contains reducing groups such as phenolic hydroxyl and methoxy groups in its molecular structure, can also serve as a reducing agent and capping agent for metal nanoparticles. It also possesses antitumor, antimicrobial, 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) hydrogels [P(AM-AA) hydrogels] and enhance the antimicrobial and antioxidant properties of the hydrogels.

[0004] In addition, the initial stage of wound healing includes hemostasis. Therefore, the development of hydrogel dressings with hemostatic function is particularly important. Current research mainly focuses on combining polymers or organic small molecules to achieve hemostasis. A new approach is to integrate some traditional Chinese medicines with hemostatic effects into hydrogels to give hydrogels hemostatic function. Callicarpa nudiflora is a traditional Chinese medicine with hemostatic effects. It has been widely used in postpartum hemorrhage, traumatic hemorrhage and gastrointestinal bleeding. It contains a large number of bioactive compounds such as phenylethyl isothiocyanate, glycosides, flavonoids and terpenes, and also has anti-inflammatory, antithrombotic and antioxidant effects. However, there have been no reports on the co-introduction of Callicarpa nudiflora and curcumin-lignin reduced silver nanoparticles into AM / AA hydrogel systems. 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. This method utilizes a one-pot preparation process under mild conditions, achieving efficient reduction of silver nanoparticles and stable crosslinking of the hydrogel network without disrupting the activity of lignin / curcumin. Furthermore, by simultaneously introducing curcumin-lignin-reduced silver nanoparticles and Callicarpa nudiflora, a single hydrogel system can achieve integrated antibacterial, antioxidant, hemostatic, and ultra-high stretchability properties. This method could potentially lead to the development of a novel, high-performance hydrogel dressing, offering new approaches for maximizing the value of natural ingredients, constructing dynamic hydrogel networks, and treating chronic wounds. The method has promising application prospects in the field of biomedical materials.

[0006] The preparation method of the multifunctional lignin hydrogel comprises the following steps:

[0007] Curcumin powder and alkali lignin powder were dissolved in 5-10 mL of sodium hydroxide solution (pH=11) in a certain proportion, and the above solution was slowly added dropwise to an equal volume of silver ammonia solution with a concentration of 7.5-15 mg / mL. The mixture was reacted at room temperature for 30-60 minutes to obtain a lignin-curcumin-Ag nanoparticle dispersion. An appropriate amount of Callicarpa nudiflora, 2.7 g acrylamide (AM), 0.3 g acrylic acid (AA), 3 mg N, N-methylenebisacrylamide (MBA), and 45 mg ammonium persulfate (APS) were then dissolved in the above nanoparticle dispersion and stirred to form a uniform solution. The solution was allowed to stand at room temperature for 20-30 minutes to obtain a Callicarpa nudiflora-loaded lignin-curcumin silver nanoparticle-P(AM-AA) hydrogel.

[0008] The present invention provides a multifunctional lignin hydrogel for skin dressing and a preparation method thereof, which has the advantages of:

[0009] (1) Hydrogel performance: For the first time, the four functional modules of antibacterial (Ag NPs), antioxidant (lignin / curcumin), hemostasis (clean bead) and tissue adhesion (quinone groups) are integrated into a single hydrogel system. Through the molecular-level synergistic effect between the components (such as curcumin enhancing the antibacterial activity of silver nanoparticles, and the joint scavenging of free radicals by naked bead and lignin), functional superposition is achieved instead of simple mixing. Therefore, it has high adhesion, antibacterial, antioxidant, hemostatic properties and multiple functions of promoting wound healing, which can significantly improve the efficiency of microenvironment regulation of chronic wounds.

[0010] (2) Preparation method: Breaking through the limitations of traditional multi-step preparation of hydrogels, a "one-pot method" is used to simultaneously complete the synthesis of silver nanoparticles. The quinone free radicals generated in this process will promote the self-gelation of P(AM-AA) hydrogel. By adding different proportions of reactant concentrations, the dynamic balance between the covalent cross-linking (quinone free radical-induced polymerization) and non-covalent interaction (hydrogen bonding, ionic bonding, π-π stacking) of Ag@Lig-Cur NPs and Callicarpa nudiflora on the P(AM-AA) hydrogel network is regulated, thereby improving the tensile strain capacity of the hydrogel, breaking through the bottleneck of the brittle fracture of traditional hydrogels, and meeting the stringent requirements for dressing flexibility in the dynamic healing process of chronic wounds.

[0011] (3) The raw materials for the preparation of this new hydrogel are widely available, among which lignin, curcumin, and Callicarpa nudiflora are all natural ingredients, so the production cost is low.

[0012] In summary, the present invention embodies a full-chain breakthrough in material design concepts, preparation processes, functional integration mechanisms, and verification methods. It can not only develop a multifunctional hydrogel dressing with adhesion, antibacterial, antioxidant, and hemostatic properties for rapid wound healing, but also provide new ideas for the high-value utilization of natural products, dynamic hydrogel network construction, and chronic wound treatment strategies. It has good application prospects and clinical transformation value in wound dressings. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 These are actual photos of the hydrogels in Example 1 and Comparative Examples 1-4.

[0014] Figure 2 The adhesion results of the hydrogels in Example 1 and Comparative Examples 1-4 are shown.

[0015] Figure 3 The antibacterial results of the hydrogels in Example 1 and Comparative Examples 1-4 are shown.

[0016] Figure 4 These are the antioxidant results of the hydrogels in Example 1 and Comparative Examples 1-4.

[0017] Figure 5 The hemostatic results of the hydrogels in Example 1 and Comparative Examples 1-4 are shown.

[0018] Figure 6 The wound healing results of the hydrogels in Example 1 and Comparative Examples 1-4 are shown.

[0019] 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 DESCRIPTION

[0020] The following examples are hydrogels with a mass ratio of curcumin to alkali lignin of 1:300 to 4:300 and a Callicarpa nudiflora content of 10 to 40 mg. Hydrogels with mass ratios outside this range are difficult to form and have poor various properties. Therefore, the present invention only presents the experimental results obtained in the examples that meet the above range.

[0021] Example 1: 2 mg of curcumin powder and 300 mg of alkali lignin powder were dissolved in 8 mL of NaOH solution (pH = 11). This solution was slowly added dropwise to an equal volume of 7.5 mg / mL silver ammonia solution and allowed to react at room temperature for 50 minutes. Then, 30 mg of Callicarpa nudiflora, 2.7 g of AM, 0.3 g of AA, 3 mg of MBA, and 45 mg of APS were dissolved in the silver-lignin-curcumin nanoparticle dispersion and stirred to form a homogeneous solution. The solution was allowed to stand at room temperature for 30 minutes to obtain a Callicarpa nudiflora-loaded Ag@Lig-Cur NPs-P(AM-AA) hydrogel. The hydrogel exhibited an adhesion strength of 130 kPa to pig skin, a tensile strain of 2100%, an antibacterial rate greater than 99%, a free radical scavenging rate of 96.47%, a hemostatic volume and time of 37 mg and 39 s, respectively, and a 12-day wound healing rate of 98.9%.

[0022] Example 2: 1 mg of curcumin powder and 300 mg of alkali lignin powder were dissolved in 5 mL of NaOH solution (pH = 11). This solution was slowly added dropwise to an equal volume of 7.5 mg / mL silver ammonia solution and allowed to react at room temperature for 30 minutes. Then, 20 mg of Callicarpa nudiflora, 2.7 g of AM, 0.3 g of AA, 3 mg of MBA, and 45 mg of APS were dissolved in the silver-lignin-curcumin nanoparticle dispersion and stirred to form a homogeneous solution. The solution was allowed to stand at room temperature for 20 minutes to obtain a Callicarpa nudiflora-loaded Ag@Lig-Cur NPs-P(AM-AA) hydrogel. The hydrogel exhibited an adhesion strength of 120 kPa to pig skin, a tensile strain of 1900%, an antibacterial rate greater than 99%, a free radical scavenging rate of 90.47%, a hemostatic volume of 48 mg and a hemostatic time of 42 seconds, respectively, and a 12-day wound healing rate of 94.8%.

[0023] Example 3: 4 mg of curcumin powder and 300 mg of alkali lignin powder were dissolved in 10 mL of NaOH solution (pH = 11). This solution was slowly added dropwise to an equal volume of 15.0 mg / mL silver ammonia solution and allowed to react at room temperature for 60 minutes. Then, 40 mg of Callicarpa nudiflora, 2.7 g of AM, 0.3 g of AA, 3 mg of MBA, and 45 mg of APS were dissolved in the silver-lignin-curcumin nanoparticle dispersion and stirred to form a homogeneous solution. The solution was allowed to stand at room temperature for 30 minutes to obtain a Callicarpa nudiflora-loaded Ag@Lig-Cur NPs-P(AM-AA) hydrogel. The hydrogel exhibited an adhesion strength of 140 kPa to pig skin, a tensile strain of 2000%, an antibacterial rate greater than 99%, a free radical scavenging rate of 97.39%, a hemostatic volume of 33 mg and a hemostatic time of 35 s, respectively, and a 12-day wound healing rate of 98.6%.

[0024] Comparative Example 1: 2 mg of curcumin powder and 300 mg of alkali lignin powder were dissolved in 8 mL of NaOH solution (pH = 11). This solution was slowly added dropwise to an equal volume of 7.5 mg / mL silver ammonia solution and allowed to react at room temperature for 40 minutes. 2.7 g of AM, 0.3 g of AA, 3 mg of MBA, and 45 mg of APS were then dissolved in the silver-lignin-curcumin nanoparticle dispersion, stirred to a uniform solution, and allowed to stand at room temperature for 30 minutes to obtain the Ag@Lig-Cur NPs-P(AM-AA) hydrogel. The hydrogel exhibited an adhesion strength of 125 kPa to pigskin, a tensile strain of 1694%, an antibacterial rate greater than 99%, a free radical scavenging rate of 82.65%, a hemostatic volume of 58 mg and a hemostatic time of 49 s, respectively, and a 12-day wound healing rate of 93.1%.

[0025] Comparative Example 2: 300 mg of alkali lignin powder was dissolved in 10 mL of NaOH solution (pH = 11). This solution was slowly added dropwise to an equal volume of 7.5 mg / mL silver ammonia solution. The mixture was allowed to react at room temperature for 60 minutes. 2.7 g of AM, 0.3 g of AA, 3 mg of MBA, and 45 mg of APS were then dissolved in the silver-lignin nanoparticle dispersion, stirred to form a homogeneous solution, and allowed to stand at room temperature for 25 minutes to produce the Ag@Lig NPs-P(AM-AA) hydrogel. The hydrogel exhibited an adhesion strength of 108.1 kPa to pigskin, a tensile strain of 1494%, an antibacterial rate exceeding 99%, a free radical scavenging rate of 52.94%, a hemostatic volume of 64 mg and a hemostatic time of 61 s. The wound healing rate was 87.4% after 12 days.

[0026] Comparative Example 3: 2 mg of curcumin powder was dissolved in 5 mL of NaOH solution (pH = 11). This solution was slowly added dropwise to an equal volume of 7.5 mg / mL silver ammonia solution. The mixture was allowed to react at room temperature for 50 minutes. 2.7 g of AM, 0.3 g of AA, 3 mg of MBA, and 45 mg of APS were then dissolved in the silver-curcumin nanoparticle dispersion, stirred to form a uniform solution, and allowed to stand at room temperature for 20 minutes to produce the Ag@Cur NPs-P(AM-AA) hydrogel. The hydrogel exhibited an adhesion strength of 62.5 kPa to pig skin, a tensile strain of 709%, an antibacterial rate greater than 99%, a free radical scavenging rate of 78.24%, a hemostatic volume of 86 mg and a hemostatic time of 78 s. The wound healing rate was 89.4% after 12 days.

[0027] Comparative Example 4: 2.7 g AM, 0.3 g AA, 3 mg MBA, and 45 mg APS were dissolved in 10 ml of deionized water and allowed to react at room temperature for 30 minutes. The hydrogel was then allowed to stand at room temperature for 2 days to produce a P(AM-AA) hydrogel. The hydrogel exhibited adhesion strength of 37 kPa to pig skin, a tensile strain of 1070%, no antibacterial activity, a free radical scavenging rate of 8.82%, a hemostatic volume of 99 mg, and a hemostatic time of 87 seconds. The 12-day wound healing rate was 81%.

[0028] Adhesion Properties: Lap shear tests were conducted on pigskin using a universal materials testing machine (ANSCMT4503, SANS, China) to measure the hydrogel-tissue adhesion strength. Two pieces of fresh pigskin purchased from a local store were cut into 50 mm long, 30 mm wide, and 2 mm thick samples. The samples were cleaned and the fat layer removed. The hydrogel (2 mm high, 20 mm diameter) was placed between the two pigskin samples. After adhesion was established, the samples were pulled apart at a crosshead speed of 5 mm / min until failure. The bond strength was calculated as the maximum load measured divided by the bond area.

[0029] Antibacterial activity determination: 100 μL of Staphylococcus aureus or Escherichia coli suspension (1×10 6 CFU / mL). After 12 h, add 1900 μL of lysogeny broth. Then, place the sample in a 37°C incubator with constant shaking. After 12 h, collect 1000 μL of bacterial suspension in a centrifuge tube. Measure the optical density of the suspension at 600 nm (OD Sample ), the absorbance without adding sample was recorded as OD Control , the inhibition rate was calculated according to the following formula.

[0030]

[0031] Antioxidant Activity: 50 mg of hydrogel was weighed and placed at the bottom of a centrifuge tube. 4 mL of deionized water was added and the mixture was allowed to soak at ambient temperature for 12 h. 1 mL of the supernatant extract was collected to assess DPPH scavenging activity: 1 mL of the supernatant extract was mixed with 1 mL of 50 mg / L DPPH in methanol to yield a 25 mg / L DPPH solution. The mixture was incubated at room temperature in the dark for 60 min. The absorbance was measured at 517 nm using a UV-visible spectrophotometer (UV-2600i, Japan). A mixture of deionized water and DPPH methanol solution was used as a control. DPPH radical scavenging activity was calculated using the following formula, where OD1 is the absorbance of the sample and OD0 is the absorbance of the control.

[0032]

[0033] Hemostatic Properties: The hemostatic properties of the hydrogel were tested using a liver bleeding model in 7-week-old male SD mice (36-40 g). First, the mice were anesthetized with 1 wt% sodium pentobarbital. The anesthetized mice were shaved of their abdominal hair and fixed on a plastic table. The abdomen was wiped with 75% alcohol, and then the abdominal skin was cut with surgical scissors. The abdominal wound was fixed with a needle to expose the liver, and the serous fluid around the liver was removed with filter paper. The liver was then placed on a pre-weighed filter paper. Liver bleeding was induced using a needle, and a hydrogel sample (18 mm diameter × 3 mm thickness) was immediately applied to the bleeding site. Blood loss was determined by weighing the hydrogel and filter paper, and the time to hemostasis was recorded.

[0034] Wound healing: Four 7-week-old male SD mice (36-40 g) were anesthetized with 1 wt% sodium pentobarbital. The dorsal hair of the anesthetized mice was shaved, and a full-thickness circular wound (8 mm in diameter) was created on the back of each mouse using an 8 mm skin biopsy punch. Sterilized hydrogels (8 mm in diameter, 2 mm thick) were implanted into the wound site. The wound area was recorded as S. 初始 The wound surface was observed every day, and the wound healing status of the mice was recorded on the 6th and 12th days. The wound area was recorded as S 愈合 The skin wound healing rate is calculated according to the following formula.

[0035]

Claims

1. A method for preparing a multifunctional lignin hydrogel for skin dressing, characterized in that Curcumin powder and alkali lignin powder were dissolved in a sodium hydroxide solution to obtain solution A, wherein the mass ratio of curcumin to alkali lignin was 1:300 to 4:

300. Solution A was slowly added dropwise to an equal volume of silver ammonia solution with a concentration of 7.5 to 15 mg / ml, and the mixture was reacted at room temperature for 30 to 60 minutes to obtain a lignin-curcumin-Ag nanoparticle dispersion. Then, Callicarpa nudiflora, acrylamide, acrylic acid, N,N-methylenebisacrylamide, and ammonium persulfate were co-dissolved in the nanoparticle dispersion, wherein the content of Callicarpa nudiflora was 10 to 40 mg, and the mixture was stirred to form a uniform solution B. The solution was allowed to stand at room temperature for 20 to 30 minutes to obtain a Callicarpa nudiflora-loaded lignin-curcumin silver nanoparticle-poly(acrylamide-acrylic acid) hydrogel.

2. The method for preparing the multifunctional lignin hydrogel for skin dressing according to claim 1, wherein 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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