A compound with type I reactive oxygen species generating performance, its synthesis method and applications

Abstract

The present invention discloses a compound with type I reactive oxygen species generation performance, its synthesis method and application. The general structural formula of the compound is as follows: Using a five-membered heteroaromatic acrylonitrile as the backbone, an electron acceptor pyridinium salt and an electron donor substituted aniline are introduced. By shortening the distance between the electron donor and the electron acceptor, the molecular redox potential is adjusted, and an intramolecular D-π-A system is constructed to significantly reduce the energy level difference between the singlet state and the triplet state, and accelerate the ISC process, reducing the energy difference from the excited triplet state to the ground state to be less than the energy required to generate singlet oxygen, thereby improving the type I ROS generation performance of the photosensitizer. The process of generating reactive oxygen species by this compound has less dependence on oxygen, and the modification of functional groups increases its penetration ability into stubborn biofilms, and it has good biofilm clearance ability in anoxic environments, effectively solving the problems of high oxygen dependence and insufficient penetration existing in existing compounds during use.

Description

Technical Field

[0001] The present invention belongs to the technical field of photosensitizers (PSs), and particularly relates to a compound with type I reactive oxygen species generating performance, a synthesis method thereof, and an application thereof. Background Art

[0002] Pathogenic bacteria resist the harsh external environment by forming biofilms, hinder the penetration efficiency of drugs, reduce the therapeutic effect of antibiotics, thereby increasing the dosage of drugs, and further generating persistent drug resistance. As a result, bacterial infectious diseases have become the second leading cause of death in humans after ischemic heart disease. The biofilm of pathogenic bacteria is a structural bacterial community composed of bacterial cells and a large amount of extracellular polymers. Among them, the extracellular matrix EPS contains 70%-80% extracellular polysaccharides and proteins. This structure constructs a good pathogenic environment for bacteria, provides a protective barrier and an anoxic environment for bacterial proliferation. Biofilms are also the main reason for the difficulty in radically curing bacterial diseases, and have strong resistance to antibiotics, harsh environments and host immune defense mechanisms. At present, clinical methods for removing biofilms adopt regular debridement and local antibacterial methods. However, regular wound cleaning brings great pain to patients and even damages tissues, while local antibacterial relies on a large amount of antibiotics and has disadvantages such as invasiveness and drug resistance. Therefore, it is of great significance to find an antibacterial method that can efficiently break stubborn biofilms and has no drug resistance.

[0003] In recent years, photodynamic therapy (PDT) has shown extensive application potential in killing bacteria due to its advantages such as non-invasiveness, no drug resistance, and small side effects. PDT is based on the ground-state electrons of photosensitizers (PSs) absorbing the energy of the excitation light and transitioning from the ground state to the excited state. After intersystem crossing to reach the excited triplet state, reactive oxygen species are generated through two pathways, which can oxidize various biomolecules such as lipids, proteins, and nucleic acids, thereby inactivating pathogenic bacteria. At the same time, when the excited electrons return from the first excited state to the ground state, fluorescence will be emitted, which can be used for fluorescence imaging. Photosensitizers can be used for both fluorescence imaging and photodynamic therapy, which provides a good basis for constructing a multifunctional platform integrating the diagnosis and treatment of bacterial infections.

[0004] Although a large number of AIE photosensitizers have been reported, currently available photosensitizers are generally type II photosensitizers that generate singlet oxygen through energy transfer to oxygen. They have poor binding performance with biofilms and high dependence on oxygen. The low permeability and hypoxic microenvironment of biofilms limit the application of these photosensitizers. Compared with type II photosensitizers with high oxygen dependence, type I photosensitizers have low reactive oxygen dependence, and the generated hydroxyl radicals are considered to be the most lethal reactive oxygen species in biology, and have excellent potential for dealing with the hypoxic conditions of bacterial biofilms. However, existing type I photosensitizers also have the problem of insufficient penetration for stubborn biofilms, which affects the bactericidal effect on bacteria. Summary of the Invention

[0005] In view of the above problems in the prior art, the present invention provides a compound with type I reactive oxygen species generating performance, its synthesis method and application. The process of generating reactive oxygen species by this compound has less dependence on oxygen, has good biofilm binding property in an anoxic environment, and can effectively solve the problem of high oxygen dependence of existing compounds during use. In addition, this compound is modified with functional groups, effectively improving the poor penetrability of existing photosensitizers, and can successfully penetrate the dense extracellular matrix to eradicate biofilms.

[0006] To achieve the above object, the technical solution adopted by the present invention to solve its technical problems is:

[0007] A compound with type I reactive oxygen species generating performance, its general chemical structure formula is as follows:

[0008]

[0009] Among them, R1 is: R` is an alkyl group with C1-C 12 and

[0010] R2 is an alkyl group with C1-C 10 and

[0011] Y is O or S.

[0012] The preparation method of the above-mentioned compound with type I reactive oxygen species generating performance includes the following steps:

[0013] (1) Add compound A and N-bromosuccinimide to the first solvent, stir at room temperature until the reaction is complete, then distill off the organic solvent under reduced pressure, and purify the obtained crude product by silica gel column chromatography to obtain compound B;

[0014] (2) Add compound B, compound C, a palladium catalyst and an inorganic base to the second solvent, heat under reflux under the protection of nitrogen N2 until the reaction is complete, then filter, wash and dry, collect the organic phase and remove the organic solvent, and purify the obtained crude product by silica gel column chromatography to obtain compound D;

[0015] (3) Add compound D, compound E and an inorganic base to ethanol, stir at room temperature until the reaction is complete, then filter, wash and dry, collect the organic phase and remove the organic solvent to obtain compound F, and then quaternize compound F to obtain a series of compounds with high-efficiency type I reactive oxygen species generating performance;

[0016] Among them, the structural formula of compound A is Y is O or S;

[0017] The structural formula of Compound B is Y is O or S;

[0018] The structural formula of Compound C is

[0019] The structure of Compound D is

[0020] The structure of Compound E is Among them, R1 is: R` is an alkyl group of C1-C 12 and

[0021] The structure of Compound F is

[0022] Furthermore, in step (1), the first solvent is N,N-dimethylformamide, ethanol, acetonitrile, isopropanol, n-butanol or tert-amyl alcohol; the eluent in the purification process is ethanol, methanol, dichloromethane, ethyl acetate or chloroform.

[0023] Furthermore, in step (1), the mass ratio of Compound A to N-bromosuccinimide is 1:1 - 1.2.

[0024] Furthermore, in step (2), the palladium catalyst is tetrakis(triphenylphosphine)palladium, bis(triphenylphosphine)palladium chloride, [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium, tris(dibenzylideneacetone)dipalladium or palladium acetate.

[0025] Furthermore, in step (2), the molar ratio of Compound B, Compound C, the palladium catalyst and the inorganic base is 1.0 - 1.5:1.0 - 8.0:1.0:0.01 - 0.05:10.

[0026] Furthermore, the inorganic base in steps (2) and (3) is sodium tert-butoxide, potassium tert-butoxide, sodium carbonate, potassium carbonate, cesium carbonate, potassium phosphate or barium hydroxide.

[0027] Furthermore, in step (2), the second solvent is a mixed solution of tetrahydrofuran and water with a volume ratio of 5 - 10:1, a mixed solution of toluene, ethanol and water with a volume ratio of 6 - 10:1:1 or a mixed solution of dioxane and water with a volume ratio of 5 - 10:1.

[0028] Furthermore, in step (3), the molar ratio of Compound D, Compound E and the inorganic base is 1.0 - 1.5:1.0 - 1.5:1.0.

[0029] Application of the above-mentioned compound with type I reactive oxygen species generating performance in fluorescence imaging and photodynamic sterilization.

[0030] The beneficial effects produced by the present invention are as follows:

[0031] 1. In this application, acrylonitrile, an electron acceptor, is connected to heterocyclic thiophene or furan ring, an electron donor, and pyridinium salt is introduced as the molecular structure backbone. By adjusting the positions of the electron acceptor cyano group and the electron donor five-membered aromatic heterocycle, the conjugated system is adjusted, the distance between the electron donor and the electron acceptor is shortened, and the energy level difference between the singlet state and the triplet state is significantly reduced by constructing an intramolecular D-π-A system, and the ISC process is accelerated, improving the performance of photosensitizer to generate type I ROS. Since electron-rich heterocyclic thiophene or furan and intramolecular charge transfer halogen anions are introduced into the D-A system, charge transfer is enhanced. Finally, by introducing different electron-donating groups R1 and connecting the electron-rich and electron-deficient sites, the distance between D and A is shortened, the reduction potential is adjusted, and the intersystem crossing efficiency is improved, effectively realizing the generation of type I reactive oxygen species by the photosensitizer.

[0032] The R1 group is different electron-donating groups, and the redox potential of the molecule is adjusted by using the difference in electron-donating ability, so as to enhance the electron transfer pathway and make the type of reactive oxygen species tend to be type I. The R2 group increases the penetration ability and targeting ability of the molecule to bacterial biofilms. Bacterial biofilms are mainly composed of 50%-90% of bacterial extracellular matrix (EPS) and 10%-50% of bacterial cells. Among them, EPS contains a large amount of negatively charged peptidoglycan, lipopolysaccharide and protein (pH>pL). Therefore, the penetration ability can be adjusted by different positive charges in the R2 group. In order to cope with 70%-80% of the extracellular polysaccharides with encapsulation effects in EPS, the hydrophilicity and hydrophobicity can be adjusted by adjusting the aromatic ring and alkyl chain in the R2 group to improve the molecular penetration ability; and the boric acid group can be used to react with polysaccharides to form borate bonds to bind the molecule to extracellular polysaccharides. By changing the electron-donating group at the R1 position and the hydrophilicity, hydrophobicity and targeting properties at the R2 position on the molecular structure backbone, a series of compounds with the performance of generating type I reactive oxygen species are obtained. This type of compound has the advantages of high type I reactive oxygen species yield and high photo-stability.

[0033] The compounds of the present invention efficiently generate type I reactive oxygen species. The low dependence on oxygen during the generation process of type I reactive oxygen species enables it to more effectively cope with the hypoxic conditions of biofilms. In order to increase its penetration ability to stubborn biofilms, a charge regulation and polysaccharide targeting strategy is designed at the molecular tail, and a series of type I photosensitizers are synthesized. It not only shows high efficiency in killing planktonic bacteria and cleaning bacterial biofilms in vitro, but also has a more efficient and rapid biofilm cleaning effect on the medical catheter with biofilm implanted in the abdomen of mice in vivo, and has a good effect on promoting the healing of tissue inflammation. This work not only provides a new strategy for the efficient removal of biofilms, but also provides a more systematic guidance for the rational design of the next generation of advanced antibacterial materials. It also provides a new idea for the treatment of diseases under hypoxic conditions, such as the tumor hypoxic microenvironment, etc.

[0034] The application potential of such photosensitizers in both fluorescence imaging and photodynamic therapy can not only achieve detection and treatment simultaneously, but also visually monitor the entire treatment process, thus providing us with more accurate and practical information and optimizing and improving the treatment effect, so as to realize the visual detection and precise treatment of bacterial biofilm infections. Brief Description of the Drawings

[0035] Figure 1 The figure shows the synthetic route diagram of the compound with type I reactive oxygen species generation performance of the present invention;

[0036] Figure 2 The figure shows the photophysical properties of the compounds prepared in Examples 1, 2, 3, and 4 in the solvent DMSO; Figure 2 A is the UV-vis absorption spectrum diagram; Figure 2 B is the fluorescence emission spectrum diagram;

[0037] Figure 3 The figure shows the test of the photosensitizers prepared in Examples 1, 2, 3, and 4 with the indicator H2DCF-DA under different illumination times and the change diagram of the fluorescence intensity (I / I0) at 525 nm;

[0038] Figure 4 The figure shows the test diagram of the reactive oxygen species type of the photosensitizer; Figure 4 As shown in A, the 1 O2 yield test diagram of the photosensitizers prepared in Examples 1, 2, 3, and 4, where I0 is the initial absorbance of ABDA at 378 nm and I is the absorbance of ABDA at 378 nm at different irradiation times; Figure 4 As shown in B, the ·OH yield test diagram of the photosensitizers prepared in Examples 1, 2, 3, and 4, where I0 is the initial emission intensity of HPF at 515 nm and I is the emission intensity of HPF at 515 nm at different irradiation times;

[0039] Figure 5 The figure shows the plate coating diagrams and bacterial counting diagrams of the photosensitizers prepared in Examples 1, 2, 3, and 4 with Gram-positive bacteria (Staphylococcus aureus S. aureus) after dark and light treatments respectively;

[0040] Figure 6 The figure shows the plate coating diagrams and bacterial counting diagrams of the photosensitizers prepared in Examples 1, 2, 3, and 4 with drug-resistant bacteria (MRSA) after dark and light treatments respectively;

[0041] Figure 7 The figure shows the plate coating diagrams and bacterial counting diagrams of the photosensitizers prepared in Examples 1, 2, 3, and 4 with Escherichia coli (E. coli) after dark and light treatments respectively;

[0042] Figure 8 The figure shows the change diagrams of the Zeta potential on the surface of bacteria after the photosensitizers ACR-DM, ACR-DME, ACR-DMB, and ACR-DMP prepared in Examples 1, 2, 3, and 4 were incubated with Gram-positive bacteria (S. aureus), Gram-positive drug-resistant bacteria (MRSA), and Gram-negative bacteria (E. coli) for 10 min respectively;

[0043] Figure 9 The figure shows the fluorescence imaging diagrams of bacteria after the photosensitizers ACR-DM, ACR-DME, ACR-DMB, and ACR-DMP prepared in Examples 1, 2, 3, and 4 were incubated with Gram-positive bacteria (S. aureus) and Gram-negative bacteria (E. coli) for 10 min respectively;

[0044] Figure 10 The figure shows the experimental petri dish diagrams and the statistical charts of the remaining amount of biofilm for the determination of the removal of bacterial biofilms by the photosensitizers prepared in Examples 1, 2, 3, and 4 against 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) by the crystal violet staining method;

[0045] Figure 11 The figure shows the 3D layer scan results of fluorescence imaging for the removal of bacterial biofilms by the photosensitizers prepared in Examples 1, 2, 3, and 4 against Gram-positive bacteria (Staphylococcus aureus S. aureus);

[0046] Figure 12 The figure shows the SEM morphology analysis diagrams for the removal of bacterial biofilms by the photosensitizers ACR-DMB and ACR-DMP prepared in Examples 3 and 4 against Gram-positive bacteria (Staphylococcus aureus S. aureus);

[0047] Figure 13 The figure shows the statistical charts of cell survival rates measured after the photosensitizers prepared in Examples 1, 2, 3, and 4 were incubated with 3T3 cells and treated with darkness and light respectively; among them, Figure 13 A shows ACR-DM, Figure 13 B shows ACR-DME, Figure 13 C shows ACR-DMB, Figure 13 D shows ACR-DMP;

[0048] Figure 14 The figure shows the rupture and hemolysis rates of red blood cells before and after photodynamic therapy with the photosensitizers ACR-DMB and ACR-DMP prepared in Examples 3 and 4; Figure 14 A shows ACR-DMB, Figure 14 B shows ACR-DMP;

[0049] Figure 15 Shown is the diagram of the clearance of the biofilm of the photosensitizer ACR-DMP prepared in Example 4 against Gram-positive drug-resistant bacteria (methicillin-resistant Staphylococcus aureus MRSA) implanted in vivo and the wound infection healing situation in the catheter; Figure 15 A is the diagram of the clearance of the MRSA biofilm in the catheter implanted in vivo, Figure 15 B is the diagram of the ultrasonic coating plate of the catheter implanted in vivo, Figure 15 C is the diagram of the wound healing situation at the catheter implantation site of the biofilm, Figure 15 D is the diagram of the wound grinding coating plate, Figure 15 E is the statistical chart of the colony survival rate of the ultrasonic coating plate of the catheter implanted in vivo, Figure 15 F is the statistical chart of the colony survival rate of the wound grinding coating plate;

[0050] Figure 16 Shown is the diagram of the results of H&E staining analysis and immunofluorescent protein labeling imaging analysis of the wound at the catheter implantation site; Figure 16 A is the diagram of the H&E staining result, Figure 16 B is the immunofluorescent protein imaging diagram (DAPI ultraviolet excitation wavelength 330 - 380nm, emission wavelength 420nm, emitting blue light; FITC excitation wavelength 465 - 495nm, emission wavelength 515 - 555nm, emitting green light. CY3 excitation wavelength 510 - 560, emission wavelength 590nm, emitting red light), Figure 16 C is the statistical chart of the immunoprotein optical density analysis of each in-vivo experimental group. Detailed implementation manners

[0051] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention, that is, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments.

[0052] Therefore, the following detailed description of the provided embodiments of the present invention is not intended to limit the scope of the claimed present invention, but merely represents selected embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0053] It should be noted that relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprising", "including" or any other variant thereof are intended to cover non-exclusive inclusion, such that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or elements inherent to such process, method, article or device. Without further limitation, an element defined by the statement "comprising an..." does not exclude the presence of additional identical elements in the process, method, article or device comprising the element.

[0054] The features and performance of the present invention will be further described in detail below in conjunction with embodiments and the accompanying drawings.

[0055] Example 1

[0056] A compound having type I reactive oxygen species generating performance (labeled ACR-DM), and its preparation method includes the following steps:

[0057] The reaction formula of this step is as follows:

[0058]

[0059] Compound B: 7.58 g of N-bromosuccinimide (40.2 mmol) was added to 30 mL of an N,N-dimethylformamide (DMF) solution containing 2-thiopheneacetonitrile (5 g, 40.0 mmol). After the mixture was stirred at room temperature for 12 h, DMF was removed by distillation under reduced pressure. The crude product was separated by silica gel column chromatography and eluted with petroleum ether / ethyl acetate (V / V = 8:1) to obtain 6.79 g of a brownish-black liquid with a yield of 84%;

[0060] Compound D: 202 mg of Compound B (1 mmol), 160 mg of 4-pyridineboronic acid (1.3 mmol), 1380 mg of K2CO3 (10 mmol) and 5.6 mg of Pd(PPh3)4 were added to a 100 mL two-necked round-bottom flask equipped with a condenser. Under N2 protection, 20 mL of tetrahydrofuran (THF) and 3 mL of water were added. The mixture was stirred and heated to reflux overnight. After the reaction solution was cooled to room temperature, most of the THF was removed by distillation under reduced pressure. It was extracted three times with 25 mL of dichloromethane (DCM), the organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, the solvent was removed by distillation under reduced pressure, and purified by silica gel column chromatography. The eluent was DCM / ethyl acetate (V:V = 10:1) to obtain 144.1 mg of a brown solid with a yield of 72%. 11H NMR (400 MHz, CDCl3) δ. 8.60 (d, J = 4.0 Hz, 2H), 7.41 (d, J = 4.0 Hz, 2H), 7.37 (d, J = 3.2 Hz, 1H), 7.086 (d, J = 2.8 Hz, 1H), 3.95 (s, 2H), 4.24 (s, 3H), 4.18 (s, 3H). 13 13C NMR (100 MHz, CDCl3) δ. 150.6, 141.9, 140.8, 132.9, 128.7, 125.6, 119.8, 116.5, 19.1.

[0061]

[0062] ACR-DM: 600 mg of compound D (1.0 mmol), 447 mg of compound E (1.0 mmol) and 30 mL of ethanol were successively added to a 50 mL round-bottom flask. 288 mg of sodium tert-butoxide (1 mmol) was added to the above solution. After stirring at room temperature for 12 h, the mixture was filtered to obtain a pale yellow precipitate, which was washed with a small amount of cold ethanol and dried under reduced pressure to obtain 537 mg of an orange-red solid with a yield of 54.3%. 1 1H NMR (400 MHz, CDCl3) δ. 8.58 (d, J = 4.0 Hz, 2H), 7.82 (d, J = 6.8 Hz, 2H), 7.44 - 7.43 (m, 3H), 7.27 - 7.25 (m, 2H), 6.70 (d, J = 6.8 Hz, 2H), 3.07 (s, 6H). 13 13C NMR (100 MHz, CDCl3) δ. 151.8, 150.4, 142.4, 141.2, 140.7, 139.4, 131.4, 126.2, 126.0, 120.8, 119.4, 117.9, 111.7, 98.5, 40.0.

[0063] Example 2

[0064] A compound with type I reactive oxygen species generating performance (labeled as ACR-DME), and its preparation method includes the following steps:

[0065] The reaction formula of this step is as follows:

[0066]

[0067] ACR-DME: Dissolve 50 mg of ACR-DM (0.151 mmol) in 5 mL of acetonitrile, then add 0.1 mL of methyl iodide. Heat the reaction mixture to 50 °C under N₂ protection and react for 12 h. After cooling the reaction solution to room temperature, remove the solvent by distillation under reduced pressure. Purify the crude product by silica gel column chromatography with an eluent of DCM / MeOH (V:V = 95:5) to obtain 73 mg of dark red solid ACR-DME, yield: 98%. 1 H NMR (400 MHz, DMSO-d6) δ. 8.86 (d, J = 5.2 Hz, 2H), 8.30 - 8.25 (m, 3H), 7.90 - 7.85 (m, 3H), 7.58 (d, J = 3.2 Hz, 1H), 6.85 (d, J = 7.2 Hz, 1H), 4.23 (s, 3H), 3.07 (s, 6H). 13 C NMR (150 MHz, DMSO-d6) δ. 152.4, 147.6, 146.8, 145.4, 143.6, 134.8, 133.4, 131.8, 125.9, 121.4, 119.80, 118.0, 111.8, 95.18, 46.8. HRMS (ESI): m / z [M - I - + calcd for C 21 H 20 N3S: 346.1372; found 346.1368.

[0068] Example 3

[0069] A compound with type I reactive oxygen species generating performance (labeled ACR-DMB), and its preparation method includes the following steps:

[0070] The reaction formula of this step is as follows:

[0071]

[0072] ACR-DMB: Dissolve 70 mg of ACR-DM (0.211 mmol) in 5 mL of acetonitrile, then add 55.12 mg of trimethylammonium bromide, and the reaction equivalent ratio is (1.0:1.0). Heat the reaction mixture to reflux at 80 °C under N₂ protection and react for 24 h. After cooling the reaction solution to room temperature, remove the solvent by distillation under reduced pressure. Purify the crude product by alumina column chromatography. First, use EA / DCM (V:V = 1:1) to wash the raw material impurities, and then use an eluent of DCM / MeOH (V:V = 5:1) to obtain 80 mg of dark red solid ACR-DMB, yield: 73%. 1 ​HNMR(400 MHz, DMSO-d6) δ. 8.96 (d, J = 5.8 Hz, 2H), 8.39 (d, J = 7.0 Hz, 2H), 8.33 (d, J = 4.1 Hz, 1H), 7.90 (t, J = 7.1 Hz, 3H), 7.65 (d, J = 4.3 Hz, 1H), 6.87 (d, J = 9.0 Hz, 2H), 4.57 (s, 2H), 3.08 (s, 15H), 2.92 (d, J = 7.4 Hz, 2H), 2.45 (s, 2H). 13 CNMR(600 MHz, DMSO-d6) δ. 174.8, 153.0, 145.3, 134.3, 132.4, 126.5, 122.2, 120.3, 118.6, 112.3, 95.6, 70.2, 62.2, 58.1, 53.0, 26.2. HRMS(ESI): m / z [M - 2Br - + calcd for C 26 H 23 N4S: 216.1168; found 216.1169.

[0073] Example 4

[0074] A compound with type I reactive oxygen species generating performance (labeled as ACR-DMP), and its preparation method includes the following steps:

[0075] The reaction formula of this step is as follows:

[0076]

[0077] ACR-DMP: Dissolve 70 mg of ACR-DM (0.211 mmol) in 5 mL of acetonitrile, then add 214.85 mg of 4-(bromomethyl)phenylboronic acid, and the reaction equivalent ratio is (1.0:5.0). Heat and reflux the reaction at 85 °C for 24 h under N2 protection. After the reaction solution is cooled to room temperature, the solvent is removed by distillation under reduced pressure. The crude product is separated and purified by silica gel column chromatography. First, use EA / DCM (V:V = 1:1) to wash the raw material impurities, and then use the eluent DCM / MeOH (V:V = 50:1), and the pH value of the eluent needs to be adjusted with a trace amount of trifluoroacetic acid to obtain 84 mg of dark red solid ACR-DMP, yield: 73%. 1 ​HNMR(600MHz, DMSO-d6) δ 9.07 (d, J = 7.2 Hz, 2H), 8.32 (d, J = 7.2 Hz, 2H), 8.28 (d, J = 4.2 Hz, 2H), 8.15 (s, 1H), 7.89 (d, J = 9.2 Hz, 2H), 7.85 (d, J = 8.5 Hz, 2H), 7.58 (d, J = 4.2 Hz 1, 1H), 7.49 (d, J = 8.2 Hz, 2H), 6.83 (d, J = 9.2 Hz, 2H), 5.78 (s, 2H), 3.06 (s, 6H). 13 CNMR(600MHz, DMSO-d6). δ 152.94, 148.61, 148,03, 144.30, 136.62, 135.31, 135.19, 134.39, 132.41, 128.00, 126.53, 122.55, 120.30, 118.44, 112.29, 95.60, 62.55, 55.39, 49.05. HRMS(ESI): m / z [M - Br - + calcd for C 27 H 25 BN3O2S: 466.1755; found 466.1757.

[0078] Example 5

[0079] A compound with type I reactive oxygen species generating performance (labeled ACR - DMA), and its preparation method includes the following steps:

[0080] The reaction formula of this step is as follows:

[0081]

[0082] ACR - DMA: Dissolve 70 mg of ACR - DM (0.211 mmol) in 5 mL of acetonitrile, then add 1114.965 mg of p - xylylene dibromide, the reaction equivalent ratio is (1.0:20.0), heat the reaction at 50 °C for 24 h. After the reaction solution is cooled to room temperature, the solvent is removed by distillation under reduced pressure. The crude product is separated and purified by silica gel column chromatography. First, use EA / DCM (V:V = 1:1) to wash away the raw material impurities, and then use the eluent DCM / MeOH (V:V = 20:1) to obtain 66.6 mg of dark purple - red solid ACR - DMA, yield: 53%. 1 ​HNMR(400MHz, DMSO-d6) δ 9.04 (d, J = 7.2 Hz, 2H), 8.33 (d, J = 7.1 Hz, 2H), 8.28 (d, J = 4.1 Hz, 1H), 7.89 (t, J = 10.3 Hz, 3H), 7.60 (d, J = 4.1 Hz, 1H), 7.53 (d, J = 1.7 Hz, 4H), 6.86 (d, J = 9.2 Hz, 2H), 5.75 (s, 2H), 4.72 (s, 2H), 3.08 (s, 6H). 13 CNMR(600MHz, DMSO-d6) δ 152.47, 148.26, 147.64, 144.61, 143.83, 134.73, 133.98, 132.00, 130.12, 129.06, 128.65, 127.17, 126.10, 122.15, 119.90, 118.01, 111.86, 95.16, 62.43, 33.69, 29.06. HRMS(ESI): m / z [M - Br - + calcd for C 28 H 25 BrN3S: 514.0947; found 514.0918.

[0083] Example 6

[0084] A compound with type I reactive oxygen species generating performance (labeled as ACR - DMT), and its preparation method includes the following steps:

[0085] The reaction formula of this step is as follows:

[0086]

[0087] ACR - DMT: Dissolve 70 mg of ACR - DM (0.211 mmol) in 5 mL of acetonitrile, then add 226.88 mg of diiodopropane, the reaction equivalent ratio is (1.0:10.0), heat the reaction at 50 °C for 24 h. After the reaction solution is cooled to room temperature, the solvent is removed by distillation under reduced pressure. The crude product is separated and purified by silica gel column chromatography. First, use EA / DCM (V:V = 1:1) to wash away the raw material impurities, and then use the eluent DCM / MeOH (V:V = 50:1) to obtain 132.4 mg of dark red solid ACR - DMP, yield: 92.5%. 1 ​HNMR(400MHz, DMSO-d6) δ. 8.92 (d, J = 7.2Hz, 2H), 8.32 (d, J = 7.1Hz, 2H), 8.33 (d, J = 4.1Hz, 1H), 7.90 (d, J = 9.2Hz, 2H), 7.86 (s, 1H), 7.61 (d, J = 4.1Hz, 1H), 6.86 (d, J = 9.1Hz, 2H), 4.54 (t, J = 6.9Hz, 2H), 3.27 (t, J = 7.1Hz, 2H), 3.08 (s, 6H). 13 CNMR(600MHz, DMSO-d6) δ. 152.56, 148.01, 147.50, 144.76, 143.86, 134.90, 133.73, 132.02, 126.15, 121.92, 119.89, 118.07, 111.91, 95.23, 60.30, 39.70, 33.94, 1.49. HRMS(ESI): m / z[M-I - + calcd for C 23 H 23 IN3S: 500.0652;found 500.0622.

[0088] Example 7

[0089] A compound with type I reactive oxygen species generating performance (labeled ACR-CBNBO), and its preparation method includes the following steps:

[0090] The reaction formula of this step is as follows:

[0091]

[0092] Compound F-1: Add 200.2 mg of compound D (1 mmol), 203.2 mg of compound E-1 (1 mmol) and 10 mL of ethanol into a 50 mL round-bottom flask, then add 96 mg of sodium tert-butoxide (1 mmol) into the mixture. After stirring at room temperature for 12 h, filter the dark yellow solid, wash it with a small amount of cold ethanol and dry it under reduced pressure to obtain 237 mg of solid, with a yield of 61.5%. 1 H NMR(400MHz, CDCl3) δ. 8.60 (d, J = 5.2Hz, 2H), 7.80 (d, J = 7.2Hz, 2H), 7.44 (d, J = 2.8Hz, 3H), 7.28 (d, J = 2.0Hz, 2H), 6.44 (d, J = 6.8Hz, 2H), 4.87 (s, 2H), 4.15 (s, 2H). 13 C NMR(100MHz, CDCl3) ​

[0093] δ.152.1,150.6,142.1,141.1,140.8,140.0,131.4,126.6,126.3,122.6,119.6,117.7,111.3,99.8,81.1,61.2,39.0.

[0094] ACR-CBNBO: Dissolve 50 mg of compound F-1 (0.130 mmol) in 5 mL of acetonitrile, then add 0.1 mL of methyl iodide. The reaction mixture is heated to 50 °C under N2 protection and reacted for 12 h. After cooling to room temperature, the solvent is removed by distillation under reduced pressure. The crude product is separated and purified by silica gel column chromatography. The eluent is DCM / methanol (V:V = 95:5), and 61 mg of dark red solid ACR-CBNBO is obtained. Yield: 89%. 1 H NMR (400 MHz, DMSO-d6) δ.8.86 (d, J = 5.2 Hz, 2H), 8.30 (d, J = 5.6 Hz, 2H), 8.25 (d, J = 3.2 Hz, 1H), 7.86 (d, J = 7.6 Hz, 1H), 7.60 (d, J = 3.2 Hz, 1H), 6.54 (d, J = 7.2 Hz, 1H), 4.74 (s, 4H), 4.24 (s, 3H), 4.18 (s, 3H). 13 C NMR (150 MHz, DMSO-d6) δ.152.6, 147.3, 146.8, 145.4, 143.8, 135.1, 133.3, 131.64, 126.20, 121.5, 120.90, 117.80, 111.0, 95.9, 79.7, 60.4, 46.80, 38.3. HRMS (ESI): m / z [M - I - + calcd for C 24 H 22 N3OS: 400.1478; found 400.1473.

[0095] Example 8

[0096] A compound with type I reactive oxygen species generating performance (labeled ACR-CBN), and its preparation method includes the following steps:

[0097] The reaction formula of this step is as follows:

[0098]

[0099] ​Compound F-2: 200.2 mg of compound D (1 mmol), 161.2 mg of compound E-2 (1 mmol) and 10 mL of ethanol were added to a 50 mL round-bottom flask, and then 96 mg of sodium tert-butoxide (1 mmol) was added to the above mixture. The mixture was stirred at room temperature for 12 h, the yellow precipitate was filtered, washed with a small amount of cold ethanol and dried under reduced pressure to obtain 204 mg of solid, with a yield of 59.4%. 1 H NMR (400 MHz, CDCl3) δ. 8.60 (d, J = 5.2 Hz, 2H), 7.80 (d, J = 6.8 Hz, 2H), 7.47 - 7.44 (m, 3H), 7.28 - 7.27 (m, 2H), 6.42 (d, J = 7.2 Hz, 2H), 4.02 (t, J = 5.6 Hz, 4H), 2.48–2.41 (m, 2H). 13 C NMR (100 MHz, CDCl3) δ. 153.0, 150.6, 142.5, 141.5, 140.8, 139.6, 131.4, 126.3, 126.2, 121.6, 119.6, 117.9, 110.7, 98.8, 51.7, 16.6.

[0100] ACR-CBN: 50 mg of compound F-2 (0.146 mmol) was dissolved in 5 mL of acetonitrile, and then 0.1 mL of methyl iodide was added. The reaction solution was heated to 50 °C under N2 protection and reacted for 12 h. After cooling to room temperature, the solvent was removed by distillation under reduced pressure. The crude product was separated and purified by silica gel column chromatography, and the eluent was DCM / methanol (V:V = 95:5) to obtain 64.5 mg of dark red solid ACR-CBN, yield: 91%. 1 H NMR (400 MHz, DMSO-d6) δ. 8.86 (d, J = 5.2 Hz, 2H), 8.30 - 8.25 (m, 3H), 7.90 - 7.84 (m, 3H), 7.60 - 7.58 (m, 1H), 6.51 (d, J = 5.6 Hz, 2H), 4.24 (s, 3H), 4.00 (t, J = 5.2 Hz, 3H), 2.41–2.36 (m, 2H). 13 C NMR (150 MHz, DMSO-d6) δ. 153.1, 147.5, 146.8, 145.4, 143.8, 134.9, 133.4, 131.7, 126.0, 121.41, 120.40, 117.9, 110.5, 95.4, 51.2, 46.8, 15.87. HRMS (ESI): m / z [M-I - + calcd for C 22 H 20 ​N3S: 358.1372; found 358.1367.

[0101] Example 9

[0102] A compound with type I reactive oxygen species generating performance (labeled as ACR-CPN), and its preparation method includes the following steps:

[0103] The reaction formula of this step is as follows:

[0104]

[0105] Compound F-3: Add 200.2 mg of compound D (1 mmol), 175.2 mg of compound E-3 (1 mmol) and 10 mL of ethanol into a 50 mL round-bottom flask, then add 96 mg of sodium tert-butoxide (1 mmol) into the above mixture. After stirring at room temperature for 12 h, filter the light dark yellow precipitate, wash it with a small amount of cold ethanol and dry it under reduced pressure to obtain 200.2 mg of solid, and the yield is 56%. 1 H NMR (400 MHz, CDCl3) δ. 8.59 (d, J = 4.8 Hz, 2H), 7.83 (d, J = 7.2 Hz, 1H), 7.45–7.44 (m, 3H), 7.27 (d, J = 8.0 Hz, 2H), 6.58 (d, J = 7.2 Hz, 1H), 3.39 (t, J = 5.2 Hz, 4H), 2.07–2.04 (m, 4H). 13 C NMR (100 MHz, CDCl3) δ. 150.5, 149.7, 142.8, 141.6, 140.9, 139.3, 131.8, 126.3, 125.8, 120.5, 119.5, 118.2, 111.9, 97.8, 47.7, 25.6.

[0106] ACR-CPN: Dissolve 50 mg of compound F-3 (0.14 mmol) in 5 mL of acetonitrile, then add 0.1 mL of methyl iodide. The reaction solution is heated to 50 °C and reacted for 12 h under N2 protection. After cooling to room temperature, the solvent is removed by distillation under reduced pressure. The crude product is separated and purified by silica gel column chromatography, and the eluent is DCM / methanol (V:V = 95:5) to obtain 65 mg of dark red solid ACR-CPN, and the yield: 93%. 1HNMR(400MHz, DMSO-d6) δ: 8.84 (d, J = 5.2Hz, 2H), 8.28 (d, J = 4.4Hz, 2H), 8.25 (d, J = 3.2Hz, 1H), 7.88 (d, J = 7.2Hz, 2H), 7.82 (d, J = 4.0Hz, 1H), 7.57 (d, J = 3.2Hz, 1H), 6.70 (d, J = 7.1Hz, 2H), 4.23 (s, 3H), 3.37 (s, 4H), 1.99 - 1.97 (m, 4H). 13 C NMR(150MHz, DMSO-d6) δ: 149.9, 147.8, 146.8, 145.3, 143.7, 134.6, 133.4, 132.0, 125.7, 121.3, 119.5, 118.1, 112.0, 94.40, 47.4, 46.8, 24.9. HRMS(ESI): m / z [M - I - + calcd for C 23 H 22 N3S: 372.1529; found 372.1526.

[0107] Example 10

[0108] A compound with type I reactive oxygen species generation performance (labeled ACR-CHN), and its preparation method includes the following steps:

[0109] The reaction formula of this step is as follows:

[0110]

[0111] Compound F-4: 200 mg of compound D (1 mmol), 189 mg of compound E-4 (1 mmol) and 10 mL of ethanol were successively added to a 50 mL round-bottom flask, then 96 mg of sodium tert-butoxide (1 mmol) was added to the above mixture. After stirring at room temperature for 12 h, a dark yellow precipitate was obtained by filtration, washed with a small amount of cold ethanol and dried under reduced pressure to obtain 216.8 mg of solid, with a yield of 58.6%. 1 HNMR(400MHz, CDCl3) δ: 8.60 (d, J = 4.8Hz, 2H), 7.81 (d, J = 7.2Hz, 2H), 7.46 - 7.44 (m, 3H), 7.28 (d, J = 6.8Hz, 2H), 6.90 (d, J = 7.2Hz, 2H), 3.37 (t, J = 4.0Hz, 4H), 1.69 - 1.68 (m, 6H). 13 ​13C NMR (100 MHz, CDCl3) δ 152.9, 150.6, 142.3, 141.0, 140.8, 139.9, 131.5, 126.4, 126.3, 122.3, 119.6, 117.8, 114.3, 99.5, 48.7, 25.5, 24.5.

[0112] ACR-CHN: 50 mg of compound F-4 (0.135 mmol) was dissolved in 5 mL of acetonitrile, then 0.1 mL of methyl iodide was added. The reaction mixture was heated to 50 °C under N2 protection and reacted for 12 h. After cooling to room temperature, the solvent was removed by distillation under reduced pressure. The crude product was separated and purified by silica gel column chromatography, and the eluent was DCM / methanol (V:V = 95:5), obtaining 73 mg of dark red solid ACR-CHN, yield: 92%. 1 1H NMR (400 MHz, DMSO-d6) δ 8.85 (d, J = 5.6 Hz, 2H), 8.30 (d, J = 4.0 Hz, 2H), 8.26 (d, J = 3.2 Hz, 1H), 7.86 (t, J = 5.6 Hz, 3H), 7.60 (t, J = 2.8 Hz, 1H), 7.06 (d, J = 5.2 Hz, 2H), 4.24 (s, 3H), 3.44 (t, J = 4.0 Hz, 4H), 1.52 - 1.57 (m, 6H). 13 13C NMR (150 MHz, DMSO-d6) δ 152.6, 147.3, 146.8, 145.4, 143.3, 135.0, 133.4, 131.9, 126.2, 121.4, 120.8, 117.8, 113.6, 95.9, 47.6, 46.8, 24.9, 23.9. HRMS (ESI): m / z [M - I - + calcd for C 24 H 24 N3S: 386.1685; found 386.1677.

[0113] Test Example

[0114] The synthesis process of the compounds with type I reactive oxygen species generating performance in this application is shown in Figure 1; The photophysical properties, photodynamic anti-planktonic bacteria, and photodynamic biofilm removal performance of the compounds prepared in Examples 1-4 were measured respectively. Since the experimental results of the compounds in this application are similar and the reaction conditions are the same, in the part of photophysical property measurement, photodynamic anti-planktonic bacteria, and biofilm removal, Examples ACR-DM, ACR-DME, ACR-DMB, and ACR-DMP of the present invention are mainly used as examples. Since the experimental results of other examples are the same as those of the examples cited, they will not be listed one by one. The specific results are as follows:

[0115] I. Photophysical Property Measurement

[0116] 1. Absorption and Emission Measurement of Photosensitizer

[0117] The absorption and emission spectra of Examples ACR-DM, ACR-DME, ACR-DMB, and ACR-DMP in the solvent DMSO were measured. As Figure 2 shown in Figures 4A and 2B, there is no obvious difference in the absorption wavelengths of the 4 compounds, while the emission wavelengths change significantly.

[0118] 2. Determination of Reactive Oxygen Species of Target Molecules (Indicator H2DCF-DA)

[0119] Using 2,7-dichlorodihydrofluorescein diacetate (H2DCF-DA) as a detection probe, the ROS yields of compounds ACR-DM, ACR-DME, ACR-DMB, and ACR-DMP in solution were tested. To convert H2DCF-DA into 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, and then stirred at room temperature for 30 min. Then, the pH of the solution was adjusted with 5 mL of PBS solution (pH 7.4). The resulting solution was stored frozen for later use. The DMSO solutions of the 4 compounds were added to the above solution, and the final concentration was 10 μM. The samples were placed in a fluorescence spectrometer, and the fluorescence intensity of the solution was measured every 2 s, (λ ex : 488 nm).

[0120] As Figure 3 shown, as the illumination time increased, the fluorescence intensities of the 4 compounds at 525 nm all increased. Compounds ACR-DM, ACR-DME, ACR-DMB, and ACR-DMP were enhanced by 153, 234, 282, and 421 times respectively. This experiment confirmed that the 4 compounds can effectively and rapidly generate ROS under light irradiation.

[0121] 3. Determination of Hydroxyl Radical Yield (Hydroxyphenyl Fluorescein HPF)

[0122] Using HPF (hydroxyphenyl fluorescein) as an indicator, the performance of the photosensitizer ACR-DM series in generating hydroxyl radicals under light irradiation was investigated. Hydroxyphenyl fluorescein itself has no fluorescence. When it reacts with hydroxyl radicals, peroxynitrite anions, and hypochlorite anions, strong green fluorescence is produced at 515 nm. Under dark conditions, the solutions of HPF (5 μM) and 4 compounds (10 μM) were shaken well and immediately measured for fluorescence intensity using a fluorescence spectrometer, which was recorded as 0 minutes. Then, the solution mixture was irradiated with a white light lamp (5 mW / cm 2 ) and the fluorescence was measured immediately after each 1-minute irradiation until the fluorescence intensity no longer increased.

[0123] As Figure 4 shown in Figure A, there was almost no emission at 515 nm at 0 minutes of light irradiation. After 10 minutes of light irradiation, the emission intensity increased. After 26 minutes of light irradiation, the I / I0 values of the photosensitizer ACR-DM increased by 23 times, 78 times, 54 times, and 122 times, respectively. This experiment confirmed that all 4 compounds could rapidly and efficiently generate hydroxyl radicals under light irradiation.

[0124] 4. Determination of singlet oxygen yield (indicator ABDA)

[0125] Using ABDA (9,10-anthrylene-bis(methylene)dimalonic acid) as an indicator, the performance of 4 photosensitizers and the commercially available photosensitizer Rose Bengal (RB) in generating singlet oxygen under light irradiation was investigated. When ABDA reacts with 1 O2, ABDA is oxidized to a peroxide bridge structure, resulting in a decrease in the absorbance value of ABDA at 378 nm. The rate of this decrease can indirectly reflect the 1 O2 yield of the photosensitizer under light irradiation. First, the absorbances of the photosensitizers ACR-DM, ACR-DME, ACR-DMB, ACR-DMP, and RB (5 μM) were set as blanks. Then, under dark conditions, the solutions of ABDA (50 μM) and 4 compounds (5 μM) were mixed and immediately measured for the absorbance value of the solution. Then, the solution mixture was irradiated with a white light lamp (5 mW / cm 2 ) and the absorbance value of the solution was immediately recorded after each 1-minute irradiation until the absorbance value no longer decreased.

[0126] As Figure 4 shown in Figure B, the absorbance values of the 4 photosensitizers at 378 nm decreased to varying degrees under light irradiation, while the absorbance value did not decrease when only the indicator ABDA was present in the control group. This experiment proved that ACR-DM, ACR-DME, and ACR-DMP hardly produced 1 O2, ACR-DMB produced a small amount of 1 O2, but less than the commercially available photosensitizer RB.

[0127] The results of the determination of photophysical properties show that the photosensitizers prepared in Examples 1, 2, 3, and 4 have good fluorescence emission properties and type I reactive oxygen species generation properties, and can generate a large amount of hydroxyl radicals under light irradiation. Among them, ACR-DME and ACR-DMP are pure type I photosensitizers, while ACR-DMB is a type I and type 2 composite photosensitizer, indicating the application potential of this type of compound in both imaging and photodynamic therapy under hypoxic conditions.

[0128] II. Photodynamic anti-planktonic bacteria

[0129] 1. Bacterial culture

[0130] Transfer the single colony on the solid medium to the liquid 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 disperse it evenly in sterile PBS as the experimental bacterial solution.

[0131] 2. Evaluation of bacterial viability by plate coating counting method

[0132] Take 1 mL of the above-prepared experimental bacterial solution in a centrifuge tube and divide it into PBS, PBS+Light, ACR-DM series (0.625 μM, 1.25 μM, 2.5 μM, 5.0 μM), ACR-DM series+Light (0.625 μM, 1.25 μM, 2.5 μM, 5.0 μM) in turn. Next, add an appropriate amount of the ACR-DM series stock solution to the ACR-DM series experimental groups, while the PBS control group is added with an equal volume of PBS solution. All groups are incubated at 37 °C for 10 min, centrifuged at 8×10 3 rpm for 5 min, the supernatant is removed, washed three times with PBS, and the bacteria are redispersed in 1 mL of PBS. PBS+Light and ACR-DM series+Light are exposed to LED white light (5 mW / cm 2 ) for 30 min, while the remaining groups are placed in the dark for 30 min, and then the bacterial viability is measured and quantified by the plate counting method.

[0133] The ACR-DM series of compounds are incubated with 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) for 10 min and irradiated for 30 min. Figure 5 and 6In vitro photodynamic results of the ACR-DM series against Gram-positive bacteria (Staphylococcus aureus S. aureus) and Gram-positive drug-resistant bacteria (methicillin-resistant Staphylococcus aureus MRSA) are presented respectively. In the two figures, A-D represent ACR-DM, ACR-DME, ACR-DMB, and ACR-DMP respectively. As the concentration of the compound increased, the number of Gram-positive bacteria colonies on the plate decreased to varying degrees. When the concentration of ACR-DM increased from 1.25 μM to 5.0 μM, the survival rates of both S. aureus and MRSA were above 80%, indicating that ACR-DM had no obvious antibacterial effect against Gram-positive bacteria. When the concentration of ACR-DME increased from 1.25 μM to 5.0 μM, the survival rate of S. aureus decreased from 45.9% to 5.2%, and the survival rate of MRSA decreased from 39.8% to 3.8%, showing obvious phototoxicity and dose-dependence. When the concentration of ACR-DMB increased from 1.25 μM to 5.0 μM, the survival rate of S. aureus decreased from 27.7% to 1.14%, and there was almost no MRSA on the plate after treatment with ACR-DMB, showing obvious phototoxicity and dose-dependence. When the concentration of ACR-DMP increased from 1.25 μM to 5.0 μM, there was almost no MRSA and S. aureus on the plate, showing very strong phototoxicity and obvious dose-dependence. The in vitro photodynamic antibacterial effects of the ACR-DM series against Gram-negative bacteria (Escherichia coli E. coli) are as Figure 7 shown, Figure 7 A shows ACR-DM, Figure 7 B shows ACR-DME, Figure 7 C shows ACR-DMB, Figure 7 D shows ACR-DMP. When the concentrations of ACR-DM, ACR-DME, and ACR-DMP increased from 1.25 μM to 5.0 μM, the survival rates of both S. aureus and MRSA were above 80%, indicating that ACR-DM, ACR-DME, and ACR-DMP had no obvious antibacterial effect against Gram-negative bacteria. When the concentration of ACR-DMB increased from 1.25 μM to 5.0 μM, the survival rate of E. coli decreased from 35.8% to 1.6%, showing obvious phototoxicity and dose-dependence.

[0134] In summary, the experimental results show that ACR-DM has no obvious phototoxicity to 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). ACR-DME and ACR-DMP have obvious phototoxicity to Gram-positive bacteria (Staphylococcus aureus S. aureus) and Gram-positive drug-resistant bacteria (methicillin-resistant Staphylococcus aureus MRSA). ACR-DMB not only has obvious phototoxicity to Gram-positive bacteria but also can efficiently inactivate Gram-negative bacteria under light illumination conditions.

[0135] 3. Experiment for determining Zeta potential.

[0136] 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 incubated with different probes of the ACR-DM series at room temperature for 10 min respectively. After centrifugation, the sample bacterial solution was obtained, resuspended with sterilized deionized water, and the Zeta potential on the bacterial surface was measured using Nano ZS (ZEN3600). Bacteria without adding probes under the same conditions were set as the blank control group.

[0137] As Figure 8 shown, the Zeta potential of S. aureus in the blank group was -29.9 mV. After incubation with 5 μM ACR-DM for 10 min, the Zeta potential on the bacterial surface increased to about -29.7 mV, showing no obvious change. After incubation with 5 μM ACR-DME, ACR-DMB, and ACR-DMP for 10 min, the Zeta potential on the bacterial surface increased to about -26.8, -27.8, and -27.9 mV, rising by about 2 - 3 units. The Zeta potential of MRSA in the blank group was about -30.9 mV. After incubation with 5 μM ACR-DM for 10 min, the Zeta potential on the bacterial surface increased to about -30.8 mV, showing no obvious change. After incubation with 5 μM ACR-DME, ACR-DMB, and ACR-DMP for 10 min, the Zeta potential on the bacterial surface increased to about -26.3, -25.5, and -27.2 mV, rising by about 3 - 4 units. The Zeta potential of E. coli in the blank group was -48.5 mV. After incubation with 5 μM ACR-DM, ACR-DME, and ACR-DMP for 10 min, the Zeta potential on the bacterial surface increased to about -48.4, -47.9, and -48.1 mV, showing no obvious change. After incubation with 5 μM ACR-DMB for 10 min, the Zeta potential on the bacterial surface increased to about -42.0 mV, rising by about 6 units.

[0138] The experimental results show that the probes ACR-DME and ACR-DMP with one positive charge can bind to the cell membranes of Staphylococcus aureus and MRSA. The probe ACR-DMB with two positive charges binds to the cell membrane of Escherichia coli, and the uncharged probe ACR-DM has no obvious binding effect on all bacteria. This conclusion is consistent with the results of photodynamic anti-planktonic bacteria.

[0139] 4. Bacterial fluorescence imaging

[0140] ACR-DM, ACR-DME, ACR-DMB, and ACR-DMP (2.5 μM) were incubated with Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (E. coli) in the logarithmic growth phase for 10 min, and then the bacterial selective imaging of ACR-DM was observed. Figure 9 A is the SEM morphology analysis diagram of Staphylococcus aureus (S. aureus) treated by two different methods: ACR-DMP and ACR-DMP + Light. Figure 9 B is Figure 9 the enlarged view of A; as Figure 9 shown in A, after the action of ACR-DME, ACR-DMB, and ACR-DMP on Gram-positive bacteria S. aureus, red fluorescence is shown, and Figure 9 from the bright field and composite field of the enlarged image of B, it can be seen that the red fluorescence is mainly concentrated in the cell membrane and cytoplasm of bacteria. This is mainly because after the hydrophobic interaction and electrostatic interaction between the ACR-DM series and the amphiphilic cell membrane of Gram-positive bacteria and the negatively charged teichoic acid on the membrane, they enter the cytoplasm. Only ACR-DMB has red fluorescence after incubation with E. coli among Gram-negative bacteria, and it mainly acts on the cell membrane of bacteria.

[0141] This experiment confirms that ACR-DM has poor binding force to all bacteria, while ACR-DME and ACR-DMP can selectively act on Gram-positive bacteria, and ACR-DMB has good selectivity for both types of bacteria. This conclusion is consistent with the results of photodynamic anti-planktonic bacteria experiments in vitro and the results of bacterial Zeta potential measurement.

[0142] III. In vitro experiment on biofilm clearance

[0143] 1. Crystal violet staining method for biofilm observation

[0144] Add 1000 μl of LB culture medium into a small dish, and inoculate 100 μl of Gram-positive bacteria (Staphylococcus aureus S. aureus) in the logarithmic growth phase. The small dish with only LB culture medium is the blank control group. Add 5 mM photosensitizer solutions prepared with different volumes of PBS to make the final concentration of each group of probes 0 μM, 5 μM, 10 μM, 15 μM, and 20 μM. After mixing, incubate for 30 min, and then place it under white light with an intensity of 5 mW / cm 2 for 30 min. After aspirating the supernatant, wash it 2 - 3 times with PBS. Add 500 μl of crystal violet ammonium oxalate solution to each small dish and stain at room temperature for 10 min; after aspirating the crystal violet ammonium oxalate staining solution in the small dish, rinse off the excess dye with running water; invert the culture plate on filter paper to remove the residual water, and dry it in an oven at 37 °C or air-dry it at room temperature; after complete drying, the remaining situation of the biofilm can be observed.

[0145] Figure 10 Figure A shows the small dish diagram for detecting biofilm by crystal violet staining method. It can be observed that there is almost no crystal violet residue in the blank group, while in the dark group of the probe and the ACR-DM light irradiation group, a complete biofilm attached to the bottom of the small dish can be seen stained with crystal violet. After ACR-DME, ACR-DMB, and ACR-DMP light treatments, it was observed that with the increase in concentration, the bacterial biofilm showed an obvious effect of being torn and removed. Among them, ACR-DMP was the closest to the blank group in removing the bacterial biofilm, and almost no biofilm residue was observed.

[0146] 2. Detection method for OD value of biofilm residue by crystal violet method

[0147] Add 100 μl of LB culture medium into each well of a 96-well polystyrene microplate, and inoculate 10 μl of 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) in the logarithmic growth phase. Set the well with only culture medium as the blank control group, and incubate statically at 37 °C for 36 h; then aspirate the culture medium, wash all the experimental wells 2 - 3 times with PBS, add 5 mM photosensitizer solutions prepared with different volumes of PBS to make the final concentration of each group of probes 0 μM, 5 μM, 10 μM, 15 μM, and 20 μM. After mixing, incubate for 30 min, and then place it under white light with an intensity of 5 mW / cm 2Irradiate under white light for 30 min, aspirate the supernatant, wash 2-3 times with PBS, add 100 μl of methanol to each well and fix for 15 min, then aspirate the methanol in the culture wells and air dry naturally; add 100 μl of 1% crystal violet solution to each well and stain for 5 min at room temperature; 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 the residual water, and dry it in an oven at 37 °C or air dry at room temperature; after complete drying, add 100 μl of 33% glacial acetic acid solution to each well and incubate in a 37 °C incubator for 30 min to dissolve the crystal violet; measure the OD value of the solution in the culture wells at 590 nm (the residual amount of this biofilm includes biomass components such as extracellular matrix EPS and viable bacteria of the bacterial biofilm).

[0148] As Figure 10 As shown in Figures B and 10D, they are the in vitro photodynamic clearance result graphs of the ACR-DM series against Gram-positive bacteria (Staphylococcus aureus S. aureus) and Gram-positive resistant bacteria (methicillin-resistant Staphylococcus aureus MRSA) bacterial biofilms, respectively. As the compound concentration increases, the residual amount of the biofilm in the well plate decreases to varying degrees. When the concentration of ACR-DM increases from 5 μM to 20 μM, the residual amounts of the S. aureus and MRSA biofilms are both above 57%, indicating that ACR-DM has no obvious effect on clearing Gram-positive bacterial biofilms. When the concentration of ACR-DME increases from 5 μM to 20 μM, the residual amount of the S. aureus biofilm decreases from 86.4% to 38.5%, and the residual amount of the MRSA biofilm decreases from 87.9% to 25.6%, showing obvious phototoxicity and dose dependence. When the concentration of ACR-DMB increases from 5 μM to 20 μM, the residual amount of the S. aureus biofilm decreases from 86.5% to 29.4%, and the residual amount of the MRSA biofilm decreases from 79.4% to 14.9%, showing obvious phototoxicity and dose dependence. When the concentration of ACR-DMP increases from 5 μM to 20 μM, there is almost no residual MRSA and S. aureus biofilm on the plate (the residual amounts are only 3.24% and 2.98%), showing very strong phototoxicity and obvious dose dependence. The in vitro photodynamic antibacterial effect of the ACR-DM series against Gram-negative bacteria (Escherichia coli E. coli) is as Figure 10As shown in Figure F, when the concentrations of ACR-DM and ACR-DME increased from 5 μM to 20 μM, the residual amounts of E. coli biofilms were both above 67%, indicating that ACR-DM and ACR-DME had no obvious effect on clearing Gram-negative bacterial biofilms. When the concentration of ACR-DMP increased from 5 μM to 20 μM, the residual amount of E. coli biofilm decreased to 56%, indicating that compared with ACR-DM and ACR-DME, ACR-DMP had a slight effect on Gram-negative bacterial biofilms. This may be due to the specific polysaccharide-binding ability of ACR-DMP, resulting in differences in the destruction of biofilm EPS. When the concentration of ACR-DMB increased from 5 μM to 20 μM, the residual amount of E. coli biofilm decreased from 66.9% to 5.23%, showing obvious phototoxicity and dose dependence.

[0149] 3. Detection of viable bacteria survival rate in biofilms by MTT method:

[0150] Add 100 μl of culture medium to each well of a 96-well polystyrene microplate, inoculate 10 μl of bacterial solution in the logarithmic growth phase, and set the well with only culture medium as the blank control group. Incubate statically at 37 °C for 36 h. Then aspirate the culture medium, wash all experimental wells 2-3 times with PBS, and add 5 mM photosensitizer solution prepared with different volumes of PBS to make the final concentration of each group of probes 0 μM, 5 μM, 10 μM, 15 μM, and 20 μM. After mixing, incubate for 30 min and then irradiate with white light at 5 mW / cm 2 for 30 min. After aspirating the supernatant, wash 2-3 times with PBS, add 10 μl of MTT solution (5 mg / ml) to each well, incubate in the dark for 2-4 h, and then add 150 μl of DMSO to each well and oscillate at low speed for 15 min. Under the condition of 490 nm, use an enzyme-linked immunosorbent assay (ELISA) reader to measure the OD value of the solution in the culture well (MTT solution preparation method: weigh 10 mg of MTT, dissolve it in 2 ml of LB liquid medium, sonicate for 3 min, filter with a 0.45 μm PES filter membrane, and use it in the dark).

[0151] As Figure 10As shown in Figures C and 10E, the results of in vitro photodynamic killing of viable biofilm bacteria by the ACR-DM series against Gram-positive bacteria (Staphylococcus aureus S. aureus) and Gram-positive drug-resistant bacteria (methicillin-resistant Staphylococcus aureus MRSA) are presented. As the compound concentration increased, the viability of viable bacteria in the Gram-positive biofilm in the well plates decreased to varying degrees. When the concentration of ACR-DM increased from 5 μM to 20 μM, the viability of viable bacteria in the biofilms of S. aureus and MRSA was above 55%, indicating that ACR-DM had no obvious antibacterial effect against Gram-positive bacteria. When the concentration of ACR-DME increased from 5 μM to 20 μM, the viability of viable bacteria in the S. aureus biofilm decreased from 52.4% to 18.5%, and the viability of viable bacteria in the MRSA biofilm decreased from 60.5% to 12.3%, showing obvious phototoxicity and dose dependence. When the concentration of ACR-DMB increased from 1.25 μM to 5.0 μM, the viability of viable bacteria in the S. aureus biofilm decreased from 59.6% to 11.8%, and the viability of viable bacteria in the MRSA biofilm decreased from 55.1% to 5.24%, showing obvious phototoxicity and dose dependence. When the concentration of ACR-DMP increased from 5 μM to 20 μM, almost no viable bacteria in the MRSA and S. aureus biofilms survived on the plate (the viability rates were only 0.97% and 0.19%), showing very strong phototoxicity and obvious dose dependence. The in vitro photodynamic antibacterial effects of the ACR-DM series against Gram-negative bacteria (Escherichia coli E. coli) are as Figure 10 shown in Figure G. When the concentrations of ACR-DM and ACR-DME increased from 5 μM to 20 μM, the viability of viable bacteria in the E. coli biofilm was above 74%, indicating that ACR-DM and ACR-DME had no obvious antibacterial effect against Gram-negative bacteria. When the concentration of ACR-DMP increased from 5 μM to 20 μM, the remaining amount of the E. coli biofilm decreased to 52.7%, indicating that compared with ACR-DM and ACR-DME, ACR-DMP had a slight effect on the survival of viable bacteria in the Gram-negative bacterial biofilm. It may be that the unique polysaccharide-binding ability of ACR-DMP caused the destruction of the biofilm EPS, affecting the vital activities of the viable bacteria and resulting in a decrease in the viability of viable bacteria compared with ACR-DME. When the concentration of ACR-DMB increased from 5 μM to 20 μM, the viability of viable bacteria in the E. coli biofilm decreased from 68.3% to 8.6%, showing obvious phototoxicity and dose dependence.

[0152] The experimental results showed that ACR-DM had no obvious ability to clear bacterial biofilms. ACR-DME and ACR-DMP had good abilities to kill live bacteria and clear biofilms against Gram-positive bacteria (Staphylococcus aureus S. aureus) and Gram-positive drug-resistant bacteria (methicillin-resistant Staphylococcus aureus MRSA). ACR-DMB had obvious effects on clearing all biofilms and killing live bacteria. However, in the experiment with Gram-negative bacteria, the ability of ACR-DMP to clear the biofilm of Escherichia coli was more obvious than that of ACR-DM and ACR-DME. It was speculated that this was due to its unique binding effect on polysaccharides, and this speculation was verified in subsequent experiments.

[0153] 4. Biofilm fluorescence imaging

[0154] The cell slides were immersed in hydrofluoric acid for five seconds, then washed with pure water and 75% ethanol, sterilized by high-pressure steam, completely dried and placed in a 24-well polystyrene microplate. 300 μl of LB culture medium was added to each well, and 30 μl of Gram-positive bacteria (Staphylococcus aureus S. aureus) in the logarithmic growth phase was inoculated and incubated statically at 37 °C for 36 h. Then the culture medium was aspirated, and ACR-DMB, ACR-DMP were incubated with the Staphylococcus aureus biofilm and then subjected to light and dark treatments. The dead cell nucleic acid dye SytoxGreen was used to observe the photodynamic clearance effect of ACR-DMB and ACR-DMP on the biofilm.

[0155] The commercially available dead cell nucleic acid dye Sytox Green was used to detect the membrane state of the two bacterial biofilms after ACR-DM series probes were treated with light and dark. Among them, the λ of the ACR-DM series probes ex = 561 nm, λ em = 570 nm - 620 nm, and the λ of SytoxGreen ex = 488 nm, λ em = 500 nm - 550 nm; The results were as Figure 11 shown. After ACR-DMB and ACR-DMP were incubated with the S. aureus biofilm for 30 min, all bacterial biofilms showed red fluorescence, while Sytox Green only stained the light-treated group. This experiment confirmed that ACR-DMB and ACR-DMP could efficiently kill the live bacteria in the bacterial biofilm under light. Among them, the fluorescent staining substance of ACR-DMP was significantly reduced compared with ACR-DMB, indicating that it had an obvious effect on clearing the biofilm biomass (extracellular matrix EPS and live bacterial cell biomass).

[0156] 5. Biofilm scanning electron microscopy

[0157] The silicon wafers sterilized by high-pressure steam were placed in a 24-well polystyrene microplate, 300 μl of LB culture medium was added to each well, and 30 μl of Gram-positive bacteria (Staphylococcus aureus S. aureus) in the logarithmic growth phase was inoculated, and then incubated statically at 37 °C for 36 h. After that, the culture medium was aspirated, and ACR-DMB and ACR-DMP with a concentration of 10 μM were incubated with the Staphylococcus aureus biofilm for 30 min, followed by light exposure (30 min) and dark treatment. After a series of operations such as fixation with 2.5% glutaraldehyde fixative, gradient dehydration with ethanol, natural drying, and ion sputtering and gold spraying, the morphological changes of the bacterial biofilm were observed.

[0158] As Figure 12 shown, after dark treatment with ACR-DMB and ACR-DMP, it was observed that S. aureus in the biofilm was round and plump with good morphology, and the extracellular matrix (EPS) between bacteria was completely wrapped and interconnected. However, the morphology of the bacteria after light treatment was poor, with obvious shrinkage, depression, collapse, and even leakage, indicating the strong biofilm cleaning ability and viable bacteria killing ability of ACR-DMB and ACR-DMP. By observing the state of the biofilm EPS, it was found that compared with the ACR-DMB group, the EPS in the ACR-DMP light-exposed group was completely destroyed, showing a stacked state of polysaccharide inactivation and protein denaturation, and the connection between bacteria was cut off, which also confirmed the super strong EPS clearance and biomass inactivation ability of ACR-DMP during the biofilm cleaning process.

[0159] IV. Biocompatibility experiment

[0160] 1. Cytotoxicity experiment

[0161] 1.1 Cell resuscitation

[0162] The cryopreserved 3T3 human epidermal fibroblasts were taken out from liquid nitrogen respectively, and shaken continuously in a 37 °C water bath to promote their melting. They were transferred into 5 mL centrifuge tubes respectively, 3 mL of preheated corresponding medium was added, centrifuged at 800 rpm for 3 min, and the supernatant was discarded. 2 mL of corresponding medium was added, gently pipetted, inoculated into a culture flask, and cultured in a cell culture incubator containing 5% CO2.

[0163] 1.2 Cell passage

[0164] After culturing 3T3 human epidermal fibroblasts for 24 h, the cells were taken out separately. After observing the cell morphology and density under a microscope, the cell surface was washed with 3 mL of PBS twice, and the PBS was discarded. 1 mL of trypsin was added to digest the cells for 2 min, and the cells were placed in a 37 °C cell culture incubator for digestion. After complete digestion, 2 mL of the corresponding medium was added to stop the digestion, and the cells were pipetted to make them uniform. The cells were transferred to a centrifuge tube and centrifuged at 1000 rpm for 5 min. After centrifugation, the supernatant was discarded, the bottle mouth was wiped with alcohol, burned, and 2 mL of the corresponding medium was added to pipette and mix the cells evenly (pipette gently along the wall about 20 times). Each 1 mL was inoculated into a culture flask, a total of two flasks, and cultured in a cell culture incubator containing 5% CO2. After the cells were passaged three generations and grew to the logarithmic growth phase, they could be plated for the cytotoxicity test.

[0165] 1.3 Testing the dark toxicity and phototoxicity (CCK8) of ACR-DM, ACR-DME, ACR-DMB, and ACR-DMP on 3T3 human epidermal fibroblasts under anti-biofilm conditions: Take cells in good growth state in the logarithmic growth phase. After digestion with trypsin, centrifuge the cells in a centrifuge to make them sediment, discard the supernatant, resuspend the cells with complete medium, and dilute the cell suspension to 50,000 cells / mL. Take a 96-well plate, add 100 μL of the above cell suspension to each well, so that the number of cells in each well is about 5000. Then continue to place the plate in a 37 °C cell culture incubator containing 5% CO2 for 24 h to make the suspended cells adhere again and make about 40,000 cells in each well. Subsequently, the culture medium was changed to 100 μL of fresh medium containing different concentrations of ACR-DM, ACR-DME, ACR-DMB, and ACR-DMP, and the incubation time was 30 min. The light condition was set to white light illumination of 5 mW / cm 2 , 30 min. Then the cells were placed in a 37 °C, 5% CO2 incubator for 24 h. After that, 10 μL of CCK8 mixture (CCK8: medium = 1:9) was added to each well, and the absorbance OD at 450 nm of each well was measured with an enzyme-linked immunosorbent assay (ELISA) reader. 450nm . The formula for calculating the relative cell viability is as follows:

[0166]

[0167] Among them, OD(treated cells): the absorbance of the well with cells, CCK8 solution, and drug solution; OD(control cells): the absorbance of the well with medium and CCK8 solution but without cells; OD(0controlcells): the absorbance of the well with cells and CCK8 solution but without drug solution. Among them, the measured value of OD is the average value measured from 3 independent parallel samples, and the result is expressed as the average value (M) ± standard deviation (SD).

[0168] As Figure 13 shown Figure 13 Among them, A - D are ACR - DM, ACR - DME, ACR - DMB, and ACR - DMP in sequence; under light - avoiding conditions, within the concentration range of 0 - 20 μM, the cell survival rates of ACR - DM, ACR - DME, ACR - DMB, and ACR - DMP are close to 90%, indicating that the probe has good biocompatibility; under light illumination conditions, with the increase of the probe concentration, the phototoxicity of ACR - DM and ACR - DME increases. After continuous white - light irradiation at 5 mW / cm 2 for 30 min, the cell survival rate remains above 80% with the increase of the concentrations of ACR - DM, ACR - DME, ACR - DMB, and ACR - DMP, indicating that ACR - DM, ACR - DME, ACR - DMB, and ACR - DMP have almost no obvious phototoxicity to normal cells under the concentration conditions for clearing biofilms and have good biocompatibility with normal cells.

[0169] 2. Hemolysis experiment

[0170] First, prepare a 2% red - blood - cell suspension: Take 10 ml of fresh mouse blood and place it in a heparin - sodium - coated vacuum blood collection tube, gently invert and mix well. After mixing, centrifuge at 1000 rcf / min for 10 min, remove the supernatant. Use a pipette to quantitatively absorb red blood cells (RBC), and dilute them with sterile phosphate - buffered saline (PBS) at approximately 10 times the volume of RBC. Wash 3 times at 1000 ref / min for 10 min, and then dilute with sterile phosphate - buffered saline (PBS) to prepare a 2% RBC suspension. Add 20 μL of the corresponding volume of the stock solutions of ACR - DMB and ACR - DMP to 180 μL of the 2% RBC suspension to obtain experimental solutions with final concentrations of 2.5, 5, 10, and 20 μM. The blank control group and the positive control group are added with 20 μL of PBS and Triton X - 100 solvent respectively. Set 3 parallel wells for each group, gently mix well, place them in a 37°C water - bath for incubation for 1 h, and then centrifuge at 1200 rcf / min for 5 min. Collect the supernatant, detect the ultraviolet absorption at 545 nm by an enzyme - linked immunosorbent assay. The calculation formula for the hemolysis percentage of RBC is as follows:

[0171]

[0172] As Figure 14 shown, in the positive control group (+), a large number of red blood cells rupture and the solution turns dark red, and the hemolysis rate is as high as 100%; the solution in the blank control group is relatively clear. After adding the photosensitizers ACR - DMB and ACR - DMP to the experimental group, the liquid is relatively clear. Even when the concentration increases to 20 μM, the liquid in the 96 - well plate is still clear, and the red - blood - cell hemolysis rate is less than 5% and shows a significant difference from the positive control group.

[0173] 5. In vivo biofilm removal experiment

[0174] 1. Establishment of a model of bacterial biofilm attached to medical devices

[0175] A medical catheter with a diameter of 2.3 mm and a length of 1 cm was placed in a 24-well plate and dripped with methicillin-resistant Staphylococcus aureus (MRSA) in the logarithmic growth phase, and cultured at 37°C for 48 hours until biofilm was formed (the culture medium was replaced every 24 hours). Four-week-old female Kunming mice (20-25 g) were kept at a room temperature of (20±2)°C, with free drinking water, and were adaptively fed for 1 week. The experiment was conducted after the mice had no abnormal activity or physical signs. Age-appropriate Kunming female mice that had been adaptively raised were selected, anesthetized by intraperitoneal injection of 1%, 50 mg / kg sodium pentobarbital, and a medical catheter infected with silicone biofilm was implanted subcutaneously in the abdomen. The wound was sutured with a 6*14 surgical needle and a 4-0 surgical thread, and then the wound was bandaged with sterile gauze and medical tape, and the medical catheter was kept in situ for 7 days; the mice were randomly divided into four groups (normal saline, fluorescent probe, fluorescent probe + light, and commercially available positive control drug), and normal saline (SPSS), commercially available antibiotic vancomycin (Van), and ACR-DMP were subcutaneously injected at the catheter implantation site, respectively. Among them, the mice with ACR-DMP were divided into two groups, the dark group was protected from light, and the light group had the catheter implantation site placed under white light (20 mW / cm 2 ) for 30 minutes (Van and ACR-DMP were prepared with normal saline, the injection concentration was 5 μM, and the day of treatment was D0). On D1, D3, D5, and D7, the catheters and muscle tissues were obtained in batches for ultrasonic coating and grinding coating, respectively, and the muscle tissues were stained with HE and protein immunostaining to investigate the treatment differences and healing conditions.

[0176] 2. Muscle tissue acquisition

[0177] 2.1 Preoperative preparation: Weigh the mice and record their weight (accurate to grams). Euthanasia is performed by intraperitoneal injection of 0.06-0.1 ml of 20% sodium pentobarbital solution per 10 grams of body weight according to the weight of the mice.

[0178] 2.2 Surgical cutting: After confirming the death of the mouse, use sharp surgical scissors to cut open the subcutaneous sutures, use ophthalmic forceps to clamp out the medical catheter, and then use forceps to pull up the infected part of the wound. Use scissors to cut off the complete infected skin area and surrounding muscles, and store them in physiological saline or formaldehyde for later use. Finally, the mouse carcasses are collected and processed uniformly.

[0179] In vivo catheter biofilm removal test results Figure 15 As shown, Figure 15It was observed that yellow MRSA biofilms and wound pus were attached to the catheters removed on the 1st - 7th day in both the SPSS group and the ACR - DMP group; the yellow color of the catheters removed in the Van group was significantly faded on the 7th day; the yellow color of the catheters removed in the ACR - DMP + L group disappeared significantly on the 1st day, and fresh blood was seen attached. The catheters remained white from the 3rd to the 7th day, and the MRSA biofilms were significantly cleared. Figure 15 B and Figure 15 E showed that a large amount of MRSA biofilms remained on the catheters in the SPSS group and the ACR - DMP group after the 7th day; the remaining amount of MRSA biofilms on the catheters in the Van group was only 20% of that on the 1st day after the 7th day, proving that vancomycin had a relatively obvious effect on clearing the bacterial biofilms on the implanted catheters; the remaining amount of MRSA biofilms on the catheters in the ACR - DMP + L group was 13% of that in the SPSS group on the 1st day, and it was completely cleared on the 5th day, proving that ACR - DMP had a high - efficiency effect on clearing the MRSA biofilms on the implanted catheters under light. Figure 15 C It was observed that the inflammatory reactions were obvious at the wounds near the implants in the SPSS group and the ACR - DMP group from the 1st to the 7th day, with the skin tissue ulcerated, suppurated, and rigid and blackened; the inflammation at the wounds near the implants in the Van group was significantly reduced on the 7th day, the white pus disappeared, and the skin tissue repair was obvious, and the granulation tissue of hyperplasia began to form; the inflammation at the wounds near the implants in the ACR - DMP + L group was significantly alleviated on the 1st day, the white pus disappeared, the inflammation disappeared significantly on the 3rd day, the skin tissue began to repair, the skin tissue repair was obvious on the 5th day, the granulation tissue of hyperplasia was completely formed, and the wound repair was completed on the 7th day, and the skin tissue was ruddy and elastic. Figure 15 D and Figure 15 F showed that a large amount of MRSA biofilms grew at the wounds in the SPSS group and the ACR - DMP group after the 7th day; the remaining amount of MRSA biofilms at the wounds in the Van group was only 16% of that on the 1st day after the 7th day, proving that Van had an obvious effect on clearing the MRSA biofilms on the skin tissue; the remaining amount of MRSA biofilms at the wounds in the ACR - DMP + L group was only 17.5% of that in the SPSS group on the 1st day, and it was completely cleared on the 7th day, proving that ACR - DMP had a high - efficiency effect on clearing the MRSA biofilms on the skin tissue under light.

[0180] 3. Experimental procedures for H&E staining

[0181] Fresh tissues are fixed with fixative for more than 24 hours. The tissues are taken out of the fixative and the target tissues are trimmed flat with a scalpel in a fume hood. The trimmed tissues and corresponding labels are placed in a dehydration box. The dehydration box is put into a hanging basket and dehydrated successively with gradient alcohol in a dehydrator. 75% alcohol for 4 hours - 85% alcohol for 2 hours - 90% alcohol for 2 hours - 95% alcohol for 1 hour - absolute ethanol I for 30 minutes - absolute ethanol II for 30 minutes - alcohol-benzene for 5 - 10 minutes - xylene I for 5 - 10 minutes - xylene II for 5 - 10 minutes - paraffin I for 1 hour - paraffin II for 1 hour - paraffin III for 1 hour. The tissues impregnated with paraffin are embedded in an embedding machine. First, the melted paraffin is put into an embedding frame. Before the paraffin solidifies, the tissues are taken out of the dehydration box and placed in the embedding frame according to the requirements of the embedding surface and the corresponding labels are pasted. Cool on a -20°C freezing table. After the paraffin solidifies, the wax block is taken out of the embedding frame and the wax block is trimmed. The trimmed wax block is placed on a microtome for sectioning, with a thickness of 3 μm. The sections float on a spreading machine at 40°C warm water to flatten the tissues, and the tissues are picked up with a glass slide and baked in an oven at 60°C. After the water is dried and the wax is melted, take it out and store it at room temperature for standby. The sections are successively placed in xylene I for 10 minutes - xylene II for 10 minutes - xylene III for 10 minutes - absolute ethanol I for 5 minutes - absolute ethanol II for 5 minutes - 75% alcohol for 5 minutes, and washed with tap water. Stain with hematoxylin for 4 minutes, differentiate with 1% hydrochloric acid alcohol solution (75% alcohol), blue with 1% ammonia water solution, and wash with water. Stain in eosin staining solution for 2 minutes and wash with water. The sections are successively placed in 75 - 95% alcohol for several seconds - absolute ethanol I for 5 minutes - absolute ethanol II for 5 minutes - absolute ethanol III for 5 minutes - dried in an oven at 37°C - xylene I for 5 minutes - xylene II for 5 minutes - xylene III for 5 minutes for dehydration and clearing. Take the sections out of the xylene and let them dry slightly, and seal the sections with neutral gum.

[0182] As Figure 16 A, in the figure, neutrophils (triangle dotted arrow), new blood vessels (triangle solid arrow), granuloma formation (arc solid arrow), lymphocytes (diamond solid arrow), fibroblasts (arc dotted arrow), tissue necrosis, cell debris (round solid arrow) are shown. The epidermal structure of the normal area of each skin sample is complete and clear, with obvious keratinization, a relatively thin stratified squamous epithelium layer, and closely arranged cells; the collagen fibers in the dermis are arranged in a crisscross pattern, irregularly, with red-stained and uniform cytoplasm, and a relatively large number of hair follicles and sebaceous glands, etc.; the subcutaneous muscle fibers and adipocytes have normal structures.

[0183] In the four experimental groups, large - area necrosis occurred in the D1 skin, with a large number of necrotic areas. The tissue structure and cell morphology were blurred. The cytoplasm gradually disintegrated or disappeared, the nucleus showed pyknosis, fragmentation or dissolution, and inflammatory cells and homogeneous eosinophilic protein - like substances exuded and deposited. In the SPSS group, at D3, the cell morphology was blurred, the cytoplasm gradually disintegrated or disappeared, the nucleus showed pyknosis, fragmentation or dissolution, and a large number of inflammatory cells exuded. At D5, granulomas formed in a small area. In the granuloma area, the proliferation of newly formed capillaries and fibroblasts was relatively obvious; there was more infiltration of lymphocytes and neutrophils in the stroma, and the staining was significantly deeper. At D7, partial histiocyte necrosis was visible, with a small amount of newly formed capillaries and fibrous tissue proliferation, and a large number of inflammatory cell infiltrations. In the Van group, at D3, the cell debris formed by necrosis decreased, and a large number of inflammatory cells infiltrated. At D5, scars formed in a local area, and granulomas formed in a small area. In the granuloma area, the newly formed capillaries were relatively obvious; there was more infiltration of lymphocytes and neutrophils in the stroma, and the staining was significantly deeper. At D7, there was relatively abundant newly formed capillaries and a large amount of fibrous tissue proliferation. The fibrous tissue mainly consisted of fibroblasts, and more inflammatory cell infiltrations were visible. In the ACR - DMP group, at D3 and D5, tissue necrosis occurred, the cell morphology was blurred, the cytoplasm gradually disintegrated or disappeared, the nucleus showed pyknosis, fragmentation or dissolution. At D7, scars formed in a local area, partial cell necrosis was visible, with a small amount of newly formed capillaries and fibrous tissue proliferation, and a large number of inflammatory cell infiltrations. In the ACR - DMP + L group, at D3, fibrous tissue proliferation was visible, the cell debris formed by necrosis decreased, and a large number of inflammatory cells infiltrated. At D5, scars formed in the area, obvious granuloma formation was visible. In the granuloma area, the proliferation of newly formed capillaries and fibroblasts was relatively obvious; there was more infiltration of lymphocytes and neutrophils in the stroma, and the staining was significantly deeper. At D7, there was relatively abundant newly formed capillaries and a large amount of fibrous tissue proliferation; the fibrous tissue mainly consisted of fibroblasts, the nuclei were mostly oval or long - oval, a small amount of collagen fibers formed in the stroma, and a small number of inflammatory cell infiltrations were visible.

[0184] According to the above pathological description results, it can be seen that different degrees of injury repair occurred in the skin tissues of each group. At D1 and D3, large - area necrosis occurred in the sample tissues of each group, with a large number of inflammatory cell infiltrations. Among them, the necrotic area and inflammatory cells in the ACR - DMP + L group were the least. At D5, obvious granuloma formation was visible in the ACR - DMP + L group. In the granuloma area, the proliferation of newly formed capillaries and fibroblasts was relatively obvious. The newly formed capillaries and fibroblasts in the Van and SPSS groups were relatively fewer, and obvious necrotic areas were still visible in the ACR - DMP group. At D7, the ACR - DMP + L group was mainly characterized by fibrous tissue proliferation, with a mild inflammatory reaction. Compared with the ACR - DMP + L group, the inflammatory reaction in the Van group was more obvious, while obvious necrotic areas and obvious inflammatory reactions were still visible in the SPSS and ACR - DMP groups.

[0185] 4. Experimental steps of immunofluorescence protein labeling

[0186] The paraffin sectioning process is the same as that of H&E staining. The tissue section is placed in a repair box filled with TRIS-EDTA (50X) antigen repair buffer (pH 9.0) and antigen repair is carried out in a microwave oven. Heat to boiling at medium heat for 8 min, stop heating and keep warm for 8 min, then turn to medium-low heat for 7 min. During this process, prevent excessive evaporation of the buffer and do not let the slice dry. After natural cooling, place the slide in PBS (pH 7.4) and wash it by shaking on a decolorizing shaker 3 times, 5 min each time. Use a histochemical pen to circle the tissue, then drop A solution of the tissue autofluorescence quencher to cover the tissue and incubate at room temperature for 30 min, and wash with pure water for 5 min. Drop 5% normal goat serum working solution for blocking (stock solution:PBS = 1:19) in the histochemical circle to evenly cover the tissue and block at room temperature for 30 min. Gently shake off the blocking solution, drop the primary antibody prepared in proportion with PBS (pre-cooled at 4°C) on the section, and place the section flat in a wet box and incubate at 4°C overnight. (Add a small amount of water in the wet box to prevent evaporation of the antibody). Place the slide in PBS (pH 7.2 - 7.4) and wash it by shaking on a decolorizing shaker 3 times, 5 min each time. After slightly drying the section by shaking, drop the secondary antibody (FITC / CY3 labeled) corresponding to the primary antibody in the circle to cover the tissue and incubate at room temperature for 50 min. Place the slide in PBS (pH 7.4) and wash it by shaking on a decolorizing shaker 3 times, 5 min each time. After slightly drying the section by shaking, drop DAPI staining solution in the circle, and incubate in the dark at room temperature for 10 min. Place the slide in PBS (pH 7.2 - 7.4) and wash it by shaking on a decolorizing shaker 3 times, 5 min each time. Drop B solution of the tissue autofluorescence quencher to cover the tissue and incubate in the dark at room temperature for 5 min, and rinse with running water for 3 min. After slightly drying the section by shaking, seal the section with an anti-fluorescence quenching mounting medium.

[0187] As Figure 16 Shown in B, for the ACR-DMP+L group, the results of CD31 and α-SMA labeling showed formed blood vessels, and significant expression of vascular endothelial growth factor and smooth muscle cell cytoskeleton protein. The MPO labeling result showed that the expression of anti-myeloperoxidase in this group was the lowest, indicating that its inflammatory reaction level was the lowest. Figure 16 The results of the optical density statistics in C also verified that the SPSS group and the ACR-DMP group had the highest inflammatory levels, and the repair of smooth muscle cells and vascular endothelial cells was poor. The Van inflammatory level and the repair of damaged cells were lower than those of the ACR-DMP+L group, but better than those of the SPSS group and the ACR-DMP group.

[0188] The results of H&E and immunofluorescent protein experiments showed that compared with the commercially available antibiotic Van, ACR-DMP had a good effect on photodynamically clearing bacterial biofilms, and its good biocompatibility promoted wound healing and tissue regeneration, effectively reducing the inflammatory reaction at the infection site, and achieving efficient clearance of bacterial biofilms on in-vivo implanted medical devices and wound tissues, etc.

Claims

1. A compound with type I reactive oxygen species generating performance, characterized in that, The general chemical structural formula is as follows: Among them, R1 is , R` is C1-C 12 alkyl; R2 is an alkyl group of C1-C 10 , or ; Y is O or S.

2. The preparation method of the compound with type I reactive oxygen species generating performance as described in claim 1, characterized in that, It includes the following steps: (1) Add compound A and N-bromosuccinimide to the first solvent, stir at room temperature until the reaction is complete, then distill off the organic solvent under reduced pressure. Purify the obtained crude product by silica gel column chromatography to obtain compound B; (2) Add compound B, compound C, a palladium catalyst, and an inorganic base to the second solvent, heat under reflux under N2 protection until the reaction is complete, then filter, wash, and dry. After collecting the organic phase, remove the organic solvent. Purify the obtained crude product by silica gel column chromatography to obtain compound D; (3) Add compound D, compound E, and sodium tert-butoxide to ethanol, stir at room temperature until the reaction is complete, then filter, wash, and dry. After collecting the organic phase, remove the organic solvent to obtain compound F, and then quaternize compound F to obtain a series of compounds with high-performance type I reactive oxygen species generation performance; Among them, the structural formula of compound A is , where Y is O or S; The structural formula of Compound B is , where Y is O or S; The structural formula of compound C is ; The structure of compound D is ; The structure of compound E is , where R1 is , and R` is an alkyl group of C1-C 12 . The structure of compound F is .

3. The preparation method of the compound with type I reactive oxygen species generating performance according to claim 2, characterized in that, In step (1), the first solvent is N,N-dimethylformamide, ethanol, acetonitrile, isopropanol, n-butanol, or tert-amyl alcohol; the eluent in the purification process is ethanol, methanol, dichloromethane, ethyl acetate, or chloroform.

4. The preparation method of the compound with type I reactive oxygen species generating performance according to claim 2, characterized in that, In step (1), the mass ratio of compound A to N-bromosuccinimide is 1:1 - 1.

2.

5. The preparation method of the compound with type I reactive oxygen species generating performance as described in claim 2, characterized in that, In step (2), the palladium catalyst is tetrakis(triphenylphosphine)palladium, bis(triphenylphosphine)palladium dichloride, [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium, tris(dibenzylideneacetone)dipalladium, or palladium acetate.

6. The preparation method of the compound with type I reactive oxygen species generating performance as described in claim 2, characterized in that, In step (2), the molar ratio of compound B, compound C, the palladium catalyst, and the inorganic base is 1.0 - 1.5:1.0 - 8.0:1.0:0.01 - 0.05:

10.

7. The preparation method of the compound with type I reactive oxygen species generating performance as described in claim 2, characterized in that, The inorganic base in step (2) is sodium carbonate, potassium carbonate, cesium carbonate, potassium phosphate, or barium hydroxide.

8. The preparation method of the compound with type I reactive oxygen species generating performance according to claim 2, characterized in that, In step (2), the second solvent is a mixed solution of tetrahydrofuran and water with a volume ratio of 5 - 10:1, a mixed solution of toluene, ethanol, and water with a volume ratio of 6 - 10:1:1, or a mixed solution of dioxane and water with a volume ratio of 5 - 10:

1.

9. The preparation method of the compound with type I reactive oxygen species generating performance according to claim 2, characterized in that, In step (3), the molar ratio of compound D, compound E, and sodium tert-butoxide is 1.0 - 1.5:1.0 - 1.5:1.

0.

10. Use of the compound with type I reactive oxygen species generation performance according to claim 1 in the preparation of a fluorescence imaging detection reagent, a photodynamic bactericidal reagent, and a photodynamic anticancer drug.

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