Preparation method and application of bionic sharp spike microparticle light-thermal antibacterial material

Abstract

The application relates to a preparation method and application of a bionic sharp particle light-heat antibacterial material, and belongs to the technical field of antibacterial materials. The application aims to solve the problem that the existing bionic sharp antibacterial material cannot simultaneously realize simple preparation process, good biocompatibility and green environmental protection. The method comprises the following steps: 1, preparing a fatty acid solution; 2, preparing a lecithin / DSPE-PEG 2000 solution; 3, preparing a silver nitrate solution; 4, preparing a hematin chloride solution; 5, heating reaction; and 6, preparing bionic sharp particles. Application: the bionic sharp particle light-heat antibacterial material is used for sterilization of gram-negative bacteria and drug-resistant gram-negative bacteria and for promoting healing of bacterial infection wounds.

Description

Technical Field

[0001] This invention belongs to the field of antibacterial materials technology. Background Technology

[0002] Bacteria are inextricably linked to human survival and social progress; their ubiquity means they are present in almost all organisms, objects, and the environment. Bacterial infections, especially those caused by drug-resistant bacteria, pose a significant threat to human health and impaired wound healing, representing a major challenge to global public health. Since the advent of antibiotics, they have become indispensable drugs for treating bacterial infections, but their widespread use in public health has led to an increase in antibiotic resistance. This worrying trend underscores the urgent need to develop novel non-antibiotic antimicrobial materials to address the growing threat of antibiotic resistance.

[0003] With the deepening exploration of microstructure research, natural micro / nanospinous structures with significant antibacterial properties have attracted widespread attention from the scientific community. For example, the regularly arranged nanospikes on cicada wings exhibit unique bactericidal functions through bacterial capture and subsequent membrane damage, achieving self-protection against microbial invasion. Similarly, the nanospikes on the surface of gecko skin also exhibit similar antibacterial mechanisms by inhibiting bacterial colonization and inducing membrane structure damage. Inspired by these natural biological surface microstructures, more and more biomimetic spiked antibacterial materials have been developed. However, existing spiked structure materials are mainly synthesized from silicon and metal compounds to form spiked surface coatings for application. Therefore, the following problems still exist:

[0004] (1) Preparation difficulty and cost: Manufacturing antibacterial materials with fine spiky structures requires high-precision processing technology, which increases the difficulty and cost of production.

[0005] (2) Biocompatibility issues: While sharp structures can kill bacteria, they can also damage cells or tissues, especially when these materials come into direct contact with the human body (such as implants). Therefore, their biocompatibility needs to be rigorously assessed and controlled.

[0006] (3) Environmental friendliness: The production and disposal of existing spiked antibacterial materials may have certain environmental impacts. In particular, when these materials contain heavy metals or other harmful components, their environmental hazards need to be carefully considered. Summary of the Invention

[0007] This invention aims to address the problem that existing biomimetic spiked antibacterial materials cannot simultaneously achieve simple preparation processes, good biocompatibility, and environmental friendliness, and thus provides a method for preparing and applying a biomimetic spiked microparticle photothermal antibacterial material.

[0008] A method for preparing a biomimetic spiked microparticle photothermal antibacterial material comprises the following steps:

[0009] 1. Dissolve lauric acid and stearic acid in methanol solution to obtain a fatty acid solution;

[0010] 2. Dissolve lecithin and distearate phosphatidylethanolamine-methoxy polyethylene glycol 2000 in an aqueous ethanol solution to obtain a lecithin / DSPE-PEG 2000 solution;

[0011] 3. Dissolve silver nitrate in dimethyl sulfoxide solution to obtain silver nitrate solution;

[0012] IV. Dissolve heme chloride in N,N-dimethylformamide solution to obtain heme chloride solution;

[0013] 5. Under heating and stirring conditions, fatty acid solution, silver nitrate solution and heme chloride solution were added dropwise to lecithin / DSPE-PEG 2000 solution to react and obtain a mixed system;

[0014] 6. Stir the mixture in an ice bath, then stir at room temperature to obtain a colloidal solution. Centrifuge the colloidal solution to remove the supernatant, then disperse it in sterile ultrapure water and centrifuge multiple times. Finally, disperse it in sterile ultrapure water to obtain a biomimetic spiked microparticle photothermal antibacterial material.

[0015] An application of a biomimetic spiked microparticle photothermal antibacterial material, which is used for the sterilization of Gram-negative bacteria and drug-resistant Gram-negative bacteria, and for promoting the healing of bacterial infected wounds.

[0016] The beneficial effects of this invention are:

[0017] (1) This invention is the first to realize the synthesis of biomimetic spike microparticles by using a mixture of lauric acid and stearic acid coated with a phospholipid layer as the material matrix and a one-pot reaction.

[0018] (2) The biomimetic spiked microparticles prepared by this invention are quick and easy to synthesize, with a mild reaction temperature, low cost, and are environmentally friendly. Heme chloride is a non-toxic organic photothermal agent and is often used in photothermal antibacterial therapy, while silver nitrate can be reduced by polyethylene glycol to form nanoparticles, thereby enhancing the photothermal properties and antibacterial ability of the material.

[0019] (3) The biomimetic spiked microparticles synthesized using the present invention can kill five orders of magnitude of Escherichia coli (E. coli) within 10 minutes, and can also kill three orders of magnitude of intestinal invasive drug-resistant Escherichia coli (E. coli EIEC) within 10 minutes. It has good antibacterial effect against Gram-negative bacteria and their drug-resistant bacteria, and meets the national Class I antibacterial standard.

[0020] (4) The biomimetic spike microparticles synthesized using the present invention can promote the healing rate of bacterial infected wounds by more than 99% within 14 days, and have no obvious toxic effects on biological cells, which greatly expands the practical application range of biomimetic spike materials.

[0021] This invention relates to a method for preparing and applying a biomimetic spiked microparticle photothermal antibacterial material. Attached Figure Description

[0022] Figure 1 The images shown are transmission electron microscope (TEM) images of SMPs prepared in Example 1. A is a transmission electron microscope image, B is a dark field transmission electron microscope image and a transmission electron element mapping image.

[0023] Figure 2 The images show UV-Vis absorption spectra. 1 is a PBS solution with pH 7.4, 2 is an AgNPs solution, 3 is a heme chloride solution, and 4 is an SMPs solution prepared in Example 1.

[0024] Figure 3 This is a fine spectrum of the Ag 3d binding energy of the SMPs prepared in Example 1;

[0025] Figure 4 The image shows the photothermal properties of the SMPs prepared in Example 1, where A is 0.75 W / cm². 2 Thermal imaging of NIR irradiation of a 200 μg / mL SMPs solution for 10 min, B = 0.75 W / cm². 2 The heating curves of different samples under NIR irradiation are shown (1 is PBS solution with pH 7.4, 2 is AgNPs solution with a concentration of 4 μg / mL, 3 is heme chloride solution with a concentration of 20 μg / mL, and 4 is SMPs solution with a concentration of 200 μg / mL). The temperature rise curves of the 200 μg / mL SMPs solution under different laser irradiation intensities are also shown (C is 0.5 W / cm²). 2 2 is 0.75W / cm 2 3 is 1W / cm 2 The heating curve under the condition that D is 0.75 W / cm² is shown. 2 Temperature rise curves of SMPs solutions with different concentrations irradiated by NIR (1 for 100 μg / mL, 2 for 200 μg / mL, and 3 for 300 μg / mL).

[0026] Figure 5 The image shows the antibacterial properties of the SMPs prepared in Example 1. A is a colony plate image of Escherichia coli, B is the survival rate of Escherichia coli after different treatments, C is a colony plate image of drug-resistant Escherichia coli, D is the survival rate of drug-resistant Escherichia coli after different treatments, 1 is without near-infrared light, and 2 is with near-infrared light.

[0027] Figure 6 The following is a graph showing the wound healing efficiency of SMPs prepared in Example 1. A shows the wound healing of mice in different treatment groups, B shows the wound healing rate of mice in different treatment groups (1 is PBS, 2 is PBS+NIR, 3 is SMPs, 4 is SMPs+NIR), and C shows the change in body weight of mice in different treatment groups over time (1 is PBS, 2 is PBS+NIR, 3 is SMPs, 4 is SMPs+NIR).

[0028] Figure 7 The results of the cytotoxicity experiment of the SMPs prepared in Example 1 are shown. Detailed Implementation

[0029] The technical solution of the present invention is not limited to the specific embodiments listed below, but also includes any combination of the specific embodiments.

[0030] Specific Implementation Method 1: This embodiment describes a method for preparing a biomimetic spiked microparticle photothermal antibacterial material, which is carried out according to the following steps:

[0031] 1. Dissolve lauric acid and stearic acid in methanol solution to obtain a fatty acid solution;

[0032] 2. Dissolve lecithin and distearate phosphatidylethanolamine-methoxy polyethylene glycol 2000 in an aqueous ethanol solution to obtain a lecithin / DSPE-PEG 2000 solution;

[0033] 3. Dissolve silver nitrate in dimethyl sulfoxide solution to obtain silver nitrate solution;

[0034] IV. Dissolve heme chloride in N,N-dimethylformamide solution to obtain heme chloride solution;

[0035] 5. Under heating and stirring conditions, fatty acid solution, silver nitrate solution and heme chloride solution were added dropwise to lecithin / DSPE-PEG 2000 solution to react and obtain a mixed system;

[0036] 6. Stir the mixture in an ice bath, then stir at room temperature to obtain a colloidal solution. Centrifuge the colloidal solution to remove the supernatant, then disperse it in sterile ultrapure water and centrifuge multiple times. Finally, disperse it in sterile ultrapure water to obtain a biomimetic spiked microparticle photothermal antibacterial material.

[0037] This embodiment uses natural fatty acids as the material matrix and heme chloride as the photothermal agent, ensuring excellent biocompatibility for humans. Lecithin encapsulation further enhances its biocompatibility, while PEG groups reduce silver nitrate to silver nanoparticles, which are then loaded into the material to enhance its photothermal antibacterial properties. Its unique sea urchin-like spike structure, combined with photothermal therapy, achieves superior bacterial killing effects. Biomimetic spike microparticles were added to bacterial cultures of *E. coli* and drug-resistant *E. coli*, and their antibacterial effect was observed using a plate coating method under near-infrared light irradiation. Simultaneously, SMPs were added to a mouse wound infection model to verify its wound-healing properties.

[0038] The beneficial effects of this embodiment are:

[0039] (1) This embodiment is the first to realize the synthesis of biomimetic spiked microparticles by using a mixture of lauric acid and stearic acid coated with a phospholipid layer as the material matrix and a one-pot reaction.

[0040] (2) The biomimetic spiked microparticles prepared in this embodiment are quick and easy to synthesize, with a mild reaction temperature, low cost, and are environmentally friendly. Heme chloride is a non-toxic organic photothermal agent and is often used in photothermal antibacterial therapy, while silver nitrate can be reduced by polyethylene glycol to form nanoparticles, thereby enhancing the photothermal properties and antibacterial ability of the material.

[0041] (3) The biomimetic spiked microparticles synthesized using this embodiment can kill five orders of magnitude of Escherichia coli (E. coli) within 10 minutes, and can also kill three orders of magnitude of intestinal invasive drug-resistant Escherichia coli (E. coli EIEC) within 10 minutes. It has good antibacterial effect against Gram-negative bacteria and their drug-resistant bacteria, and meets the national Class I antibacterial standard.

[0042] (4) The biomimetic spike microparticles synthesized using this embodiment can promote the healing rate of bacterial infected wounds by more than 99% within 14 days, and have no obvious toxic effects on biological cells, which greatly expands the practical application range of biomimetic spike materials.

[0043] Specific Implementation Method Two: This implementation method differs from Specific Implementation Method One in that the mass ratio of lauric acid to stearic acid in step one is (3~4):1; and the total concentration of lauric acid and stearic acid in the fatty acid solution in step one is 2.5mg / mL~4mg / mL. Everything else is the same as in Specific Implementation Method One.

[0044] Specific Implementation Method Three: This implementation method differs from Specific Implementation Method One or Two in that: the mass ratio of lecithin to distearate phosphatidylethanolamine-methoxy polyethylene glycol 2000 in step two is (3~4):1; the mass percentage of ethanol in the aqueous solution in step two is 4%~6%; and the total concentration of lecithin and distearate phosphatidylethanolamine-methoxy polyethylene glycol 2000 in the lecithin / DSPE-PEG2000 solution in step two is 3mg / mL~4mg / mL. Everything else is the same as in Specific Implementation Method One or Two.

[0045] Specific Implementation Method Four: This implementation method differs from Specific Implementation Methods One to Three in that the concentration of silver nitrate in the silver nitrate solution described in step three is 0.5 mg / mL to 1 mg / mL. Everything else is the same as in Specific Implementation Methods One to Three.

[0046] Specific Implementation Method Five: This implementation method differs from Specific Implementation Methods One to Four in that the concentration of heme chloride in the heme chloride solution described in step four is 1 mg / mL to 2.5 mg / mL. Everything else is the same as in Specific Implementation Methods One to Four.

[0047] Specific Implementation Method Six: This implementation method differs from Specific Implementation Methods One to Five in that, in step five, under the conditions of a heating temperature of 55℃~65℃ and a stirring speed of 4000rpm~6000rpm, the fatty acid solution, silver nitrate solution, and heme chloride solution are added dropwise to the lecithin / DSPE-PEG 2000 solution and reacted for 2min~5min. Everything else is the same as in Specific Implementation Methods One to Five.

[0048] Specific Implementation Method Seven: This implementation method differs from Specific Implementation Methods One to Six in that: the volume ratio of the lecithin / DSPE-PEG 2000 solution to the fatty acid solution in step five is (5~6):4; the volume ratio of the lecithin / DSPE-PEG 2000 solution to the silver nitrate solution in step five is (15~20):1; and the volume ratio of the lecithin / DSPE-PEG 2000 solution to the heme chloride solution in step five is (5~6):1. Everything else is the same as in Specific Implementation Methods One to Six.

[0049] Specific Implementation Method Eight: This implementation method differs from Specific Implementation Methods One to Seven in that: in step six, the mixture is stirred in an ice bath for 10 to 15 minutes at a temperature of 0℃ to 4℃ and a stirring speed of 4000 rpm to 6000 rpm. Then, it is stirred for 10 to 20 minutes at room temperature and a stirring speed of 4000 rpm to 6000 rpm to obtain a colloidal solution. The colloidal solution is then centrifuged for 5 to 10 minutes at a speed of 6000 rpm to 8000 rpm to remove the supernatant. Everything else is the same as in Specific Implementation Methods One to Seven.

[0050] Specific Implementation Method Nine: This implementation method describes the application of a biomimetic spiked microparticle photothermal antibacterial material, which is used for the sterilization of Gram-negative bacteria and drug-resistant Gram-negative bacteria, and for promoting the healing of bacterial infected wounds.

[0051] Specific Implementation Method Ten: This implementation method differs from Specific Implementation Method Nine in that the Gram-negative bacteria mentioned are *Escherichia coli*; and the drug-resistant Gram-negative bacteria mentioned are tetracycline-resistant enteroinvasive *Escherichia coli*. Everything else is the same as in Specific Implementation Method Nine.

[0052] The beneficial effects of the present invention are verified using the following embodiments:

[0053] Example 1, combined with Figure 1 Detailed explanation:

[0054] A method for preparing a biomimetic spiked microparticle photothermal antibacterial material comprises the following steps:

[0055] 1. Dissolve lauric acid and stearic acid in methanol solution to obtain a fatty acid solution;

[0056] The mass ratio of lauric acid to stearic acid is 4:1; the total concentration of lauric acid and stearic acid in the fatty acid solution is 4 mg / mL.

[0057] 2. Dissolve lecithin and distearate phosphatidylethanolamine-methoxy polyethylene glycol 2000 in an aqueous ethanol solution to obtain a lecithin / DSPE-PEG 2000 solution;

[0058] The mass ratio of lecithin to distearylphosphatidylethanolamine-methoxy polyethylene glycol 2000 is 3:1; the mass percentage of ethanol in the aqueous solution is 4%; the total concentration of lecithin and distearylphosphatidylethanolamine-methoxy polyethylene glycol 2000 in the lecithin / DSPE-PEG 2000 solution is 4 mg / mL.

[0059] 3. Dissolve silver nitrate in dimethyl sulfoxide solution to obtain silver nitrate solution;

[0060] The concentration of silver nitrate in the silver nitrate solution is 1 mg / mL;

[0061] IV. Dissolve heme chloride in N,N-dimethylformamide solution to obtain heme chloride solution;

[0062] The concentration of heme chloride in the heme chloride solution is 2.5 mg / mL;

[0063] 5. Under the conditions of heating temperature of 60℃ and stirring speed of 5000rpm, fatty acid solution, silver nitrate solution and heme chloride solution were added dropwise to lecithin / DSPE-PEG 2000 solution and reacted for 2min to obtain a mixed system;

[0064] The volume ratio of the lecithin / DSPE-PEG 2000 solution to the fatty acid solution is 5:4; the volume ratio of the lecithin / DSPE-PEG 2000 solution to the silver nitrate solution is 16.7:1; and the volume ratio of the lecithin / DSPE-PEG 2000 solution to the heme chloride solution is 5:1.

[0065] VI. Under the conditions of 4℃ and 5000rpm, the mixture was stirred in an ice bath for 10min, and then stirred for 10min at room temperature and 5000rpm to obtain a colloidal solution. The colloidal solution was centrifuged at 6000rpm for 5min to remove the supernatant, and then dispersed in sterile ultrapure water and centrifuged 5 times. Finally, it was dispersed in sterile ultrapure water to obtain a biomimetic spiked microparticle photothermal antibacterial material (SMPs) solution, i.e., SMPs solution.

[0066] The SMPs solution prepared in Example 1 was dropped onto a copper grid, dried, and then subjected to transmission electron microscopy. Figure 1 The images show transmission electron microscopy (TEM) images of the SMPs prepared in Example 1. A is a TEM image, B is a dark-field TEM image and a TEM elemental mapping diagram. As shown, TEM clearly reveals the internal structure and surface morphology of the material, characterized by high-density disordered peaks and obvious compound encapsulation within the spiked matrix. Statistical analysis of the particle size distribution in the TEM images indicates that the average diameter of the SMPs is 2.1 μm, and the average peak length is approximately 500 nm. Dark-field imaging further confirms the presence of Ag nanoparticles on the spiked matrix, while energy-dispersive X-ray (EDX) spectroscopy confirms the elemental distribution of Ag, Cl, C, N, and O.

[0067] Figure 2The figures show the UV-Vis absorption spectra: 1 is a PBS solution at pH 7.4, 2 is an AgNPs solution, 3 is a heme chloride solution, and 4 is an SMPs solution prepared in Example 1. As shown, UV-Vis-NIR spectroscopy revealed the characteristic absorption peaks of different samples. Heme chloride showed a distinct characteristic absorption peak at 400 nm, while SMPs showed a strong absorption peak at the same position, demonstrating the successful loading of heme chloride.

[0068] Figure 3 The figure shows the fine XPS spectrum of Ag 3d binding energy of the SMPs prepared in Example 1. As shown in the figure, the fine XPS spectrum of Ag 3d indicates that Ag in the SMPs exhibits the Ag(0) valence state. The peaks at 368.3 eV and 374.3 eV are attributed to the Ag 3d of Ag with valence 0. 5 / 2 and Ag 3d 3 / 2 The orbitals also indicate that silver nanoparticles were successfully modified onto SMPs. XPS spectra further confirm the successful synthesis of the SMPs.

[0069] The photothermal properties of the SMPs solution prepared in Example 1 were tested by exposing the SMPs solution to an 808nm laser for 10 minutes and recording the temperature changes using a photothermal imager. Figure 4 The image shows the photothermal properties of the SMPs prepared in Example 1, where A is 0.75 W / cm². 2 Thermal imaging of NIR irradiation of a 200 μg / mL SMPs solution for 10 min, B = 0.75 W / cm². 2 The heating curves of different samples under NIR irradiation are shown (1 is PBS solution with pH 7.4, 2 is AgNPs solution with a concentration of 4 μg / mL, 3 is heme chloride solution with a concentration of 20 μg / mL, and 4 is SMPs solution with a concentration of 200 μg / mL). The temperature rise curves of the 200 μg / mL SMPs solution under different laser irradiation intensities are also shown (C is 0.5 W / cm²). 2 2 is 0.75W / cm 2 3 is 1W / cm 2 The heating curve under the condition that D is 0.75 W / cm² is shown. 2 Temperature rise curves of SMPs solutions at different concentrations irradiated with NIR (1: 100 μg / mL, 2: 200 μg / mL, 3: 300 μg / mL). (Source: [Insert source here]) Figure 4 As can be seen from A, the sample exhibits a temperature increase that is dependent on the irradiation time. Figure 4As shown in Figure B, SMPs exhibited the strongest photothermal effect, with a solution temperature increase of 19.2℃, while hemin alone only increased by 3.5℃. The poor photothermal performance of hemin alone may be attributed to its high hydrophobicity and poor solubility, which were significantly improved by fatty acid encapsulation and DSPE-PEG surface modification, thereby enhancing its hydrophilicity and stability. Further studies on the effect of laser power density and sample concentration showed that as laser intensity ( Figure 4 C) and material concentration ( Figure 4 With the increase of D), the photothermal performance of SMPs gradually improves.

[0070] The bactericidal performance of the SMPs prepared in Example 1 was tested, and the test procedure is as follows:

[0071] Bacterial activation and treatment: E. coli and E. coli EIEC (tetracycline-resistant enteroinvading E. coli) stored at 4℃ were removed from the refrigerator and placed in a clean bench. One colony was inoculated using an inoculation loop and added to LB medium (composition: NaCl 10 g / L, yeast extract 5 g / L, tryptone 10 g / L). The culture was incubated at 37℃ and 150 rpm for 12 h in a constant temperature shaker. The obtained bacterial solution was centrifuged (6000 rpm, 5 min) and resuspended in PBS buffer (0.01 M, pH=7), adjusting the bacterial concentration to OD0.05. 600 =0.2.

[0072] Material addition: Prepare 2.5 mL sterile centrifuge tubes, and add 250 μL of the above bacterial culture to each tube. For the experimental groups, add 250 μL of AgNPs solution (concentration 8 μg / mL), 250 μL of heme chloride solution (concentration 40 μg / mL), or 250 μL of SMPs solution (concentration 400 μg / mL) to the E. coli and E. coli EIEC bacterial cultures, respectively. For the blank control group, add 500 μL of sterile water. Set the system under no near-infrared light for 10 min or at 0.75 W / cm². 2 Irradiate with 808nm near-infrared light for 10 minutes.

[0073] Plate coating: Place the prepared sterile culture dishes into the laminar flow hood, take 100 μL of bacterial solution from different experimental groups and control groups, spread it evenly on the solid culture dishes with a sterile spreader, and incubate in a constant temperature incubator at 37℃ for 24 h.

[0074] Plate count: Calculate the bactericidal efficiency of SMPs after incubation with bacteria for different times.

[0075] Figure 5The graph shows the antibacterial properties of the SMPs prepared in Example 1. A is an image of Escherichia coli colony plates, B is the survival rate of Escherichia coli after different treatments, C is an image of drug-resistant Escherichia coli colony plates, and D is the survival rate of drug-resistant Escherichia coli after different treatments. 1 represents no near-infrared irradiation, and 2 represents near-infrared irradiation. Figure 5 As shown in A and C, without near-infrared light irradiation, SMPs alone showed no significant bactericidal effect against Escherichia coli and drug-resistant Escherichia coli within 10 minutes. However, thanks to their excellent photothermal conversion capabilities, after near-infrared light irradiation, SMPs could kill five orders of magnitude of E. coli within 10 minutes, with a bactericidal efficiency exceeding 99.999% against E. coli. Simultaneously, they killed three orders of magnitude of drug-resistant intestinal E. coli (EIEC) within 10 minutes, with a bactericidal efficiency exceeding 99.9% against drug-resistant E. coli within 10 minutes. Figure 5 (B and D). These results demonstrate that SMPs enhance the bactericidal mechanism through photothermal reactions, effectively killing common Gram-negative pathogens and drug-resistant strains.

[0076] The wound healing promotion properties of the SMPs prepared in Example 1 were tested as follows:

[0077] Model construction: (1) The experimental animals were acclimatized for one week; (2) The bacterial solution was resuscitated the day before the experiment and the bacterial solution was prepared to the required concentration on the day of the experiment; (3) The experimental animals were anesthetized with isoflurane, and hair removal cream was used to remove hair from their backs after shaving. The surgical site was disinfected with povidone-iodine; (4) A circular full-thickness wound with a diameter of 8 mm was created on the back of the experimental animal. The shape of the wound was determined according to the experimental requirements. After the wound was cut, it was measured with a ruler and the diameter was 0.8 ± 0.1 cm.

[0078] Inoculate the wound with 10 μL of Escherichia coli (concentration of 1.25 × 10⁻⁶). 6 The wound was sealed with a 3M membrane (CFU / mL), and the appearance of cloudy pus in the wound 24 hours after infection was considered a successful infection model.

[0079] Photothermal therapy: Mice were divided into four groups according to the experimental design: PBS, PBS+NIR, SMPs, and SMPs+NIR. On days 1, 3, 5, 7, and 10, 100 μL of PBS solution (pH 7.4) or 100 μL of SMPs solution (concentration 200 μg / mL) was added to the infected wound of mice. The light intensity for photothermal therapy was 1 W / cm². 2 The procedure lasted 10 minutes, during which 3M film and medical tape were used to bandage the wound.

[0080] Wound healing rate: During the observation period, the wound diameter was measured with calipers, and the wound healing rate was calculated using the following formula: Wound healing rate (%) = (S1 - Sn) / S1 × 100%, where S1 is the wound area on day 1 and Sn is the wound area on day n. The area was photographed before each treatment.

[0081] Mouse weight: The weight of the mice was measured and recorded every three days.

[0082] Figure 6 The following graphs show the wound healing efficiency of SMPs prepared in Example 1: A shows the wound healing rate of mice in different treatment groups; B shows the wound healing rate of mice in different treatment groups (1 is PBS, 2 is PBS+NIR, 3 is SMPs, 4 is SMPs+NIR); C shows the change in body weight of mice in different treatment groups over time (1 is PBS, 2 is PBS+NIR, 3 is SMPs, 4 is SMPs+NIR); wound area monitoring ( Figure 6 A) and quantitative analysis Figure 6 B) Results showed that the wound healing rate was significantly accelerated in the SMPs group. It was observed that on day 5 of treatment, the wounds in both the SMPs group and the SMPs+NIR group significantly contracted, with healing rates of 60.9% and 78.0%, respectively. Under the same experimental conditions, the PBS group and the PBS+NIR control group showed only weak healing effects of 24.8% and 25.2%, respectively. With prolonged treatment, the wounds in the treated mice began to scab and continued to shrink. By day 14, the wounds in the SMPs+NIR group were almost completely healed, with a healing rate of 99.2%, fully demonstrating the excellent wound-healing properties of SMPs. Changes in mouse body weight were monitored during the experiment. Figure 6 (C) The results showed that SMP administration did not cause any abnormal weight fluctuations during treatment, thus confirming its good biocompatibility.

[0083] To further verify the cytotoxicity of the SMPs prepared in Example 1, mouse epithelial fibroblasts were used. The mouse cells were incubated with SMPs for 4 h and 8 h, and the biocompatibility was verified by the MTT assay. Figure 7 The results of the cytotoxicity experiment of the SMPs prepared in Example 1 are shown in the figure. Even when the concentration of the SMPs solution is increased to 200 μg / mL, the cell survival rate still reaches more than 80%, which indicates that the synthesized SMPs have low cytotoxicity and good biocompatibility in in vitro experiments.

Claims

1. A method for preparing a biomimetic spiked microparticle photothermal antibacterial material, characterized in that... It is done in the following steps:

1. Dissolve lauric acid and stearic acid in methanol solution to obtain a fatty acid solution; The mass ratio of lauric acid to stearic acid is (3~4):1; the total concentration of lauric acid and stearic acid in the fatty acid solution is 2.5mg / mL~4mg / mL; 2. Dissolve lecithin and distearate phosphatidylethanolamine-methoxy polyethylene glycol 2000 in an aqueous ethanol solution to obtain a lecithin / DSPE-PEG 2000 solution; The mass ratio of lecithin to distearylphosphatidylethanolamine-methoxy polyethylene glycol 2000 is (3~4):1; the mass percentage of ethanol in the aqueous solution is 4%~6%; the total concentration of lecithin and distearylphosphatidylethanolamine-methoxy polyethylene glycol 2000 in the lecithin / DSPE-PEG 2000 solution is 3mg / mL~4mg / mL; 3. Dissolve silver nitrate in dimethyl sulfoxide solution to obtain silver nitrate solution; The concentration of silver nitrate in the silver nitrate solution is 0.5 mg / mL to 1 mg / mL; IV. Dissolve heme chloride in N,N-dimethylformamide solution to obtain heme chloride solution; The concentration of heme chloride in the heme chloride solution is 1 mg / mL to 2.5 mg / mL; 5. Under heating and stirring conditions, fatty acid solution, silver nitrate solution and heme chloride solution were added dropwise to lecithin / DSPE-PEG 2000 solution to react and obtain a mixed system; The volume ratio of the lecithin / DSPE-PEG 2000 solution to the fatty acid solution is (5~6):4; the volume ratio of the lecithin / DSPE-PEG 2000 solution to the silver nitrate solution is (15~20):1; the volume ratio of the lecithin / DSPE-PEG 2000 solution to the heme chloride solution is (5~6):

1.

6. Stir the mixture in an ice bath, then stir at room temperature to obtain a colloidal solution. Centrifuge the colloidal solution to remove the supernatant, then disperse it in sterile ultrapure water and centrifuge multiple times. Finally, disperse it in sterile ultrapure water to obtain a biomimetic spiked microparticle photothermal antibacterial material.

2. The method for preparing a biomimetic spiked microparticle photothermal antibacterial material according to claim 1, characterized in that... In step five, under the conditions of heating temperature of 55℃~65℃ and stirring speed of 4000rpm~6000rpm, fatty acid solution, silver nitrate solution and heme chloride solution are added dropwise to lecithin / DSPE-PEG 2000 solution and reacted for 2min~5min.

3. The method for preparing a biomimetic spiked microparticle photothermal antibacterial material according to claim 1, characterized in that... In step six, the mixture is stirred in an ice bath for 10 to 15 minutes at a temperature of 0℃ to 4℃ and a stirring speed of 4000 rpm to 6000 rpm. Then, it is stirred for 10 to 20 minutes at room temperature and a stirring speed of 4000 rpm to 6000 rpm to obtain a colloidal solution. The colloidal solution is then centrifuged for 5 to 10 minutes at a speed of 6000 rpm to 8000 rpm to remove the supernatant.

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