Antibacterial and antioxidant composite hydrogel loaded with rhein and preparation and application thereof

Abstract

The application discloses a rhubarb acid-loaded antibacterial and antioxidant composite hydrogel as well as preparation and application thereof, and belongs to the technical field of tissue engineering. The application forms a stable three-dimensional network structure through electrostatic interaction between a positively-charged polymer and a negatively-charged protein under mild conditions, and successfully loads rhubarb acid as a single key functional component in the three-dimensional network structure. The core lies in that the pH response characteristic and multiple biological activities of the rhubarb acid molecule are utilized to realize the integration of diagnosis and treatment functions simultaneously. In the aspect of treatment, the synergistic effect of the strong antibacterial, antioxidant and anti-inflammatory activities of the sustained release of the rhubarb acid can actively improve the pathological microenvironment of an infected wound. In the aspect of diagnosis, the inherent color change characteristic of the rhubarb acid with the pH value makes the hydrogel itself a visual sensor, and the wound infection state can be monitored in real time and in situ without any additional sensing element.

Description

Technical Field

[0001] This invention belongs to the field of tissue engineering technology, specifically relating to an antibacterial and antioxidant composite hydrogel loaded with rhein, its preparation and application. Background Technology

[0002] The healing of infected wounds is a complex pathophysiological process, facing major challenges including uncontrollable bacterial contamination and biofilm formation, persistent excessive inflammatory response, and a pathological microenvironment dominated by excessive reactive oxygen species (ROS). These factors intertwine and collectively hinder the normal tissue repair process.

[0003] Ideal wound dressings should proactively address the aforementioned challenges. Hydrogel materials, due to their excellent biocompatibility, three-dimensional porous structure, and ability to mimic the extracellular matrix, are considered ideal substrates for advanced wound dressings. They effectively absorb wound exudate, keep the wound moist, and act as a physical barrier. Crucially, wound microenvironment parameters, especially pH, are important biological indicators dynamically reflecting infection status and healing progress. Healthy skin typically maintains a slightly acidic environment (pH 4-6), but the microenvironment shifts to alkaline (pH 7-8) during infection or when persistent tissue damage occurs. Therefore, real-time, in-situ monitoring of wound pH is essential for timely assessment of the condition, guiding adjustments to treatment strategies, and achieving precise management.

[0004] Existing technologies for endowing hydrogels with multifunctionality often face the challenge of low integration. To simultaneously achieve multiple therapeutic functions such as antibacterial, antioxidant, and anti-inflammatory effects, researchers often need to introduce various different active ingredients (such as various polyphenolic compounds). Furthermore, to achieve wound environment monitoring, additional independent sensing elements such as chromogenic dyes, fluorescent probes, or physical sensors are required. This "multi-component stacking" strategy directly leads to complex dressing compositions, cumbersome preparation processes, high costs, and difficulties in ensuring the compatibility, stability, and uniformity of the components.

[0005] In view of this, it is necessary to provide a new antibacterial and antioxidant composite hydrogel loaded with rhein to overcome the shortcomings of the prior art. Summary of the Invention

[0006] The purpose of this invention is to provide an antibacterial and antioxidant composite hydrogel loaded with rhein, its preparation, and its application. This addresses the problems of existing dressings, such as complex composition, cumbersome preparation processes, high costs, and poor compatibility, stability, and uniformity among the components.

[0007] In a first aspect, the present invention provides a method for preparing an antibacterial and antioxidant composite hydrogel loaded with rhein, comprising the following steps: providing a rhein solution, a protein solution, and a positively charged polymer solution respectively; mixing and stirring the rhein solution, the protein solution, and the positively charged polymer solution to obtain the antibacterial and antioxidant composite hydrogel loaded with rhein; wherein the concentration of the rhein solution is 0.5-1.5 wt%, the concentration of the protein solution is 0.1-1 wt%, and the concentration of the positively charged polymer solution is 2-4 wt%.

[0008] In this invention, the inventors discovered that by forming a stable three-dimensional network structure through electrostatic interaction between positively charged polymers and negatively charged proteins under mild conditions, rhein was successfully loaded into this three-dimensional network structure as a single key functional component. The core of this invention lies in utilizing the inherent pH-responsive properties and multiple bioactivities of rhein molecules to simultaneously achieve integrated diagnostic and therapeutic functions: In terms of treatment, the sustained release of potent antibacterial, antioxidant, and anti-inflammatory activities from rhein synergistically improves the pathological microenvironment of infected wounds; in terms of diagnosis, the inherent color change of rhein with pH value makes the hydrogel itself a visual sensor, enabling real-time, in-situ monitoring of wound infection status without any external sensing elements. Furthermore, this invention simplifies the system composition and preparation process through a single component, successfully constructing an intelligent wound management platform with simple components, high biocompatibility, and both active treatment and real-time feedback capabilities.

[0009] In some implementations, the concentration of rhein solution is 1 wt%, the concentration of protein solution is 0.5 wt%, and the concentration of positively charged polymer solution is 3 wt%.

[0010] In some embodiments, the volume ratio of rhein solution, protein solution and positively charged polymer solution is (0.5-1.5):(8-12):(8-12).

[0011] In some implementations, the volume ratio of rhein solution, protein solution, and positively charged polymer solution is 1:10:10.

[0012] In some implementations, the pH of the rhein solution is alkaline.

[0013] In some implementations, the protein in the protein solution includes bovine serum albumin.

[0014] In some embodiments, the positively charged polymer in the positively charged polymer solution includes cationic guar gum.

[0015] In some embodiments, the stirring temperature is room temperature during the step of mixing and stirring the rhein solution, protein solution, and positively charged polymer solution.

[0016] In a second aspect, the present invention provides an antibacterial and antioxidant composite hydrogel loaded with rhein, which is prepared by any of the above preparation methods.

[0017] In a third aspect, the present invention provides the application of the antibacterial and antioxidant composite hydrogel as described above in wound care and / or management.

[0018] The advantages of this invention, which differ from the prior art, are as follows: 1) Integrated diagnosis and treatment functions are achieved: The single active component rhein simultaneously endows the hydrogel with pH-responsive color-changing function and multiple therapeutic functions such as antibacterial / anti-inflammatory / antioxidant, overcoming the problems of system complexity and poor compatibility between components caused by the need to introduce sensing units and therapeutic components separately in the existing technology; 2) The preparation process is simplified and the biocompatibility is improved: the hydrogel substrate is constructed by electrostatic interaction between positively charged polymers and proteins. The cross-linking process is fast and mild, avoiding the toxic chemical cross-linking agents or complex pretreatment steps required in traditional cross-linking methods, making the preparation process simpler and more biocompatible. 3) Real-time visual monitoring and precise management of wounds: The hydrogel dressing can produce a visible color change according to the pH change of the wound, and can realize real-time, in-situ monitoring of wound infection status without relying on complicated instruments. This provides an intuitive basis for timely adjustment of treatment strategies and significantly improves the accuracy of wound management. Attached Figure Description

[0019] Figure 1 This is a flowchart illustrating the preparation method of the antibacterial and antioxidant composite hydrogel loaded with rhein in this invention. Figure 2 This is a rheological diagram of the antibacterial and antioxidant composite hydrogel loaded with rhein prepared in Example 1 of the present invention; Figure 3 The rheological diagram shows the antibacterial and antioxidant hydrogel loaded with rhein prepared in Comparative Example 1 of this invention. Figure 4 The rheological diagram of the composite hydrogel prepared in Comparative Example 2 of this invention is shown. Figure 5 This is a scanning electron microscope image of the antibacterial and antioxidant composite hydrogel loaded with rhein prepared in Example 1 of the present invention; Figure 6 The above figures show the inhibitory effects of the hydrogels prepared in Example 1, Comparative Examples 1 and 2 of this invention on Escherichia coli and Staphylococcus aureus. Figure 7 The pH curve of the antibacterial and antioxidant composite hydrogel loaded with rhein prepared in Example 1 of this invention for monitoring infected wounds; Figure 8 This is a graph showing the relationship between the color change and pH value of the antibacterial and antioxidant composite hydrogel loaded with rhein prepared in Example 1 of this invention. Figure 9 The bar chart shows the statistical analysis of the area of ​​infected wounds promoted by the hydrogels prepared in Examples 1, 1 and 2 of this invention. Detailed Implementation

[0020] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0021] Experimental methods not specified in the examples are generally performed under conventional conditions and as described in the manual, or as recommended by the manufacturer. Unless otherwise specified, the general equipment, materials, reagents, etc. used are commercially available.

[0022] Currently, existing dressings suffer from problems such as complex composition, cumbersome preparation process, high cost, and poor compatibility, stability and uniformity among the components.

[0023] To address the problems of existing dressings, such as complex composition, cumbersome preparation process, high cost, and poor compatibility, stability, and uniformity among components, this invention provides an antibacterial and antioxidant composite hydrogel loaded with rhein, as well as its preparation and application.

[0024] In a first aspect, the present invention provides a method for preparing an antibacterial and antioxidant composite hydrogel loaded with rhein, comprising the following steps: providing a rhein (Rhein, Rhe) solution, a protein solution, and a positively charged polymer solution respectively; mixing and stirring the rhein solution, the protein solution, and the positively charged polymer solution to obtain the antibacterial and antioxidant composite hydrogel loaded with rhein; wherein the concentration of the rhein solution is 0.5-1.5 wt%, for example, 0.5 wt%, 0.7 wt%, 1 wt%, 1.3 wt%, 1.5 wt%, or other values ​​within this range; the concentration of the protein solution is 0.1-1 wt%, for example, 0.1 wt%, 0.3 wt%, 0.5 wt%, 0.7 wt%, 1 wt%, or other values ​​within this range; and the concentration of the positively charged polymer solution is 2-4 wt%, for example, 2 wt%, 2.5 wt%, 3 wt%, 3.5 wt%, 4 wt%, or other values ​​within this range.

[0025] The method for preparing the antibacterial and antioxidant composite hydrogel provided by this invention involves forming a stable three-dimensional network structure by electrostatic interaction between positively charged polymers and negatively charged proteins under mild conditions, and successfully loading rhein as a single key functional component into the three-dimensional network structure. Its core lies in utilizing the inherent pH-responsive characteristics and multiple bioactivities of rhein molecules to simultaneously achieve integrated diagnostic and therapeutic functions: In terms of treatment, the potent antibacterial, antioxidant, and anti-inflammatory activities continuously released by rhein synergistically improve the pathological microenvironment of infected wounds; in terms of diagnosis, the inherent color change of rhein with pH value makes the hydrogel itself a visual sensor, enabling real-time, in-situ monitoring of wound infection status without any external sensing elements. Furthermore, this invention simplifies the system composition and preparation process through a single component, successfully constructing an intelligent wound management platform with simple components, high biocompatibility, and both active treatment and real-time feedback capabilities; effectively solving the technical challenge of separating treatment and monitoring functions in existing dressings, and possessing significant application value in the field of infected wound management.

[0026] In some implementations, the concentration of rhein solution is 1 wt%, the concentration of protein solution is 0.5 wt%, and the concentration of positively charged polymer solution is 3 wt%.

[0027] In this invention, by controlling the concentrations of rhein solution, protein solution, and positively charged polymer solution to optimal values, a high-performance antibacterial and antioxidant composite hydrogel can be obtained.

[0028] In some embodiments, the volume ratio of rhein solution, protein solution and positively charged polymer solution is (0.5-1.5):(8-12):(8-12).

[0029] In this invention, by controlling the volume ratio of rhein solution, protein solution, and positively charged polymer solution within a specific range, rhein can be completely loaded into the hydrogel, thereby improving the performance of the hydrogel.

[0030] In some implementations, the volume ratio of rhein solution, protein solution, and positively charged polymer solution is 1:10:10.

[0031] In this invention, by controlling the volume ratio of rhein solution, protein solution, and positively charged polymer solution to the optimal value, the performance of the hydrogel can be further improved.

[0032] In some embodiments, the pH of the rhein solution is alkaline, preferably 8-12, and more preferably 10.

[0033] In this invention, the pH value of the rhein solution is controlled to be alkaline, which provides a slightly alkaline reaction environment, thereby making the protein solution negatively charged, which facilitates electrostatic assembly with the positively charged polymer solution to obtain a hydrogel matrix.

[0034] In some implementations, the protein solution contains bovine serum albumin (BSA).

[0035] In this invention, bovine serum albumin, as a protein with excellent biocompatibility, has an isoelectric point of about 4.7. Under physiological neutral or alkaline conditions, it carries a net negative charge, which enables it to form a hydrogel network by electrostatic complexation with positively charged polymers. This provides a new approach for constructing a safe and efficient dressing substrate.

[0036] It is understandable that the protein can be conventionally selected according to actual usage needs, as long as it carries a net negative charge under physiological neutral or alkaline conditions. For example, in this invention, the protein in the protein solution preferably includes bovine serum albumin.

[0037] In some embodiments, the positively charged polymer in the positively charged polymer solution includes cationic guar gum (CG).

[0038] In this invention, by using positively charged cationic guar gum, the hydrogel matrix obtained by electrostatic assembly of guar gum and negatively charged protein has good sustained-release properties, further enabling the slow release of drugs and thus improving the performance of the hydrogel.

[0039] It is understandable that cationic guar gum can be conventionally selected according to actual usage needs, as long as it carries a positive charge. For example, in this invention, the positively charged polymer in the positively charged polymer solution preferably includes cationic guar gum.

[0040] In some embodiments, the stirring temperature is room temperature during the step of mixing and stirring the rhein solution, protein solution, and positively charged polymer solution.

[0041] It is understandable that the stirring temperature can be adjusted according to actual needs, as long as a hydrogel is obtained. For example, in this invention, the stirring temperature is preferably room temperature.

[0042] In a second aspect, the present invention provides an antibacterial and antioxidant composite hydrogel loaded with rhein, which is prepared by any of the above preparation methods.

[0043] In a third aspect, the present invention provides the application of the antibacterial and antioxidant composite hydrogel as described above in wound care and / or management.

[0044] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0045] Please see Figure 1 The flowchart illustrates the preparation method of the rhein-loaded antibacterial and antioxidant composite hydrogel of the present invention. Specifically, the preparation method includes the following steps: providing a rhein solution, a protein solution, and a positively charged polymer solution; mixing and stirring the rhein solution, protein solution, and positively charged polymer solution to obtain the rhein-loaded antibacterial and antioxidant composite hydrogel.

[0046] Example 1 A method for preparing an antibacterial and antioxidant composite hydrogel loaded with rhein includes the following steps: 1) Disperse 1g of rhein (Rhe) in 99g of deionized water, then add 10M sodium hydroxide solution to adjust the pH of the solution to 10 and keep stirring for 1 hour to obtain a Rhe solution with a concentration of 1wt%. 2) Dissolve 0.5g of bovine serum albumin (BSA) in 99.5g of deionized water to obtain a BSA solution with a concentration of 0.5wt%; 3) Disperse 3g of cationic guar gum (CG) in 97g of deionized water and stir at room temperature for 12 hours to obtain a CG solution with a concentration of 3wt%; 4) Take 1 mL of Rhe solution, 10 mL of CG solution and 10 mL of BSA solution and mix them at room temperature to obtain the antibacterial and antioxidant composite hydrogel dressing loaded with rhein (Rhe@CG-BSA).

[0047] Comparative Example 1 A method for preparing an antibacterial and antioxidant hydrogel loaded with rhein includes the following steps: 1) Disperse 1g of rhein (Rhe) in 99g of deionized water, then add 10M sodium hydroxide solution to adjust the pH of the solution to 10 and keep stirring for 1 hour to obtain a Rhe solution with a concentration of 1wt%. 2) Disperse 3g of cationic guar gum (CG) in 97g of deionized water and stir at room temperature for 12 hours to obtain a CG solution with a concentration of 3wt%; 3) Take 1 mL of Rhe solution and 10 mL of CG solution and mix them at room temperature to obtain the antibacterial and antioxidant hydrogel dressing loaded with rhein (Rhe@CG).

[0048] Comparative Example 2 A method for preparing a composite hydrogel includes the following steps: 1) Dissolve 0.5g of bovine serum albumin (BSA) in 99.5g of deionized water to obtain a BSA solution with a concentration of 0.5wt%; 2) Disperse 3g of cationic guar gum (CG) in 97g of deionized water and stir at room temperature for 12 hours to obtain a CG solution with a concentration of 3wt%; 3) Take 10 mL of CG solution and 10 mL of BSA solution and mix them at room temperature to obtain the composite hydrogel dressing (CG-BSA).

[0049] Performance Test 1 The rheological properties of the antibacterial and antioxidant composite hydrogel loaded with rhein prepared in Example 1, the antibacterial and antioxidant hydrogel loaded with rhein prepared in Comparative Example 1, and the composite hydrogel prepared in Comparative Example 2 were tested respectively, and the results are as follows: Figure 2-4 As shown.

[0050] from Figure 2-4 As can be seen, the storage modulus (G') of each prepared hydrogel is greater than the loss modulus (G''), indicating that the hydrogel has a robust, solid-like three-dimensional network structure that can withstand certain mechanical stress without flowing.

[0051] Furthermore, the antibacterial and antioxidant composite hydrogel loaded with rhein prepared in Example 1 was subjected to scanning electron microscopy, and the results are as follows: Figure 5 As shown.

[0052] from Figure 5As can be seen, the prepared composite hydrogel has a three-dimensional network structure and microporous features, as well as high porosity.

[0053] Performance Test 2 The antibacterial properties of the rhein-loaded antibacterial and antioxidant composite hydrogel prepared in Example 1, the rhein-loaded antibacterial and antioxidant hydrogel prepared in Comparative Example 1, and the composite hydrogel prepared in Comparative Example 2 were tested respectively.

[0054] Specifically, the hydrogel prepared above was added to a 24-well cell culture plate. It was sterilized with ultraviolet light for 24 hours. 1 mL of diluted bacterial suspension (10) was then added. 8 CFU / mL was added to a sterile hydrogel. Two groups were selected as control groups. One group had 1 mL of diluted bacterial suspension added to a hydrogel-free 24-cell culture plate. The other group had 1 mL of bacterial suspension added to a hydrogel-free 24-cell culture plate. After incubation for 2 hours at 100 rpm / min and 37°C, each group's material was washed with 1 mL of sterile PBS. Then, 100 μL of bacterial suspension was evenly dispersed on an agar plate and incubated for 12 hours in a constant temperature shaking incubator. Finally, the antibacterial properties of the material were analyzed by photographing and counting the colonies on the agar plates. Results are as follows: Figure 6 As shown.

[0055] from Figure 6 As can be seen, the hydrogel loaded with rhein has excellent antibacterial properties.

[0056] Performance Test 3 In this performance test, a mouse wound model with bacterial infection was used to test the ability of the rhein-loaded antibacterial and antioxidant composite hydrogel prepared in Example 1 of this invention to monitor the pH of infected wounds. Specifically, the pH data of the hydrogel with integrated diagnostic and therapeutic functions was recorded over 3 days (the pH value was determined based on the color change of the hydrogel; the correspondence between the hydrogel color change and the pH value is shown in [reference needed]). Figure 8 The results were compared with actual wound pH data, and the color change of the composite hydrogel was recorded. Figure 7 As shown.

[0057] from Figure 7 As can be seen, the pH data of the hydrogel test is consistent with the actual pH results. The results show that the pH of the wound can be monitored by observing the color change of the composite hydrogel prepared by this invention.

[0058] Application Test Case 1 In this application test, the wound healing performance of the antibacterial and antioxidant composite hydrogel loaded with rhein prepared in Example 1, the antibacterial and antioxidant hydrogel loaded with rhein prepared in Comparative Example 1, and the composite hydrogel prepared in Comparative Example 2 were evaluated using a bacterial wound infection model of SD rats (female, 220-250g).

[0059] Specifically, an infected wound model was first established: SD rats were anesthetized with 3% sodium pentobarbital, and the hair on the backs of the anesthetized rats was shaved. Then, a full-thickness wound with a diameter of approximately 10 mm was prepared on the back of the SD rats, and 100 μL of Staphylococcus aureus solution was added. After 24 hours of incubation, the bacterial infected wound model was successfully established. The experiment was divided into four groups. The first group used 3MTegaderm hydrocolloid dressing as a control group; the second group used Rhe@CG hydrogel prepared in Comparative Example 1 to cover the wound; the third group used CG-BSA composite hydrogel prepared in Comparative Example 2 to cover the wound; and the fourth group used Rhe@CG-BSA composite hydrogel prepared in Example 1 to cover the wound. Wound photographs were taken and the wound area was calculated on days 0, 3, 7, and 14. The results are as follows: Figure 9 As shown.

[0060] from Figure 9 As can be seen, compared with the control and comparative examples 1 and 2, the composite hydrogel prepared by the present invention can effectively promote the healing of bacterial infected wounds.

[0061] In summary, this invention forms a stable three-dimensional network structure by electrostatic interaction between positively charged polymers and negatively charged proteins under mild conditions, and successfully loads rhein as a single key functional component into the three-dimensional network structure. Its core lies in utilizing the inherent pH-responsive characteristics and multiple biological activities of rhein molecules to simultaneously achieve integrated diagnostic and therapeutic functions: In terms of treatment, the potent antibacterial, antioxidant, and anti-inflammatory activities continuously released by rhein work synergistically to actively improve the pathological microenvironment of infected wounds; in terms of diagnosis, the inherent color change of rhein with pH value makes the hydrogel itself a visual sensor, enabling real-time, in-situ monitoring of wound infection status without any external sensing elements.

[0062] It should be noted that all the above embodiments belong to the same inventive concept, and the descriptions of each embodiment have different focuses. Where the description in a particular embodiment is not detailed, please refer to the description in other embodiments.

[0063] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for preparing an antibacterial and antioxidant composite hydrogel loaded with rhein, characterized in that, Includes the following steps: Rhein solution, protein solution, and positively charged polymer solution are provided respectively; The rhein solution, the protein solution, and the positively charged polymer solution are mixed and stirred to obtain the rhein-loaded antibacterial and antioxidant composite hydrogel. The concentration of the rhein solution is 0.5-1.5 wt%, the concentration of the protein solution is 0.1-1 wt%, and the concentration of the positively charged polymer solution is 2-4 wt%.

2. The method for preparing the antibacterial and antioxidant composite hydrogel loaded with rhein according to claim 1, characterized in that, The concentration of the rhein solution is 1 wt%, the concentration of the protein solution is 0.5 wt%, and the concentration of the positively charged polymer solution is 3 wt%.

3. The method for preparing the antibacterial and antioxidant composite hydrogel loaded with rhein according to claim 1, characterized in that, The volume ratio of the rhein solution, the protein solution, and the positively charged polymer solution is (0.5-1.5):(8-12):(8-12).

4. The method for preparing the antibacterial and antioxidant composite hydrogel loaded with rhein according to claim 1, characterized in that, The volume ratio of the rhein solution, the protein solution, and the positively charged polymer solution is 1:10:

10.

5. The method for preparing the antibacterial and antioxidant composite hydrogel loaded with rhein according to claim 1, characterized in that, The pH value of the rhein solution is alkaline.

6. The method for preparing the antibacterial and antioxidant composite hydrogel loaded with rhein according to claim 1, characterized in that, The protein solution contains bovine serum albumin.

7. The method for preparing the antibacterial and antioxidant composite hydrogel loaded with rhein according to claim 1, characterized in that, The positively charged polymer solution contains cationic guar gum.

8. The method for preparing the antibacterial and antioxidant composite hydrogel loaded with rhein according to claim 1, characterized in that, In the step of mixing and stirring the rhein solution, the protein solution, and the positively charged polymer solution, the stirring temperature is room temperature.

9. A composite hydrogel loaded with rhein, characterized in that, It is prepared by the preparation method according to any one of claims 1-8.

10. The application of the antibacterial and antioxidant composite hydrogel as described in claim 9 in wound care and / or management.

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