Multifunctional medical sponge for wound treatment and method of making same

Abstract

This invention discloses a multifunctional medical sponge for wound treatment and its preparation method, relating to the field of wound treatment technology. It combines CaO2@PDA with Mn3O4 nanosheets and GSNO in a matrix formed by thiolated chitosan and oxidized sodium alginate, forming a single-layer homogeneous porous structure. Unlike the multi-layered or coating structures commonly found in existing technologies, this sponge lacks interlayer interfaces, avoiding the risk of delamination failure and simplifying the preparation process, thus facilitating large-scale production. The PDA shell regulates the hydrolysis rate of CaO2, extending oxygen release from 2 hours to over 72 hours. The Mn3O4 nanosheets further decompose H2O2 produced during CaO2 hydrolysis into O2, improving oxygen utilization efficiency and reducing cytotoxicity. Furthermore, the Mn3O4 nanosheets catalyze the decomposition of GSNO to release NO, increasing NO release by more than 5 times under simulated infection conditions, achieving on-demand NO release. This solves the contradiction of existing dressings' inability to simultaneously achieve antibacterial, anti-inflammatory, and healing-promoting effects.

Description

Technical Field

[0001] This invention relates to the field of wound treatment technology, and more specifically, to a multifunctional medical sponge for wound treatment and its preparation method. Background Technology

[0002] Wound healing is a complex biological process involving multiple stages, including hemostasis, inflammation, proliferation, and remodeling. For chronic, difficult-to-heal wounds such as diabetic foot ulcers, burns, and pressure ulcers, the healing process is often blocked due to factors such as local ischemia and hypoxia, persistent infection, excessive inflammatory response, and the accumulation of reactive oxygen species (ROS). Therefore, developing multifunctional medical sponges that can simultaneously alleviate hypoxia, clear ROS, inhibit infection, and promote tissue regeneration has significant clinical implications.

[0003] Currently, medical sponge materials used for wound treatment mainly include the following categories: 1. Traditional inert sponges, such as gelatin sponges, collagen sponges, and chitosan sponges, primarily serve to physically absorb exudate and provide isolation and protection. They lack the biological function of actively promoting healing and cannot effectively intervene in the pathological microenvironment of chronic wounds, such as hypoxia, infection, and oxidative stress. 2. Drug-loaded sponges load antibacterial agents (such as silver ions, antibiotics), growth factors, or anti-inflammatory drugs into the sponge matrix through physical adsorption or chemical cross-linking. However, when antibacterial agents and growth factors are loaded simultaneously in these sponges, they often antagonize each other. For example, silver ions can sometimes destroy the activity of growth factors, and drug release is uncontrollable, often resulting in burst release or incomplete release. 3. Oxygen-generating dressings utilize oxygen-generating agents such as calcium peroxide (CaO2) and magnesium peroxide to alleviate wound hypoxia. However, existing oxygen-generating dressings often suffer from problems such as explosive oxygen release and a sharp increase in local pH, which can be toxic to cells. Furthermore, simply supplying oxygen cannot solve the problems of infection and oxidative stress. 4. Multilayered Sponges: To endow sponges with multifunctionality, some studies have adopted layer-by-layer assembly or sandwich structures, setting different functional components in layers. However, multilayered structures have disadvantages such as weak interlayer bonding, easy delamination failure, and complex preparation processes. Furthermore, the interlayer interfaces may hinder material exchange and cell migration.

[0004] In summary, existing wound sponge materials face the following technical challenges that urgently need to be addressed: 1. Antibacterial, anti-inflammatory, and healing-promoting functions are difficult to achieve synergistically within the same system, leading to mutual inhibition. 2. Multi-layer or coating designs result in cumbersome preparation, poor stability, and are not conducive to large-scale production. 3. Existing technologies rarely alleviate hypoxia and remove ROS simultaneously, both of which often coexist in chronic wounds. Summary of the Invention

[0005] To address the aforementioned shortcomings, this invention provides a multifunctional medical sponge for wound treatment and its preparation method. The aim is to develop a multifunctional medical sponge with a simple structure, easy preparation, and the ability to intelligently release oxygen and nitric oxide according to the wound microenvironment while efficiently removing excess reactive oxygen species. This has significant clinical value and industrialization prospects for promoting the healing of chronic wounds.

[0006] The specific plan is as follows: The first aspect of this invention provides a method for preparing a multifunctional medical sponge for wound treatment, comprising the following steps: Step 1: Prepare CaO2@PDA nanoparticles; Step 2: Prepare Mn3O4 nanosheets; Step 3: Prepare thiolated chitosan; Step 4: Prepare sodium alginate oxide; Step 5, preparation of multifunctional medical sponge: Dissolve the thiolized chitosan prepared in step 3 in acetate-sodium acetate buffer solution, add the oxidized sodium alginate prepared in step 4, stir to form a prepolymer solution, add the CaO2@PDA nanoparticles prepared in step 1, the Mn3O4 nanosheets prepared in step 2 and GSNO to the prepolymer solution in sequence, disperse each component evenly to obtain a mixture, pour the mixture into a mold, and finally freeze-dry to obtain the multifunctional medical sponge.

[0007] Preferably, the specific steps for preparing CaO2@PDA nanoparticles in step one are as follows: CaO2 nanoparticles were dispersed in an alkaline buffer solution, dopamine hydrochloride was added, and the mixture was stirred at room temperature in the dark. After the reaction was completed, the nanoparticles were centrifuged, washed, and freeze-dried to obtain CaO2@PDA nanoparticles.

[0008] Preferably, the preparation steps of the CaO2 nanoparticles are as follows: CaCl2 solution and H2O2 solution were mixed, and NaOH solution was added dropwise while stirring to adjust the pH of the solution to alkaline. After continuous stirring, a precipitate was formed. The precipitate was centrifuged and washed to obtain CaO2 nanoparticles.

[0009] Preferably, the specific steps for preparing Mn3O4 nanosheets in step two are as follows: MnSO4·H2O solution and urea solution were mixed and subjected to a hydrothermal reaction. After the reaction was completed, the mixture was cooled to room temperature, centrifuged, washed and dried to obtain Mn3O4 nanosheets.

[0010] Preferably, the specific steps for preparing thiolized chitosan in step three are as follows: Chitosan was dissolved in acetic acid solution, and mercaptoacetic acid and catalyst were added in proportion. After stirring and reacting at room temperature, the reaction solution was transferred to a dialysis bag for dialysis, and finally freeze-dried to obtain thiolated chitosan.

[0011] Preferably, the catalyst is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0012] Preferably, the specific steps for preparing sodium alginate in step four are as follows: Sodium periodate was added to the sodium alginate solution, stirred at room temperature in the dark, and the reaction was terminated by dialysis. Finally, sodium alginate was obtained by freeze drying.

[0013] The second aspect of the present invention provides a multifunctional medical sponge for wound treatment, which is prepared by the method for preparing the multifunctional medical sponge for wound treatment according to the first aspect of the present invention.

[0014] The beneficial effects of this invention are as follows: The multifunctional medical sponge of the present invention is prepared by a one-step freeze-drying method. All functional components (CaO2@PDA nanoparticles, Mn3O4 nanosheets, GSNO) are uniformly dispersed in a thiolized chitosan / oxidized sodium alginate matrix to form a single-layer homogeneous porous structure. Unlike the multi-layer stacked or coating structures commonly found in the prior art (such as sandwich sponges, core-shell microparticles, etc.), the present invention does not have interlayer interfaces, avoiding the risk of delamination failure. At the same time, it simplifies the preparation process and is conducive to large-scale production. This invention integrates CaO2@PDA with Mn3O4 nanosheets and GSNO in the same system. The polydopamine (PDA) shell regulates the hydrolysis rate of CaO2, extending oxygen release from 2 hours to over 72 hours. Simultaneously, the Mn3O4 nanosheets possess catalase (CAT)-mimicking activity, further decomposing H2O2 generated during CaO2 hydrolysis into O2, improving oxygen utilization efficiency and reducing cytotoxicity. More importantly, the glutathione peroxidase (GPx)-mimicking activity of Mn3O4 responds to the wound infection microenvironment (high glutathione, high ROS), catalyzing the decomposition of GSNO to release nitric oxide (NO), increasing NO release by more than 5 times under simulated infection conditions, achieving on-demand NO release, thereby realizing precise antibacterial and angiogenesis-promoting effects. This invention does not rely on traditional antibiotics. It utilizes the broad-spectrum antibacterial activity of NO, the catalytic generation of ·OH (a peroxidase-like activity) by Mn3O4 nanosheets in a slightly acidic wound environment, and the trace alkaline environment generated by CaO2 hydrolysis to synergistically kill drug-resistant bacteria. Simultaneously, Mn3O4 nanosheets possess dual mimicry activities of superoxide dismutase (SOD) and catalase (CAT), which, combined with the free radical scavenging ability of the PDA shell and the activation of antioxidant signaling pathways by NO, can efficiently remove excess reactive oxygen species (ROS) from the wound surface and significantly reduce ROS levels in LPS-stimulated macrophages. This multi-mechanism synergy resolves the contradiction of existing dressings' inability to simultaneously achieve antibacterial, anti-inflammatory, and healing-promoting effects, providing a safe and efficient treatment option for chronic wounds (such as diabetic foot ulcers and burns). Attached Figure Description

[0015] Figure 1 This is a scanning electron microscope (SEM) image of the multifunctional medical sponge prepared in Example 2 of the present invention.

[0016] Figure 2 The curves show a comparison of the oxygen release performance of the sponges prepared in Example 2 and Comparative Example 1 of this invention.

[0017] Figure 3 The nitric oxide (NO) release performance curve of the multifunctional medical sponge prepared in Example 2 of the present invention.

[0018] Figure 4 Fluorescence micrograph of the effect of the multifunctional medical sponge prepared in Example 2 of the present invention on the scavenging effect of LPS-stimulated RAW 264.7 macrophages on reactive oxygen species (ROS).

[0019] Figure 5 The images show the antibacterial effects of the sponges prepared in Examples 2, 1, 2, and 3 of this invention. Detailed Implementation

[0020] The technical solutions of the present invention will now be clearly and completely described with reference to the accompanying drawings in the embodiments of the present invention.

[0021] Example 1: This embodiment discloses a method for preparing a multifunctional medical sponge for wound treatment, comprising the following steps: Step 1: Preparation of CaO2@PDA nanoparticles: CaCl2 solution and H2O2 solution were mixed, and NaOH solution was slowly added dropwise while stirring to adjust the pH of the solution to alkaline. After continuous stirring, a white precipitate formed. The white precipitate was centrifuged and washed to obtain CaO2 nanoparticles. The CaO2 nanoparticles were dispersed in Tris buffer, and dopamine hydrochloride was added. The reaction was carried out at room temperature in the dark with stirring. After the reaction was completed, the nanoparticles were centrifuged, washed, and freeze-dried to obtain CaO2@PDA nanoparticles.

[0022] Step 2, preparation of Mn3O4 nanosheets: MnSO4·H2O solution and urea solution were mixed and subjected to hydrothermal reaction. After the reaction was completed, the mixture was cooled to room temperature, centrifuged, washed and dried to obtain Mn3O4 nanosheets.

[0023] Step 3, preparation of thiolated chitosan: Chitosan is dissolved in acetic acid solution, and thioglycolic acid and catalyst are added in proportion. After stirring the reaction at room temperature, the reaction solution is transferred to a dialysis bag for dialysis, and finally freeze-dried to obtain thiolated chitosan.

[0024] Step 4, Preparation of oxidized sodium alginate: Add sodium periodate to the sodium alginate solution, stir at room temperature in the dark, terminate the reaction, dialyze, and finally freeze dry to obtain oxidized sodium alginate.

[0025] Step 5, preparation of multifunctional medical sponge: Dissolve the thiolized chitosan prepared in step 3 in acetate-sodium acetate buffer solution, add the oxidized sodium alginate prepared in step 4, stir to form a prepolymer solution, add the CaO2@PDA nanoparticles prepared in step 1, the Mn3O4 nanosheets prepared in step 2 and S-nitrosoglutathione (GSNO) to the prepolymer solution in sequence, disperse each component evenly to obtain a mixture, pour the mixture into a mold, freeze-dry it and take it out, cut it into the required shape, sterilize it and vacuum seal it to obtain the multifunctional medical sponge of the present invention.

[0026] The multifunctional medical sponge prepared according to the above method has antibacterial, anti-inflammatory, and healing-promoting functions. It can intelligently release oxygen and nitric oxide according to the wound microenvironment, while efficiently removing excess reactive oxygen species. The principle is as follows: 1. Long-lasting and stable oxygen supply: The CaO2@PDA nanoparticles in the sponge undergo a hydrolysis reaction upon contact with wound exudate: 2CaO2 + 2H2O → 2Ca(OH)2 + O2↑. The polydopamine (PDA) shell has the following functions: First, it limits the diffusion rate of water molecules to the CaO2 core, extending the oxygen release time from 2 hours for bare CaO2 to more than 72 hours. This allows for continuous metabolic support during the critical proliferation and remodeling phases of wound healing. Second, the PDA shell can effectively slow down the release rate of Ca(OH)2 and, with the help of the phenolic hydroxyl and amino groups in its molecules, buffer the local pH to a certain extent, significantly reducing the risk of alkaline damage. Finally, PDA itself is rich in phenolic hydroxyl groups, which can capture free radicals and synergistically resist oxidation. Meanwhile, the Mn3O4 nanosheets dispersed in the sponge have catalase (CAT) mimicry activity, which can further decompose H2O2 that may be generated during the hydrolysis of CaO2 into O2 and H2O (2H2O2→ 2H2O + O2), thereby improving oxygen utilization and eliminating the cytotoxicity of H2O2.

[0027] 2. Microenvironment-responsive nitric oxide release mechanism: S-nitrosoglutathione (GSNO) can spontaneously decompose to produce NO under physiological conditions, but the rate is slow. This invention utilizes two characteristics of the wound infection / inflammatory microenvironment to achieve on-demand accelerated NO release: First, bacterial infection and inflammatory response lead to increased glutathione (GSH) levels in the wound. GSH can promote the decomposition of GSNO and release NO through transnitrosation. Second, Mn3O4 nanosheets have glutathione peroxidase (GPx) mimicry activity, which can catalyze the oxidation of glutathione (GSH). Simultaneously, their active centers can provide electrons, efficiently catalyzing the decomposition of GSNO and significantly increasing the NO generation rate. In normal tissue (low GSH, low ROS), NO release is low, avoiding non-specific vasodilation; in infected wounds (high GSH, high ROS), NO release increases by more than 5 times, achieving precise antibacterial and angiogenesis promotion, and promoting wound healing.

[0028] 3. Principle of Reactive Oxygen Species (ROS) Scavenging: Excessive ROS (such as ·OH, O2-, H2O2) generated during the inflammatory phase of a wound can damage cells and delay healing. The multifunctional medical sponge of this invention scavenges ROS through three pathways: (1) Multi-enzyme activity of Mn3O4 nanosheets: It has superoxide dismutase (SOD) mimicking activity (catalyzing O2-→H2O2+O2) and catalase (CAT) mimicking activity (catalyzing H2O2→H2O+O2), converting harmful ROS into harmless substances; (2) Free radical scavenging ability of PDA shell: The catechol group in PDA can directly scavenge ·OH and DPPH free radicals; (3) Antioxidant effect of NO: Low concentration of NO can upregulate the expression of intracellular antioxidant enzymes (such as heme oxygenase-1), indirectly enhancing the ROS scavenging ability. The three synergistic effects enable the sponge to achieve a DPPH scavenging rate of over 92% and significantly reduce the ROS level in macrophages, inhibiting the inflammatory response.

[0029] 4. Synergistic Antibacterial Principle: The multifunctional medical sponge of this invention does not rely on traditional antibiotics. It kills bacteria, especially drug-resistant MRSA, through a multi-mechanism synergistic effect. First, NO can damage bacterial DNA, interfere with the respiratory chain, and increase cell membrane permeability, exhibiting a broad-spectrum antibacterial effect against both planktonic bacteria and biofilms. Second, in a slightly acidic wound environment (pH 6.5), Mn3O4 nanosheets exert peroxidase (POD)-like activity, catalyzing the generation of ·OH from H2O2. ·OH is a highly oxidizing free radical that can indiscriminately attack bacterial cell membranes and nucleic acids, but its lifespan is extremely short (nanoseconds), and its diffusion distance is extremely limited, acting only on bacteria adjacent to the Mn3O4 nanosheets and not spreading far to damage normal tissue. Simultaneously, the PDA and Mn3O4 nanozymes in the sponge matrix have strong background antioxidant capacity, acting as a chemical shield to remove excess ROS in the wound environment and protect normal cells. The sterilization rate of NO or Mn3O4 alone is only 50% to 70%, but the sterilization rate of the two combined is >98%, showing a significant synergistic effect.

[0030] Example 2: This embodiment further provides a method for preparing a multifunctional medical sponge for wound treatment. The difference between this embodiment and Embodiment 1 is that Embodiment 2 provides more specific material ratios and operating conditions.

[0031] Step 1: Preparation of CaO2@PDA nanoparticles: 10 mL of 0.5 mol / L CaCl2 solution (CaCl2 dissolved in anhydrous ethanol) and 10 mL of 0.5 mol / L H2O2 solution were added to a beaker. 0.5 mol / L NaOH solution was slowly added dropwise to pH 12 under magnetic stirring, and the mixture was stirred continuously for 30 minutes, resulting in a white precipitate. The white precipitate was centrifuged at 12000 rpm for 10 minutes, the supernatant was discarded, and the precipitate was washed three times with deionized water. The washed precipitate was redispersed in 50 mL of Tris buffer (pH 8.5, 10 mmol / L), and dopamine hydrochloride was added to a final concentration of 2 mg / mL. The mixture was stirred at room temperature in the dark for 24 hours. After the reaction was complete, the solid was collected by centrifugation at 12000 rpm for 10 minutes, washed three times with deionized water, and then freeze-dried at -50 °C and 0.1 mbar for 24 hours to obtain CaO2@PDA nanoparticles.

[0032] Step 2, Preparation of Mn3O4 nanosheets: Mix 50 mL of 0.1 mol / L MnSO4·H2O solution and 50 mL of 0.2 mol / L urea solution, transfer the mixture to a 200 mL polytetrafluoroethylene-lined hydrothermal reactor, seal it, and react at 180°C for 12 hours. After naturally cooling to room temperature, centrifuge the product in the hydrothermal reactor at 10,000 rpm for 10 minutes, discard the supernatant, and wash the product three times each with deionized water and anhydrous ethanol. Dry the resulting solid in a vacuum drying oven at 60°C for 12 hours to obtain Mn3O4 nanosheets.

[0033] Step 3, Preparation of thiolated chitosan: Weigh 2 g of chitosan (molecular weight 50 kDa, degree of deacetylation 90%) and dissolve it in 100 mL of 1% (v / v) acetic acid aqueous solution, stirring magnetically until completely dissolved. Add 1.5 g of mercaptoacetic acid and 2 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) sequentially, and stir at room temperature for 12 hours. Transfer the reaction solution to a dialysis bag (molecular weight cutoff 3500 Da) and dialyze with deionized water for 48 hours, changing the water every 6 hours. Freeze-dry the dialysate at -50 °C and 0.1 mbar for 24 hours to obtain thiolated chitosan.

[0034] Step 4, Preparation of sodium alginate oxide: Weigh 2 g of sodium alginate and dissolve it in 200 mL of deionized water. Add 0.5 g of sodium periodate and stir at room temperature in the dark for 6 hours. Add 1 mL of ethylene glycol to terminate the reaction. Dialyze (molecular weight cutoff 3500 Da) for 48 hours and freeze-dry for 24 hours to obtain oxidized sodium alginate.

[0035] Step 5: Prepare the multifunctional medical sponge: Dissolve 2 g of thiolated chitosan obtained in step three in 100 mL of acetate-sodium acetate buffer (pH=5.0, 0.1 mol / L) and stir magnetically until completely dissolved. Add 1 g of oxidized sodium alginate obtained in step four and continue stirring for 30 minutes to form a prepolymer solution. Add 50 mg of CaO2@PDA nanoparticles prepared in step one, 20 mg of Mn3O4 nanosheets obtained in step two, and 10 mg of S-nitrosoglutathione (GSNO) to the prepolymer solution in sequence. Use an ultrasonic cell disruptor at 200 W, working for 2 seconds and intermittent for 3 seconds to ultrasonically disperse the components for 10 minutes to obtain a uniformly dispersed mixture. Pour the mixture into a polytetrafluoroethylene mold, place it in a freeze dryer, freeze dry at -50°C and 0.1 mbar for 24 hours, remove it, cut it into the required shape, sterilize it with ethylene oxide, and vacuum seal it to obtain the multifunctional medical sponge of this invention.

[0036] Comparative Example 1 (bare CaO2 sponge): The preparation method is generally the same as in Example 2, but the following modifications are made in step five: Mn3O4 nanosheets and GSNO are not added, and in step one, CaO2 is not PDA coated; CaO2 particles with a particle size of 1–5 μm are used directly. The remaining specific steps are exactly the same as in Example 2.

[0037] Comparative Example 2 (Mn3O4 sponge alone): The preparation method is the same as in Example 2, but the following modification is made in step five: CaO2@PDA and GSNO are not added; only 20 mg of Mn3O4 nanosheets are added. The remaining specific steps are exactly the same as in Example 2.

[0038] Comparative Example 3 (GSNO sponge alone): The preparation method is the same as in Example 2, but the following modification is made in step five: CaO2@PDA and Mn3O4 nanosheets are not added, only 10 mg of GSNO is added. The remaining specific steps are exactly the same as in Example 2.

[0039] The medical sponges prepared in Example 2 and Comparative Examples 1-3 are characterized and their performance is tested in the following sections.

[0040] The morphology of the multifunctional medical sponge prepared in Example 2 was observed under a scanning electron microscope after being sputtered with gold. Figure 1 As shown, the sponge exhibits a three-dimensional interconnected porous structure with continuous pore walls and no obvious stratification.

[0041] Oxygen release performance test: Multifunctional medical sponges were cut into circular pieces with a diameter of 10 mm and a thickness of 3 mm, and immersed in 10 mL of simulated wound solution (pH=6.5, containing 140 mmol / L NaCl, 2.5 mmol / L CaCl2, and 0.1% tryptone), and incubated in a constant temperature water bath at 37°C. The dissolved oxygen concentration in the solution was recorded at regular intervals using a dissolved oxygen meter, and the pH value was monitored simultaneously. Comparative Example 1 (naked CaO2 sponge) was set up as a control. The results are as follows: Figure 2 As shown, the multifunctional medical sponge of Example 2 continuously releases oxygen over 72 hours, with the cumulative release rate increasing gradually over time, eventually reaching approximately 85%. The bare CaO2 sponge of Comparative Example 1 releases oxygen rapidly in the initial stage, releasing approximately 70% of the oxygen within 2 hours, after which the release rate slows significantly, ultimately reaching a cumulative release rate of approximately 73%. This demonstrates that the multifunctional medical sponge of the present invention can achieve long-lasting and stable oxygen release.

[0042] Nitric oxide (NO) release performance test: The multifunctional medical sponge discs from Example 2 were immersed in 10 mL of PBS (pH=7.4, normal conditions) and 10 mL of wound simulation solution (pH=6.5, containing 1 mmol / L glutathione GSH and 0.1 mmol / L H2O2), respectively, and incubated in a 37°C constant temperature water bath. The supernatant was collected at different time points, and the nitrite concentration was determined using the Griess reagent method, converted into the cumulative NO release. The results are as follows: Figure 3 As shown, in normal PBS, the cumulative NO release of the multifunctional medical sponge of Example 2 was 3.5 μmol / L after 48 hours. In wound simulation solution, the cumulative NO release reached 18.2 μmol / L after 48 hours, approximately 5.2 times that under normal conditions. This indicates that the multifunctional medical sponge of the present invention has microenvironment-responsive NO release characteristics, with significantly enhanced NO release in infected / inflammatory environments.

[0043] Intracellular reactive oxygen species (ROS) level detection: Lipopolysaccharide (LPS) was used to induce polarization in RAW 264.7 macrophages to simulate a wound inflammatory environment. RAW 264.7 macrophages produced large amounts of ROS and inflammatory factors. RAW 264.7 macrophages were seeded in DMEM complete medium (containing 10% fetal bovine serum and 1% penicillin / streptomycin) and cultured at 37°C in a 5% CO2 incubator. The experiment was divided into a blank group (no LPS, no sponge extract), an LPS model group (containing 1 μg / mL LPS), and a group cultured for 6 hours. The Example 2 group (containing 1 μg / mL LPS and 10% (v / v) of the sponge extract from Example 2) was also cultured for 6 hours. After the above treatment, the culture medium was discarded, the cells were washed twice with PBS, and 10 μmol / L DCFH-DA probe was added. The cells were incubated at 37°C in the dark for 30 minutes. After washing three times with PBS, the cells were immediately observed and photographed using a fluorescence microscope. Figure 4 As shown in the fluorescence microscopy images, the cells in the blank control group exhibited only weak green fluorescence, indicating extremely low basal ROS levels. The cells in the LPS model group showed strong green fluorescence, with a significantly higher fluorescence intensity than the blank control group, indicating that LPS successfully induced an intracellular ROS burst. The green fluorescence intensity of the cells in Example 2 group was significantly reduced, similar to the blank control group, with only a few cells showing weak fluorescence. This demonstrates that the sponge extract of the present invention can efficiently scavenge LPS-induced intracellular ROS and has good anti-inflammatory and antioxidant effects.

[0044] Antimicrobial performance test: The strain used was methicillin-resistant Staphylococcus aureus (MRSA, ATCC 43300). A single colony of MRSA was picked and inoculated into 5 mL of TSB medium, and cultured at 37°C with shaking at 200 rpm for 18 hours. The overnight culture was then diluted to 1×10⁻⁶ with sterile PBS. 6 CFU / mL was used to obtain a bacterial suspension. The multifunctional medical sponge from Example 2, the sponge from Comparative Example 1, the sponge from Comparative Example 2, and the sponge from Comparative Example 3 were all made into discs with a diameter of 10 mm and a thickness of 3 mm using a sterile punch. Three parallel plates were prepared for each sample. The sponge discs were placed in sterile 24-well plates, and 500 μL of bacterial suspension (1×10⁻⁶) was added to each well. 6 (CFU / mL), ensuring the sponge is completely submerged. After capping, co-incubate at 37°C and 100 rpm on a shaker for 6 hours. After co-incubation, repeatedly pipette the mixture from each well 10 times. Perform 10-fold serial dilutions of 100 μL of the mixture. Spread 100 μL of each dilution evenly onto TSA plates, with 3 parallel plates for each dilution. Incubate upside down at 37°C for 20 hours. After incubation, observe each group of plates. Figure 5As shown, in Comparative Example 1 (naked CaO2) plates, colonies were densely distributed. In Comparative Example 2 (Mn3O4 alone) plates, the number of colonies was further reduced compared to Comparative Example 1, but scattered colonies were still visible. In Comparative Example 3 (GSNO alone) plates, the number of colonies was similar to Comparative Example 1, and the colonies were relatively dense. In Example 2 (the present invention) plates, almost no colonies grew, indicating that the bacteria were largely killed after treatment with the sponges of Example 2. The results clearly show that the sponges of Example 2 have a very strong killing effect on MRSA, while the plates of all comparative examples showed a large number of colonies growing after treatment, and the antibacterial effect was far inferior to that of Example 2. This proves that the synergistic effect of CaO2@PDA, Mn3O4 nanosheets, and GSNO in the sponges of the present invention produces excellent antibacterial properties.

[0045] It should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a multifunctional medical sponge for wound treatment, characterized in that, Includes the following steps: Step 1: Prepare CaO2@PDA nanoparticles; Step 2: Prepare Mn3O4 nanosheets; Step 3: Prepare thiolated chitosan; Step 4: Prepare sodium alginate oxide; Step 5, preparation of multifunctional medical sponge: Dissolve the thiolized chitosan prepared in step 3 in acetate-sodium acetate buffer solution, add the oxidized sodium alginate prepared in step 4, stir to form a prepolymer solution, add the CaO2@PDA nanoparticles prepared in step 1, the Mn3O4 nanosheets prepared in step 2 and GSNO to the prepolymer solution in sequence, disperse each component evenly to obtain a mixture, pour the mixture into a mold, and finally freeze-dry to obtain the multifunctional medical sponge.

2. The method for preparing the multifunctional medical sponge for wound treatment according to claim 1, characterized in that, The specific steps for preparing CaO2@PDA nanoparticles in step one are as follows: CaO2 nanoparticles were dispersed in an alkaline buffer solution, dopamine hydrochloride was added, and the mixture was stirred at room temperature in the dark. After the reaction was completed, the nanoparticles were centrifuged, washed, and freeze-dried to obtain CaO2@PDA nanoparticles.

3. The method for preparing the multifunctional medical sponge for wound treatment according to claim 2, characterized in that, The preparation steps of the CaO2 nanoparticles are as follows: CaCl2 solution and H2O2 solution were mixed, and NaOH solution was added dropwise while stirring to adjust the pH of the solution to alkaline. After continuous stirring, a precipitate was formed. The precipitate was centrifuged and washed to obtain CaO2 nanoparticles.

4. The method for preparing the multifunctional medical sponge for wound treatment according to claim 1, characterized in that, The specific steps for preparing Mn3O4 nanosheets in step two are as follows: MnSO4·H2O solution and urea solution were mixed and subjected to a hydrothermal reaction. After the reaction was completed, the mixture was cooled to room temperature, centrifuged, washed and dried to obtain Mn3O4 nanosheets.

5. The method for preparing the multifunctional medical sponge for wound treatment according to claim 1, characterized in that, The specific steps for preparing thiolized chitosan in step three are as follows: Chitosan was dissolved in acetic acid solution, and mercaptoacetic acid and catalyst were added in proportion. After stirring and reacting at room temperature, the reaction solution was transferred to a dialysis bag for dialysis, and finally freeze-dried to obtain thiolated chitosan.

6. The method for preparing the multifunctional medical sponge for wound treatment according to claim 5, characterized in that, The catalyst is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

7. The method for preparing the multifunctional medical sponge for wound treatment according to claim 1, characterized in that, The specific steps for preparing sodium alginate in step four are as follows: Sodium periodate was added to the sodium alginate solution, stirred at room temperature in the dark, and the reaction was terminated by dialysis. Finally, sodium alginate was obtained by freeze drying.

8. A multifunctional medical sponge for wound treatment, characterized in that, The multifunctional medical sponge for wound treatment is prepared according to any one of claims 1 to 7.

Images