Hydrogel adhesive for repairing acute and chronic infected wounds and inhibiting scarring and method of making and use thereof

Abstract

The present application relates to a hydrogel adhesive for repairing acute and chronic infected wounds and inhibiting scars, and a preparation method and use thereof. Specifically, the present application provides a hydrogel adhesive comprising a) 3-aminophenylboronic acid modified hyaluronic acid (HA-PBA), and b) a short-chain molecule M containing two or more amino groups. The hydrogel adhesive has good wound adhesion, bactericidal, inhibiting scar hyperplasia and other biological properties, and can be used as a hydrogel adhesive for repairing acute and chronic infected wounds.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, specifically relating to a hydrogel adhesive for repairing acute and chronic infected wounds and inhibiting scarring, as well as its preparation method and uses. Background Technology

[0002] The skin is the largest organ in the human body. When the skin is injured, its barrier function is disrupted, potentially leading to infection and delayed wound healing. In severe cases, the infection can spread throughout the body via blood vessels or lymphatic vessels, causing systemic infection and ultimately endangering life. In recent years, the aging population, the increasing prevalence of diabetes and obesity, and the rising frequency of surgical procedures have significantly increased the risk of microbial infection and delayed wound healing, resulting in chronic wounds. Chronic wounds affect approximately 20 million people worldwide, with estimated annual costs for their treatment and management exceeding $31 billion, placing a significant clinical and economic burden on global healthcare systems. Scar formation is an inevitable result of the skin's repair process after exposure to damaging external factors, and it is related to race, sex, age, wound tension, location, and injury pattern. When damage reaches the deep reticular layer of the dermis, it can trigger an abnormal fibrous hyperplasia response, leading to hypertrophic scars. Hypertrophic scars are typically characterized by excessive collagen production and an imbalance in the ratio of type I to type III collagen. It is estimated that approximately 35% of postoperative scar patients develop hypertrophic scars within one year. Besides localized skin damage, scars often cause symptoms such as itching and pain. Severe scar contractures in functional areas can significantly impair mobility and place a considerable burden on patients' quality of life and mental well-being. Therefore, developing strategies that both inhibit bacterial infection and suppress scar hyperplasia is essential.

[0003] Macrophages are crucial immunoregulatory cells in inflammatory responses, manifesting as pro-inflammatory M1 and anti-inflammatory M2 types. M1 macrophages engulf dead cells and bacteria, secreting pro-inflammatory factors that disrupt collagen deposition. M2 macrophages secrete various anti-inflammatory factors, promoting collagen deposition and exhibiting anti-inflammatory, angiogenesis-promoting, tissue repair-promoting, and scar-inhibiting effects. During wound infection, bacteria adhere extensively to the wound site, and macrophages manifest as M1, leading to acute inflammation. However, if the body's immune system fails to eliminate bacteria promptly during the acute inflammatory phase, resulting in persistent infection, macrophages will continue to differentiate into M1 types, inducing chronic inflammation. Excessive release of various pro-inflammatory cytokines hinders the transition of tissue repair to the proliferative remodeling stage, prolonging wound healing time and transforming acute wounds into chronic wounds. At this stage, fibroblast proliferation is active, and excessive collagen deposition leads to the formation of hypertrophic scars at the wound site. Therefore, effectively regulating the temporal polarization of macrophage functional phenotypes to exert antibacterial, tissue-healing-promoting, and scar-eliminating effects is of great significance for the repair of infected wounds.

[0004] Therefore, there is an urgent need in the field to develop a simple hydrogel for repairing acute and / or chronic infected wounds and inhibiting scarring, in order to clear infection, promote repair and inhibit scar hyperplasia, and overcome the shortcomings of existing technologies. Summary of the Invention

[0005] This invention provides a hydrogel adhesive for repairing acute and chronic infected wounds and inhibiting scarring, as well as its preparation method and uses. The hydrogel is spontaneously formed by cross-linking modified hyaluronic acid with amino acids, achieving functions such as immune response regulation, antibacterial activity, tissue repair promotion, and scar inhibition. It has high clinical application value in the treatment of acute and chronic infected wounds.

[0006] In a first aspect, the present invention provides a hydrogel adhesive comprising: a) hyaluronic acid modified with 3-aminophenylboronic acid (HA-PBA), and b) a short-chain molecule M containing at least two amino groups.

[0007] In another preferred embodiment, the modification may also be performed by groups or reagents selected from the group consisting of hydrazine reagents, methacrylic anhydride, and free thiol groups.

[0008] In another preferred embodiment, the grafting rate of the modifying group or reagent onto HA is 5%-90%.

[0009] In another preferred embodiment, the grafting rate of the modifying group or reagent onto HA is 25%-35%.

[0010] In another preferred embodiment, the concentration of the modified hyaluronic acid (HA) is 0.001–5 g / mL.

[0011] In another preferred embodiment, the short-chain molecule M is selected from the group consisting of L-arginine, D-arginine, and L-lysine.

[0012] In another preferred embodiment, the content of the short-chain molecule M in the hydrogel adhesive is 5-1300 mM.

[0013] In another preferred embodiment, the short-chain molecule M is present in a content of 10-1000 mM in the hydrogel adhesive.

[0014] In another preferred embodiment, the short-chain molecule M is present in the hydrogel adhesive at a concentration of 50-800 mM.

[0015] In another preferred embodiment, the content of the short-chain molecule M in the hydrogel adhesive is 100-500 mM.

[0016] In another preferred embodiment, the main carbon chain length of the short-chain molecule M is not less than 3 carbon atoms, preferably 3-5.

[0017] In another preferred embodiment, the short-chain molecule M has at least two amino groups.

[0018] In another preferred embodiment, the short-chain molecule M has a non-coplanar amino structure.

[0019] In another preferred embodiment, the short-chain molecule M participates in gelation as a crosslinking component.

[0020] In another preferred embodiment, the hydrogel adhesive has one or more features selected from the group consisting of:

[0021] (1) It has good wound adhesion properties;

[0022] (2) Immunomodulatory effects;

[0023] (3) Bactericidal effect;

[0024] (4) Inhibit scar hyperplasia.

[0025] A second aspect of the present invention provides a method for preparing a hydrogel adhesive as described in the first aspect of the present invention, wherein the method is selected from the group consisting of:

[0026] Method 1: Dissolve the modified HA in a solution containing short-chain molecule M, mix thoroughly and evenly to obtain a hydrogel;

[0027] Method 2: Dissolve the short-chain molecule M in a solution of modified HA, mix thoroughly, and obtain a hydrogel;

[0028] Method 3: Mix the modified HA solution with a solution containing short-chain molecule M, mix thoroughly and evenly to obtain a hydrogel;

[0029] The modification refers to modification by a group or reagent selected from the group consisting of: 3-aminophenylboronic acid, hydrazine reagents, methacrylic anhydride, and free thiol groups.

[0030] In another preferred embodiment, the mass ratio of the modified HA to the solution containing the short-chain molecule M in method 1 is 10:1 to 1:20.

[0031] In another preferred embodiment, the mass ratio of the modified HA to the solution containing the short-chain molecule M in method 1 is 5:1 to 1:10.

[0032] In another preferred embodiment, the mass ratio of the modified HA and the solution containing the short-chain molecule M in method 1 is 2:1 to 1:5.

[0033] In another preferred embodiment, the solution in the step is selected from the group consisting of water, physiological saline, and phosphate buffer solution.

[0034] In another preferred embodiment, the solvent is a phosphate buffer solution.

[0035] In another preferred embodiment, the short-chain molecules in the step are used as crosslinking components to prepare the hydrogel adhesive.

[0036] A third aspect of the invention provides the use of the hydrogel adhesive as described in the first aspect of the invention, for repairing acute and chronic infected wounds and inhibiting scar hyperplasia.

[0037] A fourth aspect of the present invention provides a method for repairing acute and chronic infected wounds and inhibiting scar hyperplasia, comprising applying a hydrogel adhesive as described in the first aspect of the present invention to the wound.

[0038] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0039] Figure 1 The images show the molecular structure and gel formation of L-Arg crosslinked HA-PBA. Specifically, (a) gel formation images of 2.5% (w / v) HA-PBA solution with L-Arg powder; (b) gel formation images of 2.5% (w / v) HA-PBA solid with L-Arg solutions of different concentrations; and (c) gel formation images of 2.5% (w / v) HA-PBA solution with L-Arg solutions of different concentrations.

[0040] Figure 2 Low- and high-magnification scanning electron microscope images of the lyophilized hydrogel are shown.

[0041] Figure 3 The cell proliferation after co-incubation of hydrogel and macrophages was shown.

[0042] Figure 4 The cell proliferation after co-incubation of the hydrogel with mouse fibroblasts was shown.

[0043] Figure 5 Images of bacterial colonies and bacterial activity after co-incubation of hydrogel with Staphylococcus aureus are shown.

[0044] Figure 6 Images of live / dead bacteria and bacterial activity obtained by hydrogel immunotherapy against Staphylococcus aureus are shown.

[0045] Figure 7 Representative images of infected wounds from each group are shown on days 0, 3, 7, and 14.

[0046] Figure 8 HE staining images of the tissues surrounding the wounds in each group are shown on day 14.

[0047] Figure 9 The images show Sirius red stained polarized light images of the tissues surrounding the wounds in each group on day 14.

[0048] Figure 10 Representative α-SMA staining images of the tissues surrounding the wound in each group are shown on day 7 and day 14.

[0049] Figure 11 Immunofluorescence staining images of macrophages in the tissue surrounding the wound in each group are shown on day 7.

[0050] Figure 12 The images show the molecular structures and gel images of D-arginine (D-Arg) and L-lysine (L-Lys) crosslinked with HA-PBA, respectively.

[0051] Figure 13 Images of HA-PBA solutions at different concentrations are shown.

[0052] Figure 14 Images show unmodified PBA-containing HA mixed with L-Arg solutions of different concentrations.

[0053] Figure 15 Images show a mixture of L-histidine (L-His), glycine (Gly), and serine (Ser) solutions with HA-PBA. Detailed Implementation

[0054] Through extensive and in-depth research, the inventors unexpectedly developed a type of hydrogel adhesive capable of repairing acute and chronic infected wounds and inhibiting scarring. This hydrogel adhesive is formed spontaneously by modified hyaluronic acid, particularly hyaluronic acid modified with 3-aminophenylboronic acid (HA-PBA), and amino acid molecules containing two or more amino groups. It achieves immune response regulation, antibacterial properties, promotes tissue repair, and inhibits scarring, demonstrating high clinical application value in treating acute and chronic infected wounds. Based on this, the present invention was completed.

[0055] Hyaluronic acid (HA)

[0056] Hyaluronic acid (HA)-based hydrogels have attracted considerable attention in the treatment of chronic wounds due to their unique three-dimensional network structure, high water content, and excellent biocompatibility. Based on their high designability, researchers can further endow HA-based hydrogels with rich biological functions through doping with nanomaterials, chemical modification, and physical integration, thereby improving their clinical application prospects. However, the composition and preparation process of current hydrogels are complex, and the development of HA-based hydrogel adhesives with both antibacterial and scar-inhibiting functions is still limited. Amino acids are a class of short-chain natural compounds containing two or more non-coplanar amino groups, including L-arginine, D-arginine, and lysine. They can participate in the formation of hydrogels as cross-linking components, endowing them with antibacterial, tissue-repair-promoting, and scar-inhibiting functions.

[0057] This invention relates to a hydrogel adhesive and its preparation method.

[0058] As used herein, "hydrogel adhesive of the present invention," "HA-PBA hydrogel adhesive of the present invention," or "hydrogel of the present invention" all refer to hydrogels formed by crosslinking HA-PBA with amino acids containing at least two amino groups. Specifically, the term "HA-PBA hydrogel adhesive of the present invention with L-Arg crosslinking" refers to a hydrogel formed by crosslinking HA-PBA with L-Arg. Other similar terms have similar meanings.

[0059] As used herein, “HA-PBA” refers to hyaluronic acid (HA) modified with 3-aminophenylboronic acid (PBA).

[0060] The present invention provides a hydrogel adhesive comprising: a) hyaluronic acid modified with 3-aminophenylboronic acid (HA-PBA), and b) a short-chain molecule M containing at least two amino groups.

[0061] In another preferred embodiment, the modification may also be performed by groups or reagents selected from the group consisting of hydrazine reagents, methacrylic anhydride, and free thiol groups.

[0062] In another preferred embodiment, the grafting rate of the modifying group or reagent onto HA is 5%-90%.

[0063] In another preferred embodiment, the grafting rate of the modifying group or reagent onto HA is 25%-35%.

[0064] In another preferred embodiment, the concentration of the modified hyaluronic acid (HA) is 0.001–5 g / mL.

[0065] In another preferred embodiment, the short-chain molecule M may be selected from the group consisting of L-arginine, D-arginine, and L-lysine.

[0066] In another preferred embodiment, the content of the short-chain molecule M in the hydrogel adhesive is 5-1300 mM.

[0067] In another preferred embodiment, the short-chain molecule M is present in a content of 10-1000 mM in the hydrogel adhesive.

[0068] In another preferred embodiment, the short-chain molecule M is present in the hydrogel adhesive at a concentration of 50-800 mM.

[0069] In another preferred embodiment, the content of the short-chain molecule M in the hydrogel adhesive is 100-500 mM.

[0070] In another preferred embodiment, the main carbon chain length of the short-chain molecule M is not less than 3 carbon atoms, preferably 3-5.

[0071] In another preferred embodiment, the short-chain molecule M has at least two amino groups.

[0072] In another preferred embodiment, the short-chain molecule M has a non-coplanar amino structure.

[0073] In another preferred embodiment, the short-chain molecule M participates in gelation as a crosslinking component.

[0074] In another preferred embodiment, the hydrogel adhesive has one or more features selected from the group consisting of:

[0075] (1) It has good wound adhesion properties;

[0076] (2) Immunomodulatory effects;

[0077] (3) Bactericidal effect;

[0078] (4) Inhibit scar hyperplasia.

[0079] Specifically, the present invention provides a hydrogel adhesive formed by crosslinking L-arginine and 3-aminophenylboronic acid modified hyaluronic acid (HA-PBA), the structure of which is as follows:

[0080]

[0081] This invention also provides a method for preparing a hydrogel adhesive, wherein the method is selected from the group consisting of:

[0082] Method 1: Dissolve the modified HA in a solution containing short-chain molecule M, mix thoroughly and evenly to obtain a hydrogel;

[0083] Method 2: Dissolve the short-chain molecule M in a solution of modified HA, mix thoroughly, and obtain a hydrogel;

[0084] Method 3: Mix the modified HA solution with a solution containing short-chain molecule M, mix thoroughly and evenly to obtain a hydrogel;

[0085] The modification refers to modification by a group or reagent selected from the group consisting of: 3-aminophenylboronic acid, hydrazine reagents, methacrylic anhydride, and free thiol groups.

[0086] In another preferred embodiment, the mass ratio of the modified HA to the solution containing the short-chain molecule M in method 1 is 10:1 to 1:20.

[0087] In another preferred embodiment, the mass ratio of the modified HA to the solution containing the short-chain molecule M in method 1 is 5:1 to 1:10.

[0088] In another preferred embodiment, the mass ratio of the modified HA and the solution containing the short-chain molecule M in method 1 is 2:1 to 1:5.

[0089] Compared with the prior art, the main advantages of the present invention are:

[0090] (1) The preparation process of the hydrogel system of the present invention is simple, the raw materials are simple and the cost is low, and the gelation environment is mild (room temperature is sufficient).

[0091] (2) The hydrogel adhesive of the present invention does not introduce biotoxic organic reagents and is suitable for mass production.

[0092] (3) The composite hydrogel prepared by the present invention can time-regulate the immune response mediated by macrophages, kill bacteria, promote the healing of infected wounds, and inhibit scar hyperplasia.

[0093] (4) The hydrogel adhesive described in this invention has good biocompatibility.

[0094] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0095] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0096] Example 1: Preparation of HA-PBA

[0097] Weigh 2g of HA (20-40kDa) and dissolve it in 100mL of deionized water. After stirring and dissolving at room temperature, add 1.37g of 3-aminophenylboronic acid to the HA solution, and then add 40mL of dimethyl sulfoxide (DMSO) and stir until homogeneous. This solution is labeled as solution A. Weigh 1.92g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 1.15g of N-hydroxysuccinimide and dissolve them in 60mL of DMSO. This solution is labeled as solution B. Add solution B dropwise to solution A, stir until homogeneous, and adjust the pH to 4-5. React at room temperature for 24 hours. Dialyze the above products sequentially using 1mM NaOH for 2 days and deionized water for 3 days, and collect the products. Finally, obtain the HA-PBA solid material by rotary evaporation and lyophilization.

[0098] The above preparation method can control the grafting rate of PBA on HA to 25%-35%.

[0099] Example 2: Three methods for preparing L-Arg crosslinked HA-PBA hydrogel adhesives

[0100] Method 1: Dissolve 2.5% (w / v) HA-PBA in phosphate buffered saline (PBS) at room temperature. After complete dissolution, add L-arginine (L-Arg) powder to the solution to prepare mixed systems with final concentrations of 100mM, 200mM and 500mM, respectively.

[0101] Method 2: L-Arg was dissolved in PBS at room temperature at concentrations of 100 mM, 200 mM, and 500 mM. Then, 2.5% (w / v) HA-PBA was added to prepare a mixed solution.

[0102] Method 3: Dissolve 5% (w / v) HA-PBA in PBS at room temperature, and add equal volumes of 200mM, 400mM, and 1000mM L-Arg solutions respectively to form a mixed system with final L-Arg concentrations of 100mM, 200mM, and 500mM.

[0103] All three methods can form L-Arg crosslinked HA-PBA hydrogel adhesives, and the HA-PBA hydrogel adhesives containing three different concentrations of arginine are named HPLL, HPLM and HPLH respectively.

[0104] Experimental conclusion: Figure 1 Images show the gelation process after L-Arg and HA-PBA crosslinked. L-Arg crosslinks HA-PBA through BN coordination bonds. Figure 2As shown, all three concentrations of hydrogels have a three-dimensional porous structure, and the pore structure of the HPLH group is significantly denser than that of the HPLL and HPLM groups, indicating that increasing the L-Arg concentration can increase the degree of cross-linking of the hydrogel.

[0105] Example 3: The proliferative capacity of the hydrogel adhesive of the present invention on macrophages.

[0106] Experimental Methods: The hydrogel was placed in the lower chamber of a Transwell plate, and the lower chamber was pre-swelled by soaking in fresh culture medium for 1 hour. This was followed by a fresh culture medium replacement and continued soaking. After 4 days, the hydrogel extract from the lower chamber was collected and diluted 5-fold. Macrophages (RAW264.7) were then inoculated at a concentration of 1×10⁻⁶ cells / mL. 5 Cells were seeded at a density of 100 cells / well in 24-well plates. After culturing for 1 day, hydrogel extraction buffer was added, and incubation continued for another day. Alamar Blue was then used for further analysis. TM Cell viability was assessed using the HS cell viability assay kit. After adding fresh hydrogel extract and culturing for another 3 days, cell viability in each group was assessed on day 4.

[0107] Experimental results: such as Figure 3 As shown, the results indicate that the L-Arg crosslinked HA-PBA hydrogel adhesive of the present invention can promote the proliferation of RAW264.7 cells.

[0108] Example 4: Cytotoxicity test of the hydrogel adhesive of the present invention

[0109] Experimental Methods: The hydrogel was placed in the lower chamber of a Transwell plate, and the lower chamber was pre-swelled by soaking in fresh culture medium for 1 hour. This was followed by a fresh culture medium replacement and further soaking. After 4 days, the hydrogel extract from the lower chamber was collected and diluted 5-fold. Mouse fibroblasts (L929) were then subjected to a 1×10⁻⁶ infusion. 4 Cells were seeded at a density of 100 cells / well in 24-well plates. After culturing for 1 day, hydrogel extraction medium was added, and the cells were co-incubated for 1 day. The cells were then seeded with Alamar Blue. TM Cell viability was assessed using the HS cell viability assay kit. After adding fresh extraction medium and culturing for another 3 days, cell viability was assessed in each group on day 4. Fresh hydrogel extraction medium was then added, and cell viability was assessed in each group on day 7 after another 3 days of culturing.

[0110] Experimental results: such as Figure 4 As shown, the results indicate that the L-Arg crosslinked HA-PBA hydrogel adhesive of the present invention is non-toxic to L929 cells.

[0111] Example 5: Antibacterial activity test of the hydrogel adhesive of the present invention.

[0112] Experimental Methods: The hydrogel was placed in the lower chamber of a Transwell plate, and the lower chamber was pre-swelled by soaking in physiological saline for 1 hour. The saline solution was then replaced and soaking continued for 4 days. The hydrogel extract from the lower chamber was collected and diluted 5-fold. 900 μL of the hydrogel extract and 100 μL of 10-fold dilution solution were added to each well of a 12-well plate. 8 Staphylococcus aureus at CFU / mL was mixed and incubated at 37°C for 24 h. After 24 h, the bacterial suspension in the 12-well plate was transferred to a 1.5 mL EP tube and shaken to evenly disperse the bacterial suspension. 100 μL of the above bacterial suspension was used for a bacterial plate coating experiment. Alamar Blue was used. TM The HS kit was used to test the bacterial activity of each group.

[0113] Experimental results: Figure 5 The results showed that there was no significant difference in bacterial colony count and bacterial activity between the hydrogel adhesive group and the Control group, indicating that the L-Arg crosslinked HA-PBA hydrogel adhesive of the present invention does not have antibacterial activity.

[0114] Example 6 verifies that the hydrogel adhesive achieves sterilization through immunomodulation.

[0115] Experimental method: RAW264.7 was processed at 2×10⁻⁶. 5 Cells were seeded at a density of [number] cells / well in 12-well plates. After culturing for 1 day, hydrogel extraction buffer was added. After continuing culturing for 4 days, 100 μL of [reagent name] was added. 8 CFU / mL Staphylococcus aureus suspension was incubated for 3 hours. Subsequently, the cell-bacterial suspension in the wells was collected. After centrifugation at 400xg, the supernatant was collected, and the bacteria were isolated. Alamar Blue was used for separation. TM The HS cell viability kit was used to test bacterial activity, and the live / dead bacterial staining kit was used to observe the live / dead bacterial status.

[0116] Experimental results: Images of live and dead bacteria stained as follows Figure 6 As shown, green fluorescent markers indicate live bacteria, while red fluorescent markers indicate dead bacteria. The proportion of red fluorescence in the hydrogel adhesive group was higher than that in the Control group, and the red fluorescence increased with increasing L-Arg concentration in the hydrogel, especially in the HPLH group, indicating that the hydrogel adhesive kills bacteria through macrophages. Statistical graphs show that bacterial activity significantly decreased with increasing L-Arg concentration in the hydrogel adhesive, indicating that the L-Arg crosslinked HA-PBA hydrogel adhesive of this invention can kill Staphylococcus aureus by regulating macrophages.

[0117] Example 7: Wound healing ability test of the hydrogel of the present invention

[0118] Experimental Methods: A full-thickness wound infection model was established using 6-8 week old male SD rats. After anesthetizing with 1% sodium pentobarbital, the rats' backs were shaved and disinfected. A 20 mm long wound was made in the skin using a scalpel, and 20 μL of 10... 8 Infected wounds were formed using CFU / mL Staphylococcus aureus. In the suture group, wounds were closed with sutures. In the experimental groups, 250 μL of HPLL, HPLM, and HPLH hydrogel adhesives were added on day 0, respectively. The rat skin was kneaded for 2 minutes to stabilize the hydrogel adhesion to the wound before a medical dressing was applied. The control group received no hydrogel adhesive and only a medical dressing was applied. The fibrin group used a commercially available porcine fibrin adhesive; after adhesion to the wound, a medical dressing was applied. All dressings were removed after one day. Wound sites were photographed at days 0, 3, 7, and 14. Surrounding tissue was collected at days 7 and 14 and fixed with 4% paraformaldehyde for further research.

[0119] HE staining was used to assess wound healing in the experimental and control groups. Sirius red stained polarized light microscopy was used to determine collagen expression levels after wound healing. In the α-SMA fluorescence staining image, α-SMA-labeled myofibroblasts and smooth muscle cells showed green fluorescence. In the macrophage immunofluorescence staining experiment, iNOS labeled M1 macrophages showed red fluorescence, while Arg-1 labeled M2 macrophages showed green fluorescence.

[0120] Experimental results: Figure 7 Experimental results showed that the healing speed of the hydrogel adhesive group and the suture group was faster than that of the Control group and the Fibrin group (commercially available adhesive), and the hydrogel adhesive group healed faster with increasing L-Arg content. The L-Arg crosslinked HA-PBA hydrogel adhesive still showed good wound adhesion function under bacterial infection conditions, while the commercially available gel had poor wound adhesion effect under bacterial infection conditions.

[0121] Figure 8 HE staining results showed that the hydrogel adhesive group had better healing than the Control group and the suture group.

[0122] Figure 9 The results in (a) showed that on day 14, the expression levels of type I collagen (yellow) in the Control and Suture groups were significantly higher than those of type III collagen (green), and the type I collagen in the Suture group was coarser and more disordered. Compared with the Control and Suture groups, the hydrogel adhesive group showed more type III collagen deposition, and the proportion of type III collagen increased with increasing L-Arg content, and the arrangement gradually became more regular. Figure 9 As shown in (b), the ratio of type I collagen to type III collagen in the hydrogel adhesive group was significantly lower than that in the Control group and the suture group, which was beneficial in inhibiting scar hyperplasia, especially in the HPLH group.

[0123] Figure 10 The study showed that at days 7 and 14, the green fluorescence in the hydrogel adhesive group was significantly higher than that in the Control and suture groups, indicating increased α-SMA expression, which is beneficial for generating contractile force and accelerating wound healing. These results suggest that, compared to unsealed or sutured wounds, hydrogel adhesives, through flexible bonding and the generation of moderate tensile force, are more conducive to promoting wound repair and inhibiting scar hyperplasia, especially in the HPLH group.

[0124] Figure 11 Immunofluorescence staining images of the tissues surrounding the wound in each group on day 7. As shown in the figure, the green fluorescence of the hydrogel adhesive group gradually increased with the increase of L-Arg concentration, and was higher than that of the Control group and the suture group, indicating that the proportion of M2 macrophages in the hydrogel adhesive group was higher than that in the Control group and the suture group, inducing an anti-inflammatory response during the tissue healing stage.

[0125] Example 8: Hydrogel adhesive formed from L-lysine and D-arginine

[0126] D-arginine (D-Arg) and L-lysine (L-Lys) were dissolved in PBS solution at room temperature. The concentrations of D-Arg and L-Lys were 100 mM, 200 mM, and 500 mM, respectively. Subsequently, 2.5% (w / v) HA-PBA was dissolved in the D-Arg and L-Lys solutions, respectively, to obtain D-Arg-crosslinked HA-PBA hydrogel adhesives and L-Lys-crosslinked HA-PBA hydrogel adhesives.

[0127] Figure 12 The results show that D-Arg, L-Lys, and HA-PBA can all gel, forming D-Arg crosslinked HA-PBA hydrogel adhesives and L-Lys crosslinked HA-PBA hydrogel adhesives.

[0128] Comparative Example 1

[0129] This comparative example aims to demonstrate that HA-PBA that is not cross-linked with L-Arg cannot form a hydrogel.

[0130] HA-PBA was dissolved in PBS solution at room temperature with HA-PBA at w / v ratios of 1%, 2.5%, and 5%, and the state of the HA-PBA solution was observed.

[0131] Figure 13Images show HA-PBA solutions of different concentrations. The results indicate that 1% (w / v), 2.5% (w / v), and 5% (w / v) HA-PBA solutions could not form hydrogels.

[0132] Comparative Example 2

[0133] This comparative example aims to illustrate that unmodified HA cannot crosslink with L-Arg to form a hydrogel.

[0134] L-Arg was dissolved in PBS solution at room temperature at concentrations of 100 mM, 200 mM, and 500 mM. 2.5% (w / v) HA was dissolved in the L-Arg solution and observed at room temperature.

[0135] like Figure 14 As shown, HA without PBA cannot form a hydrogel with L-Arg.

[0136] Comparative Example 3

[0137] This comparative example aims to illustrate that HA-PBA cannot crosslink with amino acids containing one amino group to form hydrogels.

[0138] L-histidine (L-His), glycine (Gly), and serine (Ser) were dissolved in PBS solution at room temperature. The concentrations of L-His were 100 mM, 200 mM, and 268 mM (saturation concentration), and the concentrations of Gly and Ser were 100 mM, 200 mM, and 500 mM, respectively. Then, 2.5% (w / v) HA-PBA was added, and the solution was dissolved at room temperature for observation.

[0139] Figure 15 The results showed that L-His, Gly, and Ser, which contain an amino group, could not form hydrogels with HA-PBA.

[0140] discuss:

[0141] This invention unexpectedly revealed that hyaluronic acid (HA-PBA) solutions modified with 3-aminophenylboronic acid cannot form hydrogels, but HA-PBA can spontaneously crosslink with amino acid molecules M containing two amino groups to form a hydrogel adhesive. Furthermore, the hydrogel exhibits superior crosslinking degree with increasing amino acid concentration. In animal experiments, wounds coated with the hydrogel adhered well, possibly because the skin at the wound site could gather towards the center as the hydrogel's moisture evaporated. Moreover, Examples 5 and 6 demonstrate that the L-Arg crosslinked HA-PBA hydrogel adhesive of this invention does not possess antibacterial activity itself; rather, the hydrogel adhesive achieves its antibacterial effect by modulating the body's own immune system.

[0142] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A hydrogel adhesive, characterized in that, The hydrogel adhesive comprises: a) hyaluronic acid HA-PBA modified with 3-aminophenylboronic acid, and b) a short-chain molecule M containing at least two amino groups; The short-chain molecule M is selected from one or more of the following groups: L-arginine, D-arginine, and L-lysine; The adhesive is formed through spontaneous cross-linking via boron-nitrogen (BN) coordination bonds; The grafting rate of 3-aminophenylboronic acid onto hyaluronic acid (HA) is 25%-90%. The concentration of the modified hyaluronic acid is 0.001~0.0025 g / mL; The content of the short-chain molecule M in the hydrogel adhesive is 100-800 mM.

2. The hydrogel adhesive as described in claim 1, characterized in that, The short-chain molecule M has a non-coplanar amino structure.

3. The hydrogel adhesive as described in claim 1, characterized in that, The hydrogel adhesive has one or more features selected from the group consisting of: (1) It has good wound adhesion properties; (2) Immunomodulatory effects; (3) Bactericidal effect; (4) Inhibit scar hyperplasia.

4. The method for preparing the hydrogel adhesive as described in claim 1, characterized in that, The method is selected from the following group: Method 1: Dissolve the modified hyaluronic acid in a solution containing short-chain molecule M, mix thoroughly and evenly to obtain a hydrogel; Method 2: Dissolve the short-chain molecule M in the modified hyaluronic acid solution, mix thoroughly and evenly to obtain a hydrogel; Method 3: Mix the modified hyaluronic acid solution with a solution containing short-chain molecule M, mix thoroughly and evenly to obtain a hydrogel; The modification refers to modification with 3-aminophenylboronic acid.

5. The preparation method according to claim 4, characterized in that, The solutions used in the steps are selected from the following group: water, physiological saline, and phosphate buffer solution.

6. The use of a hydrogel adhesive as described in claim 1, characterized in that, It is used to prepare hydrogel adhesives for repairing acute and chronic infected wounds and inhibiting scar hyperplasia.

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