Gallate-based cationic photodynamic antibacterial agent with sterilizing, anti-infection and wound healing promoting effects and preparation method of gallate-based cationic photodynamic antibacterial agent

Abstract

The invention discloses a gallic acid-based cationic photodynamic antibacterial agent with sterilizing, anti-infection and wound healing promoting effects and a preparation method thereof, and belongs to the technical field of antibacterial agent synthetic chemistry. The cationic photodynamic antibacterial agent EY-QEGDM-MG is prepared by taking a natural compound gallic acid as a raw material and carrying out ring opening polymerization and quaternization reaction with photosensitizer eosin EY and ethylene glycol diglycidyl ether (EGDE), and the polymer shows an excellent antibacterial effect on escherichia coli and multi-drug-resistant MRSA germs under the action of illumination, and can be used for preparing a cationic photodynamic antibacterial agent EY-QEGDM-MG. The polymer has good blood compatibility and cell compatibility, in addition, the polymer can kill bacteria around infected wounds and can rapidly promote wound healing, and therefore the polymer can provide a new method for photodynamic antibacterial agents for treating the infected wounds in hospitals.

Description

Technical Field

[0001] This invention belongs to the field of antibacterial agent synthesis chemistry technology, specifically relating to gallic acid-based cationic photodynamic antibacterial agents with bactericidal, anti-infective, and wound-healing effects, as well as their preparation methods and applications. Background Technology

[0002] Bacterial infections have become one of the major threats to human health worldwide. Currently, the main treatment for bacterial infections in clinical practice is antibiotic therapy. However, in recent years, the overuse of antibiotics has led to the emergence of drug-resistant bacteria, increasing the difficulty of treating clinical infections. Faced with the increasing number of drug-resistant bacteria and the difficulties in medication caused by them, there is an urgent need to develop new antibacterial agents.

[0003] Various novel, non-traditional antibacterial agents, such as silver and gold nanoparticles, antimicrobial peptides, bacteriophages, quaternary ammonium salts, and cationic polymers, have been reported to exhibit excellent antibacterial effects. Among them, positively charged cationic antibacterial agents show highly efficient antibacterial properties against both drug-resistant and non-drug-resistant bacteria due to their disruptive effect on negatively charged bacterial membranes. However, when cationic antibacterial agents are used as antibacterial coatings, their antibacterial efficiency is significantly reduced because their insertion into biofilms is hindered.

[0004] Recently, photodynamic therapy (PDT) mediated by visible and near-infrared light has attracted widespread attention due to its advantages such as controllability, biosafety, and lack of induction of bacterial resistance. Photodynamic antibacterial therapy utilizes photosensitizers (such as eosin Y (EY) and zinc phthalocyanine) to absorb photon energy and generate large amounts of reactive oxygen species (ROS), leading to bacterial cell damage or death. ROS, primarily composed of singlet oxygen, can directly cause oxidative damage to many important cellular biomolecules such as lipids, proteins, DNA, and RNA, effectively killing bacteria without promoting the development of drug-resistant bacteria.

[0005] However, due to the different surface structures of Gram-negative and Gram-positive bacteria, photodynamic therapy (PDT) has different bactericidal effects on these two types of bacteria. Gram-negative bacteria are not sensitive to PDT and cannot be inactivated by light, while Gram-positive bacteria are highly sensitive to PDT because the singlet oxygen generated by the photosensitizer can easily penetrate their porous outer cell membrane. To address these issues, photodynamic therapy combined with cationic antibacterial agents can provide a better treatment approach for bacterial infections.

[0006] Gallic acid is a natural polyhydroxyphenolic acid compound derived from various natural plants. Studies have shown that it possesses significant antibacterial and wound-healing properties. Its mechanisms of action include inhibiting bacterial uptake of iron, polysaccharides, and amino acids, disrupting bacterial cell walls and membranes, suppressing inflammatory responses, and promoting cell proliferation. Gallic acid has great potential as a natural antibacterial agent; however, when used alone, it is difficult to penetrate cells to exert its antibacterial effect and can lead to drug resistance. Ethylene glycol diglycidyl ether (EGDE) is a compound with a diepoxy group. It can react with primary amine groups via a ring-opening reaction to form hydroxyl-rich polymers. The hydroxyl groups play a crucial role in enhancing hydrophilicity and antibacterial activity.

[0007] This thesis designs and synthesizes a novel cationic photodynamic antibacterial polymer, EY-QEGDM-MG, based on gallic acid, EGDE, and eosin Y, through reactions such as amino-epoxy ring-opening polymerization, esterification, substitution, and quaternization. This antibacterial agent contains multiple functional groups, including quaternary ammonium salt QA, photosensitizers, and abundant hydroxyl groups, exhibiting synergistic bactericidal properties. Furthermore, it can be easily coated onto various substrates through a Schiff base reaction with polydopamine. In vitro antibacterial experiments, hemolysis experiments, and cytotoxicity experiments demonstrate that this antibacterial agent possesses highly efficient antibacterial properties and good biocompatibility. Under light conditions, it exhibits strong antibacterial activity against *Escherichia coli*. E. coli The minimum inhibitory concentrations (MICs) for the antibacterial agent against methicillin-resistant Staphylococcus aureus (MRSA) were 256 and 64 µg / mL, respectively. Within the concentration range of 1.25–20 mg / mL, the antibacterial agent showed no significant cytotoxicity against L929 mouse fibroblasts, with cell viability exceeding 90%. The hemolysis rate on erythrocytes was <5%. In vivo anti-infection and wound healing assays in mice further demonstrated the excellent bactericidal and wound-healing properties of this antibacterial agent, making it a highly promising novel antibacterial agent. Furthermore, to date, there are no research reports on gallic acid-based cationic photodynamic antibacterial agents and their antibacterial and anti-infective applications. Summary of the Invention

[0008] To address the aforementioned problems in the existing technology, the first technical problem to be solved by this invention is to provide a gallic acid-based cationic photodynamic antibacterial agent with excellent bactericidal, anti-infective, and wound-healing promoting effects. The second technical problem to be solved by this invention is to provide a method for synthesizing a gallic acid-based cationic photodynamic antibacterial agent with bactericidal, anti-infective, and wound-healing promoting effects, for use in bactericides, anti-infectives, and wound-healing promoting drugs. The third technical problem to be solved by this invention is to provide an application of the gallic acid-based cationic photodynamic antibacterial agent in bactericides, anti-infectives, and wound-healing promoting drugs.

[0009] To solve the above problems, the technical solution adopted by the present invention is as follows: A gallic acid-based cationic photodynamic antibacterial agent with bactericidal, anti-infective, and wound-healing properties is named EY-QEGDM-MG, and its structural formula is as follows: .

[0010] The method for synthesizing the compound EY-QEGDM-MG includes the following steps: Step 1: Synthesis of EY-NH2 building blocks A certain amount of eosin, acidic alumina, and methanesulfonic acid were added to a round-bottom flask and stirred until homogeneous. Then, a certain amount of 3-amino-1-propanol was added dropwise while stirring. The reaction system was heated to 80 °C and stirred overnight for 12 h. After the reaction was completed, the mixture was cooled to room temperature and filtered through a sintered funnel. A certain amount of distilled water was added to the filtrate, and the precipitate was collected by centrifugation at 3500 rpm. The precipitate was washed with distilled water and centrifuged 2-3 times. Then, a certain amount of triethylamine aqueous solution was added to wash the precipitate. The precipitate was washed with distilled water and centrifuged 2-3 times. Finally, the precipitate was dried in a vacuum drying oven at 75 °C for 12-24 h to obtain eosin building blocks EY-NH2. Step 2: Synthesis of MG-Br from Gallic Acid Building Blocks A certain amount of potassium carbonate and methyl gallate were added to a Shrek flask. After purging with argon three times, a certain amount of DMF was added under argon-flushing and stirring. After stirring evenly, a certain amount of 1,6-dibromohexane was added. The reaction mixture was reacted at room temperature for 18 h under argon protection. The reaction mixture was filtered through a diatomaceous earth core funnel. A certain amount of water was added to the filtrate and mixed. The aqueous phase was then extracted with dichloromethane 3-4 times. The organic phases were combined and washed with a certain amount of water 4-5 times. The organic phases were then dried with anhydrous magnesium sulfate for 3 h. Dichloromethane was removed by rotary evaporation. Then, silica gel column chromatography was performed using petroleum ether:ethyl acetate = 10:1-5:1-3:1 as the eluent. The eluent was collected and the solvent was recovered. The product was dried at room temperature in a vacuum drying oven for 24 h to obtain gallic acid building blocks MG-Br. Step 3: Synthesis of EY-QEGDM-MG polymer A certain amount of eosin building blocks were added to a Schlenk tube, and the atmosphere was evacuated. Under argon gas, a certain amount of DMSO, dimethylaminoethylamine, and EGDE were added dropwise. The reaction mixture was heated to 80 °C and stirred for 12 h. After the reaction, the product was transferred to an MD44 3500D dialysis bag and dialyzed twice with 20-40% ethanol aqueous solution for 3-5 h each time, followed by dialysis three times with distilled water for 3-5 h each time. The dialysis product was then transferred to a centrifuge tube and centrifuged at 3500 rpm. The precipitate was washed three times with distilled water and dried in a vacuum drying oven at 75 °C to obtain the product EY-EGDM. A certain amount of EY-EGDM and gallic acid building blocks were added to a Schlenk tube, and after evacuation, a certain amount of DMSO was added under argon gas. The reaction mixture was heated to 80 °C and stirred for 12 h. The reaction system was then transferred to an MD44 3500D dialysis bag. Dialyze the product twice with 20-40% ethanol aqueous solution in a 3500D dialysis bag for 3-5 hours each time, and then dialyze three times with distilled water for 3-5 hours each time. Transfer the dialysis product to a centrifuge tube, centrifuge at 3500 rpm, wash the precipitate three times with distilled water, and dry it in a vacuum drying oven at 75℃ for 12 hours to obtain the target polymer EY-QEGDM-MG.

[0011] The reaction process of EY-QEGDM-MG is as follows:

[0012] The application of the gallic acid-based cationic photodynamic antibacterial agent with bactericidal, anti-infective, and wound-healing properties in bactericidal, anti-infective, and wound-healing drugs.

[0013] The pathogens mentioned include Escherichia coli. E. coli And methicillin-resistant Staphylococcus aureus (MRSA); the infected wound described is an infected wound caused by Staphylococcus aureus (S. aureus).

[0014] This invention first prepares gallic acid building blocks. Using methyl gallate as a raw material, a substitution reaction with 1,6-dibromohexane yields hexane-modified methyl gallate (MG-Br). Then, eosin building blocks are prepared. Using eosin EY as a raw material, an esterification reaction with 3-amino-1-propanol yields amino-modified EY (EY-NH2). Next, the eosin building block EY-NH2 undergoes a ring-opening polymerization reaction with dimethylaminoethylamine and ethylene glycol diglycidyl ether (EGDE) to obtain polymer EY-EGDM. Finally, polymer EY-EGDM undergoes a quaternization reaction with gallic acid building block MG-Br to obtain the final polymer EY-QEGDM-MG. This invention... 1 The structure, EY content, molecular weight, and degree of quaternization of the target compound were confirmed by techniques such as H-NMR, UV, GPC, and XPS.

[0015] This invention determined the antibacterial activity of the polymer EY-QEGDM-MG against *Escherichia coli* and methicillin-resistant Staphylococcus aureus (MRSA) under different concentrations. Within a concentration range of 1-2048 μg / mL, the polymer exhibited minimum inhibitory concentrations (MICs) of 1024 µg / mL against *E. coli* and 256 µg / mL under no-light conditions, while under light conditions, the MICs were 256 µg / mL and 64 µg / mL, respectively, indicating that it possesses excellent photodynamic antibacterial effects.

[0016] This invention determined the cytotoxicity and blood compatibility of the polymer EY-QEGDM-MG, and investigated its effect on the survival rate of L929 mouse fibroblasts at concentrations ranging from 1.25 to 20 mg / mL. Results showed that under light conditions, the L929 cell survival rate was 93% at a polymer concentration of 20 mg / mL. Hemolysis experiments indicated a hemolysis rate of <5%, and no significant morphological changes in erythrocytes were observed. These findings demonstrate that the polymer exhibits low cytotoxicity and good biocompatibility.

[0017] This invention determined the in vivo therapeutic effect of the polymer EY-QEGDM-MG on infected skin wounds in mice. A mouse model of Staphylococcus aureus infection was constructed, and the bactericidal and wound-healing effects of the polymer under both light-free and light-exposed conditions were investigated. The results showed that after 14 days of treatment, the EY-QEGDM-MG light-exposed group exhibited the best wound healing, with a wound closure rate of 97%. The wound closure rate in the EY-QEGDM-MG light-free group was approximately 92%, and there was virtually no bacterial growth around the wound surface. This indicates that the polymer possesses excellent bactericidal and wound-healing properties.

[0018] Beneficial effects: Compared with the prior art, the advantages of this invention are: (1) This invention uses gallic acid, a natural compound, as raw material. Through ring-opening polymerization and quaternization reaction with photosensitizer EY and ethylene glycol diglycidyl ether (EGDE), a cationic photodynamic antibacterial agent EY-QEGDM-MG was successfully prepared. This polymer has a variety of antibacterial functional groups, including abundant benzene rings, hydroxyl groups, quaternary ammonium salts and photosensitizers. The synergistic effect of multiple antibacterial functional groups has a good application prospect in the field of photodynamic antibacterial agents. (2) The antibacterial polymer EY-QEGDM-MG prepared in this invention can generate a large amount of singlet oxygen under light irradiation, and shows excellent antibacterial effect against Escherichia coli and multidrug-resistant MRSA bacteria. The polymer also has good blood compatibility and cell compatibility. In addition, EY-QEGDM-MG can significantly prevent the formation of abscesses around infected wounds, kill bacteria around wounds, and promote wound healing rapidly. Therefore, this polymer provides a new method for photodynamic antibacterial agents for the treatment of infected wounds in hospitals. Attached Figure Description

[0019] Figure 1 : Polymer EY-QEGDM-MG 1 H NMR spectrum Figure 2 Results of hemolytic and cytotoxic experiments on polymer EY-QEGDM-MG Figure 3 Results of anti-infection and wound healing experiments on polymer EY-QEGDM-MG Detailed Implementation

[0020] The present invention will be further described below with reference to specific embodiments. Example 1

[0021] 1. Synthesis of polymer EY-QEGDM-MG Step 1: Synthesis of EY-NH2 building blocks 6.5 g of eosin, 3.0 g of acidic alumina, and 20 mL of methanesulfonic acid were added to a round-bottom flask and stirred until homogeneous. Then, 760 μL of 3-amino-1-propanol was added dropwise while stirring. The reaction system was heated to 80 °C and stirred overnight for 12 h. After the reaction was completed, the mixture was cooled to room temperature and filtered through a sintered funnel. A certain amount of distilled water was added to the filtrate, and the precipitate was collected by centrifugation at 3500 rpm. The precipitate was washed twice with distilled water and centrifuged. Then, a certain amount of triethylamine aqueous solution was added to wash the precipitate. The precipitate was washed twice with distilled water and centrifuged. Finally, the precipitate was dried in a vacuum drying oven at 75 °C for 12 h to obtain the dark red solid product EY-NH2 with a yield of 80%.

[0022] Step 2: Synthesis of MG-Br from Gallic Acid Building Blocks 4.2 g of potassium carbonate and 5.5 g of methyl gallate were added to a Shrek flask. After purging with argon three times, 50 mL of DMF was added under argon-pumped stirring. After stirring evenly, 5.0 mL of 1,6-dibromohexane was added. The reaction mixture was reacted at room temperature for 18 h under argon protection. The reaction mixture was filtered through a sintered funnel lined with diatomaceous earth. A certain amount of water was added to the filtrate, and the aqueous phase was extracted three times with dichloromethane. The organic phases were combined and washed five times with a certain amount of water. The organic phases were then dried with anhydrous magnesium sulfate for 3 h. Dichloromethane was removed by rotary evaporation. The product was then subjected to silica gel column chromatography using petroleum ether:ethyl acetate = 10:1-5:1-3:1 as the eluent. The eluent was collected and the solvent was recovered. The product was dried in a vacuum drying oven at room temperature for 24 h to obtain a white, slightly yellow solid, MG-Br, with a yield of 36%.

[0023] Step 3: Synthesis of EY-QEGDM-MG polymer Add 5 mmol of eosin building blocks to a Schlenk tube, evacuate the gas, and then add 20 mL of DMSO, 5 mmol of dimethylaminoethylamine, and 10 mmol of EGDE dropwise under argon gas. Heat the reaction mixture to 80 °C and stir for 12 h. After the reaction, transfer the product to an MD44 3500D dialysis bag and dialyze twice with 20% ethanol aqueous solution for 5 h each time, followed by three dialysis cycles with distilled water for 5 h each time. Transfer the dialysis product to a centrifuge tube, centrifuge at 3500 rpm, wash the precipitate three times with distilled water, and dry in a vacuum drying oven at 75 °C to obtain the product EY-EGDM. Add the product EY-EGDM and 5 mmol of gallic acid building blocks to a Schlenk tube, evacuate the gas, and then add 20 mL of DMSO under argon gas. Heat the reaction mixture to 80 °C and stir for 12 h. Transfer the reaction system to an MD44 3500D dialysis bag. The product was dialyzed twice with 20% ethanol aqueous solution in a 3500D dialysis bag for 5 hours each time, and then dialyzed three times with distilled water for 5 hours each time. The dialysis product was transferred to a centrifuge tube and centrifuged at 3500 rpm. The precipitate was washed three times with distilled water and dried in a vacuum drying oven at 75°C for 12 hours to obtain the red target polymer EY-QEGDM-MG.

[0024] 2. Characterization of the polymer EY-QEGDM-MG use 1 The structure, EY content, molecular weight, and degree of quaternization of the polymer were confirmed using techniques such as ¹H-NMR, UV, GPC, and XPS. The results are as follows: (1) 1 H-NMR test results The polymer EY-QEGDM-MG is a red powdery solid. For example... Figure 1In EY-EGDM, methylene protons (i.e., CH2-OH and N-CH2) exhibit characteristic proton signals between δ 3.0 and 4.5 ppm; these signals are much broader and more complex than those of EY-NH2. Peaks a, b, and c (δ = 2.50, 2.78, and 2.91 ppm, respectively) show the characteristic chemical shifts of DMEN protons. Peaks d, e, f, g, and h (δ = 6.95, 7.53, 7.82, 7.89, and 8.23 ​​ppm, respectively) show the characteristic chemical shifts of EY protons. These results indicate the successful synthesis of EY-EGDM. Quaternization with MG-Br, with typical peaks between δ 1.2 and 1.9 ppm, confirms the successful preparation of EY-QEGDM-MG.

[0025] (2) UV, GPC and XPS test results Both EY-QEGDM-MG and EY exhibited typical UV absorption spectra at 545 nm, and the proportion of EY in the polymer was calculated to be approximately 49.68% based on the UV absorbance values. The polydispersity index (PDI) and number-average molecular weight (Mn) of EY-QEGDM-MG were determined by gel permeation chromatography (GPC), with Mn and PDI of 1.37 × 10³ g / mol and 1.46, respectively. XPS analysis showed that the degree of quaternization of the polymer was 62.16%. Example 2

[0026] Evaluation of the in vitro antibacterial activity of polymer EY-QEGDM-MG: This embodiment determined the antibacterial activity of the polymer EY-QEGDM-MG and compared it with the antibacterial activity of the polymer monomer components (EY, EGDE, and MG). *Escherichia coli* and methicillin-resistant Staphylococcus aureus (MRSA) were selected as test strains. The inhibitory activity of the polymer against *E. coli* and MRSA was determined under both light and dark conditions in the concentration range of 0–2048 μg / mL, and the MIC (μg / mL) was calculated.

[0027] The specific experimental steps are as follows: The bacterial strain (density 1×10⁻⁶) was placed in the container... 6 100 μL of LB medium (CFU / mL) was added to each well of a 96-well plate, followed by mixing 100 μL of LB medium containing the polymer with the bacterial suspension to obtain the final concentration range [0-2048 μg / mL]. Subsequently, the light group was treated with 60 mW cm⁻¹. −2Irradiate the plates with light at 520 ± 10 nm for 30 minutes. All plates are then incubated at 37°C with gentle shaking for 24 hours. Absorbance (A, OD) at 600 nm is measured using a microplate reader. 600 The antimicrobial activity of the polymer monomer components (EY, EGDE, and MG) was also evaluated using the same method for comparison. MIC is the lowest concentration of antimicrobial polymer at which no bacterial growth was observed.

[0028] The results are shown in Table 1. The antibacterial activity of EY-QEGDM-MG showed a concentration-dependent trend. Compared with the monomer components of the polymer (EY, EGDE, and MG), EY-QEGDM-MG exhibited stronger inhibitory effects against E. coli and MRSA. Table 2 summarizes the minimum inhibitory concentrations (MICs) of the polymer and monomer components against E. coli and MRSA. The MIC value of EY-QEGDM-MG was significantly lower than that of the monomers. Furthermore, in the light-treated group, the MIC value of the polymer was significantly lower than that in the non-light-treated group. Under light treatment, the MIC values ​​of the polymer against both E. coli and MRSA decreased by 3-fold. The MICs against E. coli and MRSA under no-light conditions were 1024 and 256 µg / mL, respectively, while those under light conditions were 256 and 64 µg / mL, respectively. These results indicate that the presence of quaternary ammonium salts, benzene rings, and abundant hydroxyl groups in the polymer enhances its antibacterial activity.

[0029] Table 1. Inhibitory activities of polymer monomers and polymers (under light and dark conditions) on E. coli and MRSA. Table 2. MIC values ​​of polymer monomers and polymers (under light and dark conditions) sample <![CDATA[MIC of E. coli (μg mL −1 )]]> <![CDATA[MIC of MRSA (μg mL −1 )]]> EY >2048 >2048 EGDE >2048 >2048 MG 2048 2048 EY-QEGDM-MG (-L) 1024 256 EY-QEGDM-MG (+L) 256 64 Example 3

[0030] Evaluation of hemolytic and cytotoxic properties of polymer EY-QEGDM-MG: This embodiment used the MTT assay to determine the effect of the polymer EY-QEGDM-MG at concentrations ranging from 1.25 to 20 mg / mL on the survival rate of L929 mouse fibroblasts. The specific experimental steps are as follows: cells were cultured at a concentration of 2 × 10⁶ cells / well. 4Cells were seeded at a density of 100 μL in 96-well plates containing 10% fetal bovine serum (FBS). The plates were then incubated in a humidified incubator with 5% CO2 for 24 hours, followed by the addition of 100 μL of EY-QEGDM-MG (1.25–20 mg / mL) to the wells, and incubated for another 48 hours. For the light-treated group, 30 minutes of light exposure (60 mW cm⁻¹) was provided. −2 The absorbance was then measured at 520 ± 10 nm. MTT (5 mg / mL; 20 μL) was added, and the mixture was incubated for another 2 hours. Finally, DMSO solution (150 μL) was added to each well. The absorbance was then measured at 570 nm using a microplate reader.

[0031] This embodiment determined the blood compatibility of the polymer EY-QEGDM-MG by co-incubating the polymer with PBS containing 2% red blood cells. The specific experimental steps are as follows: Red blood cells (RBCs) were isolated from fresh rat blood and then suspended in PBS at a 2% volume concentration. This suspension was then incubated with EY-QEGDM-MG (100 ug / mL). For positive and negative controls, the red blood cells were treated with deionized water and PBS, respectively. The obtained suspension was cultured at 37°C for 3 hours and then centrifuged at 3500 rpm for 5 minutes. The absorbance of the supernatant was measured at 545 nm using a microplate reader, and the hemolysis rate (%) was calculated. Red blood cell morphology was further investigated by suspending the red blood cell precipitate in PBS, then placing the suspension on a clean glass slide and covering it with a coverslip. Images of the red blood cells were acquired using an Olympus IX71 microscope equipped with a digital camera.

[0032] Results of hemolytic and cytotoxicity experiments are shown in Figure 2 Under light-free conditions, the polymer showed no significant cytotoxicity in the concentration range of 1.25–20 mg / mL. Although the cytotoxicity of the polymer increased slightly after light exposure, the relative survival rate of all L929 cells exceeded 90%. After light treatment at a concentration of 20 mg / mL, the survival rate of L929 cells was 93%, indicating that the polymer has low cytotoxicity and good cell compatibility. Hemolytic assays showed that the hemolysis rate of the polymer was less than 5%, and the supernatant after incubation of red blood cell suspension with the polymer showed a pale pink color, indicating good blood compatibility. Furthermore, optical images showed no significant changes in the morphology of red blood cells after polymer treatment. Example 4

[0033] Evaluation of the anti-infective and wound-healing properties of polymer EY-QEGDM-MG: This embodiment evaluated the in vivo therapeutic effect of the polymer EY-QEGDM-MG on mouse skin wound infection by constructing a mouse model of Staphylococcus aureus infection. The bactericidal and wound-healing effects of the polymer on infected wounds were investigated under both light-free and light-exposed conditions. The specific experimental steps are as follows: 20 Kunming mice were randomly divided into three groups: a control group, a polymer-containing group (with or without irradiation). To establish the wound infection model, a 7 mm diameter excision was made on the back of each mouse to form a circular, full-thickness skin wound. Each wound was covered with 10... 8 A 10 μL suspension of Staphylococcus aureus at a concentration of CFU / mL was administered. The control group was covered only with sterile gauze. After 24 hours of incubation on skin wounds containing Staphylococcus aureus, a polymeric cream was applied evenly to the wounds on days 1, 3, and 5. The light-treated group underwent light treatment as described above to assess wound healing. The wounds were photographed, and wound closure rates were measured on days 1, 3, 5, 7, 10, and 14 postoperatively. On days 7 and 14 postoperatively, the entire wound and adjacent skin were completely removed. The infected tissue was mixed with 1 mL of PBS and diluted 1000-fold. The bacterial solution (100 μL) was then sprayed onto LB agar plates and incubated at 37°C. After 18 hours of incubation, bacterial colonies grown in the culture dishes were photographed and counted.

[0034] The experimental results are shown in Figure 3On day 1 after Staphylococcus aureus inoculation, abscesses of some degree appeared in all groups. From day 3 to day 14, wounds treated with EY-QEGDM-MG(+L) and EY-QEGDM-MG(−L) regenerated faster than those in the infected group. Specifically, the wounds treated with EY-QEGDM-MG(+L) closed the fastest. By day 5, the abscesses around the wounds in the EY-QEGDM-MG(+L) group had disappeared. Furthermore, by day 7, the wound closure rate in the EY-QEGDM-MG(+L) group was approximately 81%, while the wound closure rates in the EY-QEGDM-MG(−L) group and the control group were approximately 66% and 44%, respectively. After 14 days of treatment, the EY-QEGDM-MG(+L) group showed the best quality of recovery with a wound closure rate of 97%, the EY-QEGDM-MG(−L) group had a wound closure rate of approximately 92%, while some wound areas in the control group remained unhealed. To further quantify the anti-infective therapeutic effect of EY-QEGDM-MG, bacterial cells around the wound were cultured and counted on days 7 and 14. Compared with other groups, the EY-QEGDM-MG(+L) group had the fewest bacterial colonies, and almost no bacterial growth was detected on days 7 and 14. Furthermore, compared with the control group, the EY-QEGDM-MG(−L) group showed a significant reduction in bacterial count on day 7. The bacterial count in the control group was approximately 35 times that of the EY-QEGDM-MG(−L) group. On day 14, almost no bacterial growth was detected in the EY-QEGDM-MG(−L) group, while the bacterial count in the control group remained high. These results are consistent with the results of the wound healing assay. The results indicate that the EY-QEGDM-MG(+L) group has superior bactericidal activity and wound healing effect.

Claims

1. A gallic acid-based cationic photodynamic antibacterial agent with bactericidal, anti-infective, and wound-healing properties, and its preparation method, characterized in that, Named EY-QEGDM-MG, its structure is as follows: 。 2. The preparation method of the gallic acid-based cationic photodynamic antibacterial agent with bactericidal, anti-infective, and wound-healing effects as described in claim 1, characterized in that, Includes the following steps: Step 1: Synthesis of EY-NH2 building blocks A certain amount of eosin, acidic alumina, and methanesulfonic acid were added to a round-bottom flask and stirred until homogeneous. Then, a certain amount of 3-amino-1-propanol was added dropwise while stirring. The reaction system was heated to 80 °C and stirred overnight for 12 h. After the reaction was completed, the mixture was cooled to room temperature and filtered through a sintered funnel. A certain amount of distilled water was added to the filtrate, and the precipitate was collected by centrifugation at 3500 rpm. The precipitate was washed with distilled water and centrifuged 2-3 times. Then, a certain amount of triethylamine aqueous solution was added to wash the precipitate. The precipitate was washed with distilled water and centrifuged 2-3 times. Finally, the precipitate was dried in a vacuum drying oven at 75 °C for 12-24 h to obtain eosin building blocks EY-NH2. Step 2: Synthesis of MG-Br from Gallic Acid Building Blocks A certain amount of potassium carbonate and methyl gallate were added to a Shrek flask. After purging with argon three times, a certain amount of DMF was added under argon-flushing and stirring. After stirring evenly, a certain amount of 1,6-dibromohexane was added. The reaction mixture was reacted at room temperature for 18 h under argon protection. The reaction mixture was filtered through a diatomaceous earth core funnel. A certain amount of water was added to the filtrate and mixed. The aqueous phase was then extracted with dichloromethane 3-4 times. The organic phases were combined and washed with a certain amount of water 4-5 times. The organic phases were then dried with anhydrous magnesium sulfate for 3 h. Dichloromethane was removed by rotary evaporation. Then, silica gel column chromatography was performed using petroleum ether:ethyl acetate = 10:1-5:1-3:1 as the eluent. The eluent was collected and the solvent was recovered. The product was dried at room temperature in a vacuum drying oven for 24 h to obtain gallic acid building blocks MG-Br. Step 3: Synthesis of EY-QEGDM-MG polymer A certain amount of eosin building blocks were added to a Schlenk tube, and the atmosphere was evacuated. Under argon gas, a certain amount of DMSO, dimethylaminoethylamine, and EGDE were added dropwise. The reaction mixture was heated to 80 °C and stirred for 12 h. After the reaction, the product was transferred to an MD443500D dialysis bag and dialyzed twice with 20-40% ethanol aqueous solution for 3-5 h each time, followed by dialysis three times with distilled water for 3-5 h each time. The dialysis product was then transferred to a centrifuge tube and centrifuged at 3500 rpm. The precipitate was washed three times with distilled water and dried in a vacuum drying oven at 75 °C to obtain the product EY-EGDM. A certain amount of EY-EGDM and gallic acid building blocks were added to a Schlenk tube, and after evacuation, a certain amount of DMSO was added under argon gas. The reaction mixture was heated to 80 °C and stirred for 12 h. The reaction system was then transferred to an MD443500D dialysis bag. Dialyze the product twice with 20-40% ethanol aqueous solution in a 3500D dialysis bag for 3-5 hours each time, and then dialyze three times with distilled water for 3-5 hours each time. Transfer the dialysis product to a centrifuge tube, centrifuge at 3500 rpm, wash the precipitate three times with distilled water, and dry it in a vacuum drying oven at 75℃ for 12 hours to obtain the target polymer EY-QEGDM-MG. The specific reaction formula is as follows: 。

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