A water-absorbing low-swelling bacteriostatic hydrogel dressing and a preparation method and application thereof

Abstract

The application discloses a water-absorbing low-swelling bacteriostatic hydrogel dressing and a preparation method and application thereof. The hydrogel dressing is prepared by taking chitosan, polyvinyl alcohol and oxidized chitosan as a skeleton and introducing natural extract berberine as a crosslinking agent and an antibacterial agent. The hydrogel dressing prepared by the method has good water absorption performance and low swelling performance. The hydrogel dressing can effectively manage wound exudates and provide stable interface protection for the wound. Meanwhile, the hydrogel has good anti-inflammatory, antioxidant, bacteriostatic and excellent biological safety. The hydrogel dressing can be applied to the treatment of skin infectious injury as a wound dressing, and has important significance in clinical production practice.

Description

Technical Field

[0001] This invention belongs to the field of hydrogel dressing technology, specifically relating to a water-absorbing, low-swelling, antibacterial hydrogel dressing, its preparation method, and its application. Background Technology

[0002] The skin, the largest organ in the human body, plays a crucial role in sensing external stimuli, regulating body temperature, and protecting the body. However, the skin is easily damaged by various injuries. Improper wound management can easily lead to rebleeding and infection, a common clinical problem that severely hinders the normal healing process and causes skin dysfunction. Therefore, skin injuries pose significant risks and losses to the medical field. Achieving effective wound closure and rapid repair remains a key challenge. Hydrogels are widely used in the medical field due to their similarity to the extracellular matrix, good biocompatibility, effective absorption of exudate, and ability to maintain wound moisture and promote healing. The high absorbency and porous structure of hydrogels make them prone to swelling. Swelling hydrogels can absorb wound exudate, maintain wound moisture balance, promote cell migration and proliferation, and release antibacterial agents to inhibit wound infection. However, the dramatic swelling of traditional hydrogels causes a surge in volume and weight, leading to decreased mechanical properties, drug burst release, and failure of physical barriers, severely limiting their clinical application.

[0003] Compared to traditional hydrogels, anti-swelling hydrogels can provide durable and stable interfacial protection for dynamic wounds, avoiding stress concentration caused by volume expansion and thus reducing mechanical pressure on newly formed tissue. However, common anti-swelling hydrogel strategies have encountered bottlenecks. These strategies, including hydrophobic modification, metal coordination, or chemical crosslinking agents, often rely on complex chemical crosslinking and crosslinking agents, resulting in cumbersome preparation processes, poor biocompatibility, and even loss of antibacterial properties. On the other hand, current anti-swelling hydrogels often neglect the original advantage of hydrogels—water absorption—in order to achieve high anti-swelling performance. Conversely, retaining an appropriate water absorption capacity of the hydrogel is more beneficial for absorbing wound exudate, thereby reducing bacterial growth and promoting skin wound healing.

[0004] Therefore, developing an antibacterial hydrogel dressing that is simple to prepare, green and safe, and has dynamic balance properties of water absorption and swelling is of great significance for providing long-lasting and stable interfacial protection for skin wounds, effective exudate management, sustained antibacterial properties, and promoting wound healing. Summary of the Invention

[0005] To address the shortcomings and deficiencies of existing technologies, the primary objective of this invention is to provide a method for preparing a water-absorbing, low-swelling, antibacterial hydrogel dressing.

[0006] This invention uses chitosan, polyvinyl alcohol and oxidized chitosan as the backbone, and introduces berberine, a natural extract, as a crosslinking agent and antibacterial agent to prepare a hydrogel dressing.

[0007] Another object of the present invention is to provide a water-absorbing, low-swelling, antibacterial hydrogel dressing prepared by the above method.

[0008] Another object of the present invention is to provide the application of the above-mentioned water-absorbing, low-swelling, antibacterial hydrogel dressing.

[0009] To achieve this objective, the present invention adopts the following technical solution:

[0010] In a first aspect, the present invention provides a method for preparing a water-absorbing, low-swelling, antibacterial hydrogel dressing, comprising the following steps:

[0011] (1) Mix chitosan acetic acid solution and polyvinyl alcohol aqueous solution evenly to obtain chitosan / polyvinyl alcohol mixed solution;

[0012] (2) Add an aqueous solution of oxidized chitosan to the chitosan / polyvinyl alcohol mixed solution and mix evenly to obtain a chitosan / polyvinyl alcohol / oxidized chitosan mixed solution;

[0013] (3) Add berberine aqueous solution to the chitosan / polyvinyl alcohol / oxidized chitosan mixed solution and stir to form a gel to obtain a water-absorbing, low-swelling, antibacterial hydrogel dressing.

[0014] Preferably, in the chitosan-acetic acid aqueous solution in step (1), the ratio of chitosan to water is (4-6) g: 100 mL; and the volume ratio of acetic acid to water is (1-2): 100.

[0015] Preferably, the chitosan-acetic acid aqueous solution in step (1) is obtained by dissolving chitosan in an acetic acid solution.

[0016] Preferably, in the polyvinyl alcohol aqueous solution in step (1), the ratio of polyvinyl alcohol to water is (5-8) g: 100 mL.

[0017] Preferably, the polyvinyl alcohol aqueous solution in step (1) is obtained by dissolving polyvinyl alcohol in water at a temperature of 85-95°C for 1-2 hours and a rotation speed of 300-500 rpm.

[0018] Preferably, the volume ratio of the chitosan acetic acid solution and the polyvinyl alcohol aqueous solution in step (1) is (1-1.5):(2-3).

[0019] Preferably, the mixing in step (1) is carried out by mechanical stirring at a speed of 400-600 rpm, at room temperature, and for 2-4 hours.

[0020] Preferably, in the oxidized chitosan aqueous solution in step (2), the ratio of oxidized chitosan to water is (0.1~0.3) g : (5~10) mL.

[0021] Preferably, the oxidized chitosan aqueous solution in step (2) is obtained by adding oxidized chitosan to water and then vortexing for 5 to 10 minutes to disperse it evenly.

[0022] Preferably, the volume ratio of the chitosan / polyvinyl alcohol mixed solution to the oxidized chitosan aqueous solution in step (2) is (25-30):(5-10).

[0023] Preferably, the term "mixing evenly" in step (2) refers to stirring at room temperature for 2 to 3 hours.

[0024] Preferably, in the berberine aqueous solution in step (3), the ratio of berberine to water is (0.15~0.5) g : (5~10) mL.

[0025] Preferably, the berberine aqueous solution in step (3) is obtained by adding berberine to water, vortexing for 5 to 10 minutes, and then heating at 50 to 65°C for 0.5 to 1.5 hours.

[0026] Preferably, the volume ratio of the chitosan / polyvinyl alcohol / oxidized chitosan mixed solution to the berberine aqueous solution in step (3) is (30-40):(5-10).

[0027] Preferably, the stirring speed in step (3) is 800-1000 rpm, the temperature is room temperature, and the stirring time is 24-48 hours.

[0028] Secondly, the present invention provides a water-absorbing, low-swelling, antibacterial hydrogel dressing prepared by the above-mentioned preparation method.

[0029] Thirdly, the present invention provides the application of the above-mentioned water-absorbing, low-swelling, antibacterial hydrogel dressing.

[0030] Application of the above-mentioned water-absorbing, low-swelling, antibacterial hydrogel dressing in the preparation of materials for full-thickness skin wound infection.

[0031] This invention utilizes inexpensive and abundant chitosan, green and safe polyvinyl alcohol, and oxidized chitosan to construct a hydrogel framework with high biocompatibility. At the same time, it uses berberine, a natural drug extract with excellent antibacterial properties, as a crosslinking agent and antibacterial agent. A water-absorbing, low-swelling, antibacterial hydrogel dressing is prepared by simple mechanical stirring and one-pot method.

[0032] The hydrogel dressing prepared by the method of this invention has good water absorption and low swelling properties. It effectively manages wound exudate and provides stable interfacial protection for the wound; simultaneously, the hydrogel also possesses good anti-inflammatory, antioxidant, antibacterial, and excellent biocompatibility. This hydrogel dressing can be used as a wound dressing in the treatment of infectious skin injuries and has significant implications for clinical production practice.

[0033] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0034] (1) The hydrogel dressing of the present invention uses high polymers with good biosafety and wide availability as raw materials, and uses berberine, a natural drug extract, as a crosslinking agent and antibacterial agent. No synthetic chemical crosslinking agents or metal ions are added. It is non-toxic, harmless, green and safe, and has low material cost and simple preparation.

[0035] (2) This invention utilizes chitosan and polyvinyl alcohol to form a gel network through physical entanglement and hydrogen bonding. The aldehyde groups of oxidized chitosan and the amino groups of chitosan further enhance the gel network through Schiff base bonds and hydrogen bonds. Berberine, a natural drug extract, is introduced as a crosslinking agent and antibacterial agent. A porous network structure is formed through π-π stacking, hydrophobic interaction, multiple hydrogen bonds, and Schiff base bonds. The introduction of berberine (BBR) increases the hydrophobic properties of the gel network. The hydrophobic microdomains formed by π-π stacking and hydrophobic interaction can "anchor" the polymer chains, thereby inhibiting polymer chain movement and reducing the swelling properties of the gel. Therefore, this hydrogel can maintain appropriate water absorption capacity, effectively absorb wound exudate and reduce bacterial growth, without excessive swelling, thus avoiding secondary damage to the wound caused by stress concentration and secondary contamination caused by wound exposure due to gel network collapse.

[0036] (3) The hydrogel dressing of the present invention has good anti-inflammatory, antibacterial and antioxidant properties, and has excellent therapeutic effect on skin injury infection. Attached Figure Description

[0037] Figure 1 The infrared spectra of the CPOB hydrogel and its components obtained in Example 3 are shown.

[0038] Figure 2 The images are scanning electron microscope (SEM) images of the CPO (a) and CPOB (b) hydrogels obtained in Example 3.

[0039] Figure 3 The results show the water contact angles of the CPO and CPOB hydrogels obtained in Example 3.

[0040] Figure 4 The sustained-release effect of the CPOB hydrogel obtained in Example 3 on the drug (berberine) in phosphate buffer solutions of different pH values ​​is shown.

[0041] Figure 5 The graph shows the volume change of the CPOB hydrogel obtained in Example 3 after absorbing water in physiological saline at different time points.

[0042] Figure 6 In Figure (a), the results of pigskin stretch adhesion of the CPO and CPOB hydrogels obtained in Example 3 are shown.

[0043] Figure 6 Image (b) shows the adhesion of the CPOB hydrogel obtained in Example 3 on metal (left image in the first row), glass (middle image in the first row), plastic (right image in the first row), pigskin (second and third rows), and gloves (fourth row).

[0044] Figure 7 Figures (a) and (c) show the antibacterial effects and inhibition rates of the CPO and CPOB hydrogels obtained in Example 3 against Escherichia coli.

[0045] Figure 7 Figures (b) and (d) show the antibacterial effect and inhibition rate of the CPO and CPOB hydrogels obtained in Example 3 against Staphylococcus aureus (MRSA).

[0046] Figure 8 The changes in the infected wound at 0, 3, 7, and 14 hours after treatment with the CPO and CPOB hydrogels described in Example 6.

[0047] Figure 9 The results are quantitative measurements of wound area at 3, 7, and 14 hours after treatment of infected wounds with the CPO and CPOB hydrogels described in Example 7.

[0048] Figure 10 (a) shows the HE staining results of the section described in Example 7.

[0049] Figure 10 (b) shows the results of scleroderma staining of the section described in Example 7.

[0050] Figure 11 In the middle (a), the CS / PVA / BBR mixture obtained in Comparative Example 1 is shown.

[0051] Figure 11 (b) is the OCS / PVA / BBR mixture obtained in Comparative Example 2.

[0052] Figure 11 (c) is the CMCS / PVA / OCS / BBR mixture obtained in Comparative Example 3.

[0053] Figure 11 In the middle (d), the CPOB (CS / PVA / OCS / BBR) hydrogel obtained in Example 3 is shown. Detailed Implementation

[0054] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the implementation of the present invention is not limited thereto.

[0055] Unless otherwise specified in the embodiments of this invention, the conditions shall be performed according to conventional conditions or conditions recommended by the manufacturer. All raw materials and reagents used, unless otherwise specified, are commercially available conventional products.

[0056] Example 1

[0057] (1) Preparation of chitosan / polyvinyl alcohol (CP) solution: 4 g of chitosan (CS) was dissolved in 100 mL of 1% acetic acid aqueous solution to obtain chitosan acetic acid solution; 5 g of polyvinyl alcohol was added to a round-bottom flask containing 100 mL of deionized water and placed in an oil bath at 85℃ and 300 rpm for 1 hour until completely dissolved to obtain polyvinyl alcohol solution. The chitosan acetic acid solution and polyvinyl alcohol solution were mixed at a volume ratio of 1:2 and stirred at 400 rpm at room temperature for 2 hours to obtain a homogeneous CP solution.

[0058] (2) Preparation of oxidized chitosan (OCS) solution: Add 0.1 g of oxidized chitosan to 5 mL of deionized water, vortex for 5 minutes, and disperse evenly to obtain OCS solution.

[0059] (3) Preparation of chitosan / polyvinyl alcohol / oxidized chitosan (CPO) prepolymer solution: The CP solution in step (1) and the OCS solution in step (2) are mixed in a volume ratio of 25:5, stirred at room temperature for 2 hours to obtain the CPO prepolymer solution.

[0060] (4) Preparation of berberine (BBR) solution: Add 0.15 g of berberine to 5 mL of deionized water, vortex for 5 minutes, and then heat in a water bath at 50°C for 30 minutes until completely dissolved to obtain berberine solution.

[0061] (5) Preparation of chitosan / polyvinyl alcohol / oxidized chitosan / berberine (CPOB) hydrogel: The CPO prepolymer solution from step (3) and the BBR solution from step (4) were mixed at a volume ratio of 30:5. The mixture was stirred at room temperature and mechanically at 800 rpm for 24 hours to finally obtain the CPOB hydrogel.

[0062] Example 2

[0063] (1) Preparation of chitosan / polyvinyl alcohol (CP) solution: 6 g of chitosan was dissolved in 100 mL of 2% (v / v) acetic acid aqueous solution to obtain chitosan acetic acid solution; 8 g of polyvinyl alcohol was added to a round-bottom flask containing 100 mL of deionized water and placed in an oil bath at 95℃ and 500 rpm for 2 hours until completely dissolved to obtain polyvinyl alcohol solution. The chitosan acetic acid solution and polyvinyl alcohol solution were mixed at a volume ratio of 1.5:3 and stirred at 600 rpm at room temperature for 4 hours to obtain a homogeneous CP solution.

[0064] (2) Preparation of oxidized chitosan (OCS) solution: Add 0.3 g of oxidized chitosan to 10 mL of deionized water, vortex for 10 minutes, and disperse evenly to obtain OCS solution.

[0065] (3) Preparation of chitosan / polyvinyl alcohol / oxidized chitosan (CPO) prepolymer solution: The CP solution in step (1) and the OCS solution in step (2) are mixed at a volume ratio of 30:10, stirred at room temperature for 3 hours to obtain the CPO prepolymer solution;

[0066] (4) Preparation of berberine (BBR) solution: Add 0.5g of berberine to 10 mL of deionized water, vortex for 10 minutes, and then heat in a water bath at 65℃ for 1.5 hours until completely dissolved to obtain berberine solution.

[0067] (5) Preparation of chitosan / polyvinyl alcohol / oxidized chitosan / berberine (CPOB) hydrogel: The CPO prepolymer solution from step (3) and the BBR solution from step (4) were mixed at a volume ratio of 40:10. The mixture was stirred at room temperature and mechanically at 1000 rpm for 48 hours to finally obtain the CPOB hydrogel.

[0068] Example 3

[0069] (1) Preparation of chitosan / polyvinyl alcohol (CP) solution: 5 g of chitosan was dissolved in 100 mL of 1.5% acetic acid aqueous solution to obtain chitosan acetic acid solution; 6.5 g of polyvinyl alcohol was added to a round-bottom flask containing 100 mL of deionized water and placed in an oil bath at 90℃ and 400 rpm, and stirred for 1.5 hours until completely dissolved to obtain polyvinyl alcohol solution. The chitosan acetic acid solution and polyvinyl alcohol solution were mixed at a volume ratio of 1.25:2.5 and stirred at 500 rpm at room temperature for 3 hours to obtain a homogeneous CP solution.

[0070] (2) Preparation of oxidized chitosan (OCS) solution: 0.2 g of oxidized chitosan was added to 7.5 mL of deionized water and vortexed for 7.5 minutes to disperse evenly and obtain OCS solution.

[0071] (3) Preparation of chitosan / polyvinyl alcohol / oxidized chitosan (CPO) prepolymer solution: The CP solution in step (1) and the OCS solution in step (2) are mixed in a volume ratio of 27.5:7.5, stirred at room temperature for 2.5 hours to obtain the CPO prepolymer solution.

[0072] (4) Preparation of berberine (BBR) solution: Add 0.325 g of berberine to 7.5 mL of deionized water, vortex for 7.5 minutes, and then heat in a water bath at 60℃ for 1 hour until completely dissolved to obtain berberine solution.

[0073] (5) Preparation of chitosan / polyvinyl alcohol / oxidized chitosan / berberine (CPOB) hydrogel: The CPO prepolymer solution from step (3) and the BBR solution from step (4) were mixed at a volume ratio of 35:7.5. The mixture was stirred at room temperature and mechanically at 900 rpm for 36 hours to finally obtain the CPOB hydrogel.

[0074] Example 4

[0075] (1) Preparation of chitosan / polyvinyl alcohol (CP) solution: 4 g of chitosan was dissolved in 100 mL of 2% acetic acid aqueous solution to obtain chitosan acetic acid solution; 5 g of polyvinyl alcohol was added to a round-bottom flask containing 100 mL of deionized water and placed in an oil bath at 95℃ and 300 rpm for 2 hours until completely dissolved to obtain polyvinyl alcohol solution. The chitosan acetic acid solution and polyvinyl alcohol solution were mixed at a volume ratio of 1:3 and stirred at 400 rpm at room temperature for 4 hours to obtain a homogeneous CP solution.

[0076] (2) Preparation of oxidized chitosan (OCS) solution: Add 0.1 g of oxidized chitosan to 10 mL of deionized water, vortex for 5 minutes, and disperse evenly to obtain OCS solution.

[0077] (3) Preparation of chitosan / polyvinyl alcohol / oxidized chitosan (CPO) prepolymer solution: The CP solution in step (1) and the OCS solution in step (2) are mixed at a volume ratio of 25:10, stirred at room temperature for 3 hours to obtain the CPO prepolymer solution.

[0078] (4) Preparation of berberine (BBR) solution: Add 0.15 g of berberine to 10 mL of deionized water, vortex for 5 minutes, and then heat in a water bath at 65℃ for 30 minutes until completely dissolved to obtain berberine solution.

[0079] (5) Preparation of chitosan / polyvinyl alcohol / oxidized chitosan / berberine (CPOB) hydrogel: The CPO prepolymer solution from step (3) and the BBR solution from step (4) were mixed at a volume ratio of 30:10, and stirred at room temperature for 48 hours at a mechanical stirring speed of 800 rpm to finally obtain CPOB hydrogel.

[0080] Example 5

[0081] (1) Preparation of chitosan / polyvinyl alcohol (CP) solution: 6 g of chitosan was dissolved in 100 mL of 1% acetic acid aqueous solution to obtain chitosan acetic acid solution; 8 g of polyvinyl alcohol was added to a round-bottom flask containing 100 mL of deionized water and placed in an oil bath at 85℃ and 500 rpm for 1 hour until completely dissolved to obtain polyvinyl alcohol solution. The chitosan acetic acid solution and polyvinyl alcohol solution were mixed at a volume ratio of 1.5:2 and stirred at 600 rpm at room temperature for 2 hours to obtain a homogeneous CP solution.

[0082] (2) Preparation of oxidized chitosan (OCS) solution: Add 0.3 g of oxidized chitosan to 5 mL of deionized water, vortex for 10 minutes, and disperse evenly to obtain OCS solution.

[0083] (3) Preparation of chitosan / polyvinyl alcohol / oxidized chitosan (CPO) prepolymer solution: The CP solution in step (1) and the OCS solution in step (2) are mixed in a volume ratio of 30:5, stirred at room temperature for 3 hours to obtain the CPO prepolymer solution.

[0084] (4) Preparation of berberine (BBR) solution: Add 0.15 g of berberine to 10 mL of deionized water, vortex for 5 minutes, and then heat in a water bath at 65℃ for 0.5 hours until completely dissolved to obtain berberine solution.

[0085] (5) Preparation of chitosan / polyvinyl alcohol / oxidized chitosan / berberine (CPOB) hydrogel: The CPO prepolymer solution from step (3) and the BBR solution from step (4) were mixed at a volume ratio of 40:5. The mixture was stirred at room temperature and mechanically at 1000 rpm for 24 hours to finally obtain the CPOB hydrogel.

[0086] Example 6: Antibacterial Performance Test of Hydrogel

[0087] (1) Preparation of liquid and solid culture media: Liquid culture media: Add 25 g of LB broth powder to 1 L of ultrapure water and stir well; Solid culture media: Add 25 g of LB broth powder to 1 L of ultrapure water, stir well, then add 20 g of agar powder and stir well. Put the liquid culture media, solid culture media, centrifuge tubes, and coating steel beads into an autoclave and sterilize at 120℃ for 15 minutes.

[0088] (2) Bacterial activation: Take 50 μL of each of glycerol-preserved Escherichia coli (ATCC 25922) and methicillin-resistant Staphylococcus aureus (MRSA, ATCC 43300) and add them to a 50 mL centrifuge tube containing 30 mL of liquid culture medium. Incubate at 37 °C and 120 rpm in a shaker for 12 hours.

[0089] (3) Bacterial purification: Take 3 ml of the bacterial solution obtained in step (2) and 3 ml of blank liquid culture medium, and measure the absorbance at a wavelength of 600 nm using a UV spectrophotometer. After diluting the bacterial solution, adjust the absorbance to 1 OD, and then dilute the bacterial solution 1000 times. Spread the solution on a plate using the streak plate method. Place the plate in a 37℃ incubator and incubate for 12 hours.

[0090] (4) Preparation of bacterial culture: Pick a single colony from the plate and place it in the liquid culture medium. Mix well by blowing and aspiration. Incubate at 37°C and 120 rpm in a shaker for 12 hours. Take 3 ml of the bacterial culture and 3 ml of blank liquid culture medium. Measure the absorbance at a wavelength of 600 nm using a UV spectrophotometer. Adjust the absorbance to 1 OD after diluting the bacterial culture. Then dilute the bacterial culture 100 times for later use.

[0091] (5) Co-culture: Take 5 mg of the lyophilized gel from Example 4, add the diluted bacterial solution from step (4), and co-culture at 37°C and 120 rpm for 12 hours.

[0092] (6) Plate spreading: Take the co-cultured bacterial solution, dilute it 10 times with liquid culture medium, take 100 μL of diluted bacterial solution and spread it on a plate, and place the plate in a 37℃ incubator for 12 hours.

[0093] In addition, the blank gel and control group were treated in the same way as in steps (1) to (6) above. The only difference was that the gel added in step (5) was a blank CPO gel without BBR loading and a blank control group with added physiological saline. All other methods and steps were the same.

[0094] (7) Antibacterial rate calculation: Take out the plate, take a picture and record the colony count, and calculate the antibacterial rate according to the formula:

[0095]

[0096] In the formula, W c W represents the colony count of the control group. t This represents the number of colonies in the experimental group.

[0097] Example 7: Application of hydrogel dressings in skin trauma

[0098] (1) Preparation before the experiment: Purchase Kunming mice and raise them in the animal experiment center for a week, allowing them to eat and drink freely, so that the mice can adapt to the environment.

[0099] (2) Skin preparation: After anesthetizing the mice by inhalation anesthesia with 5% isoflurane, the hair was shaved off and disinfected with povidone-iodine.

[0100] (3) Preparation of infected wound: A full-thickness skin defect model was created on the back of a mouse using a 1 cm biopsy puncture device. 100 μL of a 1×10⁻⁶ solution was dripped onto the wound. 5 A full-thickness skin wound infection model was constructed using MRSA bacterial suspension at CFU / mL.

[0101] (4) Wound treatment: The wound was covered with unloaded BBR CPO blank gel and Example 4 CPOB hydrogel, while the blank control group was not treated.

[0102] (5) Sample collection: The changes in the wound were recorded with a digital camera on days 0, 3, 7 and 14. The dressing on the skin surface was removed, and the tissue around the wound was taken out, soaked in formalin and fixed for later use.

[0103] (6) Tissue embedding: The tissue soaked in formalin was removed, cleaned with phosphate buffer solution, and then dehydrated with ethanol solutions of different concentration gradients (50%, 70%, 80%, 95%, 100% I (i.e., first dehydration with anhydrous ethanol) and 100% II (i.e., second dehydration with anhydrous ethanol)). The tissue was then cleared twice with xylene, 30 minutes each time. Next, xylene and paraffin solution were mixed in a 1:1 ratio, and the cleared tissue was immersed in the mixture for 30 minutes, followed by 30 minutes in pure paraffin solution. The paraffin-embedded tissue was then encased in paraffin, with the tissue block placed in the center of the paraffin layer.

[0104] (7) Section preparation: Fix the embedded paraffin tissue block on the microtome, adjust the section thickness, first coarsely cut to the center of the wound tissue, and then set the thickness to 5 μm. Take one section every 5 slides, and keep 5 sections for each sample. Add a drop of hot water to the glass slide, place the cut tissue paraffin section into the hot water to flatten it, tilt the glass slide, and then the tissue section will adhere flatly to the glass slide. Place the glass slide in a 60℃ slide oven for 50 min, and store at low temperature for later use.

[0105] (8) HE staining: The paraffin tissue was placed at room temperature for 30 minutes. Then, it was dewaxed to water: soaked in xylene for 30 minutes, xylene:ethanol (1:1) for 30 minutes, 95% ethanol for 10 minutes, 90% ethanol for 10 minutes, 85% ethanol for 10 minutes, 80% ethanol for 10 minutes, 75% ethanol for 10 minutes, and ultrapure water for 5 minutes. Then, the sections were immersed in a hematoxylin staining tank for 15 minutes, differentiated with 1% hydrochloric acid ethanol for 6 seconds, rinsed with running water for 20 minutes, and stained with eosin for 3 minutes. Finally, the sections were cleared and mounted, and images were acquired using an inverted microscope.

[0106] (9) Masson staining: After dewaxing the paraffin sections to water as described in the HE staining procedure above, use the Masson trichrome staining kit (purchased from Seville Biotechnology, catalog number G1006-100ML) for staining. First, add Masson A solution and soak at room temperature. Then, incubate the sections in a 65℃ oven for 30 minutes, while preheating Masson D and F solutions in a 65℃ oven. Wash with tap water for 30 seconds until the yellow color on the tissue fades. Mix equal volumes of Masson B and C solutions (prepare fresh) and immerse the sections in the mixture for 1 minute. Wash away excess staining solution with running water, drain slightly (do not dry the sections), add Masson E solution and immerse for 1 minute. Differentiate until the collagen fibers are light red and the fibers are red. After draining the Masson E solution slightly, add Masson F solution and immerse for 20 seconds. Then, rinse the sections in 1% glacial acetic acid to differentiate. Make the sections transparent, mount with neutral resin, and finally observe and acquire images using an inverted microscope.

[0107] Figure 4 As shown, the swelling ratios of CPOB hydrogel in PBS buffer solutions at pH 5.6, 7.4, and 9.5 were 438.29%, 537.80%, and 754.62%, respectively. The swelling ratio gradually increased with rising pH, a phenomenon consistent with pH changes during wound healing. Therefore, the swelling properties of CPOB hydrogel are beneficial for absorbing wound exudate.

[0108] Figure 5 As shown, after 24 hours of soaking in physiological saline, the volume of CPOB hydrogel did not increase significantly, proving that after CPOB hydrogel absorbs water, the gel network expands only slightly and does not collapse, and the gel network remains stable.

[0109] Figure 6 As shown in Figure (a), the adhesion strength of CPOB hydrogel to pigskin is 243.82 kPa, significantly higher than that of CPO gel (45.96 kPa). This indicates that the addition of berberine helps increase the stability and adhesion of the gel, effectively sealing wounds and reducing secondary microbial contamination in the environment.

[0110] Figure 6As shown in (b), the CPOB hydrogel adheres well to metal, glass, plastic, pigskin, and gloves. It also adheres firmly to the surface of pigskin and gloves by bending and twisting, demonstrating that the CPOB hydrogel has excellent adhesion properties and is widely applicable to different scenarios.

[0111] Figure 7 As shown, the CPOB hydrogel exhibited inhibition rates of 95.92% and 98.64% against Escherichia coli and Staphylococcus aureus, respectively, significantly higher than the inhibition rates of 38.45% and 53.99% in the CPO blank hydrogel group. These results indicate that the CPOB hydrogel possesses excellent antibacterial properties, demonstrating that the addition of berberine significantly enhances its antibacterial performance.

[0112] Figure 8 As shown, the wound area of ​​the CPOB hydrogel-treated wounds at days 0, 3, 7, and 14 was significantly smaller on day 3 than that of the CPO and blank control groups. The wound area reduction trend on day 7 was consistent with that on day 3. By day 14, the wounds in the CPOB hydrogel-treated group were almost completely healed, with hair regrowth around the wound and no obvious scarring. The wound area in the CPO group was significantly larger than that in the CPOB group, and there was less hair growth around the wound. The blank control group had the largest wound area, less hair growth around the wound, and obvious scarring around the wound. The results indicate that CPOB hydrogel has the best effect on promoting wound healing, and the addition of berberine is beneficial in promoting wound healing.

[0113] Figure 9 As shown, the wound area results obtained after quantifying the wounds on days 3, 7, and 14 as described in step (5) of Example 7 can be visually displayed to compare the wound areas of different treatment groups of CPO and CPOB hydrogels at the same time points. The results show that the wound area of ​​the CPOB hydrogel group was significantly smaller than that of the CPO hydrogel group on days 3, 7, and 14, and the skin wound was almost completely healed on day 14, proving that the addition of berberine can significantly promote the wound healing process, indicating that CPOB hydrogel has great potential in the field of clinical application.

[0114] Figure 10 As shown in (a), the HE staining results of the wound obtained in step (8) of Example 7 on day 14 show that the number of new blood vessels (red arrows) and glands (green arrows) formed in the skin wound treated with CPOB gel were significantly higher than those in the CPO group and the blank control group. This is attributed to the good wound sealing performance and wound exudate management ability of CPOB hydrogel, which reduces bacterial growth and has excellent antibacterial properties, reduces wound infection, and thus promotes wound healing.

[0115] Figure 10As shown in Figure (b), the wound obtained in step (9) of Example 7 was stained with Masson's stain on tissue sections on day 14. The wound treated with CPOB hydrogel almost completely healed and was covered with hair follicles and glands (green arrows). The newly formed granulation tissue dissipated, and the blood vessels (red arrows) were evenly distributed, indicating that skin function had returned to normal. The skin in the CPO hydrogel-treated group still had defects, with obvious newly formed granulation tissue and concentrated blood vessels (red arrows). Healthy tissue did not completely cover the wound, and glands and hair follicles were only distributed at the junction of healthy skin and newly formed skin. Skin function had not yet fully recovered. The skin wound in the blank control group still had large defects, had not formed complete epithelial tissue, and had no obvious granulation tissue, hair follicles, or glands at the wound site. The results show that CPOB hydrogel can significantly promote skin wound healing, reduce scar formation, and promote wound function recovery.

[0116] In summary, a water-absorbing, low-swelling, antibacterial CPOB hydrogel dressing was prepared using simple mechanical stirring and a one-pot method. The CPOB hydrogel dressing exhibits good water absorption, appropriate swelling properties, and good adhesion, while also possessing excellent antibacterial properties. This provides effective exudate management for skin wounds, long-lasting and stable interfacial protection, and significantly promotes skin wound healing.

[0117] Comparative Example 1

[0118] (1) Preparation of chitosan / polyvinyl alcohol (CP) solution: 5 g of chitosan was dissolved in 100 mL of 1.5% acetic acid aqueous solution to obtain chitosan acetic acid solution; 6.5 g of polyvinyl alcohol was added to a round-bottom flask containing 100 mL of deionized water and placed in an oil bath at 90℃ and 400 rpm, and stirred for 1.5 hours until completely dissolved to obtain polyvinyl alcohol solution. The chitosan acetic acid solution and polyvinyl alcohol solution were mixed at a volume ratio of 1.25:2.5 and stirred at 500 rpm at room temperature for 3 hours to obtain a homogeneous CP solution.

[0119] (2) Preparation of blank solvent: Take 7.5 mL of deionized water and vortex for 7.5 minutes to obtain a blank aqueous solution.

[0120] (3) Preparation of chitosan / polyvinyl alcohol (CP) prepolymer solution: The CP solution in step (1) and the blank aqueous solution in step (2) are mixed in a volume ratio of 27.5:7.5, stirred at room temperature for 2.5 hours to obtain the CP prepolymer solution.

[0121] (4) Preparation of berberine (BBR) solution: Add 0.325 g of berberine to 7.5 mL of deionized water, vortex for 7.5 minutes, and then heat in a water bath at 60℃ for 1 hour until completely dissolved to obtain berberine solution.

[0122] (5) Preparation of chitosan / polyvinyl alcohol / berberine (CPB) solution: The CP prepolymer solution from step (3) and the BBR solution from step (4) are mixed at a volume ratio of 35:7.5. The mixture is stirred at room temperature and mechanically at 900 rpm for 36 hours to finally obtain the CS / PVA / BBR (i.e., CPB) solution.

[0123] Compared to Example 3, the CPB system without added chitosan did not gel ( Figure 11 (a) The lack of aldehyde (-CHO) functional groups provided by oxidized chitosan (OCS) in the system prevents the Schiff base covalent cross-linking reaction from occurring, resulting in the absence of the main framework of the gel network and making it difficult to form a three-dimensional gel framework.

[0124] Comparative Example 2

[0125] (1) Preparation of PVA solution: 6.5 g of polyvinyl alcohol was added to a round-bottom flask containing 100 mL of deionized water and placed in an oil bath at 90 °C and 400 rpm. The mixture was stirred for 1.5 hours until completely dissolved to obtain a polyvinyl alcohol solution. A 1.5% aqueous acetic acid solution was mixed with the polyvinyl alcohol solution at a volume ratio of 1.25:2.5 and stirred at 500 rpm at room temperature for 3 hours to obtain a homogeneous PVA base solution.

[0126] (2) Preparation of oxidized chitosan (OCS) solution: 0.2 g of oxidized chitosan was added to 7.5 mL of deionized water and vortexed for 7.5 minutes to disperse evenly and obtain OCS solution.

[0127] (3) Preparation of polyvinyl alcohol / chitosan oxide (PVA-OCS) prepolymer solution: The PVA base solution of step (1) and the OCS solution of step (2) are mixed in a volume ratio of 27.5:7.5 and stirred at room temperature for 2.5 hours to obtain PVA-OCS prepolymer solution.

[0128] (4) Preparation of berberine (BBR) solution: Add 0.325 g of berberine to 7.5 mL of deionized water, vortex for 7.5 minutes, and then heat in a water bath at 60℃ for 1 hour until completely dissolved to obtain berberine solution.

[0129] (5) Preparation of polyvinyl alcohol / chitosan oxide / berberine (POB) solution: The PVA-OCS prepolymer solution from step (3) and the BBR solution from step (4) were mixed at a volume ratio of 35:7.5. The mixture was stirred at room temperature and mechanical stirring speed of 900 rpm for 36 hours to finally obtain the polyvinyl alcohol-chitosan oxide-berberine (OCS / PVA / BBR) solution.

[0130] Compared to Example 3, the POB system without chitosan did not gel ( Figure 11 (b) Chitosan is the only amino donor for Schiff base crosslinking in this system. Without chitosan, the Schiff base reaction cannot occur, and the system loses the main network framework required to form a gel; the remaining weak interactions such as hydrogen bonds and electrostatic interactions are insufficient to independently support the three-dimensional crosslinked network, so the system can only exist in solution.

[0131] Comparative Example 3

[0132] (1) Preparation of CMCS-PVA solution (CMP): 5 g of carboxymethyl chitosan was dissolved in 100 mL of deionized water to obtain a carboxymethyl chitosan aqueous solution; 6.5 g of polyvinyl alcohol was added to a round-bottom flask containing 100 mL of deionized water and placed in an oil bath at 90℃ and 400 rpm, and stirred for 1.5 hours until completely dissolved to obtain a polyvinyl alcohol solution. The carboxymethyl chitosan aqueous solution and the polyvinyl alcohol solution were mixed at a volume ratio of 1.25:2.5 and stirred at 500 rpm at room temperature for 3 hours to obtain a homogeneous solution.

[0133] (2) Preparation of oxidized chitosan (OCS) solution: 0.2 g of oxidized chitosan was added to 7.5 mL of deionized water and vortexed for 7.5 minutes to disperse evenly and obtain OCS solution.

[0134] (3) Preparation of carboxymethyl chitosan / polyvinyl alcohol / oxidized chitosan (CMPO) prepolymer solution: The CMP solution in step (1) and the OCS solution in step (2) are mixed in a volume ratio of 27.5:7.5, stirred at room temperature for 2.5 hours to obtain CMPO solution.

[0135] (4) Preparation of berberine (BBR) solution: Add 0.325 g of berberine to 7.5 mL of deionized water, vortex for 7.5 minutes, and then heat in a water bath at 60℃ for 1 hour until completely dissolved to obtain berberine solution.

[0136] (5) Preparation of carboxymethyl chitosan / polyvinyl alcohol / chitosan oxide / berberine solution (CMPOB): The CMPO prepolymer solution from step (3) and the BBR solution from step (4) are mixed at a volume ratio of 35:7.5. The mixture is stirred at room temperature and mechanically at a speed of 900 rpm for 36 hours to finally obtain the carboxymethyl chitosan-polyvinyl alcohol-chitosan oxide-berberine solution (CMCS / PVA / OCS / BBR).

[0137] Compared to Example 3, the CMPOB system in which chitosan was replaced with carboxymethyl chitosan did not gel ( Figure 11(c) The reason is that the number of amino groups on the carboxymethyl chitosan molecular chain is significantly reduced, which weakens the Schiff base crosslinking. At the same time, the strong electrostatic adsorption between the carboxyl groups on the molecular chain and the berberine quaternary ammonium groups prevents berberine from being uniformly dispersed to participate in physical crosslinking. Neither of the dual crosslinking mechanisms can be effectively established, resulting in the system failing to reach the critical crosslinking density for gelation.

[0138] Comparative Example 4

[0139] (1) Preparation of chitosan / polyvinyl alcohol (CP) solution: 5 g of chitosan was dissolved in 100 mL of 1.5% acetic acid aqueous solution to obtain chitosan acetic acid solution; 6.5 g of polyvinyl alcohol was added to a round-bottom flask containing 100 mL of deionized water and placed in an oil bath at 90℃ and 400 rpm, and stirred for 1.5 hours until completely dissolved to obtain polyvinyl alcohol solution. The chitosan acetic acid solution and polyvinyl alcohol solution were mixed at a volume ratio of 1.25:2.5 and stirred at 500 rpm at room temperature for 3 hours to obtain a homogeneous CP solution.

[0140] (2) Preparation of oxidized chitosan (OCS) solution: 0.2 g of oxidized chitosan was added to 7.5 mL of deionized water and vortexed for 7.5 minutes to disperse evenly and obtain OCS solution.

[0141] (3) Preparation of chitosan / polyvinyl alcohol / oxidized chitosan (CPO) prepolymer solution: The CP solution in step (1) and the OCS solution in step (2) are mixed in a volume ratio of 27.5:7.5, stirred at room temperature for 2.5 hours to obtain the CPO prepolymer solution.

[0142] (4) Preparation of CPO gel: CPO gel is obtained by continuously stirring the CPO prepolymer solution from step (3) for 48 hours.

[0143] Compared to Example 3, the CPO gel exhibits a significantly prolonged gelation time. The CPO gel primarily relies on the Schiff base dynamic covalent cross-linking between chitosan and oxidized chitosan as the driving force for gelation. Lacking the physical cross-linking, electrostatic, and hydrophobic interactions provided by berberine, the intermolecular forces are weakened, and the cross-linking is reduced. Therefore, the time required to reach the critical cross-linking density for gelation is significantly prolonged. The hydrophobic, adhesive, antibacterial, and wound-healing properties of the CPO gel are significantly weaker than those of the CPOB gel obtained in Example 3.

[0144] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a water-absorbing, low-swelling, antibacterial hydrogel dressing, characterized in that, Includes the following steps: (1) Mix chitosan acetic acid solution and polyvinyl alcohol aqueous solution evenly to obtain chitosan / polyvinyl alcohol mixed solution; (2) Add an aqueous solution of oxidized chitosan to the chitosan / polyvinyl alcohol mixed solution and mix evenly to obtain a chitosan / polyvinyl alcohol / oxidized chitosan mixed solution; (3) Add berberine aqueous solution to the chitosan / polyvinyl alcohol / oxidized chitosan mixed solution and stir to form a gel to obtain a water-absorbing, low-swelling, antibacterial hydrogel dressing.

2. The preparation method according to claim 1, characterized in that, In step (3), the ratio of berberine to water in the berberine aqueous solution is (0.15-0.5) g : (5-10) mL; And / or, the volume ratio of the chitosan / polyvinyl alcohol / oxidized chitosan mixed solution to the berberine aqueous solution in step (3) is (30-40):(5-10).

3. The preparation method according to claim 1 or 2, characterized in that, In step (2), the ratio of oxidized chitosan to water in the oxidized chitosan aqueous solution is (0.1-0.3) g : (5-10) mL; And / or, the volume ratio of the chitosan / polyvinyl alcohol mixed solution to the oxidized chitosan aqueous solution in step (2) is (25-30): (5-10).

4. The preparation method according to claim 1 or 2, characterized in that, In step (1), the ratio of chitosan to water in the chitosan-acetic acid aqueous solution is (4-6) g: 100 mL; the volume ratio of acetic acid to water is (1-2):

100. And / or, in the polyvinyl alcohol aqueous solution described in step (1), the ratio of polyvinyl alcohol to water is (5-8) g: 100 mL; And / or, the volume ratio of the chitosan acetic acid solution and the polyvinyl alcohol aqueous solution in step (1) is (1-1.5):(2-3).

5. The preparation method according to claim 1 or 2, characterized in that, In step (3), the stirring speed is 800-1000 rpm, the temperature is room temperature, and the stirring time is 24-48 hours.

6. The preparation method according to claim 1 or 2, characterized in that, The berberine aqueous solution in step (3) is obtained by adding berberine to water, vortexing for 5 to 10 minutes, and then heating at 50 to 65°C for 0.5 to 1.5 hours. And / or, the oxidized chitosan aqueous solution in step (2) is obtained by adding oxidized chitosan to water and then vortexing for 5 to 10 minutes to disperse it evenly; And / or, the term "mixing evenly" in step (2) refers to stirring at room temperature for 2 to 3 hours.

7. The preparation method according to claim 1 or 2, characterized in that, The chitosan-acetic acid aqueous solution in step (1) is obtained by dissolving chitosan in an acetic acid solution; And / or, the polyvinyl alcohol aqueous solution in step (1) is obtained by dissolving polyvinyl alcohol in water at a temperature of 85-95°C for 1-2 hours and a rotation speed of 300-500 rpm. And / or, the mixing in step (1) is carried out by mechanical stirring at a speed of 400-600 rpm, at room temperature, for 2-4 hours.

8. A water-absorbing, low-swelling, antibacterial hydrogel dressing prepared by the preparation method according to any one of claims 1 to 7.

9. The non-therapeutic use of the water-absorbing, low-swelling, antibacterial hydrogel dressing of claim 8.

10. The application according to claim 9, characterized in that, Application of the water-absorbing, low-swelling, antibacterial hydrogel dressing in the preparation of materials for full-thickness skin wound infection.

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