A photosensitizer with hydrogen sulfide releasing performance and its preparation method and application

Abstract

The present invention discloses a photosensitizer with hydrogen sulfide release performance and its preparation method and application, relating to the technical field of photosensitizers. The photosensitizer of the present invention uses dithiophene acrylonitrile or monothiophene acrylonitrile as a skeleton, and by changing the electron-donating group at the R position on the molecular structure skeleton, pyridinium is a strong electron-withdrawing group to construct a typical D-π-A structure, thereby reducing the energy gap between the singlet excited state and the triplet excited state, improving intersystem crossing to improve ROS generation efficiency, and its synthesis method is simple, the conditions are mild, the yield is high, and excellent fluorescence imaging and photodynamic therapy effects are shown. A donor group with hydrogen sulfide production ability is connected to the molecular tail, overcoming the pro-inflammatory side effects of photodynamic therapy and synergistically promoting blood circulation reconstruction, thereby subsiding inflammation in chronic wounds, accelerating wound repair, achieving photodynamic antibacterial therapy and H2S anti-inflammatory and promoting the synergistic effect of tissue repair, and preventing the development of chronic wounds.

Description

Technical Field

[0001] The present invention relates to the technical field of photosensitizers, and in particular to a photosensitizer with hydrogen sulfide releasing performance, a preparation method thereof and an application thereof. Background Art

[0002] Chronic wounds include diabetic ulcers, arterial insufficiency ulcers, venous ulcers, and pressure ulcers. These wounds take longer than six weeks to heal, significantly impacting patients' quality of life and leading to a high rate of lower limb amputation. Key factors contributing to chronic wounds are bacterial biofilm infection and excessive inflammatory responses. Chronic wounds are often infected with bacterial biofilms, which cause wounds to experience a prolonged inflammatory phase and inhibit skin tissue regeneration. Therefore, biofilm removal and suppression of inflammatory responses are crucial for the treatment of chronic wounds.

[0003] Currently, chronic wounds are often treated clinically with repeated debridement or long-term use of antibiotics. However, repeated debridement may spread bacteria to deeper tissues and excessively remove healthy tissue, which is invasive and causes pain to patients. Topical antibiotics have limited effectiveness against biofilm infections in chronic wounds because bacteria in biofilms are highly resistant to antibiotics, and the required antibiotic concentration is 10 to 1000 times higher than that against planktonic bacteria. Therefore, non-antibiotic antimicrobial strategies are urgently needed to combat biofilm resistance.

[0004] Photodynamic therapy (PDT) has attracted widespread attention due to its controllability, non-invasiveness, and lack of drug resistance. PDT uses photosensitizers (PS) to produce toxic reactive oxygen species (ROS) under light irradiation, which causes oxidative damage to bacterial lipids, nucleic acids, and proteins, leading to irreversible bacterial death. However, ROS in PDT is a double-edged sword. Although the transient production of large amounts of ROS has a powerful antibacterial effect and reduces the risk of drug-resistant bacteria, excessive ROS, as an exogenous stimulus, will induce oxidative stress in the surrounding tissues, triggering the release of a series of inflammatory cytokines, significantly aggravating the inflammation level of chronic wounds, and ultimately leading to cell death and tissue damage. In addition, ROS storms inevitably lead to endothelial cell dysfunction and inhibit angiogenesis. These factors have seriously hindered the clinical application and development of PDT in the treatment of chronic wounds, and there is an urgent need to improve the side effects caused by PDT.

[0005] Hydrogen sulfide (H2S), an essential endogenous gaseous transmitter, has important physiological effects, including anti-inflammatory, angiogenesis-promoting, and antioxidant activities. Previous studies have shown that H2S can stimulate endogenous antioxidant defense mechanisms, protecting cells from infection-related oxidative stress, reducing inflammation, and promoting cell proliferation, migration, and vascular formation, accelerating wound healing. As a reducing substance, H2S can act as a ROS scavenger. After PDT treatment, H2S can directly or indirectly scavenge reactive oxygen species by reducing or directly binding to the heme center of metalloproteins, thereby reducing oxidative damage to cells and promoting wound healing. The combination of photodynamic therapy and H2S therapy for the treatment of chronic wounds remains an unresolved issue. Summary of the Invention

[0006] To address the above-mentioned problems, the present invention provides a photosensitizer with hydrogen sulfide-releasing properties, as well as its preparation method and application. The photosensitizer can achieve a synergistic effect of photodynamic antibacterial therapy and H2S anti-inflammatory and tissue repair promotion, promptly remove the excess ROS generated by PDT, overcome the pro-inflammatory side effects of PDT, and synergistically promote blood circulation reconstruction, thereby alleviating inflammation in chronic wounds and accelerating the healing of chronic wounds.

[0007] The technical solution of the present invention to solve the above technical problems is as follows: providing a photosensitizer with hydrogen sulfide releasing performance, having a structure as shown in Formula I or Formula II and its corresponding salt:

[0008]

[0009]

[0010] Among them, R1 is R is C1-C 12 The alkyl group, R2 is n=1-10 or

[0011] Further, Where R is methyl or ethyl; Where n=1.

[0012] Furthermore, the specific structure of the above-mentioned photosensitizer having hydrogen sulfide releasing performance is as follows:

[0013]

[0014] The preparation method of the above-mentioned photosensitizer having hydrogen sulfide releasing performance includes the following synthesis route:

[0015] Synthesis route 1: (1) Dissolve 2-thiopheneacetonitrile and N-bromosuccinimide in a solvent and react at room temperature for 22-26 hours to obtain compound B;

[0016] (2) Compound B, compound C, a palladium catalyst, and an inorganic base are dissolved in a solvent, and reacted at 60-70° C. under an inert atmosphere for 10-14 hours to obtain compound D;

[0017] (3) Compound D and 5-bromo-2-thiophenecarboxaldehyde are dissolved in a solvent, an inorganic base is added, and the mixture is reacted at room temperature for 10 to 14 hours to obtain compound E;

[0018] (4) Compound E, 4-pyridine boronic acid, a palladium catalyst, and an inorganic base are added to a solvent, and the mixture is reacted at 60-70° C. under an inert atmosphere for 10-14 hours to obtain compound F;

[0019] (5) Dissolving 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione and compound G in a solvent and reacting at room temperature for 8 to 12 hours to obtain compound H;

[0020] (6) Compound F and Compound H are dissolved in a solvent and reacted at 50-70°C for 22-26 hours to obtain a compound of Formula I. The synthetic route is as follows:

[0021]

[0022] Synthesis route 2: (1) Dissolve 2-thiopheneacetonitrile and N-bromosuccinimide in a solvent and react at room temperature for 22 to 26 hours to obtain compound B;

[0023] (2) Compound B, 4-pyridine boronic acid, a palladium catalyst, and an inorganic base are added to a solvent, and the mixture is reacted at 60-70° C. under an inert atmosphere for 10-14 hours to obtain Compound J;

[0024] (3) Compound J and Compound K are dissolved in a solvent, an inorganic base is added, and the mixture is reacted at room temperature for 22 to 26 hours to obtain Compound L;

[0025] (4) Dissolving 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione and compound G in a solvent and reacting at room temperature for 8 to 12 hours to obtain compound H;

[0026] (5) Compound L and Compound H are dissolved in a solvent and reacted at 50-70°C for 22-26 hours to obtain a compound represented by Formula II. The synthetic route is as follows:

[0027]

[0028] Furthermore, the solvents in step (1) of synthesis route 1 and synthesis route 2 are acetone, N,N-dimethylformamide, methanol, ethanol, tetrahydrofuran, acetonitrile, cyclohexane or toluene; the solvents in step (2) and step (4) of synthesis route 1 and step (2) of synthesis route 2 are all aqueous solutions of tetrahydrofuran or aqueous solutions of dioxane; the volume ratio of tetrahydrofuran to water in the aqueous solution of tetrahydrofuran is 1 to 15:1; the volume ratio of dioxane to water in the aqueous solution of dioxane is 1 to 15:1; the solvents in step (3) of synthesis route 1 and synthesis route 2 are all methanol, ethanol, n-propanol, n-butanol, isopropanol or tert-butanol; the solvents in step (5) of synthesis route 1 and step (4) of synthesis route 2 are all N,N-dimethylformamide, acetonitrile or tetrahydrofuran; the solvents in step (6) of synthesis route 1 and step (5) of synthesis route 2 are all N,N-dimethylformamide, acetonitrile, tetrahydrofuran or acetone.

[0029] Furthermore, the reaction time in step (1) of synthetic route 1 is 24 h; the reaction temperature in steps (2) and (4) is 68 ° C. and the reaction time is 12 h; the reaction time in step (3) is 12 h; and the reaction temperature in step (6) is 60 ° C. and the reaction time is 24 h.

[0030] Furthermore, the reaction time in steps (1) and (3) of synthetic route 2 is 24 h; the reaction temperature in step (2) is 68° C. and the reaction time is 12 h; and the reaction temperature in step (5) is 60° C. and the reaction time is 24 h.

[0031] Furthermore, the molar ratio of 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione to compound G in both synthetic routes 1 and 2 is 1:1-10.

[0032] Furthermore, the palladium catalysts in synthesis route 1 and synthesis route 2 are all di(tri-tert-butylphosphine)palladium, tetrakis(triphenylphosphine)palladium, palladium acetate, palladium pivalate, di(triphenylphosphine)palladium chloride, [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium or tris(dibenzylideneacetone)dipalladium; the inorganic bases in synthesis route 1 and synthesis route 2 are all barium hydroxide, sodium hydroxide, potassium hydroxide, potassium phosphate, sodium carbonate, sodium bicarbonate or potassium carbonate.

[0033] Furthermore, the molar ratio of compound F to compound H in synthesis route 1 is 1:1-10.

[0034] Furthermore, the molar ratio of compound L to compound H in synthetic route 2 is 1:1-10.

[0035] The application of the above photosensitizer with hydrogen sulfide releasing performance in the preparation of photodynamic antibacterial drugs.

[0036] The present invention has the following beneficial effects:

[0037] (1) The photosensitizer of the present invention uses dithiophene acrylonitrile or monothiophene acrylonitrile as the skeleton. By changing the electron-donating group at the R position on the molecular structure skeleton, pyridinium is used as a strong electron-withdrawing group to construct a typical D-π-A structure, thereby reducing the energy gap between the singlet excited state and the triplet excited state, improving intersystem crossing and thereby increasing the ROS generation efficiency.

[0038] (2) The present invention designed a donor group with hydrogen sulfide-releasing ability at the molecular tail and synthesized a series of photosensitizers with hydrogen sulfide-releasing properties, which not only showed significant effects in killing planktonic bacteria and clearing biofilms in vitro, but also in the in vivo rat diabetic wound model, while quickly clearing bacterial biofilms, inhibiting inflammatory responses, promoting cell proliferation and migration, and the formation of vascular structures, thereby accelerating diabetic wound healing.

[0039] (3) The photosensitizer with hydrogen sulfide release performance provided by the present invention can achieve the synergistic effect of photodynamic antibacterial therapy and H2S anti-inflammatory and tissue repair promotion, thereby accelerating the healing of chronic wounds; due to the electronegativity of bacterial plasma membranes and extracellular polymers (EPS), this type of photosensitizer enhances the permeability of biofilms through electrostatic interactions, and produces a large amount of ROS under white light excitation, achieving the effect of photodynamic biofilm removal; in addition, this type of photosensitizer reacts with intracellular enzymes to produce H2S, which promptly removes the excess ROS generated by PDT after PDT treatment, overcomes the pro-inflammatory side effects of PDT, and synergistically promotes blood circulation reconstruction, thereby reducing inflammation in chronic wounds and accelerating wound repair; this is of great significance for improving the treatment effect of PDT and preventing the development of chronic wounds.

[0040] (4) The photosensitizer with hydrogen sulfide-releasing properties of the present invention has potential applications in fluorescence imaging and photodynamic therapy. It can not only achieve detection and treatment simultaneously, but also visually monitor the entire treatment process, thereby providing more accurate and practical information and optimizing and improving the treatment effect, thereby achieving visual detection and precise treatment of the two diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Graphs showing the photophysical properties and reactive oxygen production of the photosensitizer of Example 6 in a solvent; wherein A is a graph showing the photophysical properties of the photosensitizer of Example 6 in a solvent, B is a graph showing the fluorescence intensity change of total reactive oxygen species at 525 nm, C is a graph showing the fluorescence intensity change of singlet oxygen at 378 nm, and D is a graph showing the fluorescence intensity change of hydroxyl radicals at 515 nm;

[0042] Figure 2 The photosensitizer of Example 6 produces a hydrogen sulfide detection pattern;

[0043] Figure 3 The plate coating diagram and bacterial count diagram are shown after the photosensitizer of Example 6 is treated with Gram-positive bacteria S. aureus, Gram-positive drug-resistant bacteria MRSA, and Gram-negative bacteria E. coli, respectively; wherein A is Gram-positive bacteria S. aureus, B is Gram-positive drug-resistant bacteria MRSA, and C is Gram-negative bacteria E. coli;

[0044] Figure 4 This is a graph showing changes in the surface Zeta potential of bacteria after incubating the photosensitizer of Example 6 with Gram-positive bacteria S. aureus, Gram-positive drug-resistant bacteria MRSA, and Gram-negative bacteria (E. coli) for 10 minutes respectively;

[0045] Figure 5 Bacterial fluorescence imaging images of the photosensitizer of Example 6 after incubation with Gram-positive bacteria MRSA and Gram-negative bacteria E. coli for 10 minutes respectively;

[0046] Figure 6 Figure 1 is a small dish diagram showing the effect of the photosensitizer of Example 6 on the removal of bacterial biofilms of Gram-positive bacteria S. aureus, Gram-positive drug-resistant bacteria MRSA, and Gram-negative bacteria E. coli by the crystal violet staining method; wherein A represents Gram-positive bacteria S. aureus, B represents Gram-positive drug-resistant bacteria MRSA, and C represents Gram-negative bacteria E. coli;

[0047] Figure 7 This is a statistical graph showing the residual amount of the photosensitizer in Example 6 on the biofilm of Gram-positive bacteria S. aureus, Gram-positive drug-resistant bacteria MRSA, and Gram-negative bacteria Escherichia coli E. coli measured by the crystal violet staining method; wherein A represents Gram-positive bacteria S. aureus, B represents Gram-positive drug-resistant bacteria MRSA, and C represents Gram-negative bacteria E. coli;

[0048] Figure 8 This is a fluorescence imaging 3D layer scanning result diagram of the photosensitizer in Example 6 for the removal of Gram-positive bacteria MRSA and Gram-negative bacteria Escherichia coli E.coli bacterial biofilm;

[0049] Figure 9 These are SEM morphology analysis images of the biofilm removal effect of the photosensitizer in Example 6, wherein A is a SEM morphology analysis image of MRSA biofilm removal, and B is a SEM morphology analysis image of E. coli biofilm removal;

[0050] Figure 10 The results of red blood cell rupture and hemolysis rate before and after the photosensitizer photodynamic therapy in Example 6 are shown in FIG.

[0051] Figure 11This is a statistical graph of cell survival rates after the photosensitizer of Example 6 was incubated with HUVEC cells and treated in darkness and light respectively;

[0052] Figure 12 This is a picture of the cell scratch experiment;

[0053] Figure 13 To promote angiogenesis imaging in vitro;

[0054] Figure 14 The results of the biofilm removal experiment in diabetic rats in vivo are shown in Figure 1. A shows the rat inflammation model construction, B shows the bacterial count, C shows the wound size, and D shows the bacterial count.

[0055] Figure 15 This is a graph showing the levels of interleukin-6 and tumor necrosis factor (TNF-α) in the serum of rats after treatment with the photosensitizer of Example 6; wherein A is a graph showing the level of interleukin-6, and B is a graph showing the level of tumor necrosis factor (TNF-α);

[0056] Figure 16 These are H&E staining and Masson analysis images of infected tissue after photosensitizer treatment in Example 6; A is the H&E staining image, B is the Masson staining image, and C is the Masson analysis image;

[0057] Figure 17 These are the results of immunohistochemical analysis of the infected site after photosensitizer treatment in Example 6; wherein A is a statistical graph showing the TNF-α content, B is a statistical graph showing the interleukin-6 content, C is a statistical graph showing the transforming growth factor-β content, D is a statistical graph showing the vascular endothelial growth factor content, and E is a statistical graph showing the platelet-endothelial cell adhesion molecule content;

[0058] Figure 18 Graphs showing the results of in vivo biosafety analysis of the photosensitizer of Example 6; wherein A is a staining diagram of major organs; B is a statistical diagram of the biochemical indicator ALT content; C is a statistical diagram of the biochemical indicator AST content; D is a statistical diagram of the biochemical indicator CREA-S content; and E is a statistical diagram of the biochemical indicator UREA content. DETAILED DESCRIPTION

[0059] The following examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. In the examples, where specific conditions are not specified, the experiments were performed under conventional conditions or those recommended by the manufacturer. Reagents or instruments used, where the manufacturer is not specified, are commercially available conventional products.

[0060] Example 1:

[0061] A photosensitizer with hydrogen sulfide-releasing properties has a structure as shown in Formula I-1:

[0062]

[0063] In this embodiment, the photosensitizer having a structure as shown in Formula I-1 is labeled TPTCP-HS and is prepared by the following steps:

[0064] (1) 2-Thiopheneacetonitrile (5.0 g, 40.6 mmol) was dissolved in 30 mL of N,N-dimethylformamide solution, and N-bromosuccinimide (7.226 g, 40.6 mmol) was added and reacted at room temperature for 24 h. After the reaction, the solvent was removed by distillation under reduced pressure. The crude product was separated and purified by silica gel column chromatography with an eluent ratio of petroleum ether / ethyl acetate (V:V = 10:1) to obtain 7.274 g of brown oily compound B with a yield of 89%. The synthetic route is as follows:

[0065]

[0066] (2) Compound B (1.0 g, 4.9 mmol), 4-(diphenylamino)phenylboronic acid (2.139 g, 7.4 mmol), tetrakistriphenylphosphine palladium (12 mg, 0.2% mmol), and potassium carbonate (4.063 g, 29.4 mmol) were placed in a three-necked flask. After evacuation and introduction of nitrogen, 40 mL of tetrahydrofuran and 10 mL of water were added and the mixture was refluxed at 68°C for 12 h. After the reaction, tetrahydrofuran was removed by distillation under reduced pressure, and the mixture was extracted three times with 20 mL of dichloromethane. The organic phase was washed three times with saturated brine, the organic phase was collected and dried over anhydrous sodium sulfate, and the organic solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel column chromatography with an eluent ratio of petroleum ether / ethyl acetate (V:V=5:1) to obtain 1.4 g of solid compound D-1 with a yield of 80%. The synthetic route is as follows:

[0067]

[0068] (3) Compound D-1 (1.099 g, 3 mmol) was dissolved in 50 mL of ethanol, and 5-bromo-2-thiophenecarboxaldehyde (537.1 mg, 3 mmol) and sodium tert-butoxide (288 mg, 3 mmol) were added. The mixture was reacted at room temperature for 12 h. After the reaction, the residue was filtered and washed with a small amount of icy ethanol. After drying, 971 mg of solid compound E-1 was obtained with a yield of 60%. The synthetic route is as follows:

[0069]

[0070] (4) Compound E-1 (1.079 g, 2 mmol), 4-pyridineboronic acid (295 mg, 2.4 mmol), tetrakistriphenylphosphine palladium (4.6 mg, 0.2% mmol), and potassium carbonate (1.656 g, 12 mmol) were placed in a three-necked flask, evacuated, and 40 mL of tetrahydrofuran and 10 mL of water were added under nitrogen protection. The mixture was refluxed at 68°C for 12 h. After the reaction, tetrahydrofuran was removed by distillation under reduced pressure, and the mixture was extracted three times with 20 mL of dichloromethane. The organic phase was washed three times with saturated brine, the organic phase was collected and dried over anhydrous sodium sulfate, and the organic solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel column chromatography with an eluent ratio of dichloromethane / methanol (V:V=50:1) to obtain 860 mg of solid compound F-1 with a yield of 80%. The synthetic route is as follows:

[0071]

[0072] (5) 5-(4-Hydroxyphenyl)-3H-1,2-dithiole-3-thione [(ADT-OH) 50 mg, 0.22 mmol] was dissolved in 30 mL of DMF, potassium carbonate (164 mg, 1.2 mmol) and 1,3-dibromopropane (134 mg, 0.66 mmol) were added, and the reaction was allowed to proceed overnight at room temperature. After the reaction was completed, the organic solvent was removed by distillation under reduced pressure. The crude product was separated and purified by silica gel column chromatography with an eluent ratio of petroleum ether to ethyl acetate (V:V = 50:1) to obtain 40 mg of an orange-red solid compound H-1 with a yield of 52%. The synthetic route is as follows:

[0073]

[0074] (6) Compound F-1 (100 mg, 0.18 mmol) and compound H-1 (65 mg, 0.18 mmol) were dissolved in 30 mL of acetonitrile and reacted at 60°C for 24 h. After the reaction, the organic solvent was removed by distillation under reduced pressure. The product was separated and purified by silica gel column chromatography using a dichloromethane to methanol ratio (V:V = 20:1) as the eluent to obtain 80 mg of red solid TPTCP-HS with a yield of 50%. The synthetic route is as follows:

[0075]

[0076] Characterization data: 1H NMR(400MHz,DMSO)δ9.06–9.00(m,2H),8.41(d,J=6.5Hz,2H),8.33(d,J=4.2Hz,1H), 8.10(s,1H),7.91(t,J=4.0Hz,1H),7.85(dd,J=9.1,2.7Hz,2H),7.74(s,1H),7.64–7 .56(m,2H),7.52–7.44(m,2H),7.36(t,J=7.8Hz,4H),7.11(dd,J=17.2,7.7Hz,6H),6 .98(t,J=8.0Hz,4H),4.75(t,J=6.1Hz,2H),4.22(t,J=5.2Hz,2H),2.51-2.39(m,2H).

[0077] Example 2:

[0078] A photosensitizer with hydrogen sulfide-releasing properties has a structure as shown in Formula I-2:

[0079]

[0080] In this embodiment, the photosensitizer having a structure as shown in Formula I-2 is labeled TPTCP-Me-HS, and step (1) is the same as in Example 1. Steps (2) to (6) are specifically as follows:

[0081] (2) Compound B (1.0 g, 4.9 mmol), 4-(dimethylamino)phenylboronic acid (1.221 g, 7.4 mmol), tetrakistriphenylphosphine palladium (12 mg, 0.2% mmol), and potassium carbonate (4.063 g, 29.4 mmol) were placed in a three-necked flask. After evacuation and introduction of nitrogen, 40 mL of tetrahydrofuran and 10 mL of water were added and the mixture was refluxed at 68°C for 12 h. After the reaction, tetrahydrofuran was removed by distillation under reduced pressure, and the mixture was extracted three times with 20 mL of dichloromethane. The organic phase was washed three times with saturated brine, the organic phase was collected and dried over anhydrous sodium sulfate, and the organic solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel column chromatography with an eluent ratio of petroleum ether / ethyl acetate (V:V=5:1) to obtain 926.2 g of solid compound D-2 with a yield of 78%. The synthetic route is as follows:

[0082]

[0083] (3) Compound D-2 (0.727 g, 3 mmol) was dissolved in 50 mL of ethanol, and 5-bromo-2-thiophenecarboxaldehyde (573 mg, 3 mmol) and sodium tert-butoxide (288 mg, 3 mmol) were added. The mixture was reacted at room temperature for 12 h. After the reaction, the residue was filtered and washed with a small amount of icy ethanol. After drying, 747 mg of solid compound E-2 was obtained with a yield of 60%. The synthetic route is as follows:

[0084]

[0085] (4) Compound E-2 (0.831 g, 2 mmol), 4-pyridineboronic acid (295 mg, 2.4 mmol), tetrakistriphenylphosphine palladium (4.6 mg, 0.2% mmol), and potassium carbonate (1.656 g, 12 mmol) were placed in a three-necked flask, evacuated, and 40 mL of tetrahydrofuran and 10 mL of water were added under nitrogen protection. The mixture was refluxed at 68°C for 12 h. After the reaction, tetrahydrofuran was removed by distillation under reduced pressure, and the mixture was extracted three times with 20 mL of dichloromethane. The organic phase was washed three times with saturated brine, the organic phase was collected and dried over anhydrous sodium sulfate, and the organic solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel column chromatography with an eluent ratio of dichloromethane / methanol (V:V=50:1) to obtain 662 mg of solid compound F-2 with a yield of 80%. The synthetic route is as follows:

[0086]

[0087] (5) 5-(4-Hydroxyphenyl)-3H-1,2-dithiole-3-thione [(ADT-OH) 50 mg, 0.22 mmol] was dissolved in 30 mL of DMF, potassium carbonate (164 mg, 1.2 mmol) and 1,3-dibromopropane (134 mg, 0.66 mmol) were added, and the reaction was allowed to proceed overnight at room temperature. After the reaction was completed, the organic solvent was removed by distillation under reduced pressure. The crude product was separated and purified by silica gel column chromatography with an eluent ratio of petroleum ether to ethyl acetate (V:V = 50:1) to obtain 40 mg of an orange-red solid compound H-1 with a yield of 52%. The synthetic route is as follows:

[0088]

[0089] (6) Compound F-2 (100 mg, 0.24 mmol) and compound H-1 (84 mg, 0.24 mmol) were dissolved in 30 mL of acetonitrile and reacted at 60°C for 24 h. After the reaction, the organic solvent was removed by distillation under reduced pressure. The product was separated and purified by silica gel column chromatography using a dichloromethane to methanol ratio (V:V = 20:1) as the eluent to obtain 128 mg of red solid TPTCP-Me-HS with a yield of 70%. The synthetic route is as follows:

[0090]

[0091] Characterization data: 1 H NMR (400MHz, DMSO) δ9.06–9.00(m,2H),8.41(d,J=6.5Hz,2H),8.33(d,J=4.2 Hz,1H),8.10(s,1H),7.91(t,J=4.0Hz,1H),7.85(dd,J=9.1,2.7Hz,2H),7.7 4(s,1H),7.64–7.56(m,2H),7.11(d,J=6.71Hz,2H),6.98(t,J=8.0Hz,4H),4 .75(t,J=6.1Hz,2H),4.22(t,J=5.2Hz,2H),2.84(s,6H),2.51-2.39(m,2H).

[0092] Example 3:

[0093] A photosensitizer with hydrogen sulfide-releasing properties has a structure as shown in Formula I-3:

[0094]

[0095] In this embodiment, the metal complex having a structure as shown in Formula I-3 is labeled as TPTCP-OM-HS, and step (1) is the same as in Example 1, and steps (2) to (6) are specifically as follows:

[0096] (2) Compound B (1.0 g, 4.9 mmol), [4-[bis(4-methoxyphenyl)amino]phenyl]boronic acid (2.584 g, 7.4 mmol), tetrakistriphenylphosphine palladium (12 mg, 0.2% mmol), and potassium carbonate (4.063 g, 29.4 mmol) were placed in a three-necked flask. After evacuation and introduction of nitrogen, 40 mL of tetrahydrofuran and 10 mL of water were added, and the mixture was refluxed at 68°C for 12 h. After the reaction, tetrahydrofuran was removed by distillation under reduced pressure, and the mixture was extracted three times with 20 mL of dichloromethane. The organic phase was washed three times with saturated brine, the organic phase was collected and dried over anhydrous sodium sulfate, and the organic solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel column chromatography with an eluent ratio of petroleum ether / ethyl acetate (V:V=5:1) to obtain 1.7544 g of solid compound D-3 with a yield of 84%. The synthetic route is as follows:

[0097]

[0098] (3) Compound D-3 (1.0 g, 2.4 mmol) was dissolved in 50 mL of ethanol, and 5-bromo-2-thiophenecarboxaldehyde (458 mg, 2.4 mmol) and sodium tert-butoxide (230 mg, 2.4 mmol) were added. The mixture was reacted at room temperature for 12 h. After the reaction, the mixture was filtered and the residue was washed with a small amount of icy ethanol and dried to obtain 835 mg of solid compound E-3 with a yield of 58%. The synthetic route is as follows:

[0099]

[0100] (4) Compound E-3 (1.2 g, 2 mmol), 4-pyridineboronic acid (295 mg, 2.4 mmol), tetrakistriphenylphosphine palladium (4.6 mg, 0.2% mmol), and potassium carbonate (1.656 g, 12 mmol) were placed in a three-necked flask, evacuated, and 40 mL of tetrahydrofuran and 10 mL of water were added under nitrogen protection. The mixture was refluxed at 68°C for 12 h. After the reaction, tetrahydrofuran was removed by distillation under reduced pressure, and the mixture was extracted three times with 20 mL of dichloromethane. The organic phase was washed three times with saturated brine, the organic phase was collected and dried over anhydrous sodium sulfate, and the organic solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel column chromatography with an eluent ratio of dichloromethane / methanol (V:V = 50:1) to obtain 956 mg of solid compound F-3 with a yield of 80%. The synthetic route is as follows:

[0101]

[0102] (5) 5-(4-Hydroxyphenyl)-3H-1,2-dithiole-3-thione [(ADT-OH) 50 mg, 0.22 mmol] was dissolved in 30 mL of DMF, potassium carbonate (164 mg, 1.2 mmol) and 1,3-dibromopropane (134 mg, 0.66 mmol) were added, and the reaction was allowed to proceed overnight at room temperature. After the reaction was completed, the organic solvent was removed by distillation under reduced pressure. The crude product was separated and purified by silica gel column chromatography with an eluent ratio of petroleum ether to ethyl acetate (V:V = 50:1) to obtain 40 mg of an orange-red solid compound H-1 with a yield of 52%. The synthetic route is as follows:

[0103]

[0104] (6) Compound F-3 (100 mg, 0.18 mmol) and compound H-1 (64 mg, 0.18 mmol) were dissolved in 30 mL of acetonitrile and reacted at 60°C for 24 h. After the reaction, the organic solvent was removed by distillation under reduced pressure. The product was separated and purified by silica gel chromatography using a dichloromethane to methanol ratio (V:V = 20:1) as the eluent to obtain 119 mg of red solid TPTCP-OM-HS with a yield of 70%. The synthetic route is as follows:

[0105]

[0106] Characterization data: 1 H NMR (400MHz, DMSO) δ9.06–9.00(m,2H),8.41(d,J=6.5Hz,2H),8.33(d,J=4.2Hz,1H) ,8.10(s,1H),7.91(t,J=4.0Hz,1H),7.85(dd,J=9.1,2.7Hz,2H),7.74(s,1H),7.64 –7.56(m,2H),7.36(t,J=7.8Hz,4H),7.11(dd,J=17.2,7.7Hz,6H),6.98(t,J=8.0Hz ,4H),4.75(t,J=6.1Hz,2H),4.22(t,J=5.2Hz,2H),3.79(s,6H),2.51-2.39(m,2H).

[0107] Example 4:

[0108] A photosensitizer with hydrogen sulfide-releasing properties has a structure as shown in Formula I-4:

[0109]

[0110] In this embodiment, the metal complex having a structure as shown in Formula I-4 is labeled as TPTCP-Et-HS, and step (1) is the same as in Example 1, and steps (2) to (6) are specifically as follows:

[0111] (2) Compound B (1.0 g, 4.9 mmol), 4-(diethylamino)phenylboronic acid (1.428 g, 7.4 mmol), tetrakistriphenylphosphine palladium (12 mg, 0.2% mmol), and potassium carbonate (4.063 g, 29.4 mmol) were placed in a three-necked flask. After evacuation and introduction of nitrogen, 40 mL of tetrahydrofuran and 10 mL of water were added and the mixture was refluxed at 68°C for 12 h. After the reaction, tetrahydrofuran was removed by distillation under reduced pressure, and the mixture was extracted three times with 20 mL of dichloromethane. The organic phase was washed three times with saturated brine, the organic phase was collected and dried over anhydrous sodium sulfate, and the organic solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel column chromatography with an eluent ratio of petroleum ether / ethyl acetate (V:V=5:1) to obtain 0.9672 g of solid compound D-4 with a yield of 73%. The synthetic route is as follows:

[0112]

[0113] (3) Compound D-4 (1.352 g, 5 mmol) was dissolved in 50 mL of ethanol, and 5-bromo-2-thiophenecarboxaldehyde (955 mg, 5 mmol) and sodium tert-butoxide (479 mg, 5 mmol) were added. The mixture was reacted at room temperature for 12 h. After the reaction, the mixture was filtered, and the residue was washed with a small amount of icy ethanol and dried to obtain 1.552 g of solid compound E-4 with a yield of 70%. The synthetic route is as follows:

[0114]

[0115] (4) Compound E-4 (1.552 g, 3.5 mmol), 4-pyridineboronic acid (516 mg, 4.2 mmol), tetrakistriphenylphosphine palladium (8.1 mg, 0.2% mmol), and potassium carbonate (2.898 g, 21 mmol) were placed in a three-necked flask, evacuated, and 40 mL of tetrahydrofuran and 10 mL of water were added under nitrogen protection. The mixture was refluxed at 68°C for 12 h. After the reaction, tetrahydrofuran was removed by distillation under reduced pressure, and the mixture was extracted three times with 20 mL of dichloromethane. The organic phase was washed three times with saturated brine, the organic phase was collected and dried over anhydrous sodium sulfate, and the organic solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel column chromatography with an eluent ratio of dichloromethane / methanol (V:V=50:1) to obtain 1.08 g of solid compound F-4 with a yield of 70%. The synthetic route is as follows:

[0116]

[0117] (5) 5-(4-Hydroxyphenyl)-3H-1,2-dithiole-3-thione [(ADT-OH) 50 mg, 0.22 mmol] was dissolved in 30 mL of DMF, potassium carbonate (164 mg, 1.2 mmol) and 1,3-dibromopropane (134 mg, 0.66 mmol) were added, and the reaction was allowed to proceed overnight at room temperature. After the reaction was completed, the organic solvent was removed by distillation under reduced pressure. The crude product was separated and purified by silica gel column chromatography with an eluent ratio of petroleum ether to ethyl acetate (V:V = 50:1) to obtain 40 mg of an orange-red solid compound H-1 with a yield of 52%. The synthetic route is as follows:

[0118]

[0119] (6) Compound F-4 (100 mg, 0.23 mmol) and compound H-1 (82 mg, 0.23 mmol) were dissolved in 30 mL of acetonitrile and reacted at 60°C for 24 h. After the reaction, the organic solvent was removed by distillation under reduced pressure. The product was separated and purified by silica gel column chromatography using a dichloromethane to methanol ratio (V:V = 20:1) as the eluent to obtain 112 mg of red solid TPTCP-Et-HS with a yield of 62%. The synthetic route is as follows:

[0120]

[0121] Characterization data: 1 H NMR (400MHz, DMSO) δ9.06–9.00(m,2H),8.41(d,J=6.5Hz,2H),8.33(d,J=4.2Hz,1H),8.10(s,1 H),7.91(t,J=4.0Hz,1H),7.85(dd,J=9.1,2.7Hz,2H),7.74(s,1H),7.64–7.56(m,2H),7.36(t ,J=7.8Hz,4H),7.11(dd,J=17.2,7.7Hz,6H),6.98(t,J=8.0Hz,4H),4.75(t,J=6.1Hz,2H),4.2 2(t,J=5.2Hz,2H),3.31(dd,J=14.49,7.11Hz,4H),2.51-2.39(m,2H),1.16(t,J=6.93Hz,6H).

[0122] Example 5:

[0123] A photosensitizer with hydrogen sulfide-releasing properties has a structure as shown in Formula II-1:

[0124]

[0125] In this embodiment, the photosensitizer having a structure as shown in Formula II-1 is labeled ACR-TPA-HS and is prepared by the following steps:

[0126] (1) 2-Thiopheneacetonitrile (5.0 g, 40.6 mmol) was dissolved in 30 mL of N,N-dimethylformamide solution, and N-bromosuccinimide (7.226 g, 40.6 mmol) was added and reacted at room temperature for 24 h. After the reaction, the solvent was removed by distillation under reduced pressure. The crude product was separated and purified by silica gel column chromatography with an eluent ratio of petroleum ether / ethyl acetate (V:V = 10:1) to obtain 7.274 g of brown oily compound B with a yield of 89%. The synthetic route is as follows:

[0127]

[0128] (2) Compound B (110.5 mg, 0.547 mmol), 4-pyridineboronic acid (134.8 mg, 1.094 mmol), tetrakistriphenylphosphine palladium (1.26 mg, 0.2% mmol) and potassium carbonate (453.6 mg, 3.282 mmol) were placed in a three-necked flask, evacuated, and 40 mL of tetrahydrofuran and 10 mL of water were added under nitrogen protection. The mixture was reacted at 68°C for 12 h. After the reaction, tetrahydrofuran was removed and the mixture was extracted three times with 25 mL of dichloromethane. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and dichloromethane was removed by vacuum distillation. The crude product was separated and purified by silica gel column chromatography with an eluent ratio of dichloromethane / methanol (V:V=80:1) to obtain 88.1 mg of yellow solid compound J-1 with a yield of 80%. The synthetic route is as follows:

[0129]

[0130] (3) Compound J-1 (400 mg, 2 mmol), 4-diphenylaminobenzaldehyde (546.7 mg, 2 mmol), and sodium tert-butoxide (194.6 mg, 2 mmol) were dissolved in 50 mL of ethanol and reacted at room temperature for 24 h. After the reaction, the residue was filtered and washed with a small amount of icy ethanol. After drying, 537.6 mg of yellow solid compound L-1 was obtained with a yield of 59%. The synthetic route is as follows:

[0131]

[0132] (4) 5-(4-Hydroxyphenyl)-3H-1,2-dithiole-3-thione [(ADT-OH) 50 mg, 0.22 mmol] was dissolved in 30 mL of DMF, potassium carbonate (164 mg, 1.2 mmol) and 1,3-dibromopropane (134 mg, 0.66 mmol) were added, and the reaction was allowed to proceed overnight at room temperature. After the reaction was completed, the organic solvent was removed by distillation under reduced pressure. The crude product was separated and purified by silica gel column chromatography with an eluent ratio of petroleum ether to ethyl acetate (V:V = 50:1) to obtain 40 mg of an orange-red solid compound H-1 with a yield of 52%. The synthetic route is as follows:

[0133]

[0134] (5) Compound L-1 (100 mg, 0.22 mmol) was dissolved in 30 mL of acetonitrile, and compound H (77 mg, 0.22 mol) was added. The mixture was reacted at 60°C for 24 h. After the reaction, the acetonitrile was removed by distillation under reduced pressure. The product was separated and purified by silica gel column chromatography using a dichloromethane to methanol ratio (V:V = 20:1) as the eluent to obtain 99 mg of red solid ACR-TPA-HS with a yield of 56%. The synthetic route is as follows:

[0135]

[0136] Characterization data: 1 H NMR (400MHz, DMSO) δ9.01(d,J=6.5Hz,2H),8.34(d,J=7.1Hz,2H),8.29(d,J=4 .1Hz,1H),7.92(s,1H),7.86(t,J=8.5Hz,4H),7.74(s,1H),7.68(d,J=4.1Hz, 1H),7.43(t,J=7.8Hz,4H),7.24(d,J=6.9Hz,2H),7.19(d,J=7.8Hz,4H),6.99 –6.88(m,4H),4.72(t,J=6.6Hz,2H),4.22(t,J=5.7Hz,2H),2.51-2.39(m,2H).

[0137] Example 6:

[0138] A photosensitizer with hydrogen sulfide-releasing properties has a structure as shown in Formula II-2:

[0139]

[0140] In this embodiment, the photosensitizer having a structure as shown in Formula II-2 is labeled ACR-DM-HS, and steps (1) to (2) are the same as those in Example 5. Steps (3) to (5) are specifically as follows:

[0141] (3) Compound J-1 (400 mg, 2 mmol) was dissolved in 50 mL of ethanol, and sodium tert-butoxide (194.6 mg, 2 mmol) and 4-dimethylaminobenzaldehyde (298 mg, 2 mmol) were added. The mixture was reacted at room temperature for 24 h. After the reaction, the residue was filtered and washed with a small amount of icy ethanol and dried to obtain 491 mg of yellow solid compound L-2 with a yield of 74%. The synthetic route is as follows:

[0142]

[0143] (4) 5-(4-Hydroxyphenyl)-3H-1,2-dithiole-3-thione [(ADT-OH) 50 mg, 0.22 mmol] was dissolved in 30 mL of DMF, potassium carbonate (164 mg, 1.2 mmol) and 1,3-dibromopropane (134 mg, 0.66 mmol) were added, and the reaction was allowed to proceed overnight at room temperature. After the reaction was completed, the organic solvent was removed by distillation under reduced pressure. The crude product was separated and purified by silica gel column chromatography with an eluent ratio of petroleum ether to ethyl acetate (V:V = 50:1) to obtain 40 mg of an orange-red solid compound H-1 with a yield of 52%. The synthetic route is as follows:

[0144]

[0145] (5) Compound L-2 (100 mg, 0.3 mmol) was dissolved in 30 mL of acetonitrile, and compound H-1 (104 mg, 0.3 mmol) was added. The mixture was reacted at 60°C for 24 h. After the reaction, the acetonitrile was removed by distillation under reduced pressure. The product was separated and purified by silica gel column chromatography using a dichloromethane to methanol ratio (v:v = 20:1) as the eluent to obtain 130 mg of red solid ACR-DM-HS with a yield of 64%. The synthetic route is as follows:

[0146]

[0147] Characterization data: 1 H NMR (400MHz, DMSO) δ9.01(d,J=6.6Hz,2H),8.29(dd,J=8.6,5.3Hz,3H),7.92-7.80(m,5H),7.74(s,1H),7.59(d,J=4.0Hz ,1H),7.00-6.91(m,2H),6.89-6.78(m,2H),4.72(t,J=6.7Hz,2H),4.22(t,J=5.7Hz,2H),3.07(s,6H),2.51-2.39(m,2H).

[0148] Example 7:

[0149] A photosensitizer with hydrogen sulfide-releasing properties has a structure as shown in Formula II-3:

[0150]

[0151] In this embodiment, the photosensitizer having a structure as shown in Formula II-3 is labeled ACR-DE-HS. Steps (1) to (2) are the same as those in Example 5. Steps (3) to (5) are specifically as follows:

[0152] (3) Compound J-1 (400 mg, 2 mmol) was dissolved in 50 mL of ethanol, and sodium tert-butoxide (194.6 mg, 2 mmol) and 4-diethylaminobenzaldehyde (354 mg, 2 mmol) were added. The mixture was reacted at room temperature for 24 h. After the reaction, the residue was filtered and washed with a small amount of icy ethanol and dried to obtain 503 mg of yellow solid compound L-3 with a yield of 70%. The synthetic route is as follows:

[0153]

[0154] (4) 5-(4-Hydroxyphenyl)-3H-1,2-dithiole-3-thione [(ADT-OH) 50 mg, 0.22 mmol] was dissolved in 30 mL of DMF, potassium carbonate (164 mg, 1.2 mmol) and 1,3-dibromopropane (134 mg, 0.66 mmol) were added, and the reaction was allowed to proceed overnight at room temperature. After the reaction was completed, the organic solvent was removed by distillation under reduced pressure. The crude product was separated and purified by silica gel column chromatography with an eluent ratio of petroleum ether to ethyl acetate (V:V = 50:1) to obtain 40 mg of an orange-red solid compound H-1 with a yield of 52%. The synthetic route is as follows:

[0155]

[0156] (5) Compound L-3 (117 mg, 0.3 mmol) was dissolved in 30 mL of acetonitrile, and compound H-1 (104 mg, 0.3 mmol) was added. The mixture was reacted at 60°C for 24 h. After the reaction, the acetonitrile was removed by distillation under reduced pressure. The product was separated and purified by silica gel column chromatography using a dichloromethane to methanol ratio (V:V = 20:1) as the eluent to obtain 127 mg of a red solid with a yield of 60%. The synthetic route is as follows:

[0157]

[0158] Characterization data: 1 H NMR (400MHz, DMSO) δ9.01(d,J=6.6Hz,2H),8.29(dd,J=8.6,5.3Hz,3H),7.92-7.80(m,5H),7.74(s,1H),7.59(d,J=4.0Hz,1H),7.00-6.91(m,2 H),6.89–6.78(m,2H),4.72(t,J=6.7Hz,2H),4.22(t,J=5.7Hz,2H),3.31(dd,J=14.49,7.11Hz,4H),2.51-2.39(m,2H),1.16(t,J=6.93Hz,6H).

[0159] Example 8:

[0160] A photosensitizer with hydrogen sulfide-releasing properties has a structure as shown in Formula II-4:

[0161]

[0162] In this embodiment, the photosensitizer having a structure as shown in Formula II-4 is labeled ACR-OM-HS, and steps (1) to (2) are the same as those in Example 5. Steps (3) to (5) are specifically as follows:

[0163] (3) Compound J-1 (400 mg, 2 mmol) was dissolved in 50 mL of ethanol, and sodium tert-butoxide (194.6 mg, 2 mmol) and 4-[bis(4-methoxyphenyl)amino]benzaldehyde (667 mg, 2 mmol) were added. The mixture was reacted at room temperature for 24 h. After the reaction, the residue was filtered and washed with a small amount of icy ethanol and dried to obtain 526 mg of yellow solid compound L-4 with a yield of 51%. The synthetic route is as follows:

[0164]

[0165] (4) 5-(4-Hydroxyphenyl)-3H-1,2-dithiole-3-thione [(ADT-OH) 50 mg, 0.22 mmol] was dissolved in 30 mL of DMF, potassium carbonate (164 mg, 1.2 mmol) and 1,3-dibromopropane (134 mg, 0.66 mmol) were added, and the reaction was allowed to proceed overnight at room temperature. After the reaction was completed, the organic solvent was removed by distillation under reduced pressure. The crude product was separated and purified by silica gel column chromatography with an eluent ratio of petroleum ether to ethyl acetate (V:V = 50:1) to obtain 40 mg of an orange-red solid compound H-1 with a yield of 52%. The synthetic route is as follows:

[0166]

[0167] (5) Compound L-4 (155 mg, 0.3 mmol) was dissolved in 30 mL of acetonitrile, and compound H-1 (104 mg, 0.3 mmol) was added. The mixture was reacted at 60°C for 24 h. After the reaction, the acetonitrile was removed by distillation under reduced pressure. The product was separated and purified by silica gel column chromatography using a dichloromethane to methanol ratio (v:v = 20:1) as the eluent to obtain 116 mg of red solid ACR-DP-HS with a yield of 45%. The synthetic route is as follows:

[0168]

[0169] Characterization data: 1 H NMR (400MHz, DMSO) δ9.01 (d, J=6.6Hz, 2H), 8.29 (dd, J=8.6, 5.3Hz, 3H), 7.92-7.80 (m, 5H), 7.74 (s, 1H), 7.59 (d, J=4.0Hz,1H),7.36(d,J=7.8Hz,4H),7.11(d,J=7.7Hz,4H),7.00-6.91(m,2H),6.89–6.78(m,2H),4.72(t,J=6.7 Hz, 2H), 4.22 (t, J = 5.7 Hz, 2H), 3.79 (s, 6H), 2.51-2.39 (m, 2H).

[0170] Experimental example

[0171] The three properties of the photosensitizer prepared in Example 6, namely, photophysical properties, hydrogen sulfide production performance, photodynamic anti-plankton bacteria performance, and photodynamic biofilm removal performance, were measured. Since the experimental results of the compounds in this application are similar and the reactions are the same, the photophysical properties, hydrogen sulfide production performance, photodynamic anti-plankton bacteria performance, and biofilm removal performance of the present invention, ACR-DM-HS, are mainly used as an example. Since the experimental results and reactions of other examples are the same as those of the examples, they are not listed one by one. The specific results are as follows:

[0172] 1. Determination of photophysical properties

[0173] 1.1 Absorption and emission measurements of photosensitizers

[0174] The absorption and emission spectra of ACR-DM-HS of Example 6 in DMSO were measured. Figure 1 As shown in Figure A, the maximum absorption and emission wavelengths of ACR-DM-HS are 512 nm and 748 nm, respectively.

[0175] 1.2 Target molecule reactive oxygen species determination

[0176] 2,7-Dichlorodihydrofluorescein diacetate (H2DCF-DA) was used as a detection probe to test the ROS production of the compound ACR-DM-HS in solution. To convert H2DCF-DA to 2,7-dichlorodihydrofluorescein (H2DCF), 0.25 mL of H2DCF-DA ethanol solution (1 mM) was added to 1 mL of NaOH (10 mM) aqueous solution, then stirred at room temperature for 30 min. The pH of the solution was then adjusted with 5 mL of PBS solution (pH = 7.4), and the resulting solution was frozen for future use. ACR-DM-HS DMSO solution was added to the above solution to a final concentration of 10 μM. The sample was placed in a fluorescence spectrometer, and the fluorescence intensity of the solution was measured every 30 s (λex: 488 nm). The results are shown in Figure 2. Figure 1 As shown in Figure B, as the illumination time prolonged, the fluorescence intensity of ACR-DM-HS at 525 nm increased by 117 times, indicating that ACR-DM-HS can effectively and rapidly generate ROS under light irradiation.

[0177] 1.3 Singlet oxygen yield determination

[0178] The singlet oxygen generation performance of ACR-DM-HS and commercially available photosensitizer Rose Bengal (RB) under light irradiation was investigated using ABDA (9,10-anthryl-bis(methylene)dione) as an indicator. 1During the O2 reaction, ABDA is oxidized to form a peroxy bridge structure, which causes the absorbance of ABDA at 378 nm to decrease. The rate of decrease can indirectly reflect the photosensitizer under light. 1 O2 production rate. First, the absorbance of photosensitizer ACR-DM-HS and RB (10 μM) was set as blank; then, ABDA (50 μM) and ACR-DM-HS (10 μM) solutions were mixed under dark conditions and the absorbance of the solution was immediately measured. Then, a white light lamp (5 mW / cm 2 ) irradiate the solution mixture, and record the absorbance value of the solution immediately after each irradiation for 30 seconds until the absorbance value no longer decreases. Figure 1 As shown in Figure C, the absorbance value of ACR-DM-HS at 378 nm did not decrease under light, while the absorbance value of RB at 378 nm decreased to varying degrees under light. However, the absorbance value of the control group, which only contained the indicator ABDA, did not decrease, indicating that ACR-DM-HS produced almost no 1 O2.

[0179] 1.4 Determination of hydroxyl radical yield

[0180] HPF (hydroxyphenyl fluorescein) was used as an indicator to investigate the hydroxyl radical generation performance of the photosensitizer ACR-DM-HS under light irradiation. Hydroxyphenyl fluorescein itself is non-fluorescent, but when reacting with hydroxyl radicals, peroxynitrite anions, and hypochlorite anions, it produces strong green fluorescence at 515 nm. HPF (5 μM) and ACR-DM-HS (10 μM) were mixed in the dark and the fluorescence intensity was immediately measured using a fluorescence spectrometer, marked as 0 min. The fluorescence intensity was measured using a white light lamp (5 mW / cm 2 ) irradiate the solution mixture, and measure the fluorescence immediately after each 4-min irradiation until the fluorescence intensity no longer increases. Figure 1 As shown in Figure D, there is almost no emission at 515 nm at 0 min of illumination, the emission intensity increases after 4 min of illumination, and the I / I0 value of the photosensitizer ACR-DM-HS increases 132 times after 28 min of illumination, indicating that ACR-DM-HS can quickly and efficiently generate hydroxyl radicals under illumination.

[0181] From the measurement results of 1.1 to 1.4, it can be seen that the photosensitizer prepared in Example 6 has good fluorescence emission performance and type I reactive oxygen species generation performance, and can generate a large number of hydroxyl free radicals under light; ACR-DM-HS is a type I photosensitizer, and this type of compound has application potential in both imaging and photodynamic therapy under hypoxic conditions.

[0182] 2 Investigation of H2S release performance

[0183] 2.1 Preparation of macrophage lysate

[0184] The digested cells were resuspended and diluted to an appropriate concentration, and then a certain amount of cell solution was added to each well of a 6-well plate in turn; then, the plate was gently shaken in a cross shape and placed in a cell culture incubator for 24 h. The culture medium in the plate was discarded and washed 2-3 times with PBS. The macrophage strong lysis buffer, protease inhibitors, and phosphatase inhibitors were mixed in a 5 mL EP tube at a ratio of (V = 100:1:1), and 200 μL of macrophage lysis mixture was added to each well. The plate was placed on ice for 30 min, and the cells were scraped off with a cell scraper. The collected cells were placed in a 1.5 mL centrifuge tube and placed on ice for 30 min to fully lyse the cells. After ultrasonic lysis, the cells were centrifuged at 12000 g for 15 min and the supernatant was collected.

[0185] 2.2 Preparation of MRSA Lysate

[0186] Take 20 mL of MRSA bacterial liquid in the logarithmic growth phase and centrifuge it at 8000 rpm / min for 10 minutes; discard the supernatant and freeze it in liquid nitrogen for 10 seconds, then dissolve it in a 37°C water bath, repeat this process 5 times; centrifuge and collect the supernatant for later use.

[0187] 2.3H2S detection

[0188] The test was performed using a hydrogen sulfide detection kit, and the specific steps were carried out according to the instructions. Figure 2 As shown, H2S released from ACR-DM-HS was detectable in macrophage lysates. As the concentration of ACR-DM-HS increased from 5 μM to 20 μM, the H2S concentration increased from 14.8 μM to 26.3 μM. These results demonstrate that ACR-DM-HS has the ability to selectively and autonomously release H2S, demonstrating the potential of this compound for anti-inflammatory effects and promoting tissue repair.

[0189] 3. Photodynamic anti-planktonic bacteria

[0190] 3.1 Bacterial culture

[0191] Transfer the single colony on the solid culture medium to the liquid culture medium and incubate it in a shaker at 37°C for 15 hours to obtain a bacterial suspension; take a certain volume of the bacterial suspension, centrifuge it and discard the supernatant, and then evenly disperse it in sterile PBS as the experimental bacterial solution.

[0192] 3.2 Evaluation of bacterial viability by plate spread counting method

[0193] 1 mL of the prepared experimental bacterial solution was placed in a centrifuge tube and divided into PBS, PBS + Light, ACR-DM-HS (2.5 μM, 5 μM, 10 μM), and ACR-DM-HS + Light (2.5 μM, 5 μM, 10 μM). An appropriate amount of ACR-DM-HS stock solution was then added to the ACR-DM-HS experimental group, while an equal volume of PBS solution was added to the PBS control group. All groups were incubated at 37°C for 10 min and 8 × 10 3 rpm centrifugation for 3 min, the supernatant was removed, and the cells were washed three times with PBS. The bacteria were redispersed in 1 mL of PBS and the PBS+Light and ACR-DM-HS+Light cells were exposed to LED white light (5 mw / cm 2 ) for 30 minutes, while the remaining groups were placed in a dark environment for 30 minutes, and then the bacterial activity was determined and quantified by the plate count method. ACR-DM-HS was incubated with Gram-positive bacteria (S. aureus) and Gram-positive drug-resistant bacteria (methicillin-resistant Staphylococcus aureus MRSA) for 10 minutes and then exposed to light for 30 minutes; ACR-DM-HS was incubated with Gram-negative bacteria (E. coli) for 30 minutes and then exposed to light for 30 minutes. The results are shown in Figure 2. Figure 3 shown.

[0194] Figure 3 Figures A and B show the in vitro photodynamic effects of ACR-DM-HS on Gram-positive bacteria (S. aureus) and Gram-positive resistant bacteria (methicillin-resistant Staphylococcus aureus MRSA). When the concentration of ACR-DM-HS increased from 2.5 μM to 10 μM, the survival rate of S. aureus decreased from 18% to 1.3%, and the survival rate of MRSA decreased from 34.9% to 1.5%. Figure 3 As can be seen in Figure C, the survival rate of E. coli decreased from 32.7% to 3.4%, showing obvious phototoxicity and dose dependence.

[0195] In summary, the experimental results show that ACR-DM-HS not only has obvious phototoxicity to Gram-positive bacteria, but also can efficiently inactivate Gram-negative bacteria under light conditions.

[0196] 3.3 Zeta potential measurement experiment

[0197] Gram-positive bacteria (S. aureus), Gram-positive resistant bacteria (methicillin-resistant Staphylococcus aureus MRSA), and Gram-negative bacteria (E. coli) were incubated with ACR-DM-HS at room temperature in the dark for 10 minutes. After centrifugation, the sample solution was obtained and resuspended in sterile deionized water. The Zeta potential of the bacterial surface was measured using Nano S (ZEN3600). Bacteria without probes under the same conditions were set as blank controls. The results are shown in Figure 2. Figure 4 shown.

[0198] from Figure 4 As can be seen, the Zeta potential of S. aureus in the blank group was -23.8 mV. After incubation with 10 μM ACR-DM-HS for 10 minutes, the Zeta potential on the bacterial surface increased to -16.8 mV, an increase of about 7 units. The Zeta potential of MRSA in the blank group was approximately -24.5 mV. After incubation with 10 μM ACR-DM-HS for 10 minutes, the Zeta potential on the bacterial surface increased to approximately -15.6 mV, an increase of about 9 units. The Zeta potential of E. coli in the blank group was -33.2 mV. After incubation with 10 μM ACR-DM-HS for 30 minutes, the Zeta potential on the bacterial surface increased to approximately -29.3, an increase of about 4 units.

[0199] The results showed that the probe ACR-DM-HS could bind to the plasma membrane of Staphylococcus aureus, MRSA and Escherichia coli, which was consistent with the results of photodynamic therapy against planktonic bacteria.

[0200] 3.4 Bacterial fluorescence imaging

[0201] ACR-DM-HS (10 μM) was incubated with Gram-positive bacteria (S. aureus) in the logarithmic growth phase in the dark for 10 minutes and Gram-negative bacteria (E. coli) in the dark for 30 minutes, and then the bacteria were observed for fluorescence imaging. Figure 5 As shown. Figure 5 It can be seen that ACR-DM-HS shows red fluorescence after interacting with Gram-positive bacteria S. aureus and Gram-negative bacteria E. coli, and the bright field and composite field of the magnified image show that the red fluorescence is mainly concentrated in the bacterial cell membrane and cytoplasm. This is mainly based on the hydrophobic and electrostatic interactions between ACR-DM-HS and the amphiphilic cell membrane and the negatively charged teichoic acid on the membrane, and mainly acts on the bacterial cell membrane.

[0202] The experiment confirmed that ACR-DM-HS acts on Gram-positive and Gram-negative bacteria. This conclusion is consistent with the results of the photodynamic in vitro anti-plankton experiment and the bacterial Zeta potential measurement results.

[0203] 4 In vitro biofilm removal experiments

[0204] 4.1 Crystal violet staining biofilm observation method

[0205] 1000 μL LB culture medium was added to a small dish, and 100 μL of Gram-positive bacteria (Staphylococcus aureus) in the logarithmic growth phase was inoculated. The small dish containing only LB culture medium was used as the blank control group. Different volumes of 5 mM ACR-DM-HS solution prepared in PBS were added to make the final concentration of the probe 20 μM. After mixing and incubation for 30 minutes, the probe was placed under 5 mW / cm 2 Irradiate under white light for 30 minutes. After aspirating the supernatant, wash with PBS 2 to 3 times, fix with 1 mL of methanol for 15 minutes and air dry naturally. Add 1 mL of crystalline ammonium lithospermate solution to each dish and stain at room temperature for 10 minutes. After aspirating the crystalline ammonium lithospermate staining solution in the dish, rinse the excess dye with running water, invert the culture plate on the filter paper to remove the residual water, and dry it in a 37°C oven or cool it at room temperature. After it is completely dry, the biofilm residue can be observed. The results are as follows: Figure 6 shown.

[0206] Figure 6 Figures A and B in the middle are pictures of the small dishes for detecting S. aureus and MRSA biofilms using the crystal violet staining method. It can be seen that there is almost no crystal violet residue in the blank group, while in the dark group of ACR-DM-HS, intact biofilms attached to the bottom of the small dish are stained with crystal violet. Figure 6 In Figure C, the crystal violet staining method was used to detect E. coli biofilm. Less crystal violet residue was observed in the small dish. The results showed that after ACR-DM-HS light treatment, a significant effect of clearing bacterial biofilm was observed, and almost no biofilm residue was observed.

[0207] 4.2 Crystal violet method for detecting residual biofilm OD value

[0208] 100 μL LB culture medium was added to each well of a 96-well polystyrene microplate, and 10 μL of logarithmic growth phase Gram-positive bacteria (Staphylococcus aureus S. aureus), Gram-positive drug-resistant bacteria (methicillin-resistant Staphylococcus aureus MRSA) and Gram-negative bacteria (Escherichia coli E. coli) were inoculated. The wells containing only culture medium were set as blank controls and incubated at 37°C for 36 h. After that, the culture medium was aspirated, and all experimental wells were washed 2-3 times with PBS. Then, different volumes of 5 mM photosensitizer solution prepared in PBS were added to make the final concentration of each probe group 0 μM, 5 μM, 10 μM, 15 μM, and 20 μM. After mixing and incubation for 30 min, the plates were placed under 5 mW / cm 2Irradiate under white light for 30 minutes, aspirate the supernatant and wash with PBS 2 to 3 times, add 100 μL of methanol to each well and fix for 15 minutes, then aspirate the methanol in the culture wells and let it air dry naturally; add 100 μL of 1% crystal violet solution to each well and stain at room temperature for 5 minutes; after aspirating the crystal violet staining solution in the culture wells, rinse the excess dye with running water, invert the culture plate on filter paper to remove residual water, and dry it in a 37°C oven or cool it at room temperature; after drying, add 100 μL of 33% glacial acetic acid solution to each well and incubate it in a 37°C incubator for 30 minutes to dissolve the crystal violet; under the condition of 590 nm, use an enzyme marker to measure the OD value of the solution in the culture wells (this biofilm residue includes biomass components such as the extracellular matrix EPS of the bacterial biofilm and live bacteria). The results are as follows Figure 7 shown.

[0209] Figure 7 Figures A and B in the middle are the results of in vitro photodynamic removal of Gram-positive (S. aureus) and (methicillin-resistant Staphylococcus aureus MRSA) bacterial biofilms by ACR-DM-HS; as the concentration of ACR-DM-HS increased, the residual biofilm in the well plate decreased to varying degrees. When the concentration of ACR-DM-HS increased from 5μM to 20μM, the residual amount of S. aureus biofilm decreased from 75.2% to 7.5%, and the residual amount of MRSA biofilm decreased from 71.9% to 6.2%, showing obvious phototoxicity and dose dependence. Figure 7 As can be seen in Figure C, when the concentration of ACR-DM-HS increased from 5μM to 20μM, the residual amount of E. coli biofilm decreased from 64.8% to 10.1%, showing obvious phototoxicity and dose dependence. The results show that ACR-DM-HS has a significant effect in clearing biofilms.

[0210] 4.3 Biofilm Fluorescence Imaging

[0211] 1 mL of LB culture medium was added to a glass-bottomed dish, and 30 μL of Gram-positive bacteria (MRSA) and Gram-negative bacteria (E.coili) in the logarithmic growth phase were inoculated and incubated at 37°C for 36 hours. The culture medium was then aspirated, and the ACR-DM-HS was incubated with the biofilm and then treated with light and darkness. The dead cell nucleic acid dye Sytox Green was used to observe the photodynamic removal effect of the biofilm by ACR-DMB-HS. The commercially available dead cell nucleic acid dye Sytox Green was used to detect the status of the two bacterial biofilms after the ACR-DM-HS probe was treated with light and darkness. The λ ex =512nm,λ em =520nm-560nm, λ of Sytox Green ex=488nm,λ em =500nm-550nm.

[0212] The results are as follows Figure 8 As shown in the figure, after incubation of ACR-DM-HS with MRSA biofilm and Ecoli biofilm for 30 minutes, all bacterial biofilms showed red fluorescence, while Sytox Green only stained the illumination group, demonstrating that ACR-DM-HS has a significant effect in clearing biofilm biomass (extracellular matrix EPS and live bacterial cell biomass).

[0213] 4.4 Biofilm Scanning Electron Microscopy

[0214] The silicon wafer sterilized by high-pressure steam was placed in a 24-well polystyrene microwell culture plate, 300 μL of LB culture medium was added to each well, and 30 μL of Gram-positive bacteria (MRSA) and Gram-negative bacteria (E.coili) in the logarithmic growth phase were inoculated respectively. The plates were incubated at 37°C for 36 hours. After that, the culture medium was aspirated and ACR-DM-DM-HS with a concentration of 20 μM was incubated with Staphylococcus aureus biofilm for 30 minutes. After that, the plates were exposed to light (30 minutes) and dark. After that, the plates were fixed with 2.5% glutaraldehyde fixative, dehydrated with ethanol gradient, dried naturally, and sprayed with gold by ion sputtering. The morphological changes of the bacterial biofilm were observed. The results are shown in Figure 2. Figure 9 shown.

[0215] from Figure 9 It can be seen that after dark treatment with ACR-DM-HS, MRSA and E. coli in the biofilm were observed to be round and plump, with good morphology, and the bacteria were completely wrapped in the extracellular matrix (EPS) and interconnected; while after light treatment, the bacteria had poor morphology, with obvious wrinkling, collapse, and even leakage, indicating that ACR-DM-HS has a strong biofilm removal ability.

[0216] 5 Biocompatibility test

[0217] 5.1 Hemolysis test

[0218] First, a 2% red blood cell suspension was prepared: 10 mL of fresh mouse blood was placed in a sodium heparin-coated vacutainer tube and gently inverted to mix thoroughly. After mixing, the tube was centrifuged at 1000 rcf / min for 10 minutes. The supernatant was removed, and a quantitative amount of red blood cells (RBCs) was aspirated using a pipette. The tubes were washed three times with sterile phosphate-buffered saline (PBS) (approximately 10 times the volume of RBCs) at 1000 rcf / min for 10 minutes. The RBC suspension was then diluted with sterile phosphate-buffered saline (PBS) to prepare a 2% RBC suspension. To 180 μL of the 2% RBC suspension, 20 μL of the corresponding volume of ACR-DM-HS stock solution was added to yield final concentrations of 2.5, 5, 10, and 20 μM. For the blank and positive controls, 20 μL of PBS and Triton X-100 solvent were added, respectively. Three replicate wells were prepared for each group. The tubes were gently mixed, incubated in a 37°C waterbath for 1 hour, and then centrifuged at 1200 rcf / min for 5 minutes. The supernatant was collected and the ultraviolet absorption at 545 nm was detected by enzyme labeling. The hemolysis percentage of RBC was calculated as follows:

[0219] Hemolysis percentage = (OD sample - OD negative control) / (OD positive control - OD negative control) × 100%

[0220] The results are as follows Figure 10 As shown, a large number of erythrocytes in the positive control group (+) ruptured and the solution was dark red, with a hemolysis rate as high as 100%; the solution in the blank control group was relatively clear, and the liquid in the experimental group was clearer after adding the photosensitizer ACR-DM-HS. Even when the concentration was increased to 20 μM, the liquid in the 96-well plate remained clear, and the erythrocyte hemolysis rate was less than 5%, which was significantly different from the positive control group.

[0221] 5.2 Cytotoxicity assay

[0222] 5.2.1 Cell recovery

[0223] Thaw the cells taken out from -80°C environment in a water bath (37°C), transfer the solution into a centrifuge tube containing complete culture medium (DMEM, 1 mL), and centrifuge at low speed (800 rpm, 3 min); discard the supernatant, resuspend the cells in fresh DMEM, and then evenly transfer them into a culture flask containing DMEM (5 mL) for culture (37°C, 5% CO2).

[0224] 5.2.2 Cell culture and passaging

[0225] During the cell culture process, observe cell morphology promptly. When the cell density reaches approximately four-fifths, passage the cells. First, discard the old DMEM and wash the cells twice with PBS. Then, add trypsin (1 mL) to digest the cells. Stop digestion when the cells become round and partially suspended. Transfer the digested cells to a centrifuge tube and centrifuge (1000 rpm, 5 min). Discard the supernatant, resuspend the cells in fresh DMEM, and transfer them to a culture flask containing 5 mL of DMEM for culture (37°C, 5% CO2).

[0226] 5.2.3 Cell plating

[0227] Resuspend the digested cells and dilute the cells to an appropriate concentration according to the number of wells plated. Then add a certain amount of cell solution to each well in turn; then gently shake in a cross shape and place in a cell culture incubator for 24 hours.

[0228] 5.2.4 Testing the dark toxicity and phototoxicity of ACR-DM-HS on HUVEC human umbilical vein endothelial cells under anti-biofilm conditions (CCK8)

[0229] Cells in good growth condition in the logarithmic growth phase were taken, digested with trypsin, and centrifuged to sediment the cells. The supernatant was discarded, the cells were resuspended in complete culture medium, and the cell suspension was diluted to 50,000 cells / mL; 100 μL of the above cell suspension was added to each well of a 96-well plate to make the number of cells in each well approximately 5,000. The plate was then placed in a 37°C cell culture incubator containing 5% CO2 for 24 h to allow the suspended cells to reattach to the wall and to have approximately 40,000 cells per well. The culture medium was then replaced with 100 μL of fresh ACR-DM-HS culture medium containing different concentrations. The incubation time was 30 min, and the illumination condition was set to white light at 5 mW / cm 2 After 30 minutes of illumination, the cells were placed in a 37°C, 5% CO2 incubator for 24 hours. 100 μL of CCK8 mixed solution (CCK8; culture medium = 1:9) was added to each well, and the absorbance of each well at 450 nm was measured using a microplate reader (OD). 450nm The formula for calculating the relative cell survival rate is as follows:

[0230] Relative cell viability = [OD(treated cells)-OD(control cells)] / OD(treated cells)

[0231] Wherein, OD (treated cells) is the absorbance of the wells with cells, CCK8 solution and drug solution; OD (control cells) is the absorbance of the wells with culture medium and CCK8 solution but no cells; wherein, the measured OD value is the average value measured by 3 independent parallel samples, and the results are expressed as the mean (M) ± standard deviation (SD).

[0232] The results are as follows Figure 11 As shown in the figure, under dark conditions, the cell survival rate of ACR-DM-HS was over 100% in the concentration range of 0-20 μM, indicating that the probe had good biocompatibility and cell proliferation ability; under light conditions, ACR-DM-HS was under 5 mW / cm 2 After continuous white light irradiation for 30 minutes, the cell survival rate exceeded 100% with the increase of probe concentration, indicating that ACR-DM-HS has good biocompatibility and potential cell proliferation effect on normal cells at the concentration required to clear biofilm.

[0233] 5.2.5 Cell scratch assay

[0234] Use a Maker pen to evenly draw lines on the back of a 6-well plate, approximately every 0.5-1 cm, across the holes, with at least 5 lines across each well; take L929 cells in the logarithmic growth phase, digest them with 0.25% trypsin-EDTA, make a single cell suspension with DMEM complete medium containing 10% FBS, and then inoculate them into a 6-well plate. Use a 200μL pipette tip to reach as low as possible to the horizontal lines on the back of the 6-well plate. Wash the cells 3 times with PBS, administer 20μMACR-DM-HS and NaHS respectively, and continue incubation; select appropriate time points to take pictures under a fluorescence inverted microscope. The results are as follows Figure 12 shown.

[0235] from Figure 12 As can be seen, after 24 h of co-incubation, the cells in the ACR-DM-HS group first achieved 100% coverage of the scratch space, and cell proliferation was significantly enhanced compared with the PBS group, confirming that the compound ACR-DM-HS prepared in Example 6 had good cell proliferation potential.

[0236] 5.2.6 In vitro angiogenesis assay

[0237] The angiogenesis-promoting effect of ACR-DM-HS was studied by in vitro angiogenesis assay.

[0238] HUVEC cells were starved and cultured overnight in 0.2% DMEM medium, washed twice with DPBS, and digested for later use. ACR-DM-HS and NaHS groups were set up, and 20 μM ACR-DM-HS or NaHS was added to each well. An equal amount of DMEM was used as a control. Matrigel (150 μL / well) dissolved at 4°C was added to a 48-well plate and solidified at 37°C for 30 minutes. The diluted HUVEC cells were inoculated into the above 48-well plate (300 μL / well) and cultured at 37°C, 5% CO2 for 8 hours. The tubular formation of HUVEC was observed under an inverted optical microscope. The results are shown in Figure 2. Figure 13 shown.

[0239] from Figure 13 It can be seen that after co-incubation with ACR-DM-HS for 2h, 6h and 8h, tubular networks of HUVECs were formed, and the tubular networks formed by cells in the ACR-DM-HS group were the most abundant, indicating that the production of H2S positively triggered the angiogenic potential of HUVECs.

[0240] 6 In vivo biofilm removal experiment in diabetic rats

[0241] 6.1 Preparation of diabetic rat model

[0242] Three-month-old Wistar rats (200-250g) were adaptively housed for one week and subjected to this experiment after their activity and physical signs were normal. Adaptively housed rats were fasted for 12 hours but not water, and then intraperitoneally injected with 65mg / kg of streptozotocin (STZ). Because STZ is easily inactivated, it was quickly weighed according to the rat's total weight and placed in a dry, sterile bottle. The bottle was wrapped with tin foil and placed in an ice bath. Immediately before use, a 1% solution was prepared in 0.1mol / L, pH 4.4 sodium citrate buffer. Because STZ is unstable and easily decomposed, it should be prepared immediately before use. A diabetic rat model was successfully established when the blood glucose level was ≥16.7mmol / L. After blood glucose stabilized for one week, a wound model was prepared.

[0243] 6.2 Wound Model Preparation

[0244] Rats with a blood glucose level ≥16.7 mmol / L were selected, their backs shaved, and their weights weighed. Anesthesia was performed by intraperitoneal injection of 50 mg / kg sodium pentobarbital. Under sterile conditions, a circular hole 1 cm in diameter was made on each side of the spine in the middle of the rat's back using a special puncher. 50 μL of bacterial suspension (2 × 10 8 CFU / mL) were inoculated on each wound and covered with sterile gauze for 1 day until the wound infection model was successful.

[0245] Rats were randomly divided into 4 groups (Control group, ACR-DM-HS group, ACR-DME light group, ACR-DM-HS light group), and PBS, ACR-DME or ACR-DM-HS (20 μM) were applied to the wounds. 30 minutes after administration, the light group was illuminated with LED white light (20 mW / cm 2 ) irradiated the wound for 30 minutes, while the remaining groups were kept in the dark as controls. After treatment, the rats were moved to a dark room and housed in the dark. The day of treatment was designated D0. Skin tissue from the wound surface was obtained in batches on D1, D3, D7, and D12, and ground and smeared. Tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) levels in serum samples from rats in each group on day 3 were measured using ELISA kits. Skin tissues from rats on day 12 were subjected to H&E, Masson staining, and immunohistochemistry to examine treatment differences and healing outcomes.

[0246] The rats were weighed and recorded. Based on their weight, euthanasia was performed by intraperitoneal injection of 0.06-0.1 mL of 20% sodium pentobarbital solution per 10 g of body weight. After the rats were confirmed dead, the infected wound site was lifted with forceps, and the intact infected skin site was cut off with scissors and placed in physiological saline or paraformaldehyde for later use. Finally, the rat carcasses were collected and processed uniformly.

[0247] 6.3 Results of in vivo biofilm removal experiments in diabetic rats

[0248] The results of biofilm removal in diabetic rats were as follows Figure 14 As shown, Figure 14 As shown in Figures A and C, the wounds of rats in all four groups developed severe suppuration on the first day, demonstrating the successful establishment of a rat inflammatory model of MRSA infection. On the third day, the wounds in the control group remained large. By the seventh day, due to the rats' autoimmune response, the wounds slowly healed, with the remaining wounds accounting for over 30% of the rats by day 12. Rats treated with ACR-DM-HS showed similar infection status to the control group on day 1, but showed slightly better infection on day 12. This may be because the H₂S released by ACR-DM-HS promotes skin tissue repair and accelerates wound healing. Compared to the first two groups, the infection in the ACR-DME+L group was milder. In vivo experiments demonstrated that the photosensitizer ACR-DME exhibits robust photodynamic antimicrobial activity. However, due to the ROS storm and inflammatory response, the wounds did not fully heal, with the remaining wounds accounting for over 15% of the rats by day 12. Compared with the ACR-DME+L group, the wounds of rats treated with ACR-DM-HS+L had completely healed on the 12th day, indicating that the ACR-DM-HS+L group not only had a better photodynamic antibacterial effect, but also the H2S released after interacting with cells had a better anti-inflammatory effect and promoted wound healing.

[0249] The number of viable bacteria in the infected tissues at 1, 3, 7, and 12 days after treatment was further determined by the plate spread method to evaluate the bactericidal effect of ACR-DM-HS and ACR-DME in vivo. Figure 14 As shown in Figures B and D, on day 1, the control group had a large number of bacteria, with the number reaching 9.6×10 6 Over time, due to the rats' own immune system, the bacterial count gradually decreased. A large number of bacteria remained on the 7th day, and the bacteria on the plates had not been completely eliminated by the 12th day. Compared to the control group, the ACR-DM-HS group also had a large number of bacteria in the infected tissues, with a similar number to that of the control group. The ACR-DME+L group had a significantly reduced bacterial count, with approximately 40% remaining on the first day after treatment. The number of bacteria on the plates decreased rapidly by the 7th day, falling below 1%, and was nearly eliminated by the 12th day. Compared to the ACR-DME+L group, the ACR-DM-HS+L group had fewer bacteria in the infected tissues on both days 1 and 3, with bacteria almost eliminated by the 7th day and completely eliminated by the 12th day.

[0250] 6.4 Determination of TNF-α and IL-6 levels in rat serum on day 3 after treatment

[0251] The levels of tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) in rat serum were determined using ELISA kits. Figure 15 As shown in the results, the TNF-α and IL-6 levels in the ACR-DM-HS+L group were significantly lower than those in the ACR-DME+L group and the Control group. The results showed that the ACR-DM-HS+L group not only reduced the excessive ROS produced by photodynamic therapy by producing H2S, but also exerted the anti-inflammatory effect of H2S itself, effectively eliminating the inflammation in diabetic rats.

[0252] 6.5 Histological H&E staining and Masson analysis of tissues at the infected site on day 12 after treatment

[0253] The therapeutic effect of ACR-DM-HS on bacterial abscess infection was further evaluated by performing histological H&E staining and Masson analysis on the tissues of the infected site on the 12th day after treatment. Figure 16 As shown. Figure 16As shown in Figures A and B, the control and ACR-DM-HS groups showed epidermal loss and varying degrees of necrosis of dermal collagen fibers, with loss of collagen fiber structure, pale cytoplasm, and pyknosis or loss of nuclei. Necrotic areas were accompanied by fibrous tissue proliferation, primarily composed of spindle-shaped fibrocytes and oval-shaped fibroblasts with fine collagen fibers. Necrotic areas were also infiltrated with numerous inflammatory cells, primarily neutrophils with lobed nuclei. Extensive fibrous proliferation was observed in the ACR-DME+L and ACR-DM-HS+L groups. The ACR-DM-HS group showed more collagen fiber proliferation and hair follicle structures, while the ACR-DME+L group showed inflammatory cell infiltration and focal collagen fiber proliferation in the necrotic areas. Of the four animal experimental groups, the ACR-DM-HS+L group showed the best wound repair, followed by the ACR-DME+L group. The control group showed the worst wound repair, and the ACR-DM-HS group showed relatively poor wound repair. Figure 16 As shown in Figure C, the area of ​​fibrous tissue in the skin tissue was quantitatively analyzed by Masson. The proportion of fibrous tissue area in the Control group was 16.56%, the proportion in the ACR-DM-HS group was 18.91%, the proportion in the ACR-DME+L group was 29.58%, and the proportion in the ACR-DM-HS+L group was 48.03%. The results showed that the ACR-DM-HS+L group not only had a good photodynamic antibacterial effect, but also had a good effect in promoting wound repair due to the release of H2S.

[0254] 6.6 Immunohistochemical analysis of infection sites on day 12 after treatment

[0255] Further evaluation of the therapeutic effect of ACR-DM-HS on infected tissues. Figure 17 As shown in the results of quantitative analysis, the levels of TNF-α and IL-6 in the control and ACR-DME+L groups were significantly higher than those in the ACR-DM-HS+L group. Platelet-endothelial cell adhesion molecule (CD31) labeling revealed more formed blood vessels in the ACR-DM-HS+L group, with significant expression of vascular endothelial growth factor (VEGF) and transforming growth factor-β (TGF-β). These results demonstrate that the ACR-DM-HS+L group not only exhibits photodynamic antibacterial effects but also effectively reduces inflammatory responses at the infection site and promotes wound healing and tissue repair by producing H2S.

[0256] 6.7 In vivo biosafety analysis

[0257] like Figure 18As shown in the results, there were no significant changes in the biochemical indicators (ALT, AST, CREA-S, UREA) and the histological structure of major organs (heart, liver, spleen, lung, and kidney) between the treatment group and the untreated group. The results showed that ACR-DM-HS has good biosafety in vivo.

[0258] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A photosensitizer having hydrogen sulfide releasing properties, characterized in that: The photosensitizer has a structure as shown in Formula II: ； Among them, R1 is , Where R is methyl or ethyl; R2 is , Where n=1.

2. The photosensitizer having hydrogen sulfide releasing performance according to claim 1, characterized in that: The specific structure is as follows: 、 。 3. The method for preparing the photosensitizer having hydrogen sulfide releasing performance according to any one of claims 1 to 2, characterized in that: The following synthetic pathways are included: (1) Dissolve 2-thiopheneacetonitrile and N-bromosuccinimide in a solvent and react at room temperature for 22-26 hours to obtain compound B; (2) Compound B, 4-pyridine boronic acid, palladium catalyst, and inorganic base are added to a solvent and reacted at 60-70°C under an inert atmosphere for 10-14 hours to obtain compound J; (3) Compound J and compound K are dissolved in a solvent, an inorganic base is added, and the mixture is reacted at room temperature for 22-26 hours to obtain compound L; (4) Dissolving 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione and compound G in a solvent and reacting at room temperature for 8-12 h to obtain compound H; (5) Compound L and compound H are dissolved in a solvent and reacted at 50-70°C for 22-26 hours to obtain a compound represented by Formula II. The synthetic route is as follows: 。 4. The method for preparing a photosensitizer having hydrogen sulfide releasing performance according to claim 3, characterized in that: The solvent in step (1) is acetone, N,N-dimethylformamide, methanol, ethanol, tetrahydrofuran, acetonitrile, cyclohexane or toluene; the solvent in step (2) is an aqueous solution of tetrahydrofuran or an aqueous solution of dioxane; the volume ratio of tetrahydrofuran to water in the aqueous solution of tetrahydrofuran is 1 to 15:1; the volume ratio of dioxane to water in the aqueous solution of dioxane is 1 to 15:1; the solvent in step (3) is methanol, ethanol, n-propanol, n-butanol, isopropanol or tert-butanol; the solvent in step (4) is N,N-dimethylformamide, acetonitrile or tetrahydrofuran; the solvent in step (5) is N,N-dimethylformamide, acetonitrile, tetrahydrofuran or acetone.

5. The method for preparing a photosensitizer having hydrogen sulfide releasing performance according to claim 3, characterized in that: The molar ratio of the 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione to compound G is 1:1-10.

6. The method for preparing a photosensitizer having hydrogen sulfide releasing performance according to claim 3, characterized in that: The palladium catalyst is bis(tri-tert-butylphosphine)palladium, tetrakis(triphenylphosphine)palladium, palladium acetate, palladium pivalate, bis(triphenylphosphine)palladium chloride, [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride or tris(dibenzylideneacetone)dipalladium; the inorganic base is barium hydroxide, sodium hydroxide, potassium hydroxide, potassium phosphate, sodium carbonate, sodium bicarbonate or potassium carbonate.

7. The method for preparing a photosensitizer having hydrogen sulfide releasing performance according to claim 3, characterized in that: The molar ratio of compound L to compound H is 1:1-10.

8. Use of the photosensitizer having hydrogen sulfide-releasing properties according to any one of claims 1 to 2 in the preparation of photodynamic antibacterial drugs.

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