Cyclodextrin antibacterial hydrogel capable of accelerating wound healing as well as preparation method and application of cyclodextrin antibacterial hydrogel

Abstract

The invention discloses cyclodextrin antibacterial hydrogel capable of accelerating wound healing as well as a preparation method and application of the cyclodextrin antibacterial hydrogel. The preparation method comprises the following steps: mixing a hydrophobic drug with antibacterial and anti-inflammatory effects with an octa-[6-deoxy-(3-sodium thiohydracrylate)]-gamma-cyclodextrin (SD) solution to obtain a cyclodextrin inclusion compound solution; mixing the cyclodextrin inclusion compound solution with a chitosan (CS) solution to obtain a mixed solution; and adding a NaHCO3 solution into the mixed solution to adjust the pH value to 5.8-6.5, and standing for reaction to obtain the cyclodextrin antibacterial hydrogel capable of accelerating wound healing. According to the invention, the anionic compound SD and the cationic polymer CS form the hydrogel through electrostatic interaction, and meanwhile, a hydrophobic drug with antibacterial and anti-inflammatory effects is loaded into a cavity of the SD, so that the solubility of the drug is increased, the bioavailability of the drug is improved, and the antibacterial and anti-inflammatory properties of the hydrogel are enhanced; the hydrogel disclosed by the invention can be used as a wound dressing for bacterial infection, and the wound healing condition is accelerated.

Description

Technical Field

[0001] The present invention relates to the technical field of chemistry and biomedicine, and in particular to a cyclodextrin antibacterial hydrogel capable of accelerating wound healing, and a preparation method and application thereof. Background Art

[0002] Wound healing is an important and quite complex process, involving multiple stages such as hemostasis, inflammation, cell proliferation and tissue remodeling. Due to the continuous exposure of the wound, bacteria can easily invade and colonize the wound, leading to varying degrees of wound infection. In the clinical treatment of chronic wounds, wound dressings play a vital role. Hydrogel dressings have attracted widespread attention in the field of wound repair because they can create a moist microenvironment, promote the exchange of nutrients and metabolites, promote cell migration and ultimately help wound healing. However, the antibacterial and anti-inflammatory effects of hydrogel dressings themselves are very limited. When used alone, they can only play a limited role in accelerating wound healing.

[0003] In order to improve the antibacterial and anti-inflammatory properties and further accelerate wound healing, hydrogel dressings can be used in combination with antibacterial drugs, such as taking oral antibacterial drugs while using hydrogel dressings or constructing hydrogel dressings based on antibacterial drugs. At present, the use of antibiotics has alleviated the treatment of bacterial infections to a certain extent, but the use of antibiotics will cause bacteria to develop drug resistance, making wounds more difficult to heal. Drugs such as pentacyclic triterpenoids have multiple pharmacological effects such as antibacterial, anti-inflammatory, anti-allergic, and immunomodulatory, and can be used as antibacterial drugs to replace antibiotics. However, due to the poor water solubility of drugs such as pentacyclic triterpenoids, low oral bioavailability, greater cytotoxicity at high concentrations, and obvious hemolytic effects, their commercial application value is limited. Moreover, since drugs such as pentacyclic triterpenoid compounds have a rigid skeleton and a strong hydrophobic structure, their molecular structures may contain more non-polar carbon rings and non-polar substituents, such as alkyl chains, and lack hydrophilic groups. Hydrogels in hydrogels are mainly composed of hydrophilic polymer networks and contain a large amount of water, so there are many hydrophilic groups, such as hydroxyl, carboxyl, amino, etc. Therefore, it is difficult for drugs such as pentacyclic triterpenoid compounds to directly combine with hydrogels and be encapsulated in hydrogels.

[0004] Therefore, if hydrophobic drugs with antibacterial and anti-inflammatory effects such as pentacyclic triterpenoid compounds can be combined with hydrogels to prepare a wound dressing that can effectively accelerate wound healing, it will have great application prospects. Summary of the Invention

[0005] The present invention aims to provide a cyclodextrin antibacterial hydrogel capable of accelerating wound healing, as well as its preparation method and application, to address the aforementioned problems in the prior art. The cyclodextrin antibacterial hydrogel capable of accelerating wound healing of the present invention has excellent antibacterial properties and good biocompatibility. It can be used as a dressing for bacterially infected wounds, effectively inhibiting inflammatory reactions in wounds, reducing bacterial infection in wounds, and accelerating wound healing.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] One of the technical solutions of the present invention is a method for preparing a cyclodextrin antibacterial hydrogel capable of accelerating wound healing, comprising the following steps:

[0008] A hydrophobic drug with antibacterial and anti-inflammatory effects is mixed with an octa-[6-deoxy-(sodium 3-mercaptopropionate)]-γ-cyclodextrin (SD) solution to obtain a cyclodextrin inclusion complex solution; the cyclodextrin inclusion complex solution is mixed with a chitosan (CS) solution to obtain a mixed solution; a NaHCO3 solution is added to the mixed solution to adjust the pH value to 5.8-6.5, and the solution is allowed to react to obtain the cyclodextrin antibacterial hydrogel capable of accelerating wound healing.

[0009] Chitosan (CS), as a natural cationic polymer, is mainly derived from shrimp and crab shells. It is low-cost and abundant in resources. It not only has excellent biocompatibility, biodegradability and mucus adhesion, but also has multiple physiological functions such as antibacterial, anti-cancer, lipid-lowering, and immune enhancement. There are carboxylate groups on the side chains of the outer surface of SD, which is negatively charged as a whole and can form a hydrogel with cationic polymers through electrostatic interaction. The present invention forms a hydrogel by electrostatic interaction between the anionic compound SD and the cationic polymer CS, which can make the hydrogel matrix itself have certain antibacterial and anti-inflammatory effects. Moreover, the structure of SD has a cavity, and with the help of its cavity, it can achieve the embedding and encapsulation of hydrophobic drugs with antibacterial and anti-inflammatory effects. Through molecular encapsulation, a host-guest supramolecular complex with controlled release characteristics is obtained, thereby improving the water solubility and bioavailability of hydrophobic drugs with antibacterial and anti-inflammatory effects, effectively combining the hydrogel with the hydrophobic drugs with antibacterial and anti-inflammatory effects, and enhancing the antibacterial and anti-inflammatory properties of the hydrogel. The cyclodextrin antibacterial hydrogel capable of accelerating wound healing of the present invention is applied to wounds of mice infected with bacteria, which can inhibit wound inflammation, reduce wound bacterial infection, and thus accelerate wound healing.

[0010] In the preparation method of the present invention, the pH of the mixed solution is adjusted to control the electrostatic interaction between SD and chitosan to form a hydrogel with a 3D grid structure and a porous structure. The inventors creatively discovered that only by adding NaHCO3 solution to adjust the pH value of the mixed solution can the successful preparation of the porous hydrogel be achieved. When other substances such as sodium phosphate and sodium citrate are used as pH regulators, the preparation of the porous hydrogel cannot be achieved even when the pH value is adjusted to the same or the concentration and dosage are used. This may be because the HCO3 in NaHCO3 - It is a monovalent anion and is related to NH4 in chitosan. + The electrostatic effect is weak, and HCO3 - Can react with H in chitosan solution + Combined to achieve pH control; however, the anions in sodium phosphate and sodium citrate are multivalent anions and will react with NH4 + The strong Coulomb force binds the molecules and neutralizes the charge quickly, causing the molecular chains to aggregate directly and form a white flocculent precipitate.

[0011] Furthermore, the hydrophobic drug with antibacterial and anti-inflammatory effects is a pentacyclic triterpenoid compound.

[0012] Furthermore, the pentacyclic triterpenoid compound includes glycyrrhizic acid (GL), glycyrrhetinic acid (GA), ursolic acid or oleanolic acid.

[0013] Furthermore, the mass ratio of the hydrophobic drug having antibacterial and anti-inflammatory effects to the octa-[6-deoxy-(sodium 3-mercaptopropionate)]-γ-cyclodextrin contained in the octa-[6-deoxy-(sodium 3-mercaptopropionate)]-γ-cyclodextrin solution is 1:1.25-10.

[0014] Furthermore, the mass ratio of octa-[6-deoxy-(sodium 3-mercaptopropionate)]-γ-cyclodextrin in the octa-[6-deoxy-(sodium 3-mercaptopropionate)]-γ-cyclodextrin solution to the chitosan in the chitosan solution is 1:5-50.

[0015] Optionally, the concentration of the NaHCO3 solution is 0.6-1.0M.

[0016] Furthermore, the temperature of the static reaction is 0-40° C., and the time is 36-72 hours.

[0017] Furthermore, the solvent of the octa-[6-deoxy-(sodium 3-mercaptopropionate)]-γ-cyclodextrin solution includes water.

[0018] Furthermore, the solvent of the chitosan solution includes an acid solution.

[0019] Furthermore, the concentration of the octa-[6-deoxy-(sodium 3-mercaptopropionate)]-γ-cyclodextrin solution is 5-20 mg / mL.

[0020] Furthermore, the concentration of the chitosan solution is 40-60 mg / mL.

[0021] The present invention first uses octa-[6-deoxy-(sodium 3-mercaptopropionate)]-γ-cyclodextrin to include a hydrophobic drug with antibacterial and anti-inflammatory effects to obtain a cyclodextrin inclusion compound, and then mixes the cyclodextrin inclusion compound with chitosan. Then, a NaHCO3 solution is used to adjust the pH to form a stable hydrogel. The preparation method is simple.

[0022] The second technical solution of the present invention: a cyclodextrin antibacterial hydrogel capable of accelerating wound healing prepared by the above-mentioned preparation method of the cyclodextrin antibacterial hydrogel capable of accelerating wound healing.

[0023] Furthermore, the loading amount of the hydrophobic drug with antibacterial and anti-inflammatory effects in the cyclodextrin antibacterial hydrogel capable of accelerating wound healing is 10-100 μg / mg.

[0024] The loading amount means: the mass of the hydrophobic drug contained in the cyclodextrin antibacterial hydrogel / the mass of the cyclodextrin antibacterial hydrogel after freeze-drying.

[0025] The third technical solution of the present invention: an application of the above-mentioned cyclodextrin antibacterial hydrogel capable of accelerating wound healing in the preparation of a medicine or dressing for accelerating wound healing.

[0026] Technical solution 4 of the present invention: A medicine or dressing for accelerating wound healing, comprising the above-mentioned cyclodextrin antibacterial hydrogel capable of accelerating wound healing.

[0027] Optionally, the medicine or dressing for accelerating wound healing further comprises a pharmaceutically acceptable excipient.

[0028] The present invention selects SD with a cavity structure to include pentacyclic triterpenoid compounds, but not all substances with a cavity structure can achieve the inclusion of pentacyclic triterpenoid compounds, that is, the SD in the present invention cannot be replaced by other substances with a cavity structure, such as other cyclodextrins.

[0029] Cyclodextrins are a class of substances with a cavity structure resembling a truncated cone, derived from the chair conformation of the glucopyranose units. Hydroxyl groups are located on the outer surface of the cavity, with secondary hydroxyl groups located at the wider edge and primary hydroxyl groups at the narrower edge. Within the cavity, hydrogen atoms and glycosidic oxygen form a layer of bridges, imparting lipophilicity. Due to the unique three-dimensional structure of cyclodextrins, their cavity has a hydrophilic exterior and a relatively hydrophobic interior, resulting in water solubility and the ability to fully or partially incorporate hydrophobic molecules of appropriate size, fostering the formation of inclusion complexes. Inclusion complexes exist in dynamic equilibrium in aqueous solution, characterized by the absence of covalent bonds and defined by stoichiometric host-guest relationships.

[0030] Cyclodextrins are primarily classified into α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin. Due to the varying number of glucose units they contain, their cavity diameters and volumes differ, with γ-cyclodextrin being the largest. Pentacyclic triterpenoids like GL, whose basic structure consists of a pentacyclic triterpenoid core and a glycoside group, are relatively large in size. Therefore, the cavity of γ-cyclodextrin is most likely to accommodate pentacyclic triterpenoids. However, natural γ-cyclodextrin has limited hydrophobic regions and catalytic activity, and the strong intramolecular hydrogen bonds between secondary hydroxyl groups reduce their ability to form hydrogen bonds with surrounding water molecules. Experiments have shown that its ability to incorporate pentacyclic triterpenoids is actually very poor.

[0031] The SD selected in the present invention is equivalent to replacing the secondary hydroxyl group with sodium deoxy-3-mercaptopropionate while maintaining the basic skeleton of the cyclodextrin macrocycle unchanged, thereby reducing the possibility of intramolecular hydrogen bond formation and forming sodium carboxylate, thereby increasing water solubility.

[0032] Through research, the inventors discovered that SD is a derivative of γ-cyclodextrin. SD can include pentacyclic triterpenoids such as GL, while γ-cyclodextrin (γ-CD) cannot. This is mainly due to the following key differences in molecular design:

[0033] (1) Differences in structural adaptability

[0034]

[0035] Mechanism: SD completely encapsulates the pentacyclic triterpene rings and glycoside groups of pentacyclic triterpene compounds such as GL through its expanded cavity, while the γ-CD cavity can only partially accommodate pentacyclic triterpene compound molecules such as GL and cannot stably bind.

[0036] (2) Difference in binding strength

[0037] Hydrophobic interaction: The sulfide chain methyl groups on the inner wall of the SD cavity form dense hydrophobic interactions with the hydrophobic surface of the pentacyclic triterpenoid core of pentacyclic triterpenoid compounds such as GL, while the hydrophobic interaction of γ-CD is weaker;

[0038] The conformationally induced side chains of SD can be dynamically adjusted to form a "glove-hand" inclusion complex, while the rigid structure of γ-CD cannot adapt to the stereoconfiguration of pentacyclic triterpenoids such as GL.

[0039] The present invention discloses the following technical effects:

[0040] This invention forms a hydrogel by electrostatically interacting anionic compound SD and cationic polymer CS. A hydrophobic drug with antibacterial and anti-inflammatory properties is simultaneously loaded into the cavity of SD, increasing the drug's solubility and bioavailability, thereby enhancing the hydrogel's antibacterial and anti-inflammatory properties. The hydrogel not only exhibits excellent antibacterial properties, promotes cell migration, and reduces inflammatory factors, but also exhibits good biocompatibility and robust, stable mechanical properties.

[0041] The hydrogel of the present invention has good antibacterial properties, and the hydrophobic drugs with antibacterial and anti-inflammatory effects encapsulated in SD can be sustained-released under physiological conditions, and the bioavailability is effectively improved. The present invention uses in vitro cell experiments and red blood cell hemolysis experiments to prove that the hydrogel of the present invention has good biocompatibility and promotes the migration of rat fibroblasts (NIH / 3T3) cells, and the released natural drugs synergize with the hydrogel to reduce the expression of inflammatory factors (NO, TNF-α and IL-6) in Raw cells. The hydrogel was applied to bacterial wounds in mice, and the results showed that it can significantly reduce the number of bacteria in the mouse wounds, inhibit inflammatory reactions, and accelerate wound healing.

[0042] The hydrogel strategy of the present invention can be used to develop new dressings for treating bacterially infected wounds, which not only has important practical application value in the medical field, but also provides new ideas and possibilities for future research in materials science and biomedicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0044] Figure 1 Scanning electron micrographs of CS / SD hydrogels prepared in Example 1 under different pH conditions (i.e., with the addition of different concentrations of NaHCO3 solution), where (A), (B), (C), and (D) represent the pH values of the gel-forming mixed solution of 4.7, 5.8, 6.2, and 6.5, respectively;

[0045] Figure 2The infrared spectra of CS / SD hydrogel and raw materials CS and SD (A), the infrared spectra of CS / SD hydrogel and CS / SD / GL hydrogel and raw material GL (B), and the UV-visible absorption spectra of CS / SD hydrogel and CS / SD / GL hydrogel and raw material GL (C);

[0046] Figure 3 The rheological properties test results of CS / SD hydrogels obtained by adding 1M NaHCO3 solution, where A is the dynamic frequency rheological test result, B is the strain oscillation shear rheological test result, and C is the cyclic strain rheological test result;

[0047] Figure 4 Figure 3 is a diagram of the viscosity and self-healing performance test process of CS / SD hydrogel, where A shows the CS / SD hydrogel inverted in a plastic container, B shows the CS / SD hydrogel placed on a finger joint and bent, CD shows the CS / SD hydrogel being stretched, EF shows the CS / SD hydrogel being dyed and separated, and G shows the hydrogel being stretched after being combined into one.

[0048] Figure 5 The swelling performance test results and degradation performance test results of CS / SD hydrogel obtained by adding 1M NaHCO3 solution, where A is the swelling performance test result and B is the degradation performance test result;

[0049] Figure 6 GL release efficiency curve of CS / SD / GL hydrogel at different pH values;

[0050] Figure 7 Figure 3 shows the cytotoxicity test results of GL, CS / SD hydrogel, and CS / SD / GL hydrogel on RAW 264.7 and NIH / 3T3 cells, where (A) is the cell viability of RAW 264.7 after 24 h of culture, (B) is the cell viability of RAW 264.7 after 48 h of culture, (C) is the cell viability of NIH / 3T3 after 24 h of culture, and (D) is the cell viability of NIH / 3T3 after 48 h of culture.

[0051] Figure 8 Hemolysis status of erythrocytes treated with water, NaCl, GL, CS / SD hydrogel, and CS / SD / GL hydrogel;

[0052] Figure 9The in vitro antibacterial effect test results of CS / SD hydrogel and CS / SD / GL hydrogel, where (A) is a photograph of S. aureus and E. coli colonies after treatment with CS / SD and CS / SD / GL hydrogel extracts at different concentrations, (B) is a statistical graph of the inhibitory effect on S. aureus, (C) is a statistical graph of the inhibitory effect on E. coli, and (D) is a scanning electron micrograph of bacteria after treatment with different materials.

[0053] Figure 10 The migration of NIH / 3T3 cells after being treated with different materials at different time periods;

[0054] Figure 11 The results of in vitro anti-inflammatory effect tests are shown, where (a) shows RAW 264.7 cells in normal growth state (i.e., negative control), (b) shows RAW 264.7 cells after LPS stimulation (i.e., positive control), (c)-(e) show RAW 264.7 cells after LPS stimulation with GL, CS / SD, and CS / SD / GL, respectively, and (f)-(h) show the analysis results of NO, TNF-α, and IL-6 levels in cells under the above treatment conditions;

[0055] Figure 12 Photos of mouse wounds in different treatment groups and at different treatment times (A), wound area trace analysis (B), and wound healing rates (C);

[0056] Figure 13 The colony photos (A) and colony count results (B) of bacteria cultured in the skin around the wounds of mice in different treatment groups and at different treatment times;

[0057] Figure 14 The pH changes of mouse wounds in different treatment groups and at different treatment times;

[0058] Figure 15 These are H&E staining images of regenerated skin tissue at the wound site of mice in different treatment groups and at different treatment times;

[0059] Figure 16 The analysis results of TNF-α and IL-6 in the wounds of mice in different treatment groups and at different treatment times, among which (A) and (C) are the staining results of TNF-α and IL-6, respectively, and (B) and (D) are the content analysis results of TNF-α and IL-6, respectively;

[0060] Figure 17 α-SMA and CD31 staining of the skin of mice in different treatment groups on the 14th day after wound injury (A), as well as the relative fluorescence area of CD31 positivity (B) and the relative fluorescence area of α-SMA positivity (C). DETAILED DESCRIPTION

[0061] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0062] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0063] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0064] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0065] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0066] It should be pointed out that the matters not described in detail in the present invention are conventional operating means in this field and are not the focus of the present invention.

[0067] When referring to room temperature or normal temperature in the specific embodiments of the present invention, it specifically refers to 20-30°C.

[0068] Unless otherwise specified, the raw materials used in the specific embodiments of the present invention are common commercial products. Among them, the CAS number of octa-[6-deoxy-(sodium 3-mercaptopropionate)]-γ-cyclodextrin (SD, also known as sodium sugammadex) is 343306-79-6, and the deacetylation degree of chitosan is 90%.

[0069] Example 1

[0070] The preparation steps of CS / SD hydrogel are as follows:

[0071] Accurately weigh 50 mg of SD and add 5 mL of ultrapure water to fully dissolve it to obtain a 10 mg / mL SD solution. Dissolve chitosan in 2% (v / v) acetic acid solution to obtain a 50 mg / mL chitosan solution. Take 5 mL of the 50 mg / mL chitosan solution in a beaker, then slowly add 0.5 mL of the above SD solution and stir magnetically for 5 hours to mix it evenly to obtain a mixed solution. Then add 1 mL of 1 M NaHCO3 solution to the above mixed solution to adjust the pH value of the mixed solution to 6.5. Then, the mixed solution after adjusting the pH value is kept at 37°C for 48 hours to react to obtain CS / SD hydrogel.

[0072] Through the above preparation steps, CS / SD hydrogels with different concentrations of NaHCO3 solution added (i.e., at different pH values) were obtained by only changing the concentration of NaHCO3 solution (0M, 0.6M, 0.8M, 1.0M, 1.2M, and the corresponding adjusted pH values of the mixed solution were 4.7, 5.8, 6.2, 6.5, and 6.7). Among them, when the concentration of the added NaHCO3 solution was 0M, the final product was a viscous fluid and no hydrogel could be obtained.

[0073] Figure 1These are scanning electron micrographs of the CS / SD hydrogels prepared in Example 1 under different pH conditions (i.e., with the addition of different concentrations of NaHCO3 solution) (before testing, the hydrogels were freeze-dried at -40°C for 48 h). (A), (B), (C), and (D) represent the pH values of the gel-forming mixed solutions of 4.7, 5.8, 6.2, and 6.5, respectively, and the concentrations of the added NaHCO3 solution are 0 M, 0.6 M, 0.8 M, and 1.0 M. As can be seen from the figure, when the pH is 4.7, that is, no NaHCO3 is added, the product (viscous fluid) is distributed in layers after freeze-drying. After adding NaHCO3 to adjust the pH, the morphology of the product (hydrogel) after freeze-drying gradually changes from a layered distribution to a porous three-dimensional network structure, and the pores are cross-linked with each other. As the concentration of the NaHCO3 solution increases to 1.0M to raise the pH to 6.5, the pore structure gradually becomes uniform and the pore size becomes larger, which is conducive to the release of drugs. It can also absorb coagulation factors and wound exudates in wound applications, and transfer metabolites and nutrients, thereby playing an important role in hemostasis and wound healing. Since the concentration of the added NaHCO3 solution was 1.2M, the synthesized hydrogel had too many bubbles when the pH value reached 6.7, and it was difficult to defoam, so scanning electron microscopy test was not performed on it. The test results showed that a pH value of the mixed solution between 5.8-6.5 (corresponding to a concentration of the added NaHCO3 solution of 0.6-1.0M) was more conducive to obtaining a porous structured CS / SD hydrogel.

[0074] The infrared spectra of the CS / SD hydrogel prepared in Example 1 (the concentration of the added NaHCO3 solution is 1.0 M) and the raw materials CS and SD are as follows: Figure 2 As shown in (A). In the figure, 3500cm -1 The peak at 1652cm is caused by OH stretching vibration, and the change in peak width is caused by the interaction of hydrogen bonds. -1 Represents the C=O stretching vibration in acetylamino (-NHCOCH3), 3469 cm -1 The peaks at 3402 cm correspond to NH stretching vibration and OH stretching vibration; for SD, the peaks at 3402 cm -1 is OH stretching vibration, at 1567cm -1 and 1405cm -1 The absorption peaks of 3500cm in the figure correspond to the asymmetric and symmetric stretching vibrations of carboxylates. -1 The change in peak width at is caused by the interaction of hydrogen bonds. At the same time, the amino group of CS (positively charged) may be electrostatically attracted to the sodium carboxylate of SD (negatively charged), which weakens the peak signal and forms a complex.

[0075] The above results indicate that CS / SD hydrogel was successfully synthesized.

[0076] Figure 3 The rheological properties of CS / SD hydrogels obtained by adding 1M NaHCO3 solution are shown, where A is the dynamic frequency rheological test result, B is the strain oscillation shear rheological test result, and C is the cyclic strain rheological test result (1M in the figure represents the concentration of the added NaHCO3 solution is 1M, G' represents the storage modulus, and G" represents the loss modulus). Figure 3 As can be seen from Figure A, when the concentration of the added NaHCO3 solution is 1.0 M, the storage modulus (G') is greater than the loss modulus (G") in the entire frequency range of 0.1-100 rad / s, showing elastic deformation, indicating that the CS / SD hydrogel forms an elastic cross-linked network. Figure 3 As can be seen from Figure B, when the concentration of the added NaHCO3 solution is 1M, the gel point (intersection of G' and G") is 21.79%. Therefore, the CS / SD hydrogel has appropriate viscoelasticity and can maintain a stable state during daily activities to promote wound healing. The self-healing ability of the CS / SD hydrogel was evaluated by strain alternating scanning. After determining the linear viscoelastic range, a strain value of 0.1% was selected within the linear viscoelastic region, and a strain value of 500% was selected after the hydrogel structure collapsed. Alternating strain scanning was performed, with each scan time of 100s. A total of five strain conversions were performed alternately. The results are shown in Figure 3. Figure 3 As shown in Figure C, when the concentration of the added NaHCO3 solution is 1.0 M, the G' and G" of the CS / SD hydrogel can still return to their initial state after 5 cycles, proving that the CS / SD hydrogel has the ability to self-repair after fracture and the CS / SD hydrogel structure is relatively stable. Figure 4 The viscosity and self-healing properties of CS / SD hydrogel were further demonstrated. Among them, A shows that CS / SD hydrogel can adhere to the bottom of the container without falling when it is inverted in a plastic container, indicating that it has good viscosity; B shows that when CS / SD hydrogel is placed on the finger joint, when the finger joint is bent 90°, CS / SD hydrogel still maintains excellent viscosity and will not fall off with the movement of the joint; CD shows that when CS / SD hydrogel is stretched, it deforms but does not break; EF shows that the newly prepared CS / SD hydrogel is stained with rhodamine and Coomassie Brilliant Blue, and then placed separately. After a period of time, the boundary between the two CS / SD hydrogels disappears, indicating that the two CS / SD hydrogels have merged into one; G shows that the hydrogel merged into one in E is then stretched with tweezers, and the hydrogel does not break at the boundary, indicating that CS / SD hydrogel has good self-healing properties.

[0077] The CS / SD hydrogel obtained by adding 1M NaHCO3 solution was subjected to swelling performance test and degradation performance test. The specific operations are as follows: the swelling performance test was carried out on the hydrogel after freeze-drying (-40°C, 48h), and the degradation performance test was carried out on the freshly prepared hydrogel. The hydrogel was placed in a PBS solution at room temperature and different pH values (5.0, 7.0, 8.3). The hydrogel was taken out at regular intervals, and the water on the surface of the hydrogel was wiped dry with filter paper. The weight of the hydrogel was weighed, and then placed in the solution again until equilibrium was reached or a certain time was reached. The swelling rate and degradation rate of the hydrogel were calculated. The test results are as follows. Figure 5 As shown, A is the swelling performance test result and B is the degradation performance test result. It can be seen that the generated CS / SD hydrogel has good swelling performance. The swelling rate in the PBS solution with a pH value of 7.4 is the highest, reaching 400%, and the swelling rate in the PBS solution with a pH value of 5.0 is the lowest. This may be related to the pH value when the CS / SD hydrogel is formed. At the same time, the CS / SD hydrogel degrades the most in the PBS solution with a pH value of 5.0, which may be because the cross-linking density of the CS / SD hydrogel is low at a pH value of 5.0, and therefore the degradation rate is relatively fast. This shows that the CS / SD hydrogel has good degradation ability and can respond to the slightly acidic environment of the wound site, which can meet the requirements of the new wound dressing.

[0078] Example 2

[0079] The preparation steps of cyclodextrin antibacterial hydrogel (i.e., GL-loaded CS / SD hydrogel) capable of accelerating wound healing are as follows:

[0080] Accurately weigh 50 mg of SD and dissolve thoroughly in 5 mL of ultrapure water to obtain an SD solution. Then, accurately weigh 40 mg of GL and add it to the SD solution. Stir thoroughly to dissolve, resulting in an SD / GL solution (i.e., a cyclodextrin inclusion complex solution) with a GL concentration of 8 mg / mL. Dissolve chitosan in 2% (v / v) acetic acid to obtain a 50 mg / mL chitosan solution. Place 5 mL of the 50 mg / mL chitosan solution in a beaker, then slowly add 0.5 mL of the SD / GL solution. Mix thoroughly under magnetic stirring for 5 hours to obtain a mixed solution. Add 1 M NaHCO3 solution to the mixed solution until the pH reaches 6.5. The pH-adjusted mixed solution is then incubated at 37°C for 48 hours to obtain a GL-loaded CS / SD hydrogel, designated as the CS / SD / GL hydrogel. The prepared CS / SD / GL hydrogel was washed three times with distilled water to wash away the free GL adhering to the surface of the hydrogel. The free GL concentration in the solution was then tested by UV-visible spectrophotometry to calculate the mass of GL loaded into the hydrogel. Combined with the mass of the hydrogel after freeze-drying (-40°C, 48h), the GL loading in the hydrogel was calculated to be 13.8μg / mg.

[0081] Figure 2 (B) is the infrared spectra of the CS / SD hydrogel prepared in Example 1 (the concentration of the added NaHCO3 solution is 1.0 M), the CS / SD / GL hydrogel prepared in Example 2, and the raw material GL. For GL, 1729 cm -1 and 1652cm -1 The peak at 3500 cm represents the carboxyl and carbonyl groups in GL. -1 The change in peak width at may be caused by hydrogen bonding interaction. Figure 2 Panel (C) shows the UV-visible absorption spectra of the CS / SD hydrogel prepared in Example 1 (the concentration of the added NaHCO solution was 1.0 M), the CS / SD / GL hydrogel prepared in Example 2, and the raw material GL at 200-500 nm. As can be seen from the figure, the absorption peak of GL is at 257 nm, while the CS / SD hydrogel does not have an absorption peak at this location. However, when the CS / SD hydrogel is loaded with GL, a characteristic peak at 275 nm is displayed. Therefore, it can be seen that GL is successfully loaded into the CS / SD hydrogel, confirming the successful synthesis of the CS / SD / GL hydrogel.

[0082] Example 3

[0083] The preparation steps of cyclodextrin antibacterial hydrogel (i.e., glycyrrhetinic acid-loaded CS / SD hydrogel) capable of accelerating wound healing are as follows:

[0084] Accurately weigh 50 mg of SD and dissolve thoroughly in 5 mL of ultrapure water to obtain an SD solution. Then, accurately weigh 10 mg of glycyrrhetinic acid (GA) and add it to the SD solution. Stir thoroughly to dissolve, resulting in an SD / GA solution (i.e., a cyclodextrin inclusion complex solution) with a GA concentration of 2 mg / mL. Dissolve chitosan in 2% (v / v) acetic acid to obtain a 50 mg / mL chitosan solution. Place 5 mL of the 50 mg / mL chitosan solution in a beaker, then slowly add 0.5 mL of the SD / GA solution. Mix thoroughly under magnetic stirring for 5 hours to obtain a mixed solution. Add 1 M NaHCO3 solution to the mixed solution until the pH reaches 6.5. The pH-adjusted solution is then incubated at 37°C for 48 hours to obtain a GA-loaded CS / SD hydrogel, designated as the CS / SD / GA hydrogel. Testing and calculations indicate that the GA loading in the hydrogel is 17 μg / mg.

[0085] Comparative Example 1

[0086] Accurately weigh 50 mg of SD and dissolve thoroughly in 5 mL of ultrapure water to obtain a 10 mg / mL SD solution. Dissolve chitosan in 2% (v / v) acetic acid to obtain a 50 mg / mL chitosan solution. Place 5 mL of the 50 mg / mL chitosan solution in a beaker, then slowly add 0.5 mL of the SD solution. Mix thoroughly under magnetic stirring for 5 hours to obtain a mixed solution. Add 1 M sodium citrate solution to the mixed solution. Upon addition of the 1 M sodium citrate solution, floccules immediately form in the mixed solution, preventing the formation of a hydrogel.

[0087] Comparative Example 2

[0088] Accurately weigh 50 mg of SD and add 5 mL of ultrapure water to fully dissolve it to obtain a 10 mg / mL SD solution. Dissolve chitosan with 2% (v / v) acetic acid solution to obtain a 50 mg / mL chitosan solution. Take 5 mL of the 50 mg / mL chitosan solution in a beaker, then slowly add 0.5 mL of the above SD solution and stir magnetically for 5 hours to mix it evenly to obtain a mixed solution. Then add 1 M sodium phosphate solution to the above mixed solution until the pH value of the mixed solution reaches 6.5, and then let the mixed solution stand at a constant temperature of 37°C for 48 hours to react. As a result, flocs appeared in the mixed solution and no hydrogel was obtained.

[0089] Comparative Example 3

[0090] Accurately weigh 50 mg of γ-CD and add 5 mL of ultrapure water to fully dissolve it to obtain a γ-CD solution with a concentration of 10 mg / mL. Dissolve chitosan with 2% (v / v) acetic acid solution to obtain a chitosan solution with a concentration of 50 mg / mL. Take 5 mL of the 50 mg / mL chitosan solution in a beaker, then slowly add 0.5 mL of the above γ-CD solution, and stir magnetically for 5 hours to mix it evenly to obtain a mixed solution. Then add 1 M NaHCO3 solution to the above mixed solution until the pH value of the mixed solution reaches 6.5, and then let the mixed solution stand at a constant temperature of 37°C for 48 hours to react. As a result, flocs appeared in the mixed solution and no hydrogel was obtained.

[0091] Separately, 50 mg of γ-CD was accurately weighed and thoroughly dissolved in 5 mL of ultrapure water to obtain a γ-CD solution. 10 mg of GL was then accurately weighed and added to the SD solution. After thorough stirring, very little GL dissolved, indicating that the γ-CD encapsulated very little GL and failed to effectively improve GL solubility. This suggests that replacing SD with γ-CD failed to form a hydrogel and effectively encapsulate GL.

[0092] Comparative Example 4

[0093] Accurately weigh 50 mg of sulfobutyl-β-cyclodextrin (SBE-β-CD) and dissolve thoroughly in 5 mL of ultrapure water to obtain a 10 mg / mL SBE-β-CD solution. Dissolve chitosan in 2% (v / v) acetic acid to obtain a 50 mg / mL chitosan solution. Place 5 mL of the 50 mg / mL chitosan solution in a beaker, then slowly add 0.5 mL of the SBE-β-CD solution. Mix thoroughly under magnetic stirring for 5 hours to obtain a mixed solution. Add 1 M NaHCO3 solution to the mixed solution until the pH reaches 6.5. The mixed solution is then incubated at 37°C for 48 hours to produce the CS / SBE-β-CD hydrogel.

[0094] Separately, 50 mg of SBE-β-CD was accurately weighed and thoroughly dissolved in 5 mL of ultrapure water to obtain an SBE-β-CD solution. 10 mg of GL was then accurately weighed and added to the SBE-β-CD solution. After thorough stirring, very little GL dissolved, indicating that the SBE-β-CD encapsulated very little GL and failed to effectively improve its solubility. This suggests that while replacing SD with SBE-β-CD allows hydrogel formation, it fails to effectively encapsulate GL. Specifically, the sulfobutyl-β-cyclodextrin in the CS / SBE-β-CD hydrogel has little effect on encapsulating small hydrophobic drugs such as GL, resulting in a low drug loading and poor subsequent efficacy.

[0095] Test Example 1

[0096] The CS / SD / GL hydrogel prepared in Example 2 was subjected to an in vitro release test of GL. The test method was as follows: 5.0 g of CS / SD / GL hydrogel was placed in 15 mL of PBS solution with pH values of 5.0, 7.4, and 8.3, respectively. The solution was shaken at 37°C and 200 rpm. 3 mL of the solution was taken out at regular intervals to detect the amount of GL released. Then, 3 mL of PBS solution with the same pH value was added, keeping the total volume of the release solution unchanged. The amount of GL released was measured using a UV spectrophotometer. The test results are shown in Figure 2. Figure 6 As shown in the figure, the release curves show that GL can be rapidly released within 12 hours, after which GL release slows and gradually reaches a release equilibrium. Within the first 8 hours, the release rate of the CS / SD / GL hydrogel at an acidic pH of 5.0 is higher than that at pH 7.4 and 8.3. By 12 hours, the final release of all three hydrogels reaches 60%. The release amount under weak acid conditions is higher than that under neutral and weak alkaline conditions. This is likely because the hydrogel degrades more rapidly under acidic conditions, which facilitates the rapid release of GL molecules from the hydrogel, thereby accelerating drug release. The results show that the construction of a gel drug delivery system can prolong the drug's action time, achieve a sustained drug delivery effect, and improve the disadvantages of frequent drug delivery. The prepared CS / SD / GL hydrogel drug delivery system achieves the expected release effect in a simulated wound environment and has good application prospects in the field of wound healing drug delivery.

[0097] Effect Verification Example 1

[0098] Cytotoxicity assay

[0099] Cytotoxicity is an important issue in the application of antibacterial hydrogels. The MTT assay was used to determine the cytotoxicity of GL, CS / SD hydrogels (the concentration of the added NaHCO3 solution was 1.0 M), and CS / SD / GL hydrogels to RAW 264.7 and NIH / 3T3 cells. The specific procedure was as follows: NIH / 3T3 cells and RAW 264.7 cells were cultured to the logarithmic growth phase, and then 5×10 3Cells were seeded into 96-well plates. After the cells adhered, 100 μL of free GL solution (25 μg / mL, 50 μg / mL, 75 μg / mL, 100 μg / mL, in PBS) with different concentrations, CS / SD / GL hydrogel extract loaded with equal GL concentration (obtained by extracting the CS / SD / GL hydrogel prepared in Example 2 in PBS at room temperature for 24 h, and by changing the mass of the CS / SD / GL hydrogel and the volume of PBS used, the concentration of GL contained in the CS / SD / GL hydrogel in the PBS solvent, i.e., the mass of GL contained in the CS / SD / GL hydrogel / the volume of PBS were 25 μg / mL, 50 μg / mL, 75 μg / mL, 100 μg / mL, respectively) or CS / SD hydrogel with different concentrations were added. The gel extract (obtained by extracting the CS / SD hydrogel in PBS at room temperature for 24 hours, wherein the concentrations of the CS / SD hydrogel extract of 25 μg / mL, 50 μg / mL, 75 μg / mL, and 100 μg / mL respectively represent the concentrations of the CS / SD hydrogel in PBS solvent, which are consistent with the concentrations of the hydrogel carrier portion in the CS / SD / GL hydrogel extract loaded with equal GL concentrations of 25 μg / mL, 50 μg / mL, 75 μg / mL, and 100 μg / mL in PBS solvent) was cultured for 24 hours or 48 hours, and then 20 μL of 5 mg / mL MTT solution was added. After further culture for 4 hours, the supernatant in the well was discarded, 150 μL DMSO was added to dissolve the crystals, and finally the OD value at 492 nm was detected by a microplate reader to calculate the cell survival rate. The results are shown in FIG. Figure 7As shown, (A) shows the cell viability of RAW 264.7 cells after 24 hours of culture, (B) shows the cell viability of RAW 264.7 cells after 48 hours of culture, (C) shows the cell viability of NIH / 3T3 cells after 24 hours of culture, and (D) shows the cell viability of NIH / 3T3 cells after 48 hours of culture. It can be seen that after 24 hours and 48 hours of co-culture with RAW 264.7 and NIH / 3T3 cells, respectively, GL, CS / SD hydrogel, and CS / SD / GL hydrogel showed good survival rates, exceeding 100%, even at a concentration of 100 μg / mL, promoting cell proliferation. This indicates that CS / SD hydrogel and CS / SD / GL hydrogel have good biocompatibility with these two cell types, laying the foundation for their wound healing applications. Hydrogel wound dressings come into direct contact with red blood cells during application, making it crucial to study the blood-related properties of hydrogel materials. Using secondary water as a positive control and 0.9 wt% NaCl solution as a negative control, 50 μg / mL free GL solution, CS / SD / GL extract loaded with equal GL concentration, and CS / SD extract with a concentration of 50 μg / mL (the preparation methods and concentrations of CS / SD / GL extract and CS / SD extract are the same as above) were incubated with red blood cells for 4 hours. The hemolysis of red blood cells treated with different experimental groups was as follows: Figure 8 The results show that the hemolysis rates of GL, CS / SD hydrogel, and CS / SD / GL hydrogel were 2.39%, 1.79%, and 1.49%, respectively, all below the international standard of 5%. Therefore, both CS / SD and CS / SD / GL hydrogels have good blood compatibility and demonstrate significant application value in biomedical fields such as hemostasis and skin regeneration. The CS / SD / GL hydrogel's ability to significantly reduce the hemolysis rate may be due to the inclusion of SD with chitosan to form a hydrogel, which imparts a sustained-release mechanism. This means that the hydrogel does not produce a sudden release of GL, which could lead to excessive local concentrations of GL, thereby reducing its toxicity. The slow release of GL prolongs its effect, enhancing its effectiveness.

[0100] Effect Verification Example 2

[0101] (1) In vitro antibacterial effect of hydrogel

[0102] Wound infection is characterized by the presence of proliferating bacteria in living tissue. These bacteria damage tissue and hinder wound healing. Therefore, the primary function of wound dressings is to have antibacterial effects. The plate count method was used to investigate the in vitro antibacterial effects of CS / SD hydrogel (with a 1.0 M NaHCO3 solution) and CS / SD / GL hydrogel. The specific operation was as follows: a single colony was picked in PBS using a disposable inoculating loop in an ultra-clean workbench, evenly dispersed, and then diluted in PBS in successive steps. Finally, the colony concentration was determined to be 2×10 7 CFU / mL. Take 50μL of the diluted bacterial solution and add different concentrations of hydrogel extract (the preparation method of the extract and the meaning of the concentration are the same as above) or GL solution to 500μL, and culture at 37℃ for 30min. Then take 100μL of the cultured bacterial solution and spread it on the culture plate, and culture it in a constant temperature incubator at 37℃ for 18h. Finally, record the number of colonies and calculate the inhibition rate. The results are as follows Figure 9 As shown, (A) is a colony photo of S. aureus and E. coli after being treated with CS / SD and CS / SD / GL hydrogel extracts of different concentrations, (B) is a statistical diagram of the inhibitory effect on S. aureus, (C) is a statistical diagram of the inhibitory effect on E. coli, and (D) is a scanning electron microscope image of bacteria after being treated with different materials, wherein the normal group bacteria were treated with PBS, the GL group was treated with a free GL solution with a concentration of 1 mg / mL, and the CS / SD / GL group and the CS / SD group were treated with extracts with a concentration of 50 μg / mL (the preparation methods and concentrations of the CS / SD / GL extracts and CS / SD extracts are the same as above). Figure 9As shown, after co-incubating extracts of CS / SD and CS / SD / GL hydrogels at different concentrations with S. aureus and E. coli for 30 minutes, the antibacterial effects of the hydrogels against S. aureus and E. coli gradually increased with increasing CS / SD and CS / SD / GL hydrogel concentrations. At concentrations below 10 μg / mL, the CS / SD hydrogel showed no antibacterial effect against S. aureus, but achieved a 60% antibacterial effect against E. coli. At a concentration of 35 μg / mL, the antibacterial effects against S. aureus and E. coli reached 90% and 100%, respectively. When GL was loaded into the CS / SD hydrogel, the CS / SD / GL hydrogel exhibited even stronger antibacterial activity. This indicates that the combined action of GL and CS / SD hydrogel imparts enhanced antibacterial activity to the hydrogel. Furthermore, after treatment with the CS / SD and CS / SD / GL hydrogels, Staphylococcus aureus bacteria were destroyed, shrinking and deforming. The cell membranes of Escherichia coli cells were severely concave and deformed, making it difficult for the bacteria to grow. These changes in bacterial morphology demonstrate that the CS / SD and CS / SD / GL hydrogels can kill bacteria, demonstrating their superior antibacterial efficacy.

[0103] (2) Hydrogels promote cell migration in vitro

[0104] NIH / 3T3 cells in the logarithmic growth phase were cultured at 2×10 5 The cells were seeded at a density of 100 cells per well in a six-well plate. After the cells were fully grown, they were marked with a marker, and a 200μL sterile pipette tip was used to draw a line vertically along the marked line. The old culture medium was aspirated, and the suspended cells were washed with PBS until they were clean. The normal group (ie Normal) was cultured with a culture medium containing 10wt% serum, and the control group (ie Control) was cultured with a culture medium containing 2wt% serum. The experimental groups were added with 100μL of 50μg / mL free GL solution, CS / SD / GL extract, and CS / SD hydrogel extract (the preparation method of the extract and the meaning of the concentration are the same as above), and continued to culture in 2wt% serum culture medium for 12h and 24h. The cell migration status was observed under a microscope and photographed and recorded. The effects of GL, CS / SD hydrogel (the concentration of the added NaHCO3 solution was 1.0M) and CS / SD / GL hydrogel on the migration of NIH / 3T3 cells are shown in Figure 2. Figure 10As shown in the figure, over time, the cell scratches in each group showed a trend of gradually narrowing. This phenomenon shows that GL, CS / SD hydrogel and CS / SD / GL hydrogel all have the ability to promote cell migration. Compared with the control group, after being treated with CS / SD and CS / SD / GL hydrogels for 12 hours, the cells were able to migrate rapidly, and the migration speed was greater than that of the GL-treated group. After 24 hours, the scratch healing was more obvious. Compared with the normal group, the ability of CS / SD and CS / SD / GL hydrogels to promote cell migration can achieve the same effect as the normal group, and CS / SD / GL hydrogel is slightly better than CS / SD hydrogel, indicating that the released GL has the ability to cooperate with CS / SD hydrogel to accelerate the migration of fibroblasts (NIH / 3T3 cells) to the scratch area and promote cell proliferation.

[0105] (3) In vitro anti-inflammatory effect of hydrogel

[0106] RAW 264.7 cells in good growth condition were plated at 3×10 5 The cells were seeded at a density of 100 cells per well in a six-well plate. After the cells adhered to the wall, they were cultured for 4 hours using DMEM high-glucose medium (the meaning of the concentration is the same as above) containing 50 μg / mL free GL, CS / SD / GL hydrogel loaded with an equal amount of GL concentration, or 50 μg / mL CS / SD hydrogel (the concentration of the added NaHCO3 solution was 1.0 M). LPS was then added to make the final LPS concentration 1 μg / mL, and the culture was continued for 24 hours. Cells cultured in DMEM high-glucose medium without adding drugs and LPS were used as negative controls, and cells cultured without adding drugs but with LPS were used as positive controls. Finally, the cells were photographed to observe the cell morphology and the cell supernatant was taken to detect the content of NO, TNF-α and IL-6 in RAW 264.7 cells using an ELISA kit. The test results are shown in the figure. Figure 11 shown.

[0107] The second stage of the wound healing process is the inflammatory phase. Inflammation is crucial in regulating the wound healing process. In infected wounds, bacterial invasion and the release of proinflammatory cytokines such as interleukin 1-β and tissue necrosis factor-ɑ inhibit the function of fibroblasts, leading to an increase in matrix metalloproteinases (MMPs), thereby reducing the production of growth factors and ultimately preventing entry into the proliferation phase. Therefore, the expression of cellular proinflammatory factors TNF-α, IL-6, and NO was detected to demonstrate the anti-inflammatory ability of the hydrogel. Figure 11As shown, (a) is the RAW 264.7 cells in the normal growth state (ie, negative control), and the cells can be seen growing in round aggregates attached to the wall. (b) is the RAW 264.7 cells after LPS stimulation (ie, positive control), and it can be seen that the cells have become larger and tentacles have appeared, indicating that the cell modeling is successful. (c)-(e) are RAW 264.7 cells after LPS stimulation and the addition of GL, CS / SD, and CS / SD / GL. It can be seen that when RAW 264.7 cells are co-cultured with GL, CS / SD, or CS / SD / GL and LPS, the cell tentacles are reduced, and the morphology of some cells becomes round, gradually restoring the normal RAW 264.7 cell morphology. This shows that GL, CS / SD, and CS / SD / GL can effectively inhibit cell differentiation and relieve inflammatory responses. The supernatant of each group of cells was taken to measure the NO content, and the results are shown as follows Figure 11 As shown in (f), the NO levels after GL, CS / SD, and CS / SD / GL treatments were 62.78 μM, 53.14 μM, and 43.96 μM, respectively, compared to 170.48 μM in the LPS group, indicating that NO secretion was effectively inhibited. The TNF-α and IL-6 levels in the supernatant of each cell were detected using ELISA kits, as shown in Figure 5. Figure 11 As shown in (g) and (h), after LPS stimulation of RAW 264.7 cells, the secretion of TNF-α and IL-6 in the LPS group (i.e., positive control group) was significantly higher than that in the negative control group (i.e., Normal). After treatment with GL, CS / SD, and CS / SD / GL hydrogels, the contents of TNF-α and IL-6 decreased. Among them, CS / SD / GL hydrogel had the best inhibitory effect on TNF-α and IL-6, indicating that CS / SD / GL hydrogel showed a stronger anti-inflammatory effect due to the addition of GL.

[0108] (4) Hydrogel promotes wound healing in mice

[0109] The therapeutic effect of the hydrogel on mouse wounds was evaluated using a Staphylococcus aureus infected mouse skin wound model. After KM mice were adaptively fed for one week, the back hair was removed and the mice were anesthetized. A circular full-thickness skin wound with a diameter of 7 mm was created on the back using a hole punch. Then, 50 μL of a 2×10 8A 500 μg / mL Staphylococcus aureus liquid was applied. After the liquid was absorbed, the wound was fixed with medical gauze and medical tape. After 24 hours of incubation, the wound was considered successfully established if it was red, swollen, and purulent. After successful modeling, the mice were randomly divided into five groups (10 mice per group). The first group served as a positive control and was smeared with a commercially available medical hydrogel wound dressing (ofloxacin hydrogel, Wanchuan Pharmaceutical). The second group served as a negative control (treated with 0.9 wt% saline and no other treatment). The third group was treated with a 50 μg / mL GL solution in PBS. The fourth group was smeared with the CS / SD hydrogel prepared in Example 1 (with a 1.0 M NaHCO solution added). The fifth group was smeared with the CS / SD / GL hydrogel dressing prepared in Example 2. The same amount of hydrogel was used in each application for the first, fourth, and fifth groups. All groups received normal water and food, and the dressing was changed every 48 hours.

[0110] The wounds on the back of the mice were photographed and recorded on days 0, 3, 7, 10, and 14, and the wound healing rates of the mice were analyzed. Figure 12 As shown, (A) shows photographs of mouse wounds treated with different materials at different times, (B) shows wound area trace analysis (from left to right, groups 1 to 5), and (C) shows wound healing rate statistics. As can be seen from the figure, the wound area in each group gradually decreased over time. The wounds in the CS / SD / GL hydrogel group healed the fastest, while those treated with 0.9wt% saline healed more slowly. On day 14 of treatment, scabs in the wounds of all groups, except those treated with 0.9wt% saline, fell off naturally. The wounds treated with ofloxacin hydrogel (positive control) and CS / SD / GL hydrogel had the smallest wound surfaces.

[0111] On the 7th and 14th days of treatment, the mice were euthanized, and the skin around the wound was placed in a tissue homogenizer and thoroughly ground with 1 mL of normal saline. The homogenate was then diluted a certain number of times and 100 μL was added to the bacterial culture medium and evenly spread. The cells were then placed in a 37°C constant temperature incubator for 18 h, and photographed and the number of bacterial colonies was calculated. Figure 13 Figure 2 shows a colony image (A) and a colony count (B). Treatment with the CS / SD / GL hydrogel significantly reduced the number of Staphylococcus aureus colonies, achieving the same efficacy as the commercially available antibacterial hydrogel ofloxacin. Furthermore, the number of colonies in wounds treated with the CS / SD hydrogel was lower than that in the GL hydrogel, consistent with wound healing. These results demonstrate that the CS / SD / GL hydrogel significantly promotes wound skin healing.

[0112] The pH change during skin wound healing is caused by a variety of factors, including inflammatory response, cell metabolism and external environment. The pH value is a key indicator of wound status, and it is closely related to many physiological processes, especially bacterial infection defense and angiogenesis. For normal skin or healing wounds, the pH value is usually in the slightly acidic range, between 4 and 6. This slightly acidic environment is crucial for wound healing because it can inhibit the growth of harmful bacteria while promoting normal cell repair. In the case of bacterial infection, the pH value around the wound tends to increase. The pH value of normal skin was measured with a portable pH meter before mouse modeling. After the mouse modeling was successful, the pH value of the wound skin was measured again with a pH meter. After the mice were randomly divided into groups, the pH changes in each group were monitored on days 0, 1, 2, 3, 7, 10, and 14 of treatment. The results are shown in the figure. Figure 14 As shown in the figure, the pH of normal mice without wound modeling is approximately 6.25. After bacterial infection, the pH of the wound rises sharply to around 8.0. This is likely due to Staphylococcus aureus breaking down urea to produce ammonia, which in turn increases the pH of the skin wound. During treatment, because wound healing is accompanied by inflammation, the pH of the wound decreases, facilitating the remodeling phase of wound proliferation. As wound healing progresses, the pH around the wound gradually returns to normal after 14 days of treatment.

[0113] On the 3rd, 7th, and 14th days of treatment, the mice were euthanized and skin tissue with a diameter of approximately 1 cm was cut around the wound. The skin tissue was fixed in a tissue fixative and then embedded in paraffin. Tissue sections were prepared and stained with H&E for histological analysis. Figure 15(As shown, the white dashed line indicates the epithelial-dermal boundary; red arrows indicate inflammatory cells; black arrows indicate fibroblasts; brown arrows indicate capillaries; and green arrows indicate hair follicles. Scale bar: 50 μm.) As can be seen, on the third day of treatment, new granulation tissue began to regenerate. All groups exhibited significant inflammation, with a high infiltration of inflammatory cells and the production of numerous fibroblasts, accelerating wound healing. On the seventh day, the negative control group still exhibited inflammation, while the inflammatory response in the other groups was less pronounced, accompanied by the formation of hair follicles and capillaries. The epidermis began to form in the positive control group and the CS / SD / GL groups, but the epidermis-dermis connection was weak and easily separated. By the 14th day of treatment, inflammation in all groups had subsided, with inflammatory cells largely disappearing. During this process, a significant number of hair follicles were observed, indicating the initial formation of characteristic skin structures within the wound. The connective tissue in the dermis was relatively mature, with numerous hair follicles and oil glands developing. Furthermore, numerous new blood vessels were formed in the dermis. In comparison, the wound recovery effects of the positive control group and the CS / SD / GL group were more significant, with nearly complete wound healing, revealing a continuous and intact epidermis and more developed granulation tissue. Furthermore, wounds treated with the positive control group and the CS / SD / GL group also demonstrated good therapeutic effects in regenerating skin appendages, such as hair follicles. Therefore, the CS / SD / GL hydrogel has potential application value as a wound dressing.

[0114] The expression of inflammatory factors TNF-α and IL-6 during wound healing reflects the level of inflammation in the wound microenvironment. Immunohistochemical staining was used to assess their levels. The mice were euthanized on the 3rd, 7th, and 14th days of treatment, and their skin tissues were taken for immunohistochemical staining analysis. The staining results of TNF-α and IL-6 are shown in Figure 2. Figure 16 As shown in (A) and (C), with the increase of treatment time, the expression of inflammation in each group decreased. Compared with the negative control group, the expression of TNF-α and IL-6 in CS / SD / GL was lower than that in GL and CS / SD. The staining results were then quantitatively analyzed. The analysis results of TNF-α and IL-6 contents are shown in Figure 3. Figure 16 (B) and (D) (*P<0.05, **P<0.01, ***P<0.001). Figure 16 It can be seen that the TNF-α and IL-6 levels in the CS / SD / GL group were lower than those in the other groups, which is consistent with the wound healing process. This indicates that the addition of GL can enhance the anti-inflammatory activity of the hydrogel and further effectively inhibit the expression of inflammatory factors in the wound tissue, thereby accelerating wound healing.

[0115] New blood vessels play a very important role in the wound healing process, so immunofluorescence staining of vascular endothelial cell markers was performed. CD31 is a core marker of endothelial cells and is used in vascular-related research; α-SMA is a key marker of smooth muscle and myofibroblasts, showing the vascular wall. On the 14th day of treatment, immunofluorescence staining of the new skin was performed to analyze the content of α-SMA and CD31 and comprehensively evaluate the regeneration of new blood vessels in the granulation tissue. The staining results are as follows Figure 17 As shown in (A) (scale bar 20 μm, Merge is a figure in which α-SMA and CD31 are merged together), except for the negative control group, the other groups can significantly promote angiogenesis on the 14th day. Among them, the fluorescence expression of α-SMA and CD31 is the strongest in the CS / SD / GL group, followed by the positive control group and CS / SD group, and the weakest in the negative control group. The fluorescence expression results of α-SMA and CD31 were quantitatively determined by calculating the relative fluorescence area of positive expression, as shown in Figure 2. Figure 17 As shown in (B) and (C), the CS / SD / GL group had the highest expression levels of α-SMA and CD31 fluorescence, indicating increased neovascularization in skin tissue. Therefore, GL-loaded CS / SD hydrogels can promote angiogenesis in bacterial wounds by regulating α-SMA and CD31 levels, thereby accelerating wound healing.

[0116] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing a cyclodextrin antibacterial hydrogel capable of accelerating wound healing, characterized in that: The following steps are involved: A hydrophobic drug with antibacterial and anti-inflammatory effects is mixed with an octa-[6-deoxy-(sodium 3-mercaptopropionate)]-γ-cyclodextrin solution to obtain a cyclodextrin inclusion complex solution; the cyclodextrin inclusion complex solution is mixed with a chitosan solution to obtain a mixed solution; a NaHCO3 solution is added to the mixed solution to adjust the pH value to 5.8-6.5, and the solution is allowed to react to obtain the cyclodextrin antibacterial hydrogel capable of accelerating wound healing.

2. The method for preparing the cyclodextrin antibacterial hydrogel capable of accelerating wound healing according to claim 1, wherein: The hydrophobic drug with antibacterial and anti-inflammatory effects is a pentacyclic triterpenoid compound.

3. The method for preparing the cyclodextrin antibacterial hydrogel capable of accelerating wound healing according to claim 2, wherein: The pentacyclic triterpenoid compounds include glycyrrhizic acid, glycyrrhetinic acid, ursolic acid or oleanolic acid.

4. The method for preparing the cyclodextrin antibacterial hydrogel capable of accelerating wound healing according to claim 1, wherein: The mass ratio of the hydrophobic drug having antibacterial and anti-inflammatory effects to the octa-[6-deoxy-(sodium 3-mercaptopropionate)]-γ-cyclodextrin contained in the octa-[6-deoxy-(sodium 3-mercaptopropionate)]-γ-cyclodextrin solution is 1:1.25-10; And / or, the mass ratio of octa-[6-deoxy-(sodium 3-mercaptopropionate)]-γ-cyclodextrin in the octa-[6-deoxy-(sodium 3-mercaptopropionate)]-γ-cyclodextrin solution to chitosan in the chitosan solution is 1:5-50.

5. The method for preparing the cyclodextrin antibacterial hydrogel capable of accelerating wound healing according to claim 1, wherein: The temperature of the static reaction is 0-40° C., and the time is 36-72 hours.

6. The method for preparing the cyclodextrin antibacterial hydrogel capable of accelerating wound healing according to claim 1, wherein: The solvent of the octa-[6-deoxy-(sodium 3-mercaptopropionate)]-γ-cyclodextrin solution includes water; And / or, the solvent of the chitosan solution includes an acid solution.

7. The method for preparing the cyclodextrin antibacterial hydrogel capable of accelerating wound healing according to claim 1, wherein: The concentration of the octa-[6-deoxy-(sodium 3-mercaptopropionate)]-γ-cyclodextrin solution is 5-20 mg / mL; And / or, the concentration of the chitosan solution is 40-60 mg / mL.

8. A cyclodextrin antibacterial hydrogel capable of accelerating wound healing prepared by the method for preparing a cyclodextrin antibacterial hydrogel capable of accelerating wound healing according to any one of claims 1 to 7.

9. Use of the cyclodextrin antibacterial hydrogel capable of accelerating wound healing as claimed in claim 8 in the preparation of a medicine or dressing for accelerating wound healing.

10. A medicine or dressing for accelerating wound healing, characterized in that: The invention comprises the cyclodextrin antibacterial hydrogel capable of accelerating wound healing as claimed in claim 8.

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