Preparation method of pH-sensitive antibacterial hydrogel spray for wound inflammation repair based on anthocyanin and Cu < 2 + > complexation

Abstract

The invention relates to a preparation method of a pH-sensitive antibacterial hydrogel spray for wound inflammation repair based on anthocyanin and Cu < 2 + > complexation. The pH-sensitive antibacterial hydrogel spray is used for promoting wound inflammation repair and dynamic monitoring. The preparation method comprises the following steps: mixing a polyvinyl alcohol (PVA) solution and a CuSO4 solution according to a certain volume ratio to prepare a solution 1; mixing a sodium alginate (SA) solution with an anthocyanin solution to prepare a solution 2; then, the solution 1 and the solution 2 are mixed for use at normal temperature, and the hydrogel with soft texture is formed through a cross-linking reaction of polyvinyl alcohol (PVA) and sodium alginate (SA). The hydrogel prepared by the method has the characteristics of softness and skin fitting, and the time for preparing the hydrogel by the method is short. The hydrogel disclosed by the invention shows good application potential in wound care and inflammation monitoring, and provides a beneficial reference for developing a novel multifunctional wound inflammation repair material.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of polymer materials, and particularly relates to a preparation method of a pH-sensitive antibacterial hydrogel spray based on anthocyanin and Cu 2+ complexation for wound inflammation repair. BACKGROUND

[0002] In recent years, with the increasing attention to the demand for microenvironment regulation in the wound healing process, the development of multifunctional smart dressings has become a hot research direction (Liang et al. ACS Nano 2021, 15, 8, 12687-12722). During the wound healing process, the changes in the local microenvironment (such as pH value) play a crucial role in tissue repair. Studies have shown that the pH value of infected wounds usually changes from weakly acidic to alkaline, and dynamic monitoring of the pH value change can not only evaluate the wound state in real time, but also provide scientific guidance for clinical treatment. At the same time, wound infection is a common complication in the wound healing process, and the use of antibacterial materials can significantly reduce the incidence of infection, thereby accelerating the healing process. Therefore, the development of smart materials with antibacterial properties, pH response characteristics and dynamic monitoring capabilities is of great significance in the field of wound care.

[0003] Traditional hydrogels have been widely used in wound care due to their excellent moisturizing properties, flexibility and biocompatibility, but there is still room for breakthrough in functionalization. Based on this, the present study proposes a pH-sensitive antibacterial hydrogel based on anthocyanin and Cu 2+ complexation. SUMMARY

[0004] The purpose of the present application is to provide a method for rapidly preparing a hydrogel spray for promoting wound inflammation repair and dynamically monitoring skin wounds, i.e. a preparation method of a pH-sensitive antibacterial hydrogel spray based on anthocyanin and Cu 2+ complexation for wound inflammation repair, which can be rapidly prepared. The obtained hydrogel spray can be applied to any wound shape to form a gel, and the shape of the formed gel can be automatically adjusted according to the morphological characteristics of the wound. The antibacterial property is strong and the skin wound inflammation repair situation can be observed in real time. At the same time, it can be placed in a small spray bottle, which is convenient to carry and can cope with emergency medical rescue. Therefore, it has great application potential in the medical field.

[0005] The purpose of the present application is achieved by the following technical solutions:

[0006] The application forms a hydrogel by cross-linking of polyvinyl alcohol (PVA) and sodium alginate (SA), the formed hydrogel has certain antibacterial function, can be used for monitoring pH change around the wound in real time, and can promote repair of wound inflammation.

[0007] An antibacterial hydrogel spray for wound inflammation repair based on anthocyanin and Cu 2+ complexation of pH-sensitive type, a polyvinyl alcohol (PVA) solution is mixed with a CuSO4 solution according to a certain volume ratio to prepare solution 1; a sodium alginate (SA) solution is mixed with an anthocyanin solution to prepare solution 2; then, solution 1 and solution 2 are mixed at room temperature for use, and a hydrogel with soft texture is formed through cross-linking reaction of polyvinyl alcohol (PVA) and sodium alginate (SA).

[0008] Solution 1 and solution 2 can be filled into a small spray bottle and sprayed on the wound according to equal volume ratio, and the hydrogel can be quickly formed. The hydrogel realizes significant antibacterial effect by virtue of the antibacterial property of Cu 2+ , and realizes dynamic visual monitoring of the wound state relying on the pH-sensitive color change property of anthocyanin, and shows potential application value in the field of wound care.

[0009] The antibacterial hydrogel spray preparation method has the following specific operation steps:

[0010] (1) a PVA solution with a mass percentage of 10% is mixed with a 100mM CuSO4 solution according to a volume ratio of 1:1 at room temperature to obtain solution 1;

[0011] (2) a SA solution with a solution mass percentage of 4% is mixed with an anthocyanin solution according to a volume ratio of 1:1 at room temperature to obtain solution 2;

[0012] (3) solution 1 obtained in step (1) and solution 2 obtained in step (2) are respectively placed in a small spray bottle for standby;

[0013] When used, solution 1 and solution 2 are sprayed on the wound according to a volume ratio of 1:1, cross-linking reaction of polyvinyl alcohol (PVA) and sodium alginate (SA) occurs, and a hydrogel with soft texture, i.e. the antibacterial hydrogel, is formed.

[0014] The preparation method of the PVA solution in step (1) is as follows: 10.0g of PVA is dissolved in 100mL of deionized water, and stirred and dissolved at 90℃ to obtain the PVA solution.

[0015] The method for preparing the CuSO4 solution in step (1) is as follows: Weigh 1.5961g of CuSO4 and dissolve it in 100mL of deionized water. Stir and dissolve at room temperature to obtain the CuSO4 solution.

[0016] The method for preparing the SA solution in step (2) is as follows: Weigh 4.0g of SA and dissolve it in 100mL of deionized water at room temperature to obtain the SA solution;

[0017] The anthocyanin solution in step (2) is prepared by dissolving 150 mg of anthocyanin in 10 mL of deionized water to obtain the anthocyanin solution.

[0018] The antibacterial hydrogel spray prepared by the aforementioned method comprises solution 1 and solution 2.

[0019] At room temperature, the standby solutions (i.e., solutions 1 and 2) have the characteristics of low viscosity and high fluidity.

[0020] The aforementioned antibacterial hydrogel spray forms a hydrogel through the cross-linking of polyvinyl alcohol (PVA) and sodium alginate (SA). This hydrogel possesses antibacterial properties, can be used to monitor pH changes around the wound in real time, and can promote wound inflammation repair. The resulting spray is a soft, skin-adhering antibacterial hydrogel spray.

[0021] The application of the antibacterial hydrogel spray in the preparation of antibacterial and anti-inflammatory agents for skin wounds.

[0022] In the aforementioned application, solution 1 and solution 2 are placed in spray bottles respectively, and solution 1 and solution 2 are sprayed onto the wound at a volume ratio of 1:1 to quickly form a hydrogel.

[0023] Compared with the prior art, the advantages of the present invention are as follows:

[0024] In preparing the antibacterial hydrogel spray, this invention uses anthocyanins as a raw material. Anthocyanins, as a natural pigment, possess excellent biocompatibility and significant pH-sensitive color-changing properties, enabling real-time visual monitoring of the wound microenvironment; copper ions (Cu... 2+ It has been extensively studied for its broad-spectrum antibacterial properties and healing-promoting effects. This is achieved by combining anthocyanins and Cu... 2+ Introducing a hydrogel network system not only endows the hydrogel with excellent antibacterial properties, but also enables wound inflammation repair and dynamic monitoring.

[0025] Furthermore, this hydrogel spray is characterized by its simple preparation and rapid response, forming quickly through the cross-linking of polyvinyl alcohol (PVA) and sodium alginate (SA). When applied to a wound, a hydrogel film forms within 20 seconds. This rapid film formation indicates that its physical properties can be adjusted according to the wound's morphological characteristics, allowing it to conform to irregular wound surfaces. This multifunctional hydrogel spray not only effectively addresses the functional limitations of traditional wound dressings but also offers convenient portability in a spray bottle, enabling immediate response to emergency medical care. In practical applications, it demonstrates excellent ease of use and clinical applicability. Therefore, this pH-sensitive antibacterial hydrogel spray possesses significant application potential in the medical field, particularly in the development of intelligent wound care materials, providing a valuable reference for the development of novel multifunctional wound inflammation repair materials.

[0026] In summary, the present invention has the following advantages:

[0027] (1) This invention uses PVA / SA / anthocyanin / Cu 2+ The preparation of hydrogel sprays is simple and quick.

[0028] (2) The hydrogel spray prepared in this invention has good skin adhesion properties and can be customized according to the wound morphology to fit irregular wound surfaces.

[0029] (3) The hydrogel formed by the spray prepared by the present invention has good anti-inflammatory and antibacterial effects and promotes inflammation repair. The formed hydrogel can monitor the wound condition through color.

[0030] (4) The hydrogel prepared by this invention has the characteristic of visualizing wound changes.

[0031] (5) The hydrogel prepared by the present invention can be placed in a small spray bottle, which is convenient to carry and can be used for emergency medical care.

[0032] (6) PVA solution, CuSO4, and SA must coexist to form a hydrogel. When solution 1 and solution 2 are mixed and sprayed, a hydrogel can be formed quickly at room temperature within a short time. Moreover, the hydrogel spray can be applied to any wound shape, and the shape will automatically adjust according to the morphological characteristics of the wound during gel formation.

[0033] (7) The hydrogel prepared by this invention is soft and conforms well to the skin, and the preparation time is short. The hydrogel of this invention shows good application potential in wound care and inflammation monitoring, and provides a useful reference for the development of novel multifunctional wound and inflammation repair materials. Attached Figure Description

[0034] Figure 1This is a schematic diagram of the hydrogel prepared according to an embodiment of the present invention.

[0035] Figure 2 The PVA / SA / anthocyanin / Cu prepared in the embodiments of the present invention 2+ The color appearance of the hydrogel at different pH values ​​(simulating changes in skin wound inflammation).

[0036] Figure 3 The inhibitory effect of the PVA / SA / anthocyanin / Cu2+ hydrogel prepared in the embodiments of the present invention on different bacteria.

[0037] Figure 4 This is a schematic diagram illustrating the downregulation of intracellular NO levels by the PVA / SA / anthocyanin / Cu2+ hydrogel prepared in an embodiment of the present invention.

[0038] Figure 5 This is a schematic diagram illustrating the downregulation of intracellular TNF-α inflammatory factor levels by the PVA / SA / anthocyanin / Cu2+ hydrogel prepared in an embodiment of the present invention. Detailed Implementation

[0039] The present invention will now be described in detail with reference to the accompanying drawings and embodiments:

[0040] Example 1:

[0041] A method based on anthocyanins and Cu 2+ Complexed pH-sensitive antibacterial hydrogel for wound inflammation repair (hereinafter referred to as PVA / SA / anthocyanin / Cu) 2+ The preparation method of the hydrogel spray, and its specific operation steps are as follows:

[0042] (1) Preparation of PVA solution: Weigh 10.0g PVA and dissolve it in 100mL of deionized water. Stir and dissolve at 90℃.

[0043] (2) Prepare 100 mL of CuSO4 solution with a concentration of 100 mM: Weigh 1.5961 g of CuSO4 and dissolve it in 100 mL of deionized water.

[0044] (3) Preparation of SA solution: Weigh 4.0g of SA and dissolve it in 100mL of deionized water at room temperature.

[0045] (4) Prepare 15mg / mL anthocyanin: Weigh 150mg of anthocyanin and dissolve it in 10mL of deionized water.

[0046] (5) Mix the PVA solution obtained in step (1) and the CuSO4 solution obtained in step (2) in a volume ratio of 1:1 in spray bottle 1 to obtain solution 1.

[0047] (6) Mix the solutions obtained in steps (3) and (4) at a volume ratio of 1:1 in spray bottle 2 to obtain solution 2.

[0048] The hydrogel spray includes solution 1 and solution 2. When using, simply spray the small spray bottle mentioned in steps (5) and (6) onto the wound at a 1:1 volume ratio.

[0049] The schematic diagram of the preparation of hydrogel spray in Example 1 of this invention is shown below. Figure 1 As shown. From Figure 1 The actual operation steps and synthesis principle of this preparation method can be derived.

[0050] This invention optimizes the structure and properties of hydrogels by adjusting key experimental parameters in the above method (such as the concentration ratio of PVA to SA, CuSO4 concentration, and anthocyanin concentration), giving them soft and skin-adhering properties. At the same time, the hydrogels prepared by this method are quick to prepare. The experimental steps in Example 1 are the final optimized steps.

[0051] The inventors conducted experiments on simulated skin wound inflammation changes, inhibition effect, NO expression level test, and TNF-α expression level test on the hydrogel spray prepared according to the method described in Example 1 of this invention. The specific methods are described in Examples 2-5.

[0052] Example 2: Simulating Inflammatory Changes in Skin Wounds

[0053] 1. Weigh 10.0g of PVA and dissolve it in 100mL of deionized water, stirring at 90℃.

[0054] 2. Prepare 100 mL of 100 mM CuSO4 solution: Weigh 1.5961 g of CuSO4 and dissolve it in 100 mL of deionized water.

[0055] 3. Weigh 4.0g of SA and dissolve it in 100mL of deionized water at room temperature.

[0056] 4. Prepare 15mg / mL anthocyanin: Weigh 150mg of anthocyanin and dissolve it in 10mL of deionized water.

[0057] 5. Mix the solutions from steps 1 and 2 in a volume ratio of 1:1 in spray bottle 1 to obtain solution 1.

[0058] 6. Mix the solutions from steps 3 and 4 above in a volume ratio of 1:1 in spray bottle 2 to obtain solution 2.

[0059] 7. Add 4 mL of PBS buffer solution with different pH values ​​(pH 4.0–9.0) to each of the 6-well culture dishes.

[0060] 8. Spray 1 mL of each of the solutions 1 and 2 obtained in steps 5 and 6 onto the surface of the buffer solution in step 7, and let stand for 5 minutes to observe the color change of the hydrogel.

[0061] 9. Use a camera to record the color development of the formed hydrogel under different pH conditions.

[0062] Figure 2 The PVA / SA / anthocyanin / Cu prepared in the embodiments of the present invention 2+ The color appearance of the hydrogel spray at different pH values ​​(simulating changes in skin wound inflammation). Figure 2 As can be seen, the hydrogel will exhibit different colors at different pH values, and changes in wound inflammation can be observed through visualization.

[0063] Example 3: Inhibition Effect Experiment

[0064] 1. Weigh 10.0g of PVA and dissolve it in 100mL of deionized water, stirring at 90℃.

[0065] 2. Prepare 100 mL of 100 mM CuSO4 solution: Weigh 1.5961 g of CuSO4 and dissolve it in 100 mL of deionized water.

[0066] 3. Weigh 4.0g of SA and dissolve it in 100mL of deionized water at room temperature.

[0067] 4. Prepare 15mg / mL anthocyanin: Weigh 150mg of anthocyanin and dissolve it in 10mL of deionized water.

[0068] 5. Mix the solutions from steps 1 and 2 in a volume ratio of 1:1 in spray bottle 1 to obtain solution 1.

[0069] 6. Mix the solutions from steps 3 and 4 above in a volume ratio of 1:1 in spray bottle 2 to obtain solution 2.

[0070] 7. Pour the solutions 1 and 2 obtained in steps 5 and 6 into molds, mix and shape them, cut them into round sheets (hydrogel sheets) with a diameter of 6 mm and a thickness of about 2 mm, and store them under sterile conditions for later use.

[0071] 8. Resuscitate E. coli, S. aureus and MRSA from -80℃ glycerol strains, inoculate them into LB liquid medium and culture at 37℃ and 180 rpm for 12-16 hours with shaking.

[0072] 9. Dilute the bacterial culture to 1×10⁻⁶ using PBS buffer. 6 CFU / mL was used as the experimental bacterial suspension.

[0073] 10. Take the bacterial suspensions obtained in step 9 (1 mL, 1×10⁻⁶) and place them in a container.6 Add the CFU / mL hydrogel sheet prepared in step 7 to a centrifuge tube and let it stand at 37°C for 24 hours.

[0074] 11. Take the supernatant from the centrifuge tube in step 10, serially dilute it 10-fold with PBS buffer, take 100 μL and spread it on an LB plate. After incubating at 37°C for 16-18 h, count the number of colonies (CFU).

[0075] Figure 3 The PVA / SA / anthocyanin / Cu prepared in the embodiments of the present invention 2+ The inhibitory effect of hydrogels on different bacteria. Figure 3 As can be seen, compared with the control group, the hydrogel has a good inhibitory effect on E. coli, S. aureus and MRSA.

[0076] Example 4: NO expression level test

[0077] 1. Weigh 10.0g of PVA and dissolve it in 100mL of deionized water, stirring at 90℃.

[0078] 2. Prepare 100 mL of 100 mM CuSO4 solution: Weigh 1.5961 g of CuSO4 and dissolve it in 100 mL of deionized water.

[0079] 3. Weigh 4.0g of SA and dissolve it in 100mL of deionized water at room temperature.

[0080] 4. Prepare 15mg / mL anthocyanin: Weigh 150mg of anthocyanin and dissolve it in 10mL of deionized water.

[0081] 5. Mix the solutions from steps 1 and 2 in a volume ratio of 1:1 in spray bottle 1 to obtain solution 1.

[0082] 6. Mix the solutions from steps 3 and 4 above in a volume ratio of 1:1 in spray bottle 2 to obtain solution 2.

[0083] 7. Quickly spray the solutions 1 and 2 obtained in steps 5 and 6 above onto the surface of a glass slide and allow them to gel to form a hydrogel film for use in cell culture experiments.

[0084] 8. RAW 264.7 macrophages were seeded into 6-well plates at a density of 1 × 10⁻⁶ cells per well. 5 Cells / wells were incubated overnight at 37°C and 5% CO2 to allow them to adhere to the culture vessel.

[0085] 9. In step 8, add lipopolysaccharide (LPS) stimulation solution (final concentration 1 μg / mL) to the cell wells. At the same time, add the hydrogel membrane sample prepared in step 7 to the experimental group. The blank group is not treated. The LPS group is a positive control. Incubate overnight at 37℃ and 5% CO2.

[0086] 10. Collect the supernatant from the culture medium in step 9, and use the Griess assay kit according to the instructions to determine the extracellular NO content.

[0087] 11. Use a UV-Vis spectrophotometer to read the absorbance at a wavelength of 540 nm, and calculate the NO concentration using a standard curve.

[0088] Figure 4 The PVA / SA / anthocyanin / Cu prepared in the embodiments of the present invention 2+ A schematic diagram illustrating the downregulation of intracellular NO levels by hydrogels. Figure 4 As can be seen, comparing the NO expression levels in the hydrogel-treated group and the control group, the hydrogel significantly reduced NO expression and had a good anti-inflammatory effect.

[0089] Example 5: Assay of TNF-α expression level

[0090] 1. Weigh 10.0g of PVA and dissolve it in 100mL of deionized water, stirring at 90℃.

[0091] 2. Prepare 100 mL of 100 mM CuSO4 solution: Weigh 1.5961 g of CuSO4 and dissolve it in 100 mL of deionized water.

[0092] 3. Weigh 4.0g of SA and dissolve it in 100mL of deionized water at room temperature.

[0093] 4. Prepare 15mg / mL anthocyanin: Weigh 150mg of anthocyanin and dissolve it in 10mL of deionized water.

[0094] 5. Mix the solutions from steps 1 and 2 in a volume ratio of 1:1 in spray bottle 1 to obtain solution 1.

[0095] 6. Mix the solutions from steps 3 and 4 above in a volume ratio of 1:1 in spray bottle 2 to obtain solution 2.

[0096] 7. Quickly spray the solutions 1 and 2 obtained in steps 5 and 6 above onto the surface of a glass slide and allow them to gel to form a hydrogel film for use in cell culture experiments.

[0097] 8. RAW 264.7 macrophages were seeded into 6-well plates at a density of 1 × 10⁻⁶ cells per well. 5Cells / wells were incubated overnight at 37°C and 5% CO2 to allow them to adhere to the culture vessel.

[0098] 9. In step 8, add lipopolysaccharide (LPS) stimulation solution (final concentration 1 μg / mL) to the cell wells. At the same time, add the hydrogel membrane sample prepared in step 7 to the experimental group. The blank group is not treated. The LPS group is a positive control. Incubate overnight at 37℃ and 5% CO2.

[0099] 10. Collect the supernatant from the culture medium in step 9. Using the mouse TNF-α enzyme-linked immunosorbent assay kit, follow the instructions to detect the TNF-α concentration in the cell culture supernatant of each group. Measure the absorbance (450 nm) and convert the concentration using a standard curve.

[0100] Figure 5 The PVA / SA / anthocyanin / Cu prepared in the embodiments of the present invention 2+ A schematic diagram illustrating the downregulation of intracellular inflammatory factor TNF-α levels by hydrogels. Figure 5 As can be seen, comparing the TNF-α expression levels of the hydrogel-treated group and the control group, it was found that the hydrogel significantly reduced the expression of TNF-α and had a good anti-inflammatory effect.

[0101] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method based on anthocyanins and Cu 2+ A method for preparing a complexed, pH-sensitive antibacterial hydrogel spray for wound inflammation repair, characterized in that: Solution 1 was prepared by mixing polyvinyl alcohol (PVA) solution and CuSO4 solution in a certain volume ratio; solution 2 was prepared by mixing sodium alginate (SA) solution and anthocyanin solution; then, solution 1 and solution 2 were mixed and used at room temperature, and a hydrogel with a soft texture was formed through the cross-linking reaction of polyvinyl alcohol (PVA) and sodium alginate (SA).

2. The method for preparing the antibacterial hydrogel spray according to claim 1, characterized in that: The specific operating steps are as follows: (1) Mix a 10% PVA solution by mass with a 100mM CuSO4 solution at a volume ratio of 1:1 at room temperature to obtain solution 1; (2) Mix the SA solution with a mass percentage of 4% and the anthocyanin solution at a volume ratio of 1:1 at room temperature to obtain solution 2; (3) Place the solution 1 obtained in step (1) and the solution 2 obtained in step (2) into small spray bottles for later use; When used, solution 1 and solution 2 are sprayed onto the wound at a volume ratio of 1:

1. Polyvinyl alcohol (PVA) and sodium alginate (SA) undergo a cross-linking reaction to form a hydrogel with a soft texture, namely the antibacterial hydrogel.

3. The method for preparing the antibacterial hydrogel spray according to claim 2, characterized in that: The method for preparing the PVA solution in step (1) is as follows: Weigh 10.0g of PVA and dissolve it in 100mL of deionized water, stir and dissolve at 90℃ to obtain the PVA solution.

4. The method for preparing the antibacterial hydrogel spray according to claim 2, characterized in that: The method for preparing the CuSO4 solution in step (1) is as follows: Weigh 1.5961g of CuSO4 and dissolve it in 100mL of deionized water. Stir and dissolve at room temperature to obtain the CuSO4 solution.

5. The method for preparing the antibacterial hydrogel spray according to claim 2, characterized in that: The method for preparing the SA solution in step (2) is as follows: Weigh 4.0g of SA and dissolve it in 100mL of deionized water at room temperature to obtain the SA solution; The anthocyanin solution in step (2) is prepared by dissolving 150 mg of anthocyanin in 10 mL of deionized water to obtain the anthocyanin solution.

6. An antibacterial hydrogel spray prepared by the method described in claim 1, characterized in that: It includes solution 1 and solution 2.

7. The application of the antibacterial hydrogel spray as described in claim 6 in the preparation of antibacterial agents and anti-inflammatory agents for skin wounds.

8. The application according to claim 7, characterized in that: Place solutions 1 and 2 into separate spray bottles, and spray them onto the wound at a volume ratio of 1:1 to quickly form a hydrogel.

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