A hydrogel dressing with photothermal / photodynamic synergistic antibacterial activity and a preparation method thereof

Abstract

The present application provides a kind of hydrogel dressing with photothermal / photodynamic synergistic antibacterial activity and its preparation method, belong to biological medical tissue engineering material technical field, the present application is by the indocyanine green (ICG) with photodynamic (PDT) treatment ability and the hollow prussian blue nanoparticle Pt-HPB with photothermal treatment ability doped with platinum as photosensitizer, combined with the hydrogel made of oxidized sodium alginate and polylysine, these antibacterial nanoparticles are supported by the hydrogel with good water dispersibility, a kind of composite hydrogel (Pt-HPB-BSA & ICG@Gel) with photothermal photodynamic synergistic antibacterial ability is prepared, as the medical dressing of wound infection antibacterial treatment and promoting wound repair.

Description

Technical Field

[0001] This invention relates to a method for preparing a hydrogel dressing based on photothermal / photodynamic synergistic antibacterial properties, in order to solve the problem of difficult healing of bacterial infections at the wound site, and belongs to the field of biomedical tissue engineering materials technology. Background Technology

[0002] Infectious diseases caused by pathogens (especially multidrug-resistant bacteria) and their global spread have become a serious public health problem. Bacterial infections can cause varying degrees of cell degeneration, leading to tissue necrosis, and can even be life-threatening if not treated promptly. Antibiotics, as the star drugs for treating bacterial infections, have been widely used to treat infections caused by many pathogens, such as Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. However, antibiotic resistance is inevitable with frequent use, affecting treatment efficacy. Therefore, there is an urgent need to develop new antimicrobial strategies to kill bacteria without the emergence of drug-resistant strains.

[0003] Photothermal therapy (PTT) converts light energy into heat energy, using this heat to kill bacteria and other pathogens. It offers advantages such as controllability, non-drug resistance, and minimal invasiveness. It cleverly bypasses the substance-dependent pathways that lead to bacterial resistance, and photothermal antibacterial therapy is gradually gaining popularity. Compared to other wavelengths, near-infrared light has a greater penetration depth, allowing it to penetrate tissue directly to the lesion, reducing damage to the skin and tissues. Near-infrared photothermal antibacterial therapy not only achieves bactericidal effects but also addresses the problems of traditional antibiotics, such as narrow antibacterial spectrum, drug resistance, and drug toxicity. It also promotes tissue regeneration and wound healing. Prussian blue nanocubes (PB) are a clinical antidote approved by the US Food and Drug Administration (FDA) for the treatment of thallium poisoning, exhibiting excellent biocompatibility and safety. PB has strong absorption in the near-infrared (NIR) region and excellent photothermal conversion efficiency; it has been used as a photothermal agent in PTT. In addition, as an inorganic nanomaterial, PB also has catalase (CAT)-like enzyme activity and superoxide dismutase (SOD)-like enzyme activity, which can resist oxidation and reduce the inflammatory response that may be induced after PTT treatment.

[0004] Photodynamic therapy (PDT) is based on the principle that photosensitizers absorb photon energy at specific wavelengths and convert surrounding oxygen molecules into highly toxic reactive oxygen species (ROS), leading to damage to bacterial DNA and membranes. Photosensitizers can rapidly generate ROS to kill and induce bacterial death, thus preventing bacteria from developing resistance. Indocyanine green (ICG), a photosensitizer with strong near-infrared (NIR) light absorption at 808 nm, has attracted researchers' attention due to its excellent biocompatibility and photothermal properties. Under NIR irradiation, ICG transfers energy to surrounding oxygen molecules, generating chemically reactive ROS. These ROS interact with biological membranes, disrupting the structure for bacterial replication through oxidative stress, damaging the integrity of the bacterial membrane, and ultimately killing the bacteria.

[0005] In addressing the issue of antibiotic resistance, current methods often employ either photothermal therapy or photodynamic therapy (PDT) alone. However, photothermal therapy alone reaches temperatures as high as 70°C, potentially damaging normal tissue during treatment. PDT alone requires large amounts of reactive oxygen species (ROS) to kill bacteria, but excessive ROS can induce inflammation around the infected tissue, leading to necrosis of normal tissue. Furthermore, ROS production requires oxygen, which depletes oxygen in the wound tissue, causing hypoxia and a decrease in ROS concentration, thus reducing the effectiveness of PDT.

[0006] If photothermal therapy is combined with photodynamic therapy for antibacterial purposes, near-infrared light irradiation can lower the treatment temperature, reduce the demand for reactive oxygen species, kill bacteria and reduce inflammation, and promote wound healing and repair. However, there are few reports on the application of photothermal and photodynamic therapy in wound dressings, and nanoparticles are easily lost from the treated wound surface, leading to a significant decrease in antibacterial efficacy. Summary of the Invention

[0007] To address the aforementioned problems, this invention provides a hydrogel dressing with synergistic photothermal / photodynamic antibacterial activity and its preparation method. This invention uses indocyanine green (ICG), which has photodynamic (PDT) therapeutic capabilities, and platinum-doped hollow Prussian blue nanoparticles (Pt-HPB), which have photothermal therapeutic capabilities, as photosensitizers, combined with a hydrogel. The hydrogel, with its good water dispersibility, supports these antibacterial nanoparticles, thus preparing a composite hydrogel with synergistic photothermal and photodynamic antibacterial capabilities. This hydrogel can be used as a medical dressing for antibacterial treatment of wound infections and for promoting wound healing.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A method for preparing a hydrogel dressing with synergistic photothermal / photodynamic antibacterial activity, comprising the following steps:

[0010] Step 1: Preparation of polylysine-modified platinum-doped hollow Prussian blue nanoparticles (PL&Pt-HPB);

[0011] Step 2: Preparation of BSA-modified ICG nanoparticles (BSA & ICG);

[0012] Step 3: Preparation of oxidized sodium alginate (OSA);

[0013] Step 4: Prepare a hydrogel dressing with photothermal / photodynamic synergistic antibacterial activity (Pt-HPB-BSA&ICG@Gel).

[0014] In step one, the specific steps for preparing polylysine-modified platinum-doped hollow Prussian blue nanoparticles are as follows:

[0015] 1) Preparation of Prussian blue metal-organic framework nanoparticles (PB)

[0016] Weigh 3g of polyvinylpyrrolidone (PVP) into a 100mL flask, add 40mL of 0.01M HCl solution and stir until the liquid is clear. Add K3[Fe(CN)6]·3H2O and stir at room temperature for 1h. Then, heat the mixture in an oil bath to 80℃ for 20 hours. After the reaction is complete, cool to room temperature, centrifuge, wash and freeze-dry to obtain Prussian blue metal-organic framework nanoparticles (PB), which are stored at 4℃ for later use. The mass ratio of K3[Fe(CN)6]·3H2O to polyvinylpyrrolidone is 1:(20-23).

[0017] 2) Preparation of hollow Prussian blue nanoparticles (HPB)

[0018] Weigh 100 mg of polyvinylpyrrolidone (PVP) and place it in a 100 mL beaker. Add 20 mL of 1 M HCl solution to dissolve it and stir until clear. Weigh 20 mg of Prussian blue metal-organic framework nanoparticles (PB) prepared in step 1) and add them to the mixed solution. After stirring for 1 h, transfer the mixture to a 60 mL muffle furnace liner and calcine at 140 °C for 4 h. After natural cooling, centrifuge, wash, and freeze-dry to obtain hollow Prussian blue nanoparticles (HPB), which are then stored at 4 °C for later use.

[0019] 3) Preparation of Pt-doped hollow Prussian blue nanoparticles (Pt-HPB)

[0020] In a 100 mL flask, 50 mg of HPB nanoparticles obtained in step 2) and chloroplatinic acid were weighed and mixed with 20 mL of ethanol solution. The mixture was stirred at room temperature for 1 h. The mixture was then placed in an oil bath and refluxed at 80 °C for 4 h. After centrifugation, washing and drying, Pt-doped hollow Prussian blue nanoparticles (Pt-HPB) were obtained. The molar ratio of Pt in chloroplatinic acid to Fe in hollow Prussian blue nanoparticles was 0.28–0.32.

[0021] 4) Preparation of polylysine-modified platinum-doped hollow Prussian blue nanoparticles (PL&Pt-HPB)

[0022] Weigh 1 mg of Pt-HPB and 200 μg of polylysine (PL), dissolve them in distilled water, stir at 800 rpm for 1 h at room temperature. After the reaction is terminated, centrifuge, wash and freeze dry to obtain polylysine-modified platinum-doped hollow Prussian blue nanoparticles (PL & Pt-HPB), and store at 4 °C for later use.

[0023] Step 2, the steps for preparing BSA-modified ICG nanoparticles (BSA & ICG) are as follows:

[0024] 10 mg of bovine serum albumin (BSA) was weighed and placed in a 10 mL round-bottom flask, and dissolved in 1 mL of phosphate-buffered saline (PBS). Indocyanine green (ICG) was weighed according to a mass ratio of 10:1 (BSA to ICG) and dissolved in dimethyl sulfoxide (DMSO) at a ratio of 1 mg: 200 μl. The solution was then slowly added dropwise to the BSA solution. The mixture was stirred at 4 °C in the dark for 12 h. After the reaction was completed, the mixture was dialyzed at 4 °C in the dark for 24 h. After freeze-drying, BSA-modified ICG nanoparticles (BSA & ICG) were obtained.

[0025] Light-protected dialysis is preferably performed using a dialysis bag with a molecular weight of 3500 Da.

[0026] Step 3, the steps for preparing oxidized sodium alginate (OSA) are as follows:

[0027] 1.0 g of sodium alginate was dissolved in 60 mL of water to obtain a sodium alginate solution. An equimolar amount of sodium periodate was dissolved in 20 mL of water. After complete dissolution, the sodium periodate solution was slowly added to the solution, and water was added to bring the total volume to 100 mL. The oxidation reaction was carried out at room temperature in the dark with stirring for 6 hours. Ethylene glycol was then added to stop the oxidation for 1 hour. After the reaction was complete, the resulting solution was dialyzed in the dark for 48 hours and then freeze-dried to obtain oxidized sodium alginate (OSA).

[0028] The preferred ratio of ethylene glycol to sodium alginate is 1.5:1, and the light-protected dialysis is preferably performed using a dialysis bag with a capacity of 8000-14000 Da.

[0029] Step four, the steps for preparing the hydrogel dressing (Pt-HPB-BSA&ICG@Gel) with photothermal / photodynamic synergistic antibacterial activity are as follows:

[0030] Weigh 200 mg of sodium alginate prepared in step 3, add 1 mL of distilled water to dissolve it, and obtain sodium alginate solution. Weigh 80 μg of PL & Pt-HPB prepared in step 1 and 2 μg of BSA & ICG prepared in step 2 and add them to the sodium alginate solution. Adjust the pH to 7.5-8.0 with HCl. Weigh 200 mg of polylysine and dissolve it in 1 mL of distilled water. After it is completely dissolved, mix the polylysine solution with the OSA mixture by vortexing and stir thoroughly for 20 s to obtain a hydrogel dressing with photothermal / photodynamic synergistic antibacterial activity (Pt-HPB-BSA & ICG@Gel hydrogel).

[0031] The beneficial effects of this invention are:

[0032] This invention uses Prussian blue nanoparticles, a clinical antidote for thallium poisoning approved by the U.S. Food and Drug Administration (FDA), as a raw material. The invention prepares platinum-doped hollow Prussian blue nanoparticles (Pt-HPB) photothermal agents by etching and platinum doping Prussian blue metal-organic framework nanoparticles (PB). Verification has shown that Pt-HPB possesses excellent photothermal conversion efficiency and can provide good photothermal capabilities under near-infrared laser irradiation, achieving thermal killing of bacteria. As an inorganic material, it also possesses various enzyme activities; platinum doping endows it with stronger catalase-like activity, effectively supplying oxygen to infected wound sites and promoting wound healing.

[0033] Indocyanine green (ICG) has attracted researchers' attention due to its excellent biocompatibility and photothermal properties. Currently, it is widely used in the field of nanomedicine for near-infrared excited photothermal therapy (PDT) and fluorescence imaging. This invention synthesizes an indocyanine green (BSA&ICG) photosensitizer with good dispersibility and stability using bovine serum albumin (BSA) as a carrier. Under near-infrared laser irradiation, it can convert surrounding oxygen molecules into highly toxic reactive oxygen species (ROS), effectively killing bacteria.

[0034] This invention combines the two, utilizing the photothermal activity of platinum-doped hollow Prussian blue nanoparticles and the photodynamic activity of bovine serum albumin-modified indocyanine green. Under near-infrared light irradiation, the two work synergistically to disinfect bacteria, reduce wound damage, and improve antibacterial and wound-healing properties.

[0035] Oxidized sodium alginate (OSA) is produced by oxidizing naturally sourced sodium alginate. The aqueous solution of oxidized sodium alginate has high viscosity and has been used as a thickener, stabilizer, and emulsifier in food. This makes it a candidate for various medical applications, including drug delivery and wound dressings. ε-Polylysine is a polypeptide with antibacterial properties. This biopreservative was first used in food preservation in the 1980s. ε-Polylysine can be broken down into lysine in the human body, and lysine is one of the eight essential amino acids and is permitted fortification in food worldwide. Moreover, ε-Polylysine is a nutritional antibacterial agent with higher safety than other chemical preservatives and is an excellent natural biocrosslinking agent. In this invention, sodium alginate with a dialdehyde structure is obtained by oxidizing sodium alginate, which readily crosslinks with polylysine to form a natural polymer-based hydrogel.

[0036] Building upon this, Pt-HPB and BSA & ICG were further combined with natural polymer-based hydrogels to form photosensitive hydrogels, endowing the material with synergistic photothermal and photodynamic conversion effects under near-infrared light irradiation, while also exhibiting good biocompatibility, tissue affinity, and adhesion. Attached Figure Description

[0037] Figure 1 The images show TEM images and particle size distribution histograms of PB and Pt-HPB prepared in Example 1 of this invention; wherein, A is the TEM image of PB, B is the TEM image of Pt-HPB, C is the particle size distribution of PB, and D is the particle size distribution of Pt-HPB.

[0038] Figure 2 To verify the photothermal performance results of Pt-HPB and PB in Experiment 1 of this invention, where A represents the photothermal properties of different concentrations of Pt-HPB at 808 nm (1 W / cm²). 2 A) Photothermal heating curves under irradiation, B) Photothermal heating curves of 40 μg / mPt-HPB under irradiation at 808 nm with different powers, C) Photothermal heating curves of Pt-HPB and PB with the same dose, and D) Photothermal heating curves of Pt-HPB thermal stability.

[0039] Figure 3 The results of the oxygen dissolution performance study of PB and Pt-HPB in Experiment 2 were used to verify the present invention.

[0040] Figure 4 The results of the photodynamic performance study of ICG and BSA&ICG in Experiment 3 were used to verify the present invention.

[0041] Figure 5 This is a gel morphology diagram of Pt-HPB-BSA&ICG@Gel in Example 4 of the present invention.

[0042] Figure 6 This is a diagram illustrating the synergistic antibacterial effect of photothermal and photodynamic therapy on Escherichia coli in Experiment 4 of this invention.

[0043] Figure 7 This is a diagram illustrating the synergistic antibacterial effect of photothermal and photodynamic therapy against Staphylococcus aureus in Experiment 4 of this invention.

[0044] Figure 8 This is a diagram illustrating the effect of Pt-HPB-BSA&ICG@Gel on hemolysis of erythrocytes in Experiment 5 of this invention.

[0045] Figure 9 The graph shows the biosafety evaluation results of Pt-HPB-BSA&ICG@Gel in Experiment 6 of this invention. Detailed Implementation

[0046] The technical solution of the present invention will be further explained and described below with reference to specific embodiments.

[0047] Example 1 (Preparation of platinum-doped hollow Prussian blue nanoparticles)

[0048] 1) Preparation of Prussian blue metal-organic framework nanoparticles (PB): 3 g of PVP was weighed and placed in a 100 mL flask. 40 mL of 0.01 M HCl solution was added and stirred until the liquid was clear. 131.7 mg of potassium ferricyanide (K3[Fe(CN)6]·3H2O) was added and stirred at room temperature for 1 h. The mixture was then heated to 80 °C in an oil bath for 20 h. After the reaction was completed, the mixture was cooled to room temperature, centrifuged at 18000 rpm for 15 min, washed three times with ethanol and distilled water, and then freeze-dried to obtain PB nanoparticles, which were stored at 4 °C.

[0049] 2) Preparation of hollow Prussian blue nanoparticles (HPB): Weigh 100 mg of PVP and place it in a 100 mL beaker. Add 20 mL of 1 M HCl solution to dissolve it and stir until clear. Weigh 20 mg of the PVP nanoparticles prepared in step 1) and add them to the mixed solution. After stirring for 1 h, transfer the mixture to a 60 mL muffle furnace liner and calcine at 140 °C for 4 h. After natural cooling, centrifuge at 18000 rpm for 15 min and wash three times with ethanol and distilled water to obtain HPB particles. Freeze-dry the particles and store them at 4 °C for later use.

[0050] 3) Preparation of Pt-doped hollow Prussian blue nanoparticles (Pt-HPB): In a 100 mL flask, weigh 50 mg of HPB nanoparticles obtained in step 2) and 50 mg of chloroplatinic acid (molar ratio Pt / Fe≈0.3) and mix with 20 mL of ethanol solution. Stir at room temperature for 1 h, place the mixture in an oil bath and reflux at 80 °C for 4 h, centrifuge at 18000 rpm for 15 min, and wash three times with ethanol and distilled water to obtain Pt-doped hollow Prussian blue nanoparticles Pt-HPB.

[0051] 4) Preparation of polylysine-modified platinum-doped hollow Prussian blue nanoparticles (PL&Pt-HPB): Weigh 1 mg Pt-HPB and 200 μg polylysine PL, dissolve them in distilled water, stir at 800 rpm for 1 h at room temperature, after the reaction is terminated, centrifuge at 18000 rpm for 15 min, and wash three times with distilled water to obtain polylysine-modified platinum-doped hollow Prussian blue nanoparticles (PL&Pt-HPB), freeze-dry and store at 4℃ for later use.

[0052] Example 2 (Preparation of BSA-modified ICG nanoparticles: BSA & ICG)

[0053] 10 mg of bovine serum albumin (BSA) was weighed and placed in a 10 mL round-bottom flask. 1 mL of phosphate-buffered saline (PBS) was added to dissolve it. 1 mg of indocyanine green (ICG) was weighed and dissolved in 200 μL of dimethyl sulfoxide (DMSO). The solution was then slowly added dropwise to the PBS solution. The mixture was stirred at 4 °C in the dark for 12 h. After the reaction was completed, the mixture was dialyzed at 4 °C in the dark for 24 h using a 3500 Da molecular weight dialysis bag. After freeze-drying, BSA-modified ICG nanoparticles (BSA & ICG) were obtained.

[0054] Example 3 (Preparation of Oxidized Sodium Alginate (OSA))

[0055] 1.0 g of sodium alginate was dissolved in 60 mL of water to obtain a sodium alginate solution. 1.08 g of sodium periodate was dissolved in 20 mL of water. After complete dissolution, the sodium alginate solution was slowly added, and water was added to bring the total volume to 100 mL. The oxidation reaction was carried out at 800 rpm in the dark for 6 hours at room temperature. Then, 3.5 mL of ethylene glycol (equimolar amount with sodium periodate) was added to stop the oxidation for 1 hour. After the reaction was completed, the resulting solution was dialyzed through an 8000-14000 Da dialysis bag in the dark for 48 hours and then freeze-dried to obtain oxidized sodium alginate (OSA).

[0056] Example 4 (Preparation of a hydrogel dressing with photothermal / photodynamic synergistic antibacterial activity, namely Pt-HPB-BSA&ICG@Gel hydrogel)

[0057] Weigh 200 mg of OSA prepared in Example 3, add 1 mL of distilled water to dissolve it, and obtain sodium alginate oxidized solution. Weigh 80 μg of PL & Pt-HPB prepared in Example 1 and 2 μg of BSA & ICG prepared in Example 2 and add them to the sodium alginate oxidized solution. Adjust the pH to 7.5-8.0 with HCl. Weigh 200 mg of polylysine and dissolve it in 1 mL of distilled water. After it is completely dissolved, mix the polylysine solution with the OSA mixture by vortexing and stir thoroughly for 20 s to obtain Pt-HPB-BSA & ICG@Gel hydrogel.

[0058] Effect verification

[0059] Verification Experiment 1: Photothermal Performance of PB and Pt-HPB

[0060] Different concentrations (10, 20, 40, 80 μg / mL) of Pt-HPB were placed in centrifuge tubes. The laser power was set to 1W. Near-infrared light at 808 nm was used for 6 min, followed by irradiation with the near-infrared light turned off. Temperature was recorded every 30 s using an infrared imager, and the resulting temperature-time curve is shown below. Figure 2 As shown in Figure A.

[0061] Take 40 μg / mL of Pt-HPB and irradiate it with near-red light at 808 nm for 6 min. The laser power is set to (0.25, 0.5, 1, 1.5 W / cm²). 2 Then the near-infrared light was turned off. Using an infrared imager, the temperature was recorded every 30 seconds, and the resulting temperature-time curve is shown below. Figure 2 As shown in B.

[0062] Place 40 μg / mL of PB and Pt-HPB in a centrifuge tube. Set the laser power to 1W. Irradiate with near-infrared light at 808 nm for 6 min, then turn off the near-infrared light. Record the temperature every 30 s using an infrared imager. The temperature-time curve is shown below. Figure 2 As shown in C. Perform four cycles and plot the temperature-time cycle curve as shown. Figure 2 As shown in D.

[0063] Under 808nm laser irradiation, Pt-HPB can reach the treatment temperature range within 5 minutes. Figure 2 As shown by C, Pt-HPB has a higher photothermal conversion efficiency than PB. The heating rate and maximum temperature under near-infrared light irradiation both indicate the excellent photothermal performance of this nanoparticle, which also possesses temperature-stable photothermal properties. Since a temperature of 50℃ is required for better antibacterial activity, and excessively high temperatures may damage healthy tissue, a concentration of 40 μg / mL was chosen for subsequent experiments.

[0064] Verification Experiment 2: Oxygen Production Capacity and Catalase-like Activity of PB and Pt-HPB

[0065] Dissolve PB and Pt-HPB (1.0 mg) separately in 10 mL of PBS (pH 7.2-7.4), and add 10 μl of 30% H2O2. Monitor the O2 concentration in the solution in real time for 20 min using a portable dissolved oxygen meter.

[0066] Depend on Figure 3 It is evident that, compared to undoped Prussian blue nanoparticles, platinum-doped Prussian blue nanoparticles significantly enhance their oxygen production capacity, effectively providing oxygen to the infected microenvironment and promoting wound healing.

[0067] Validation Experiment 3: Photodynamic Capabilities of ICG and BSA & ICG

[0068] To investigate the photodynamic properties of ICG and BSA&ICG under near-infrared irradiation at 808 nm, 3 mL (1 μg / mL) of ICG and 30 μL (1 mg / mL) of DPBF solution were mixed with 30 μL of DPBF solution and placed in quartz cuvettes. The laser power was set to 1 W, and the mixture was irradiated with near-infrared light at 808 nm for 10 min, after which the near-infrared light was turned off. The ultraviolet absorption spectra of the mixtures in the range of 350–550 nm were recorded every 1 min using a UV spectrophotometer. The plotted curves are shown below. Figure 4 As shown in A and 4B. Quantitative comparison data were also prepared as follows: Figure 4 As shown in C.

[0069] Depend on Figure 4 As can be seen from C, under laser irradiation at a wavelength of 808nm, it is possible that the dispersion and stabilization effect of BSA on ICG effectively prevents the aggregation of ICG, resulting in BSA-modified ICG having a higher singlet oxygen production capacity than pure ICG, and thus having a superior bactericidal ability.

[0070] Experiment 4 verified the sterilization ability of the hydrogel dressing component with synergistic photothermal / photodynamic antibacterial activity.

[0071] To investigate the antibacterial capabilities of each component, four samples of each of the following were prepared: PBS, Gel, Pt-HPB, BSA&ICG, Pt-HPB-BSA&ICG, and Pt-HPB-BSA&ICG@gel. These were used to set up control groups for Escherichia coli, laser-irradiated groups for Escherichia coli, control groups for Staphylococcus aureus, and laser-irradiated groups for Staphylococcus aureus.

[0072] Each sample was co-cultured with bacteria for 12 hours and then placed in a six-well plate for 5 minutes at 808 nm (1 W / cm²). 2Laser irradiation was performed, and thermal imaging cameras were used to take pictures during the process. Fresh liquid culture medium was then added to each well to keep the material in suspension, and the mixture was incubated at 37°C for 12 hours.

[0073] Finally, take 10. -5 The diluted bacterial suspension was plated. The plated cultures were then incubated at 37°C for 12 hours, photographed, and colony counted. The antibacterial rate was calculated, and the results are as follows: Figure 6 and Figure 7 As shown.

[0074] Depend on Figure 6 , 7 It can be seen that the antibacterial effect of the gel group is due to the antibacterial effect of polylysine. Pt-HPB, BSA & ICG can significantly reduce Escherichia coli and Staphylococcus aureus. Simultaneous loading of Pt-HPB, BSA & ICG achieves a more significant bactericidal effect, with an antibacterial rate of 97.98% against Escherichia coli and 93.43% against Staphylococcus aureus. Furthermore, after co-loading in the colloid, the antibacterial effect was not significantly affected, and it still exhibited a significant bactericidal effect. This result fully demonstrates that the hydrogel dressing with photothermal / photodynamic synergistic antibacterial activity has a significant bactericidal effect in low-power photothermal-photodynamic synergistic therapy.

[0075] Verification Experiment 5 Biosafety

[0076] Studies on hemolysis of red blood cells. All animal experiments were conducted in accordance with the Animal Ethics Committee Guidelines and were approved by the Tianjin Institute of Radiation Medicine.

[0077] Hemolysis evaluation: Fresh blood was collected from SD rats anesthetized with EDTA anticoagulant. Whole blood was centrifuged and washed, then diluted 10-fold with PBS to obtain clean red blood cells. 200 μL of different solutions (1, 5, and 10 mg / mL) were added to 1 mL of red blood cells and incubated at room temperature for 4 hours. Red blood cells in PBS served as a negative control, and red blood cells in water served as a positive control. Subsequently, the supernatant was collected by centrifugation, and the absorbance was measured at 540 nm using a microplate reader. The hemolysis rate of red blood cells was calculated as follows: Figure 8 As shown. By Figure 8 It can be seen that the gel has good biocompatibility.

[0078] The percentage of hemolysis is calculated using the formula: Hemolysis% = (Absorbance of sample group - Absorbance of PBS group) / (Absorbance of water group - Absorbance of PBS group) × 100%.

[0079] Verification Experiment 6: Cytotoxicity Assay (CCK-8)

[0080] The cytotoxic effects of different materials treated in vitro were evaluated using the CCK-8 assay. L929 cells were seeded in 96-well plates at a density of 1 × 10⁶ cells / well. 4 Cells were cultured for 24 hours. After incubation with Pt-HPB-BSA & ICG@Gel (0.5, 1, 5, 10, 15 mg / mL) for 12 hours, CCK-8 was added to each well, and incubation was continued for another 90 minutes. The absorbance at 450 nm was measured using a microplate reader. Each condition was performed in triplicate. Cell viability was analyzed using the CCK-8 assay. Cell viability was calculated as follows: Figure 9 As shown. By Figure 9 It is known that colloids have good biocompatibility.

[0081] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a hydrogel dressing with synergistic photothermal / photodynamic antibacterial activity, characterized in that, The specific steps of this method are as follows: Step 1: Preparation of polylysine-modified platinum-doped hollow Prussian blue nanoparticles; Step 2: Preparation of bovine serum albumin-modified indocyanine green nanoparticles; Step 3: Prepare sodium alginate oxide; Step 4: Prepare a hydrogel dressing with synergistic photothermal / photodynamic antibacterial activity; Step four, preparing a hydrogel dressing with synergistic photothermal / photodynamic antibacterial activity, is as follows: Weigh 200 mg of sodium alginate prepared in step 3, add 1 mL of distilled water to dissolve it, and obtain a sodium alginate solution. Weigh 80 μg of polylysine-modified platinum-doped hollow Prussian blue nanoparticles prepared in step 1 and 2 μg of bovine serum albumin-modified indocyanine green nanoparticles prepared in step 2, add them to the sodium alginate solution, and adjust the pH to 7.5-8.0 with HCl. Weigh 200 mg of polylysine and dissolve it in 1 mL of distilled water. After it is completely dissolved, mix the polylysine solution and sodium alginate mixture by vortexing and stir thoroughly for 20 s to obtain a hydrogel dressing with photothermal / photodynamic synergistic antibacterial activity.

2. The method for preparing the hydrogel dressing with photothermal / photodynamic synergistic antibacterial activity according to claim 1, characterized in that, In step one, the specific steps for preparing polylysine-modified platinum-doped hollow Prussian blue nanoparticles are as follows: 1) Preparation of Prussian blue metal-organic framework nanoparticles Weigh 3g of polyvinylpyrrolidone into a 100mL flask, add 40mL of 0.01M HCl solution and stir until the liquid is clear. Add K3[Fe(CN)6]·3H2O and stir at room temperature for 1h. Then place the mixed solution in an oil bath and heat to 80℃ for 20 hours. After the reaction is complete, cool to room temperature, centrifuge, wash and freeze-dry to obtain Prussian blue metal-organic framework nanoparticles, which are stored at 4℃ for later use. The mass ratio of K3[Fe(CN)6]·3H2O to polyvinylpyrrolidone is 1:(20~23). 2) Preparation of hollow Prussian blue nanoparticles Weigh 100 mg of polyvinylpyrrolidone and place it in a 100 mL beaker. Add 20 mL of 1 M HCl solution to dissolve it and stir until clear. Weigh 20 mg of the Prussian blue metal-organic framework nanoparticles prepared in step 1) and add them to the mixed solution. After stirring for 1 h, transfer the mixture to a 60 mL muffle furnace liner and calcine at 140 °C for 4 h. After natural cooling, centrifuge, wash and freeze dry to obtain hollow Prussian blue nanoparticles, which are stored at 4 °C for later use. 3) Preparation of Pt-doped hollow Prussian blue nanoparticles In a 100 mL flask, 50 mg of the hollow Prussian blue nanoparticles obtained in step 2) and chloroplatinic acid were weighed and mixed with 20 mL of ethanol solution. The mixture was stirred at room temperature for 1 h. The mixture was then placed in an oil bath and refluxed at 80 °C for 4 h. After centrifugation, washing and drying, Pt-doped hollow Prussian blue nanoparticles were obtained. The molar ratio of Pt in chloroplatinic acid to Fe in hollow Prussian blue nanoparticles was 0.28–0.

32. 4) Preparation of polylysine-modified platinum-doped hollow Prussian blue nanoparticles Weigh 1 mg of Pt-HPB and 200 μg of polylysine, dissolve them in distilled water, and stir at 800 rpm for 1 h at room temperature. After the reaction is terminated, centrifuge, wash and freeze dry to obtain polylysine-modified platinum-doped hollow Prussian blue nanoparticles, which are then stored at 4 °C for later use.

3. The method for preparing the hydrogel dressing with photothermal / photodynamic synergistic antibacterial activity according to claim 1, characterized in that, Step two, the steps for preparing bovine serum albumin-modified indocyanine green nanoparticles are as follows: 10 mg of bovine serum albumin was weighed and placed in a 10 mL round-bottom flask, and dissolved in 1 mL of phosphate buffer. Indocyanine green was weighed according to a mass ratio of bovine serum albumin to indocyanine green of 10:1, and dissolved in dimethyl sulfoxide at a ratio of 1 mg: 200 μl. The solution was then slowly added dropwise to the bovine serum albumin solution. The mixture was stirred at 4 °C in the dark for 12 h. After the reaction was completed, the mixture was dialyzed at 4 °C in the dark for 24 h. After freeze-drying, bovine serum albumin-modified indocyanine green nanoparticles were obtained.

4. The method for preparing the hydrogel dressing with photothermal / photodynamic synergistic antibacterial activity according to claim 3, characterized in that, In step two, light-protected dialysis is preferably performed using a 3500 Da molecular weight dialysis bag.

5. The method for preparing the hydrogel dressing with photothermal / photodynamic synergistic antibacterial activity according to claim 1, characterized in that, Step 3, the steps for preparing sodium alginate oxide are as follows: 1.0 g of sodium alginate was dissolved in 60 mL of water to obtain a sodium alginate solution. Sodium periodate of equal molar amount to sodium alginate was dissolved in 20 mL of water. After complete dissolution, the sodium alginate solution was slowly added, and water was added to bring the mixture to 100 mL. The oxidation reaction was carried out at room temperature in the dark for 6 h with stirring. Then, ethylene glycol was added to stop the oxidation for 1 h. After the reaction was completed, the resulting solution was dialyzed in the dark for 48 h and then freeze-dried to obtain oxidized sodium alginate.

6. The method for preparing the hydrogel dressing with photothermal / photodynamic synergistic antibacterial activity according to claim 5, characterized in that, In step three, the mass ratio of ethylene glycol to sodium alginate is 1.5:

1.

7. The method for preparing the hydrogel dressing with photothermal / photodynamic synergistic antibacterial activity according to claim 5, characterized in that, The light-protected dialysis described in step three is performed using a dialysis bag with a capacity of 8000-14000 Da.

8. A hydrogel dressing with photothermal / photodynamic synergistic antibacterial activity prepared by the method according to any one of claims 1 to 7.

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