An anaerobic hydrogel dressing loaded with clostridium butyricum, and a preparation method and application thereof

Abstract

This invention provides an anaerobic hydrogel dressing loaded with Clostridium butyricum, its preparation method, and its application, belonging to the field of biomedical materials and wound repair technology. The invention designs an anaerobic hydrogel dressing loaded with Clostridium butyricum, utilizing an enzymatic oxygen-consuming system of vanillin oxidation catalyzed by laccase to continuously consume residual oxygen within the hydrogel, constructing and maintaining a local anaerobic microenvironment, thereby ensuring the long-term activity and function of Clostridium butyricum. The anaerobic hydrogel dressing achieves injectability and self-healing through Schiff base bonds, conforming to irregular wounds. The porous network structure formed within the anaerobic hydrogel dressing facilitates probiotic loading and hydrogen diffusion. The anaerobic hydrogel dressing achieves a dual synergistic effect of selective ROS scavenging and selective antibacterial activity, effectively promoting the healing of diabetic infectious wounds and demonstrating excellent practicality.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and wound repair technology, specifically relating to an anaerobic hydrogel dressing loaded with Clostridium butyricum, its preparation method, and its application. Background Technology

[0002] Diabetic ulcers, especially diabetic foot ulcers, are one of the most common and difficult-to-treat complications of diabetes. Epidemiological data show that approximately 15%-25% of diabetic patients will develop foot ulcers in their lifetime, of which more than 50% will become infected. The infection-related amputation rate within one year is as high as 17.4%, and the mortality rate is 15.1%. The pathological mechanisms of persistent diabetic ulcers are extremely complex, involving multiple aspects such as the accumulation of reactive oxygen species (ROS), bacterial infection and biofilm formation, persistent inflammatory response, and impaired angiogenesis. These factors promote each other and form a vicious cycle, preventing the wound from entering the normal healing process for a long time.

[0003] Currently, the main clinical treatments for diabetic ulcers include debridement, systemic or local antibiotics, growth factors, negative pressure suction, and functional dressings, but all have significant limitations. Functional hydrogel dressings, in particular, while providing a moist healing environment and loading drugs or growth factors, cannot provide the survival conditions for strictly anaerobic bacteria, thus limiting the application of anaerobic probiotics in wound treatment. Clostridium butyricum, a strictly anaerobic probiotic, has attracted attention in recent years due to its unique biological functions. Firstly, under anaerobic conditions, Clostridium butyricum metabolizes to produce hydrogen gas (H2), which has been shown to selectively scavenge two highly toxic reactive oxygen species: hydroxyl radicals (•OH) and peroxynitrite anions (ONOO⁻), without affecting physiologically functional reactive oxygen species such as nitric oxide (NO•), superoxide anions (O2⁻•), and hydrogen peroxide (H2O2), thereby avoiding redox imbalance. Secondly, *Clostridium butyricum* can secrete antibacterial substances such as butyric acid and bacteriocins, selectively inhibiting harmful bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and *Escherichia coli*, without harming beneficial symbiotic bacteria such as Bifidobacterium and Lactobacillus. Furthermore, *Clostridium butyricum* and its metabolites can also regulate immune cell function, inducing M1 macrophages to polarize towards M2, thus exerting anti-inflammatory and pro-repair effects. Therefore, *Clostridium butyricum* shows great potential for treatment of diabetic ulcers. However, *Clostridium butyricum* is highly sensitive to oxygen and loses its activity within hours under normoxic conditions, making it unsuitable for direct application to open wounds. Therefore, the key technical bottleneck for its application in the treatment of diabetic ulcers lies in how to construct and maintain an anaerobic microenvironment within wound dressings, maintain the activity of *Clostridium butyricum* in normoxic environments, and continuously release hydrogen and metabolites. Summary of the Invention

[0004] To address some shortcomings in existing technologies, this invention provides an anaerobic hydrogel dressing loaded with Clostridium butyricum, its preparation method, and its application. This invention designs an anaerobic hydrogel dressing loaded with Clostridium butyricum, utilizing an enzymatic oxygen-consuming system of vanillin oxidation catalyzed by laccase to continuously consume residual oxygen within the hydrogel, constructing and maintaining a local anaerobic microenvironment, thereby ensuring the long-term activity and function of Clostridium butyricum. The anaerobic hydrogel dressing achieves injectability and self-healing through Schiff base bonds, conforming to irregular wounds. The porous network structure formed within the anaerobic hydrogel dressing facilitates probiotic loading and hydrogen diffusion. The anaerobic hydrogel dressing achieves a dual synergistic effect of selective ROS removal and selective antibacterial activity, effectively promoting the healing of diabetic infectious wounds and demonstrating excellent practicality.

[0005] To achieve the above-mentioned technical objectives, the present invention employs the following technical means:

[0006] This invention first provides a method for preparing an anaerobic hydrogel dressing loaded with Clostridium butyricum, the preparation method comprising:

[0007] Clostridium butyricum was cultured anaerobically to the logarithmic phase, centrifuged, washed, and resuspended to obtain Clostridium butyricum bacterial suspension.

[0008] The Clostridium butyricum bacterial culture was mixed with a vanillin-coupled aldehyde-modified sodium alginate solution to obtain precursor solution A;

[0009] Laccase was mixed with carboxymethyl chitosan solution to obtain precursor solution B;

[0010] Precursor solution A and precursor solution B were mixed in equal volumes and subjected to cross-linking reaction. After the reaction was completed, an anaerobic hydrogel dressing loaded with Clostridium butyricum was obtained.

[0011] Preferably, the preparation steps of the vanillin-coupled aldehyde-modified sodium alginate include:

[0012] S1. Mix the aqueous solution of sodium alginate and the aqueous solution of sodium periodate, stir the mixture to react, add ethylene glycol after the reaction is complete and continue stirring, dialysis the reaction solution after stirring is complete, and dry it to obtain aldehyde-modified sodium alginate.

[0013] S2. Aldehyde-conjugated sodium alginate and vanillin are dissolved in deionized water and reacted. After the reaction is completed, the mixture is purified by dialysis and dried to obtain the vanillin-coupled aldehyde-conjugated sodium alginate.

[0014] Preferably, in step S1, the mixed solution contains: 1.0 g sodium alginate, 1.08 g sodium periodate, and 100 mL deionized water;

[0015] The conditions for the stirring reaction were: magnetic stirring reaction at 25°C in the dark for 5 hours;

[0016] The amount of ethylene glycol added is 1.5 mL;

[0017] The conditions for continuing the reaction are: to continue the reaction with magnetic stirring for 1 hour under dark conditions.

[0018] Preferably, in step S2, the ratio of sodium aldehyde ester, vanillin and deionized water is 400 mg: 10 mg: 10 mL;

[0019] The reaction conditions were: 25°C for 2 hours.

[0020] Preferably, during the preparation of the anaerobic hydrogel dressing loaded with Clostridium butyricum, the final concentration of Clostridium butyricum is 5 × 10⁻⁶. 9 CFU·mL -1 The final concentration of vanillin-coupled aldehyde-modified sodium alginate was 20 mg / mL. -1 The final concentration of laccase was 0.5 U·mL. -1 The final concentration of carboxymethyl chitosan was 20 mg·mL. -1 .

[0021] Preferably, the crosslinking reaction is carried out at 37°C for 30 minutes.

[0022] The present invention also provides an anaerobic hydrogel dressing loaded with Clostridium butyricum prepared by the above preparation method, wherein Clostridium butyricum is encapsulated inside the hydrogel, and the hydrogel has a porous network structure.

[0023] The present invention also provides the application of the above-mentioned anaerobic hydrogel dressing loaded with Clostridium butyricum in the preparation of products for the treatment of chronic infectious wounds in diabetic patients.

[0024] The present invention also provides the application of the above-mentioned anaerobic hydrogel dressing loaded with Clostridium butyricum in the preparation of antibacterial products; the bacteria include Staphylococcus aureus and Escherichia coli.

[0025] The present invention also provides a product for the treatment of chronic infectious wounds in diabetic patients, the product comprising the above-mentioned anaerobic hydrogel dressing loaded with Clostridium butyricum.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] This invention is the first to achieve topical application of anaerobic probiotics. It maintains the activity of *Clostridium butyricum* by creating an anaerobic microenvironment through internal oxygen consumption, thus solving the problem of *Clostridium butyricum* inactivation under normoxic conditions. The *Clostridium butyricum*-loaded anaerobic hydrogel dressing of this invention achieves a dual synergistic effect of selectively clearing ROS and selectively inhibiting bacteria, effectively promoting the healing of diabetic infectious wounds. This *Clostridium butyricum*-loaded anaerobic hydrogel dressing can be directly applied or injected to fill irregular wounds. Upon contact with tissue fluid, it slowly releases hydrogen gas and antibacterial substances without causing secondary damage.

[0028] The anaerobic hydrogel dressing loaded with Clostridium butyricum described in this invention selectively removes highly toxic reactive oxygen species (•OH and ONOO) by releasing hydrogen gas. - It does not disrupt the redox balance and protects beneficial symbiotic bacteria by killing methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli. The anaerobic hydrogel dressing loaded with Clostridium butyricum can selectively remove highly toxic reactive oxygen species, selectively inhibit pathogenic bacteria, promote the polarization of pro-inflammatory macrophages M1 to anti-inflammatory M2, promote angiogenesis, and accelerate the healing of diabetic ulcers. Attached Figure Description

[0029] Figure 1 The preparation and characterization of C. butyricum@Anaerobic-Gel are shown in the figure. Figure A shows the formation process of C. butyricum@Anaerobic-Gel, and Figure B shows a scanning electron microscope (SEM) image of C. butyricum@Anaerobic-Gel. Scale bar: 200 μm. Figure C shows a scanning electron microscope (SEM) image of C. butyricum@Anaerobic-Gel. Scale bar: 5 μm.

[0030] Figure 2 The results validate the oxygen-consuming reaction in the C. butyricum@Anaerobic-Gel precursor solution.

[0031] Figure 3 The figure shows the hydrogen production performance of C. butyricum@Anaerobic-Gel in a normal oxygen environment; A in the figure is the hydrogen production image, and B is the statistical analysis result of dissolved hydrogen.

[0032] Figure 4 The results show the in vitro selective antibacterial activity of C. butyricum@Anaerobic-Gel. In the figure, A is the plate colony image of MRSA and E. coli, B is the statistical analysis of the plate colony of MRSA and E. coli, C is the plate colony image of Bifidobacterium and Lactobacillus, and D is the statistical analysis of the plate colony of Bifidobacterium and Lactobacillus.

[0033] Figure 5 Results of selective removal of intracellular ROS by C. butyricum@Anaerobic-Gel.

[0034] Figure 6 To promote the healing of diabetic infected wounds with C. butyricum@Anaerobic-Gel; Figure A shows representative images of diabetic wounds from day 0 to day 12 after different treatments, and Figure B shows the curve of wound area versus time. Detailed Implementation

[0035] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0036] In the embodiments of the present invention, unless otherwise described, conventional experimental methods were used. The processes involved in the embodiments are all understandable and easily implemented by those skilled in the art based on the product instructions or basic knowledge in the field. The reagents and materials involved are not specifically described and are all obtained through commercial means.

[0037] Example 1:

[0038] (1) Preparation of vanillin-coupled aldehyde-modified sodium alginate (Van-ALG-CHO):

[0039] 1.0 g of sodium alginate (ALG, 5 mmol) was dissolved in 90 mL of deionized water, and 1.08 g of sodium periodate (NaIO4, 5 mmol) was dissolved in 10 mL of deionized water. The mixture was thoroughly combined and magnetically stirred at 25 °C in the dark for 5 h. Then, 1.5 mL of ethylene glycol was added to terminate the reaction, and the mixture was magnetically stirred again in the dark for 1 h. After the reaction was complete, the reaction solution was purified by dialyzing in a dialysis bag (14000 MWCO) for 3 days. Finally, the product was freeze-dried to remove moisture, yielding aldehyde-modified sodium alginate (ALG-CHO).

[0040] 400 mg of ALG-CHO and 10 mg of vanillin (Van) were weighed and dissolved simultaneously in 10 mL of deionized water, and reacted at 25 °C for 2 h. Subsequently, the reaction solution was purified by dialyzing in a dialysis bag (14000 MWCO) for 3 days. Finally, the product was dehydrated using a vacuum freeze dryer to obtain vanillin-coupled aldehyde-modified sodium alginate (Van-ALG-CHO).

[0041] (2) Preparation of anaerobic hydrogel dressing loaded with Clostridium butyricum:

[0042] C. butyricum in the logarithmic growth phase was collected by centrifugation (6000 rpm, 5 min), washed three times with 0.02 M NaCl solution, and set aside for later use.

[0043] Weigh 40 mg of vanillin-conjugated aldehyde-modified sodium alginate (Van-ALG-CHO) and dissolve it in 1 mL of DMEM culture medium, then add to a final concentration of 10. 10 CFU•mL -1 Clostridium butyricum was mixed to obtain precursor solution A. 40 mg of carboxymethyl chitosan (CMCS) was dissolved in 1 mL of solution containing a final concentration of 1 U / mL. -1 The precursor solution B was obtained by mixing the laccase in DMEM culture medium until homogeneous.

[0044] Precursor solution A and precursor solution B were mixed uniformly at a volume ratio of 1:1 and allowed to stand at 37°C for 0.5 h to obtain the anaerobic hydrogel dressing loaded with Clostridium butyricum (C. butyricum@Anaerobic-Gel).

[0045] Using the same method without adding Clostridium butyricum, a hydrogel free of Clostridium butyricum was prepared, denoted as Anaerobic-Gel.

[0046] Figure 1 Here are the gelation process and electron micrographs of C. butyricum@Anaerobic-Gel, as shown below. Figure 1 As shown in Figure A, the mixed precursor solution initially exhibited fluidity, gradually transforming into a gel state after 0.5 h, and showed no signs of flow even when inverted, demonstrating successful hydrogel formation. SEM images show (…). Figure 1 (B) This hydrogel has a typical porous network structure, which is beneficial for probiotic loading and gas delivery. The probiotic loading of C. butyricum@Anaerobic-Gel was further observed by SEM (Biographies of Infectious Diseases). Figure 1 C), the presence of bacteria was clearly visible under the microscope, proving that C. butyricum was successfully encapsulated in the hydrogel.

[0047] Example 2:

[0048] This embodiment uses a portable dissolved oxygen meter to evaluate the oxygen consumption reaction of laccase and vanillin in a hydrogel precursor solution. 200 mg of Van-ALG-GHO was weighed and dissolved in 10 mL of phenol red-free DMEM high-glucose medium, and the dissolved oxygen electrode probe was inserted. Then, liquid paraffin was slowly and completely covered the culture medium surface using a pipette to isolate it from air. After the instrument reading stabilized, laccase was added to the culture medium to a final concentration of 0.5 U / mL. -1Record the changes in dissolved oxygen content in the culture medium. The results are as follows: Figure 2 As shown. From Figure 2 It can be seen that the dissolved oxygen in the hydrogel precursor solution decreased to 0 mg•L within 30 min. -1 This demonstrates that the precursor solution can achieve an oxygen-free state before gelation, thereby maintaining an oxygen-free state inside the hydrogel and ensuring the survival and hydrogen production needs of C. butyricum.

[0049] Subsequently, in this embodiment, a hydrogen electrode was used to measure the oxygen production of *C. butyricum*@Anaerobic-Gel in a normoxic environment. 1 mL of *C. butyricum*@Anaerobic-Gel* was immersed in 2 mL of phenol red-free DMEM high-glucose culture medium and cultured at 37 °C under normoxic conditions for 12 h. The dissolved hydrogen content in the culture medium was measured using a hydrogen electrode at 0 h and 12 h, respectively. The results are as follows: Figure 3 As shown.

[0050] from Figure 3 It can be seen that after culturing in a normal oxygen environment for 12 hours, a large number of bubbles can be seen above the culture medium, and the dissolved hydrogen concentration measured by the hydrogen electrode is >800 ppb, proving that C. butyricum@Anaerobic-Gel can produce hydrogen gas in a normal oxygen environment.

[0051] In summary, this hydrogel maintains an anaerobic state internally through the oxygen-consuming reaction of laccase and vanillin. C. butyricum@Anaerobic-Gel can provide an anaerobic microenvironment for C. butyricum, maintaining its activity and ensuring its hydrogen production capacity under normoxic conditions, thus laying the foundation for the application of C. butyricum@Anaerobic-Gel in diabetic ulcer wounds.

[0052] Example 3:

[0053] This embodiment uses MRSA (ATCC 43300, Shanghai Luwei Technology Co., Ltd.), Escherichia coli (ATCC25922, Shanghai Biological Preservation Center), Bifidobacterium (SHBCC D24728, Shanghai Biological Preservation Center), and Lactobacillus (SHBCCD24867, Shanghai Biological Preservation Center) to evaluate the ability of C. butyricum@Anaerobic-Gel prepared in Example 1 to selectively inhibit harmful bacteria. The specific steps are as follows:

[0054] One mL of Anaerobic-Gel or C. butyricum@Anaerobic-Gel prepared in Example 1 was immersed in 2 mL of phenol red-free DMEM high-glucose culture medium and cultured at 37 °C under normal oxygen conditions for 12 h. The supernatant (i.e., hydrogel extract) was collected for later use.

[0055] Take bacterial suspensions (MRSA, E. coli) in the logarithmic growth phase and measure the optical density (OD) of the suspensions at 600 nm using a UV spectrophotometer. 600 The bacterial culture was diluted to 1×10⁻⁶. 4 CFU•mL -1 The diluted bacterial suspension was uniformly mixed with phenol red-free DMEM, Anaerobic-Gel, and C. butyricum@Anaerobic-Gel extracts at a volume ratio of 1:1. Using the DMEM group as a control, the mixtures were incubated at 37 ℃ under normal aerobic conditions for 6 h. The control group was then redissolved to 1×10⁻⁶. 4 CFU • mL -1 The other two groups were diluted by the same factor. 50 μL of bacterial culture was taken from each of the three groups, spread evenly on LB agar plates, and incubated at 37 ℃ under normal aerobic conditions until colonies grew. The colonies were then photographed and counted.

[0056] Collect bacterial suspensions (Bifidobacterium, Lactobacillus) in the logarithmic growth phase and measure the OD of the suspensions using a UV spectrophotometer. 600 The value was then diluted to 1×10⁻⁶. 4 CFU•mL -1 The diluted bacterial suspension was uniformly mixed with phenol red-free DMEM, Anaerobic-Gel, and C. butyricum@Anaerobic-Gel extracts at a volume ratio of 1:1, with the DMEM group serving as the control. After incubation at 37 ℃ under anaerobic conditions for 6 h, the control group was redissolved to 1×10⁻⁶. 4 CFU•mL -1 The other two groups were diluted by the same factor. 50 μL of bacterial culture was taken from each of the three groups, spread evenly on agar plates, and incubated at 37 ℃ under anaerobic conditions until colonies grew. The colonies were then photographed and counted.

[0057] The results showed that in the dilution coating plate experiment ( Figure 4 A) There was no significant difference in the inhibition of harmful bacteria between the Anaerobic-Gel group and the Control group. In contrast, the number of bacterial colonies on the agar plates in the C. butyricum@Anaerobic-Gel treatment group was significantly reduced. Statistical results are as follows: Figure 4As shown in Figure B, the number of colonies on the plates in the C. butyricum@Anaerobic-Gel group was close to 10, significantly less than the other two groups. This indicates that C. butyricum@Anaerobic-Gel can inhibit the growth of harmful bacteria. Meanwhile, the beneficial bacteria group ( Figure 4 (C and 4D) The number of bacterial colonies on the plates of the three treatment groups was relatively consistent, indicating that C. butyricum@Anaerobic-Gel does not affect the growth of beneficial bacteria.

[0058] Therefore, C. butyricum@Anaerobic-Gel can selectively inhibit harmful bacteria, laying the foundation for its application in diabetic ulcer wound infection.

[0059] Example 4:

[0060] ROS levels were assessed using mouse mononuclear macrophages (RAW264.7) to evaluate whether C. butyricum@Anaerobic-Gel possesses the ability to precisely and selectively scavenge highly toxic reactive oxygen species (•OH and ONOOˉ) and reduce oxidative stress. The specific steps are as follows:

[0061] (1) Experimental materials:

[0062] Experimental cells: mouse mononuclear macrophages (RAW264.7), obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences;

[0063] Experimental materials: HPF probe (Shanghai Maokang Biotechnology Co., Ltd.); DAF-2 DA probe (Shanghai Maokang Biotechnology Co., Ltd.); MitoSOX probe (MedChemExpress, USA); CM-H2DCFDA probe (Shanghai Maokang Biotechnology Co., Ltd.)

[0064] (2) Experimental steps:

[0065] The ability of C. butyricum@Anaerobic-Gel to selectively scavenge intracellular ROS was qualitatively analyzed using fluorescent probe labeling. The specific steps are as follows:

[0066] RAW264.7 cells from passages 3 to 5 were selected and cultured at a concentration of 5 × 10⁻⁶ cells / cell. 4Cells were seeded at a density of 1:1 in confocal microscopy dishes. After cell adhesion, the culture medium was discarded, and cells were induced to produce ROS by adding 200 μL of phenol red-free DMEM containing 200 μM H2O2 for 1 h. The culture medium was removed again, and the cells were washed three times with PBS. Then, 200 μL of the hydrogel extract obtained in Example 3 or phenol red-free DMEM culture medium was added, and the cells were cultured for another 24 h. After culture, •OH / ONOOˉ, NO•, O2⁻•, and H2O2 were labeled with fluorescent probes HPF (10 µM), DAF-2 DA (1 µM), MitoSOX (2.5 µM), and CM-H2DCFDA (0.5 µM), respectively. The cells were incubated at 37 °C for 30 min, and the fluorescence intensity of different ROS was observed under a laser confocal microscope. Cells not induced by H2O2 were used as a negative control group. The results are as follows: Figure 5 As shown.

[0067] from Figure 5 As can be seen, compared with uninduced R2O2 cells (Control(-)), the fluorescence intensity of different types of ROS in H2O2-induced RAW264.7 cells (Control(+)) was significantly enhanced, indicating that H2O2 successfully induced intracellular ROS production. However, after treatment with C. butyricum@Anaerobic-Gel, the fluorescence intensity of •OH and ONOOˉ free radicals in RAW264.7 cells was significantly reduced, while the fluorescence intensity of O2⁻•, NO•, and H2O2 was not different from other groups. This indicates that C. butyricum@Anaerobic-Gel selectively scavenges these two highly toxic free radicals, •OH and ONOOˉ, in RAW264.7 cells, achieving precise and selective scavenging of highly toxic reactive oxygen species (ROS) and reducing oxidative stress.

[0068] Example 5:

[0069] This embodiment uses male C57BL / 6J mice (6 weeks old) purchased from Nanjing Jicui Yaokang Biotechnology Co., Ltd. as experimental subjects to evaluate the efficacy of C. butyricum@Anaerobic-Gel in promoting wound healing at the animal level. The specific steps are as follows:

[0070] (1) Construction of an MRSA-infected diabetic mouse ulcer model:

[0071] S1. Prepare streptozotocin (STZ) solution:

[0072] Weigh 2.1 g of citric acid and dissolve it in 100 mL of deionized water to obtain solution A. Weigh 2.94 g of sodium citrate and dissolve it in 100 mL of deionized water to obtain solution B. Mix solutions A and B uniformly at a volume ratio of 1:1, and adjust the pH of the mixture to 4.2-4.5 using a pH meter. Filter the mixture through a 0.45 μm filter to remove impurities, obtaining STZ buffer solution.

[0073] Streptozotocin (STZ, Sigma-Aldrich, USA) was administered at a dose of 10 mg / mL. -1 The concentration of the solution is dissolved in buffer solution to obtain STZ solution.

[0074] S2. Establish a diabetic mouse model:

[0075] A diabetic mouse model was established by injecting the STZ solution obtained in step S1 into male C57BL / 6J mice. At 21:00 the day before STZ injection, the mice were fasted but allowed free access to water. Twelve hours later, the mice were intraperitoneally injected with STZ solution (50 mg / kg). -1 The mice were then fasted and deprived of water for 2 hours. After 2 hours, their diet was resumed. After 5 consecutive days of injections, random blood glucose levels were measured using a glucometer. Mice with blood glucose concentrations higher than 16.8 mM (diabetic mice) were selected as experimental subjects.

[0076] S3. Establish a diabetic mouse ulcer model induced by MRSA infection:

[0077] Diabetic mice were anesthetized using an isoflurane anesthesia machine, and their dorsal hair was removed using a shaver and depilatory cream. The skin on the back of the mice was disinfected, and a circular, full-thickness skin defect was created on the back of each diabetic mouse using an 8 mm diameter biopsy puncture device. Subsequently, 100 μL of activated MRSA (1×10⁻⁶) was collected. 9 CFU•mL -1 The suspension was dropped onto the wound to induce wound infection in mice, thus establishing a diabetic mouse ulcer model of MRSA infection.

[0078] (2) Examination of the therapeutic effect of C. butyricum@Anaerobic-Gel:

[0079] Diabetic mice were randomly divided into three groups of five mice each: a saline group (control group), an Anaerobic-Gel group, and a C. butyricum@Anaerobic-Gel group. The control group received saline treatment, while the experimental groups had Anaerobic-Gel or C. butyricum@Anaerobic-Gel treated with medical tape. Treatment was administered every two days for 12 consecutive days. During treatment, each mouse was kept in a separate cage. Photographs of the wounds on the mice's backs were taken periodically, and the wound area was calculated using ImageJ software. The results are shown below. Figure 6 As shown.

[0080] from Figure 6 As can be seen, the wound area of ​​all three groups of mice decreased over time. The wound healing rate in the *C. butyricum*@Anaerobic-Gel group was significantly faster than the other two groups, and the wound was completely closed by day 12. ImageJ software was used to analyze the wound photographs and plot the wound healing rate curves. The results are as follows: Figure 6 As shown in Figure B, on day 0, the wound areas of the three groups were basically the same. As treatment progressed, the wounds in each group gradually healed, with the C. butyricum@Anaerobic-Gel group showing the fastest healing rate, its curve consistently lower than the other two groups. By day 12, the wound area of ​​the C. butyricum@Anaerobic-Gel group was only 7.01%, significantly different from the other two groups, indicating that C. butyricum@Anaerobic-Gel had the best effect on promoting wound healing.

[0081] In summary, this invention presents an anaerobic hydrogel dressing loaded with Clostridium butyricum. Through an enzymatic oxygen-consuming system catalyzing vanillin oxidation using laccase, residual oxygen is continuously consumed within the hydrogel, constructing and maintaining a local anaerobic microenvironment, thereby ensuring the long-term activity and function of Clostridium butyricum. The anaerobic hydrogel dressing achieves injectability and self-healing through Schiff base bonds, conforming to irregular wound surfaces. The porous network structure formed within the anaerobic hydrogel dressing facilitates probiotic loading and hydrogen diffusion. Furthermore, the anaerobic hydrogel dressing achieves a dual synergistic effect of selective ROS removal and selective antibacterial activity, effectively promoting the healing of diabetic infectious wounds and demonstrating significant practicality.

[0082] The embodiments described above are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for preparing an anaerobic hydrogel dressing loaded with Clostridium butyricum, characterized in that, The preparation method includes: Clostridium butyricum was cultured anaerobically to the logarithmic phase, centrifuged, washed, and resuspended to obtain Clostridium butyricum bacterial suspension. The Clostridium butyricum bacterial culture was mixed with a vanillin-coupled aldehyde-modified sodium alginate solution to obtain precursor solution A; Laccase was mixed with carboxymethyl chitosan solution to obtain precursor solution B; Precursor solution A and precursor solution B were mixed in equal volumes and subjected to cross-linking reaction. After the reaction was completed, an anaerobic hydrogel dressing loaded with Clostridium butyricum was obtained.

2. The preparation method according to claim 1, characterized in that, The preparation steps of the vanillin-coupled aldehyde-modified sodium alginate include: S1. Mix the aqueous solution of sodium alginate and the aqueous solution of sodium periodate, stir the mixture to react, add ethylene glycol after the reaction is complete and continue stirring, dialysis the reaction solution after stirring is complete, and dry it to obtain aldehyde-modified sodium alginate. S2. Aldehyde-conjugated sodium alginate and vanillin are dissolved in deionized water and reacted. After the reaction is completed, the mixture is purified by dialysis and dried to obtain the vanillin-coupled aldehyde-conjugated sodium alginate.

3. The preparation method according to claim 2, characterized in that, In step S1, the mixed solution contains: 1.0 g sodium alginate, 1.08 g sodium periodate, and 100 mL deionized water; The conditions for the stirring reaction were: magnetic stirring reaction at 25°C in the dark for 5 hours; The amount of ethylene glycol added is 1.5 mL; The conditions for continuing the reaction are: to continue the reaction with magnetic stirring for 1 hour under dark conditions.

4. The preparation method according to claim 2, characterized in that, In step S2, the ratio of sodium aldehyde ester, vanillin and deionized water is 400 mg: 10 mg: 10 mL. The reaction conditions were: 25°C for 2 hours.

5. The preparation method according to claim 1, characterized in that, In the preparation of the anaerobic hydrogel dressing loaded with Clostridium butyricum, the final concentration of Clostridium butyricum was 5 × 10⁻⁶. 9 CFU·mL -1 The final concentration of vanillin-coupled aldehyde-modified sodium alginate was 20 mg / mL. -1 The final concentration of laccase was 0.5 U·mL. -1 The final concentration of carboxymethyl chitosan was 20 mg·mL. -1 .

6. The preparation method according to claim 1, characterized in that, The crosslinking reaction conditions are: crosslinking at 37°C for 30 min.

7. The anaerobic hydrogel dressing loaded with Clostridium butyricum prepared by the preparation method according to any one of claims 1-6, characterized in that, In the anaerobic hydrogel dressing loaded with Clostridium butyricum, Clostridium butyricum is encapsulated inside the hydrogel, which has a porous network structure.

8. The use of the anaerobic hydrogel dressing loaded with Clostridium butyricum as described in claim 7 in the preparation of a product for the treatment of chronic infectious wounds in diabetic patients.

9. The use of the anaerobic hydrogel dressing loaded with Clostridium butyricum according to claim 7 in the preparation of antibacterial products; wherein the bacteria include Staphylococcus aureus and Escherichia coli.

10. A product for the treatment of chronically infected wounds in diabetic patients, the product comprising the anaerobic hydrogel dressing loaded with Clostridium butyricum as described in claim 7.

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