A mo s2-probiotic exosome hydrogel dressing for photothermal treatment of infected wounds and a preparation method thereof

Abstract

The present application relates to a kind of MoS2-probiotic exosome hydrogel dressing for photothermal treatment infected wound surface and its preparation method, comprising: step 1, preparation HA-PBA freeze-dried powder;Step 2, HA-PBA freeze-dried powder is dissolved with PBS buffer solution, obtain HA-PBA solution, prepare hydrogel pre-polymer A;Step 3, PVA powder is dissolved with PBS buffer solution, obtain PVA solution, prepare hydrogel pre-polymer B;Step 4, preparation MoS2 nanoflower powder;Step 5, MoS2 nanoflower powder is added to hydrogel pre-polymer B, obtain the hydrogel pre-polymer B containing MoS2;Step 6, hydrogel pre-polymer A is mixed with the hydrogel pre-polymer B containing MoS2, and LEVs are added, stirring, prepare MHPP hydrogel.Under the action of NIR irradiation, MoS2 nanoflower has efficient peroxidase and photothermal response capacity, can catalyze low concentration hydrogen peroxide to produce a large number of hydroxyl radicals, combined with photothermal effect, realize the broad-spectrum bactericidal effect to a variety of bacteria.

Description

Technical Field

[0001] This invention relates to the field of wound healing technology, and in particular to a MoS2-probiotic exosome hydrogel dressing for photothermal treatment of infected wounds and its preparation method. Background Technology

[0002] As the outermost protective barrier of the human body, the skin not only plays a vital role in resisting external physical, chemical, and biological attacks, but also in maintaining homeostasis, regulating body temperature, and sensing external stimuli. When the integrity of the skin is compromised, the body immediately initiates a series of sophisticated and complex repair mechanisms. However, bacterial infection is highly likely during this repair process. Infected wounds not only delay healing, but bacterial colonization can also stimulate excessive inflammatory responses, exacerbating tissue damage. Furthermore, toxins and metabolic products produced by bacteria can interfere with normal cell proliferation and migration, affecting the formation of new tissue. More seriously, in patients with metabolic diseases such as diabetes, infected wounds are highly prone to developing into chronic infections, eventually forming chronic wounds that are difficult to heal. This not only increases the patient's suffering and financial burden but may also lead to serious complications such as systemic infections.

[0003] Hydrogel dressings are often used in medical procedures to help wounds heal quickly. However, existing hydrogel dressings have insufficient antibacterial and anti-inflammatory effects, resulting in unstable wound healing and potentially leading to further inflammation and scarring. Summary of the Invention

[0004] The purpose of this invention is to provide a MoS2-probiotic exosome hydrogel dressing for photothermal treatment of infected wounds and its preparation method, thereby solving the above-mentioned problems existing in the prior art.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: A method for preparing a MoS2-probiotic exosome hydrogel dressing for photothermal therapy of infected wounds, comprising: Step 1: Prepare HA-PBA (hyaluronic acid-phenylboronic acid) lyophilized powder; Step 2: Dissolve the HA-PBA lyophilized powder in PBS buffer (phosphate buffered saline) to obtain HA-PBA solution, and prepare hydrogel prepolymer A; Step 3: Dissolve PVA (polyvinyl alcohol) powder in PBS buffer to obtain PVA solution, thus preparing hydrogel prepolymer solution B; Step 4: Prepare MoS2 (molybdenum disulfide) nanoflower powder; Step 5: Add MoS2 nanoflower powder to hydrogel prepolymer solution B to obtain hydrogel prepolymer solution B containing MoS2. Step 6: Mix hydrogel prepolymer A with hydrogel prepolymer B containing MoS2, add LEVs (exosomes), stir, and obtain MHPP (MoS2 / LEVs / HA-PBA-PVA) hydrogel.

[0006] The beneficial effects of this invention are as follows: The MoS2-probiotic exosome hydrogel dressing has dual antibacterial and anti-inflammatory effects, and the hydrogel matrix exhibits high glucose-responsive degradation. Under NIR irradiation, the 1T phase MoS2 nanoflowers in this hydrogel possess highly efficient peroxidase and photothermal response capabilities, catalyzing the generation of a large number of hydroxyl radicals (·OH) from low concentrations of hydrogen peroxide. Combined with the photothermal effect, this achieves a broad-spectrum bactericidal effect against various bacteria. As the hydrogel degrades, the released LEVs can promote the polarization of macrophages towards the M2 anti-inflammatory phenotype, thereby inhibiting the inflammatory response and promoting tissue repair and stable wound healing. The hydrogel of this invention is non-cytotoxic, can be directly applied to the wound surface, is convenient to use, and has an effective therapeutic effect. This invention combines inorganic nanomaterials with organic organisms to exert their combined effects, thus providing a new treatment method for infected wounds or chronic, difficult-to-heal wounds.

[0007] Based on the above technical solution, the present invention can be further improved as follows.

[0008] Further, step 1, the preparation of HA-PBA lyophilized powder, specifically includes the following steps: PBA (phenylboronic acid) and HA (hyaluronic acid) are added to PBS buffer, and after stirring for a period of time, dialysis is performed. The dialysis product is then freeze-dried to obtain HA-PBA lyophilized powder.

[0009] Further, in step 1: the PBS buffer is 1×PBS buffer; the mixture is stirred at 400-1000 RPM for 48 h at 50 °C using a constant temperature magnetic stirrer; the mixture is dialyzed in pure water for 48 h, with the water changed every 12 h during dialyz; the dialyzed product is then dried in a freeze dryer for 48 h.

[0010] Another technical solution of the present invention is as follows: A MoS2-probiotic exosome hydrogel dressing for photothermal treatment of infected wounds is prepared by the above-mentioned preparation method of MoS2-probiotic exosome hydrogel dressing for photothermal treatment of infected wounds. Attached Figure Description

[0011] Figure 1 This is a schematic diagram showing the synthesis steps of 1T phase MoS2 nanoflowers and the characterization of MoS2 nanoflowers with different 1T phase contents in the MoS2-probiotic exosome hydrogel dressing for photothermal treatment of infected wounds and its preparation method according to the present invention. Figure 2XPS images and 1T phase content analysis diagrams of MoS2 nanoflowers with different 1T phase contents in a photothermal treatment of infected wounds according to the present invention and its preparation method. Figure 3 This invention relates to a MoS2-probiotic exosome hydrogel dressing for photothermal treatment of infected wounds and its preparation method. The peroxidase activity detection and TMB steady-state kinetic diagram of MoS2 nanoparticles with different 1T phase contents of MoS2 nanoparticles were obtained. Figure 4 TEM images and particle size distribution diagrams of LEVs in a MoS2-probiotic exosome hydrogel dressing for photothermal treatment of infected wounds and its preparation method according to the present invention. Figure 5 Optical images, rheological properties tests, and drug release curves under high glucose conditions of the MHPP hydrogel of the MoS2-probiotic exosome hydrogel dressing for photothermal treatment of infected wounds and its preparation method are shown in the present invention. Figure 6 This invention presents a comparative study of the antibacterial effects of three components—MoS2, LEVs, and HPP—in a MoS2-probiotic exosome hydrogel dressing for photothermal treatment of infected wounds and its preparation method.

[0012] Figure 7 This image shows the antibacterial activity of the MHPP hydrogel of a photothermal therapy dressing for infected wounds and its preparation method, under the combined action of H2O2 and NIR irradiation, against various bacteria. Figure 8 This image shows the in vitro biocompatibility test of the MHPP hydrogel used in the photothermal therapy of infected wounds and its preparation method, according to the present invention. Figure 9 This is a comparative diagram of the in vitro anti-inflammatory effects of the three components MoS2, LEVs and HPP in the MHPP hydrogel of the MoS2-probiotic exosome hydrogel dressing for photothermal treatment of infected wounds and its preparation method, according to the present invention. Figure 10 This is a statistical chart showing the in vivo wound healing effect of the MHPP hydrogel of the MoS2-probiotic exosome hydrogel dressing for photothermal treatment of infected wounds and its preparation method, according to the present invention. Figure 11 This image shows the in vivo antibacterial and anti-inflammatory effects of the MHPP hydrogel in a photothermal therapy dressing for infected wounds and its preparation method, according to the present invention. Figure 12 This image shows the in vivo biocompatibility results of the MHPP hydrogel used in the photothermal treatment of infected wounds according to the present invention. Detailed Implementation

[0013] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0014] Example 1 like Figures 1 to 12 As shown, a method for preparing a MoS2-probiotic exosome hydrogel dressing for photothermal treatment of infected wounds includes: Step 1: Prepare HA-PBA lyophilized powder; Step 2: Dissolve the HA-PBA lyophilized powder in PBS buffer to obtain HA-PBA solution, and prepare hydrogel prepolymer solution A. Step 3: Dissolve PVA powder in PBS buffer to obtain PVA solution and prepare hydrogel prepolymer solution B; Step 4: Prepare MoS2 nanoflower powder; Step 5: Add MoS2 nanoflower powder to hydrogel prepolymer solution B to obtain hydrogel prepolymer solution B containing MoS2. Step 6: Mix hydrogel prepolymer A with hydrogel prepolymer B containing MoS2, add LEVs, stir, and obtain MHPP hydrogel.

[0015] MoS2-probiotic exosome hydrogel dressing possesses dual antibacterial and anti-inflammatory effects, and the hydrogel matrix exhibits high glucose-responsive degradation. Under NIR irradiation, the 1T phase MoS2 nanoflowers in this hydrogel possess highly efficient peroxidase and photothermal response capabilities, catalyzing the generation of a large number of hydroxyl radicals (·OH) from low concentrations of hydrogen peroxide. Combined with the photothermal effect, this achieves a broad-spectrum bactericidal effect against various bacteria. As the hydrogel degrades, the released LEVs promote macrophage polarization towards the M2 anti-inflammatory phenotype, thereby inhibiting the inflammatory response and promoting tissue repair and stable wound healing. The hydrogel of this invention is non-cytotoxic, can be directly applied to the wound surface, is convenient to use, and has an effective therapeutic effect. This invention combines inorganic nanomaterials with organic organisms to exert their combined effects, thus providing a new treatment method for infected wounds or chronic, difficult-to-heal wounds.

[0016] Example 2 This embodiment is a further improvement on embodiment 1, as detailed below: Step 1, the preparation of HA-PBA lyophilized powder, specifically includes the following steps: PBA and HA are added to PBS buffer, stirred for a period of time and then dialyzed, and the dialyzed product is freeze-dried to obtain HA-PBA lyophilized powder.

[0017] In step 1: the PBS buffer is 1×PBS buffer; the mixture is stirred at 50°C for 48 hours at a speed of 400-1000 RPM using a thermostatic magnetic stirrer; the mixture is dialyzed in pure water for 48 hours, with the water changed every 12 hours during dialysis; the dialysis product is then dried in a freeze dryer for 48 hours. Specifically, 200 mL of 1×PBS buffer, 0.5478 g of PBA, and 1.51728 g of HA are added to a round-bottom flask.

[0018] Example 3 This embodiment is a further improvement on embodiment 1, as detailed below: In step 2: the PBS buffer is 1×PBS buffer; it is dissolved at room temperature to obtain a 2wt% HA-PBA solution.

[0019] In step 3: the PBS buffer is 1×PBS buffer; it is dissolved at room temperature to obtain a PVA solution with a mass fraction of 10 wt%.

[0020] Example 4 This embodiment is a further improvement on embodiment 3, as detailed below: Step 4, the preparation of MoS2 nanoflower powder specifically includes the following steps: dissolve ammonium molybdate and thiourea in deionized water, sonicate until completely dissolved, then add glacial acetic acid, and then add deionized water to make up the solution to the preset volume; dry the solution at a constant temperature for a period of time and then cool it, centrifuge the contents, wash it multiple times with deionized water and anhydrous ethanol, and dry the precipitate for a period of time to obtain black MoS2 nanoflower powder. The concentration range of the black MoS2 nanoflower powder is 250-1000 µg / mL.

[0021] In step 4: The solution is ultrasonically dissolved for 30 minutes at 100% power in an ultrasonic cleaner; the solution is then transferred to a high-pressure reactor and heated at 200°C for 24 hours in a constant-temperature oven; the contents are centrifuged at 13000 rpm for 10 minutes; and the precipitate is dried in a freeze dryer for 24 hours. Specifically, 1.9155 g of ammonium molybdate and 4.1208 g of thiourea are dissolved in 50 mL of deionized water in a beaker; 5-20 mL of glacial acetic acid is added; and then deionized water is added to bring the volume to 70 mL.

[0022] Example 5 This embodiment is a further improvement on embodiment 4, as detailed below: In step 6: Hydrogel prepolymer A and MoS2-containing hydrogel prepolymer B are mixed at a volume ratio of 3:1; LEVs are derived from, but are not limited to, probiotic species including *Lactobacillus reuteri*, *Bifidobacterium animali*, *Lactobacillus rhamnosu*, *Lactobacillus paracasei*, and *Lactobacillus plantarum*, with LEV concentrations ranging from 10-200 µg / mL. It has anti-inflammatory effects.

[0023] The extraction of LEVs from Lactobacillus reuteri specifically includes: thawing the cryopreserved Lactobacillus reuteri strain, streaking it onto an MRS solid plate using an inoculation loop, placing the plate in an anaerobic incubator, culturing for a period of time, then picking a single colony with an inoculation loop and inoculating it into MRS liquid medium, continuing anaerobic culture for a period of time; centrifuging the MRS liquid medium for a period of time, collecting the supernatant after centrifugation, and removing residual bacteria from the supernatant through a filter membrane; centrifuging the obtained supernatant for a period of time, washing the centrifuged product with PBS; centrifuging again, finally summing the separated LEVs, adding sterile PBS to bring the total volume to the preset value; aliquoting the obtained LEVs and storing them in a refrigerator for later use.

[0024] In practice, the *L. reuteri* strain stored at -80℃ was rapidly thawed and streaked onto MRS solid plates using an inoculation loop. The plates were then placed in an anaerobic chamber and incubated at 37℃ for 48 hours. A single colony was then picked and inoculated into MRS liquid medium, and the medium was further incubated anaerobicly at 37℃ for another 48 hours. The *L. reuteri* culture was collected and centrifuged at 10,000g for 30 minutes at 4℃. The supernatant was collected and filtered through a 0.22μm filter to remove any remaining bacteria. The supernatant was then centrifuged at 10,000g for 70 minutes at 4℃, and the centrifuged product was washed with 1×PBS. The process was repeated using the same parameters. Finally, the isolated LEVs were collected in cryovials, and sterile 1×PBS was added to bring the total volume to 1 mL. The obtained LEVs were aliquoted and stored at -80℃ for later use.

[0025] experiment: LEVs were prepared using the pendant drop method for transmission electron microscopy (TEM) and their morphology was characterized by observation under an electron microscope. The main procedures were as follows: 10 μL of LEV solution was slowly added dropwise onto a 200-mesh carbon-copper grid; the LEVs were negatively stained with uranium acetate-hydrogen peroxide solution for 1 min; and finally, the exosome morphology was observed and images were acquired under TEM. The results are as follows: Figure 4 As shown in Figure A.

[0026] The particle size and concentration of LEVs were determined using nanoparticle tracking analysis (NTA, NanoSight NS300, UK). Specifically, the obtained LEVs were diluted to an appropriate concentration and then analyzed using the NTA analyzer. The results are as follows: Figure 4 As shown in B.

[0027] Experimental results: The above steps successfully yielded probiotic exosomes, and the particle size of the experimental product conformed to the exosome particle size distribution.

[0028] Example 6 A MoS2-probiotic exosome hydrogel dressing for photothermal treatment of infected wounds is prepared by the method for preparing MoS2-probiotic exosome hydrogel dressings for photothermal treatment of infected wounds according to any one of Examples 1 to 5.

[0029] Specific Example 1: Preparation of MoS2-probiotic exosome hydrogel dressing for photothermal treatment of infected wounds.

[0030] Step 1: Add 200 mL of 1×PBS buffer, 0.5478 g of PBA, and 1.51728 g of HA to a round-bottom flask. Stir with a thermostatic magnetic stirrer at 50°C for 48 h at a speed of 400-1000 RPM. Transfer the resulting product to a dialysis bag and dialyze it in pure water for 48 h, changing the water every 12 h. Place the dialyzed product in a freeze dryer and dry for 48 h to obtain HA-PBA lyophilized powder.

[0031] Step 2: Dissolve the HA-PBA lyophilized powder obtained in Step 1 in 1×PBS buffer at room temperature to obtain a 2wt% HA-PBA solution, i.e., hydrogel prepolymer solution A.

[0032] Step 3: Dissolve the PVA powder in 1×PBS buffer at room temperature to obtain a 10wt% PVA solution, i.e., hydrogel prepolymer solution B.

[0033] Step 4: MoS2 nanoflowers were synthesized using a hydrothermal method. In a beaker, 1.9155 g of ammonium molybdate and 4.1208 g of thiourea were dissolved in 50 mL of deionized water. The solution was ultrasonically cleaned at 100% power for 30 min until completely dissolved. 5-20 mL of glacial acetic acid was added to the solution, followed by deionized water to bring the volume to 70 mL. The solution was transferred to a 100 mL stainless steel high-pressure reactor lined with polytetrafluoroethylene (PTFE). The reactor was placed in a constant temperature oven and heated at 200 °C for 24 h. After the reactor cooled, it was opened, and the contents were centrifuged at 13000 RPM for 10 min. The mixture was washed three times with deionized water and three times with anhydrous ethanol. The precipitate was then dried in a freeze dryer for 24 h to obtain a black powder of 1T phase MoS2 nanoflowers.

[0034] Step 5: Add the obtained 1T phase MoS2 powder with a concentration of 250-1000 μg / mL to the hydrogel prepolymer solution B.

[0035] Step 6: When the hydrogel prepolymer A and the hydrogel prepolymer B containing 1T phase MoS2 are mixed at a volume ratio of 3:1, LEVs with a concentration of 10-50µg / mL are added and the mixture is stirred rapidly to obtain 1T phase MoS2 / LEVs / HA-PBA-PVA (MHPP) hydrogel.

[0036] experiment: Adhesion and rheological tests were performed on the MHPP hydrogel, such as... Figure 5 As shown in Figure A.

[0037] Adhesion and extensibility tests were performed on the MHPP hydrogel. The hydrogel was placed on a latex glove and a pigskin surface, and photographs were taken with a digital camera. Figure 5 As shown in Figure A.

[0038] Injectability testing was performed on the MHPP hydrogel by placing it in syringes with different needle diameters (21G, 23G, 26G) and conducting a squeeze-push test. The results are as follows: Figure 5 As shown in Figure A, the storage modulus (G') and loss modulus (G'') of the hydrogel at different times and strain rates were measured using a rheometer, as shown in Figure A. Figure 5 As shown in B, 5C, 5D, and 5E.

[0039] Glucose can competitively bind to phenylboronic acid inside the hydrogel, leading to the disruption of dynamic boron ester bonds, promoting the disintegration of the hydrogel network structure and drug release.

[0040] To verify the responsiveness of the hydrogel system to a high-glucose environment, the drug release rate in the MHPP hydrogel was tested using glucose solutions of different concentrations. Indocyanine green (ICG) was used as a drug simulant, and ICG release within the hydrogel was detected at different glucose concentrations (0, 5, and 10 mg / mL). The release rate of ICG significantly increased in the presence of glucose, and this rate increased with increasing glucose concentration. At a glucose concentration of 5 mg / mL, the cumulative release after 48 hours was approximately 70%, while at a glucose concentration of 10 mg / mL, the cumulative release after 48 hours reached 79%. These results demonstrate that the prepared MHPP hydrogel possesses excellent glucose-responsive properties, and the drug release rate is as follows: Figure 5 As shown in F.

[0041] Experimental results: MHPP hydrogels exhibit excellent rheological and tissue adhesion properties; MHPP hydrogels are injectable; and the drug release rate from MHPP hydrogels is glucose concentration-dependent.

[0042] Specific Example 2: Synthesis of MoS2 nanoflowers with different 1T phases.

[0043] By changing only the volume of glacial acetic acid added through the following steps, four different 1T phase MoS2 nanoflowers were obtained.

[0044] Step 1: Dissolve 1.9155g of ammonium molybdate and 4.1208g of thiourea in 50mL of deionized water in a beaker. Sonicate the solution at 100% power for 30 minutes until completely dissolved. Add 0, 5, 10, and 20mL of glacial acetic acid to the solution, then add deionized water to bring the volume to 70mL. Transfer the solution to a 100mL stainless steel high-pressure reactor lined with polytetrafluoroethylene (PTFE). Place the reactor in a constant temperature oven and heat at 200℃ for 24 hours. After the reactor cools, open the reactor and centrifuge the contents at 13000RPM for 10 minutes. Wash the contents three times with deionized water and anhydrous ethanol, respectively. Dry the precipitate in a freeze dryer for 24 hours to obtain a black powder of 1T phase MoS2 nanoflowers. Store at room temperature.

[0045] Among them, the 1T phase MoS2 nanoflowers prepared by adding 0 mL of glacial acetic acid are designated as sample MoS2-0; the 1T phase MoS2 nanoflowers prepared by adding 5 mL of glacial acetic acid are designated as sample MoS2-1; the 1T phase MoS2 nanoflowers prepared by adding 10 mL of glacial acetic acid are designated as sample MoS2-2; and the 1T phase MoS2 nanoflowers prepared by adding 20 mL of glacial acetic acid are designated as sample MoS2-3.

[0046] experiment: The surface morphology of MoS2-0, MoS2-1, MoS2-2 and MoS2-3 samples was observed using field emission scanning electron microscopy (SEM, TescanMIRA, Czech Republic).

[0047] The crystal structures of MoS2-0, MoS2-1, MoS2-2 and MoS2-3 samples were observed using a high-resolution transmission electron microscope (TEM, JEM-200, Japan).

[0048] The crystal phase content of MoS2-0, MoS2-1, MoS2-2 and MoS2-3 samples was determined by X-ray photoelectron spectroscopy (XPS, ESCALABXi+, USA).

[0049] Experimental results: MoS2 nanoflowers with high 1T phase content can be successfully prepared by the above steps, and the MoS2-2 sample has the highest 1T phase content.

[0050] Specific Example 3: The peroxidase activity of different MoS2 nanoflowers was detected.

[0051] Nanomaterials with peroxidase activity can catalyze the production of large amounts of ·OH from H₂O₂. In this invention, TMB is used as a ·OH detection probe. TMB can react with ·OH to produce a chromogenic substance with a specific absorption peak. The peroxidase activity of MoS₂ nanoflowers is detected by measuring the absorbance of the oxidation product at a wavelength of 652 nm.

[0052] Add 50 μL of MoS2-0, MoS2-1, MoS2-2, and MoS2-3 samples (25 μg / mL), 10 μL of H2O2 solution (10 mM), and 50 μL of TMB (20 mM) to 890 μM acetate-sodium acetate buffer (0.2 M, pH=3.6), respectively. Mix well and react at 37 °C for 3 min. After centrifugation, absorb the supernatant and analyze it using a UV spectrophotometer (UV-3600IPLUS, Japan). Figure 3 As shown in Figure A.

[0053] Different concentrations of 50 μL MoS2-2 sample (25 μg / mL, 50 μg / mL, 75 μg / mL, and 100 μg / mL), 10 μL H2O2 solution (10 mM), and 50 μL TMB (20 mM) were added to 890 μM acetate-sodium acetate buffer (0.2 M, pH=3.6). After mixing, the mixture was reacted at 37 °C for 3 min. After centrifugation, the supernatant was absorbed and analyzed using a UV spectrophotometer. Figure 3 As shown in B.

[0054] 50 μL of MoS2-2 sample (25 μg / mL) was added to 890 μM acetate-sodium acetate buffer (0.2 M, pH=3.6), followed by 10 μL of H2O2 solution (2.5 mM, 5 mM, 7.5 mM, 10 mM) and 50 μL of TMB (20 mM), respectively. After mixing, the mixture was incubated at 37 °C for 3 min. The supernatant was absorbed after centrifugation and analyzed using a UV spectrophotometer. Figure 3 As shown in C.

[0055] 50 μL of MoS2-2 sample (25 μg / mL) was added to 890 μM acetate-sodium acetate buffer (0.2 M, pH=3.6), followed by 10 μL of H2O2 solution (2.5 mM). After mixing, the mixture was incubated at 37 °C for 3 min. The supernatant was then collected after centrifugation. The presence or absence of NIR irradiation (1 W / cm²) was then determined. 2 (5 min), and perform tests and analyses using a practical ultraviolet spectrophotometer, such as Figure 3 As shown in D.

[0056] 10 μg of MoS2-2 sample was dissolved in 2 mL of HAc-NaAc buffer (0.2 M, pH=3.6). Different volumes of TMB solution (10 mg / mL dissolved in DMSO) were added to the reaction solution. 2 μL of H2O2 solution (30% concentration) was added to the reactants. The absorbance change curve of the reaction system at 652 nm was recorded using a UV spectrophotometer (UV-3600IPLUS, Japan) with a time scan mode of 10 s for 5 min. The initial absorbance change rate was recorded. The Michaelis-Menten constant (K) was calculated by fitting the data to the graphpadprism software. m ) and maximum reaction rate (V max Michaelis's equations are as follows: V=V max ×[S] / (K m +[S]), Where V is the reaction rate; V max [S] represents the maximum reaction rate; [S] represents the substrate concentration; K represents the maximum reaction rate. m For example, the Michaelis constant, Figure 3 As shown in E.

[0057] Following the above procedure, the steady-state dynamic characteristics of the MoS2-0 sample were detected, such as... Figure 3 As shown in F.

[0058] Experimental results: 1T phase MoS2 nanoflowers exhibit peroxidase activity, which shows a concentration-dependent relationship with the concentrations of H2O2 and MoS2 nanoflowers. The MoS2-2 sample with the highest 1T phase content has the best peroxidase activity.

[0059] Specific Example 4: Experiment on the in vitro antibacterial properties of MHPP hydrogel.

[0060] 1) The in vitro antibacterial activities of three components—MoS2, LEVs, and HPP—in MHPP hydrogel against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa were investigated using the plate coating method. Five groups were set up for this test: control group (500 μL sterile saline), HPP (without LEVs) group (500 μL HA-PBA-PVA hydrogel), HPP (with LEVs) group (500 μL LEVs / HA-PBA-PVA hydrogel), MHPP (without LEVs) group (500 μL MoS2 / HA-PBA-PVA hydrogel), and MHPP group (500 μL MHPP hydrogel).

[0061] 2) Irradiate each group of hydrogels under ultraviolet light for 30 minutes to effectively sterilize them.

[0062] 3) In step 1), two more treatment groups are set up among the three groups: control group (no treatment) and H2O2 group + NIR group (0.5mM H2O2 + 1W / cm). 2 Irradiate with NIR for 10 min). Add 500 μL (1.0 × 10⁻⁶) of bacterial suspension to each well of a 24-well plate. 6 CFU·mL -1 The bacterial suspension was treated according to the parameters above. The bacteria included E. coli, S. aureus, and P. aeruginosa.

[0063] 4) Incubate the well plates at 37°C for 4 hours. Serially dilute the bacterial suspensions from each group and spread them onto LB agar plates using disposable spreaders. Incubate at 37°C for 24 hours. Photograph the plates with a digital camera and use ImageJ software to count bacterial colonies and perform statistical analysis, such as... Figure 6 As shown.

[0064] Experimental results: MoS2 nanoflowers play a major role in the antibacterial effect of MHPP hydrogel, and they have the strongest antibacterial efficacy under the combined action of H2O2 and NIR irradiation.

[0065] Specific Example 5: Based on Specific Example 4, the in vitro antibacterial properties of MHPP hydrogel were further tested.

[0066] 1) The in vitro antibacterial activity of MHPP hydrogel against E. coli, S. aureus, and P. aeruginosa under H2O2 and NIR irradiation was investigated using the plate coating method. Three groups were set up for this test: control group (500 μL sterile saline), HPP group (500 μL L EVAs / HA-PBA-PVA hydrogel), and MHPP group (500 μL L HPP hydrogel).

[0067] 2) Irradiate each group of hydrogels under ultraviolet light for 30 minutes to effectively sterilize them.

[0068] 3) In step 1), four more treatment groups are set up from the three groups: control group (no treatment), H2O2 group (concentration of 0.5 mM), NIR group (1 W / cm²). 2 (10 min), H2O2 group + NIR group (0.5 mM H2O2 + 1 W / cm) 2 Irradiate with NIR for 10 min). Add 500 μL (1.0 × 10⁻⁶) of bacterial suspension to each well of a 24-well plate. 6 CFU·mL -1 The bacterial suspension was treated according to the above four parameters; the bacteria included E. coli, S. aureus and P. aeruginosa.

[0069] 4) Incubate the well plates at 37°C for 4 hours. Serially dilute the bacterial suspensions from each group and spread them onto LB agar plates using disposable spreaders. Incubate at 37°C for 24 hours. Photograph the plates with a digital camera and use ImageJ software to count bacterial colonies and perform statistical analysis, such as... Figure 7 As shown.

[0070] Experimental results: MHPP hydrogels exhibit certain antibacterial effects under the action of H2O2 or NIR, and have the strongest antibacterial efficacy under the combined action of H2O2 and NIR irradiation.

[0071] Specific Example 6: Experimental in vitro biosafety of MHPP hydrogel.

[0072] 1) HPP hydrogel and MHPP hydrogel containing different concentrations of 1T phase MoS2 (250 μg / mL, 500 μg / mL, and 1000 μg / mL, respectively) were added to DMEM complete medium and incubated at 37°C for 48 h to obtain hydrogel extract.

[0073] 2) NIH-3t3 cells were added at a rate of 1 × 10⁶ cells per well. 4 The cells were seeded at a density in 96-well plates and cultured at 37°C for 12 hours in a cell culture incubator.

[0074] 3) Remove the original culture medium from the 96-well plate in step 2), add 100 μL of hydrogel extract obtained in step 1) to the wells, and incubate at 37°C for 24 h in a cell culture incubator.

[0075] 4) Remove the hydrogel leachate from the 96-well plate in step 3), add 100 μL of cell viability / death staining working solution containing Calcein-AM and PI dye, incubate at 37°C for 30 min in a cell culture incubator, and observe the cells using a fluorescence microscope and take photographs. Viable cells will be excited with green fluorescence, while dead cells will be excited with red fluorescence, as shown below. Figure 8 As shown in Figure A.

[0076] 5) Remove the hydrogel leachate from the 96-well plate in step 3), add 110 μL of CCK-8 working solution (10% CCK-8 concentration) to each well, and incubate at 37°C for 2 hours. Measure the absorbance of each well at 450 nm using a microplate reader and calculate the cell viability. The calculation formula is: OD0 = OD0 of the experimental group. 450 / Control group OD 450 ×100%, such as Figure 8 As shown in B.

[0077] 6) Assess the blood compatibility of MHPP hydrogel. Take 8 mL of anticoagulated rabbit blood into a 25 mL centrifuge tube, add 10 mL of sterile physiological saline, and gently mix well, taking care to prevent cell rupture.

[0078] 7) Take 0.2 mL of the diluted rabbit blood obtained in step 6) and add it to a 1.5 mL EP tube, then add 0.8 mL of sterile physiological saline as a negative control group. Separate positive control groups were set up: (0.2 mL diluted rabbit blood + 0.8 mL distilled water), HPP hydrogel group (0.2 mL diluted rabbit blood + 0.8 mL HPP hydrogel extract), and MHPP hydrogel group (0.2 mL diluted rabbit blood + 0.8 mL MHPP hydrogel extract, wherein the concentration of 1T phase MoS2 is 1000 μg / mL). Each group was incubated at 37℃ for 1 h.

[0079] 8) Centrifuge each mixture from step 7) at 1000 rpm for 10 minutes, and record the data using a digital camera, such as... Figure 8 As shown in C.

[0080] 9) Take 100 μL of the supernatant obtained in step 8) and add it to a 96-well plate. Measure the absorbance of the solution at 562 nm and calculate the hemolysis rate for each treatment group. The formula for calculating the hemolysis rate is: Hemolysis rate = (OD0)0 t –OD - ) / (OD + –OD -)×100% (OD) t The absorbance and OD of the experimental group - The absorbance and OD of the negative control group + (Absorbance of the positive control group), hemolysis rate calculation results are as follows: Figure 8 As shown in D.

[0081] Experimental results: MHPP hydrogel has excellent biocompatibility.

[0082] Specific Example 7: Experimental in vitro anti-inflammatory activity of MHPP hydrogel.

[0083] 1) The in vitro anti-inflammatory effects of three components, MoS2, LEVs and HPP, in MHPP hydrogel were investigated using the mouse macrophage cell line Raw264.7. Lipopolysaccharide (LPS, 1 μg / mL) was used to induce an inflammatory response in Raw264.7 cells.

[0084] 2) Seed the cells from step 1) into 35 mm culture dishes and randomly grouped them. The following groups were established: blank control group (no LPS treatment), LPS treatment group, HPP (LEVs-free) group [1 μg / mL LPS treatment + cell medium changed to HPP (LEVs-free) hydrogel extract], HPP (LEVs-containing) group [1 μg / mL LPS treatment + cell medium changed to HPP (LEVs-containing) hydrogel extract], MHPP (LEVs-free) group [1 μg / mL LPS treatment + cell medium changed to MHPP (LEVs-free) hydrogel extract], and MHPP (LEVs-containing) group (1 μg / mL LPS treatment + cell medium changed to MHPP hydrogel extract containing LEVs). All treatment groups were incubated at 37°C for 12 h in a cell culture incubator.

[0085] 3) The levels of IL-6 and TNF-α in the cell supernatant of different treatment groups in step 2) were detected using an ELISA kit, such as... Figure 9 As shown in A and 9B.

[0086] 4) Extract cellular RNA from different treatment groups in step 2), and detect the expression levels of IL-6 and TNF-α relative to the internal reference gene β-actin in different treatment groups using reverse transcription-qPCR. Figure 9 As shown in C and 9D.

[0087] Experimental results: The LEVs component plays a major role in the anti-inflammatory effect of MHPP hydrogel.

[0088] Specific Implementation Example 8: In vivo therapeutic evaluation of experimental MHPP hydrogel.

[0089] Note: All mice were housed in the specific pathogen-free ABSL-2 animal facility. All experiments were conducted in accordance with the relevant regulations of the Animal Experiment Ethics Committee of Wuhan University.

[0090] 1) Establishment of a type 1 diabetes animal model. Male BALB / c mice aged 6-8 weeks and weighing 18-25g were selected and acclimatized in the animal room for one week.

[0091] 2) Fast the mice from step 1) for 12 hours. Dissolve urezocin (STZ) in citrate-sodium citrate buffer (0.1M, pH=4.4) to prepare a 10 mg / mL STZ solution. Filter the STZ solution through a 0.22 μm filter to sterilize it, protect it from light, and store it on ice. Administer the STZ solution intraperitoneally to the mice at a dose of 50 mg / kg. Wait 1 hour after injection before feeding the mice.

[0092] 3) Repeat the steps in step 2) and inject STZ solution continuously for 5 days.

[0093] 4) One week after modeling, remove the tail tip of the mouse, take a small amount of blood, and measure the fasting blood glucose of the mouse with a blood glucose meter. If the blood glucose concentration is >16.7mmol / L for a continuous week, the type 1 diabetes model is considered to have been successfully established.

[0094] 5) Disinfect the shaved area on the back of the mice that successfully developed type 1 diabetes in step 4) with an alcohol swab. Use a skin pore opener to remove the skin from the mouse's back, creating a circular, full-thickness skin wound approximately 10 mm in diameter. Take a 1×10⁻⁶ solution... 7 CFUmL -1 50 µL of S. aureus bacterial suspension was evenly applied to the wound site of the mouse and then bandaged with a sterile bandage.

[0095] 6) Twenty-four hours after infection in the mice from step 5), the mouse wounds were wiped with a routine swab, and a serially diluted bacterial suspension was spread onto LB agar plates using a spreader and incubated for 24 hours. The bacterial colonies on the plates were analyzed to confirm the infection status. Mice that were not successfully infected were euthanized after being anesthetized with CO2. The mice were placed in a sealed container containing an appropriate amount of CO2 gas, and the CO2 concentration was slowly increased until the mice were completely unconscious and unable to breathe. Then, the cervical dislocation method was used to dispose of the unfit mice.

[0096] 7) After confirming successful infection in the mice from step 6), observe and record the mice's living conditions, weight, wound infection status, and other basic information every two days. Take photos of the wounds, measure the size of the wounds using calipers, and calculate the wound area using ImageJ software.

[0097] 8) Simultaneously, the mice successfully infected in step 6) were randomly divided into five groups: control group, HPP hydrogel group, HPP hydrogel + NIR group, MHPP hydrogel group, and MHPP hydrogel + NIR group. The control group mice received 100 μL of sterile saline at the wound site, while the hydrogel-treated groups received 100 μL of hydrogel corresponding to their group name applied to the wound site. The NIR irradiation power was 1 W / cm². 2 The irradiation time was 6 minutes for each patient, and the temperature rise of the wound site was observed using a near-infrared thermometer during the irradiation period.

[0098] 9) Based on the grouping in step 8), administer different treatments to each group of mice on days 0, 2, and 4. Take photographs of the wound sites on days 0, 2, 4, 6, 8, 10, and 12 using a digital camera and record the mice's weight. Figure 10 As shown in A, 10B, and 10C.

[0099] 10) Based on the grouping in step 8), wipe the mouse wounds with a routine swab on days 0, 2, 4, and 7. Spread the serially diluted bacterial suspension onto LB agar plates using a spreader and incubate for 24 hours. Analyze the bacterial colonies on the plates to confirm the antibacterial effect of each group. Figure 11 As shown in Figure A.

[0100] 11) Based on the grouping in step 8), all mice were euthanized on day 12 for histological analysis. Skin tissue around the wound was removed, fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, and sectioned. Pathological sections were treated with immunohistochemical staining (IL-6, IL-10, CD31). Observation and photography were performed using an optical microscope to assess in vivo anti-inflammatory and angiogenesis activity, such as... Figure 11 As shown in B.

[0101] 12) Based on the grouping in step 8), all mice were euthanized on day 12. Skin tissue around the wounds was removed, fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, and sectioned. Pathological sections were stained with hematoxylin and eosin (H&E), observed and photographed under an optical microscope, and the biocompatibility after treatment was assessed. Results showed no visceral damage or inflammation, such as... Figure 12 As shown.

[0102] Note: All data above are presented as mean ± standard deviation. GraphPadPrism and Origin were used for experimental data analysis and plotting. Statistical significance was determined using unpaired t-tests or one-way ANOVA. The significance level was set as follows: (*) p <0.05, (**) is p<0.01, (***) is p <0.001, (****) is p <0.0001.

[0103] Experimental results: MHPP hydrogel has a significant wound healing effect under the action of NIR, and shows significant bacterial inhibition after 7 days of treatment. After 12 days of treatment, it shows significant anti-inflammatory and angiogenesis effects, and no in vivo toxicity was observed in the short-term treatment.

[0104] In conclusion, in an animal model of type 1 diabetic wound infection, MHPP hydrogel dressing exhibits excellent antibacterial and anti-inflammatory effects compared to the control group, and significantly promotes wound healing with good biocompatibility.

[0105] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a MoS2-probiotic exosome hydrogel dressing for photothermal treatment of infected wounds, characterized in that, include: Step 1: Prepare HA-PBA lyophilized powder; Step 2: Dissolve the HA-PBA lyophilized powder in PBS buffer to obtain HA-PBA solution, and prepare hydrogel prepolymer solution A. Step 3: Dissolve PVA powder in PBS buffer to obtain PVA solution and prepare hydrogel prepolymer solution B; Step 4: Prepare MoS2 nanoflower powder; Step 5: Add MoS2 nanoflower powder to hydrogel prepolymer solution B to obtain hydrogel prepolymer solution B containing MoS2. Step 6: Mix hydrogel prepolymer A with hydrogel prepolymer B containing MoS2, add LEVs, stir, and obtain MHPP hydrogel.

2. The method for preparing the MoS2-probiotic exosome hydrogel dressing for photothermal treatment of infected wounds according to claim 1, characterized in that, Step 1, the preparation of HA-PBA lyophilized powder, specifically includes the following steps: PBA and HA are added to PBS buffer, stirred for a period of time, and then dialyzed. The dialyzed product is then freeze-dried to obtain HA-PBA lyophilized powder.

3. The method for preparing the MoS2-probiotic exosome hydrogel dressing for photothermal treatment of infected wounds according to claim 2, characterized in that, In step 1: the PBS buffer is 1×PBS buffer; the mixture is stirred at 400-1000 RPM for 48 h at 50 °C using a constant temperature magnetic stirrer; the mixture is dialyzed in pure water for 48 h, with the water changed every 12 h during dialyz; the dialyzed product is then dried in a freeze dryer for 48 h.

4. The method for preparing the MoS2-probiotic exosome hydrogel dressing for photothermal treatment of infected wounds according to claim 1, characterized in that, In step 2: the PBS buffer is 1×PBS buffer; it is dissolved at room temperature to obtain a HA-PBA solution with a mass fraction of 2 wt%.

5. The method for preparing the MoS2-probiotic exosome hydrogel dressing for photothermal treatment of infected wounds according to claim 1, characterized in that, In step 3: the PBS buffer is 1×PBS buffer; it is dissolved at room temperature to obtain a PVA solution with a mass fraction of 10 wt%.

6. The method for preparing the MoS2-probiotic exosome hydrogel dressing for photothermal treatment of infected wounds according to claim 1, characterized in that, Step 4, the preparation of MoS2 nanoflower powder, specifically includes the following steps: dissolving ammonium molybdate and thiourea in deionized water, sonicating until completely dissolved, then adding glacial acetic acid, and then adding deionized water to make up the solution to the preset volume; drying the solution at a constant temperature for a period of time and then cooling it, centrifuging the contents, washing it multiple times with deionized water and anhydrous ethanol, and drying the precipitate for a period of time to obtain MoS2 nanoflower powder.

7. The method for preparing the MoS2-probiotic exosome hydrogel dressing for photothermal treatment of infected wounds according to claim 6, characterized in that, In step 4: the solution is ultrasonically cleaned at 100% power for 30 minutes until completely dissolved; the solution is transferred to a high-pressure reactor and heated at 200°C for 24 hours in a constant temperature oven; the contents are centrifuged at 13000 rpm for 10 minutes; and the precipitate is dried in a freeze dryer for 24 hours.

8. The method for preparing the MoS2-probiotic exosome hydrogel dressing for photothermal treatment of infected wounds according to claim 1, characterized in that, In step 6: hydrogel prepolymer A and hydrogel prepolymer B containing MoS2 are mixed at a volume ratio of 3:1; the sources of LEVs include, but are not limited to, probiotic species such as Lactobacillus reuteri, Bifidobacterium lactis, Lactobacillus rhamnosus, Lactobacillus paracasei, and Lactobacillus plantarum.

9. The method for preparing the MoS2-probiotic exosome hydrogel dressing for photothermal treatment of infected wounds according to claim 8, characterized in that, The extraction of LEVs from Lactobacillus reuteri specifically includes: thawing the cryopreserved Lactobacillus reuteri strain, streaking it onto an MRS solid plate using an inoculation loop, placing the plate in an anaerobic incubator, culturing for a period of time, then picking a single colony with an inoculation loop and inoculating it into MRS liquid medium, continuing anaerobic culture for a period of time; centrifuging the MRS liquid medium for a period of time, collecting the supernatant after centrifugation, and removing residual bacteria from the supernatant through a filter membrane; centrifuging the obtained supernatant for a period of time, washing the centrifuged product with PBS; centrifuging again, finally summing the separated LEVs, adding sterile PBS to bring the total volume to the preset value; aliquoting the obtained LEVs and storing them in a refrigerator for later use.

10. A MoS2-probiotic exosome hydrogel dressing for photothermal treatment of infected wounds, characterized in that, Prepared by the method for preparing MoS2-probiotic exosome hydrogel dressing for photothermal treatment of infected wounds as described in any one of claims 1 to 9.

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