Photodynamic antibacterial material taking fluorene and benzimidazole as frameworks and preparation method thereof

Abstract

The invention discloses a preparation method of a photodynamic antibacterial material with fluorene and benzimidazole as frameworks, and relates to the field of fluorescent and antibacterial materials.The preparation method comprises the steps that benzimidazole and fluorene are polymerized to obtain a polymer PFTIM, then the polymer PFTIM serves as a repetitive unit to be subjected to conjugated polymerization to obtain conjugated polyelectrolyte (+)-PFTIM, and the conjugated polyelectrolyte (+)-PFTIM is subjected to photodynamic polymerization to obtain the photodynamic antibacterial material with fluorene and benzimidazole as the frameworks. The conjugated polyelectrolyte (+)-PFTIM can excite fluorescence in a visible light range, can inhibit the activity of gram-negative bacteria and gram-positive bacteria, and can effectively inhibit the growth of a biological membrane of a bacterial strain.

Description

Technical Field

[0001] The invention relates to the field of fluorescent and antibacterial materials, and in particular to a method for preparing a photodynamic antibacterial material with fluorene and benzimidazole as a skeleton. Background Art

[0002] With the increase of bacterial resistance, it is urgent to find new antibacterial methods. Photodynamic therapy has gradually become a popular choice due to its non-toxicity and high efficiency. In this field, fluorescent conjugated polymers have attracted widespread attention due to their unique optical properties and good biocompatibility. These polymers can absorb light energy of specific wavelengths and convert it into reactive oxygen species, such as singlet oxygen and free radicals, which lead to the death of bacteria. Their unique chemical structure and adjustable optical properties provide diversity and flexibility in their mechanism of action in the antibacterial process. As a new type of photodynamic therapy photosensitizer, fluorescent conjugated polymers have shown great potential in photodynamic antibacterial. With the in-depth understanding of its mechanism of action and the continuous improvement of technology, it is believed that fluorescent conjugated polymers will become one of the important strategies for future antibacterial treatment.

[0003] In the prior art, benzimidazole is a good antibacterial group, but the benzimidazole group can only inhibit Gram-positive bacteria and has a narrow antibacterial spectrum. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides a photodynamic antibacterial material with fluorene and benzimidazole as a skeleton, which has good active inhibitory effect on Gram-positive and Gram-negative bacteria under light excitation, and a preparation method thereof.

[0005] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0006] Provided is a method for preparing a photodynamic antibacterial material with fluorene and benzimidazole as a skeleton, comprising the following steps:

[0007] S1: 2,7-bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-9,9-bis-(6-bromohexyl)fluorene, 5-bromo-1-(6-bromohexyl)-2-(5-bromothien-2-yl)-1H-benzo[d]imidazole, toluene and K 2 CO 3 Mix, then add methyl trioctyl ammonium chloride dropwise, and stir for 20 min under argon atmosphere;

[0008] S2: Continue adding Pd(OAc) 2 and tricyclohexylphosphine, reacted at 90°C for 9 hours under argon atmosphere, and then extracted and filtered with methanol to obtain a neutral polymer PFTIM, the structure of which is:

[0009]

[0010] S3: PFTIM is conjugated and polymerized using THF and trimethylamine aqueous solution to obtain a photodynamic antibacterial material. The structure of the photodynamic antibacterial material is:

[0011]

[0012] Further, the preparation method of 2,7-bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)-9,9-bis-(6-bromohexyl)fluorene is specifically as follows:

[0013] A1: Add 2,7-dibromo-9,9-bis-(6-bromohexyl)fluorene, bis-pinacol and potassium acetate into a reaction bottle, add DMF to dissolve, stir for 20 minutes, and fully dissolve;

[0014] A2: Add Pd(dppf)Cl2 to the mixed solution and react at 85°C for 12h under argon atmosphere;

[0015] A3: After the reaction is completed, the product is extracted with ethyl acetate, washed and dried to obtain 2,7-bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)-9,9-bis-(6-bromohexyl)fluorene.

[0016] Further, the molar ratio of 2,7-dibromo-9,9-bis-(6-bromohexyl)fluorene, bis-pinacol and potassium acetate is 4.54:27.2:27.2;

[0017] The usage ratio of 2,7-dibromo-9,9-bis-(6-bromohexyl)fluorene and DMF is 1.4 g:10 mL;

[0018] The molar ratio of Pd(dppf)Cl2 to 2,7-dibromo-9,9-bis-(6-bromohexyl)fluorene is 0.274:4.54.

[0019] Furthermore, step A3 is specifically as follows:

[0020] A31: The reaction product of A2 is added to water and mixed, and ethyl acetate and NaCl are added;

[0021] A32: Take the organic phase, wash it three times with NaCl solution, and then wash it with anhydrous MgSO 4 Drying, filtration, rotary evaporation, vacuum drying;

[0022] A33: Using ethyl acetate:petroleum ether as a solvent of 20:1, purify the dried solid by silica gel column chromatography, and then dry in vacuo to obtain 2,7-bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)-9,9-bis-(6-bromohexyl)fluorene.

[0023] Further, the preparation method of 2,7-dibromo-9,9-bis-(6-bromohexyl)fluorene is specifically as follows:

[0024] B1: Under argon atmosphere, add 2,7-dibromofluorene, 1,6-dibromohexane and tetrabutylammonium bromide in a molar ratio of 7.7:156:0.78 into the reaction bottle in sequence and wait for complete dissolution;

[0025] B2: Add 50% KOH and stir at 75°C for 1.5h to wait for the reaction to complete; the ratio of KOH to 2,7-dibromofluorene is 8mL:7.7mmol;

[0026] B3: After the reaction was completed, deionized water was added and extracted with dichloromethane. The organic layer was washed with water three times and then washed with anhydrous MgSO 4 dry;

[0027] B4: Perform rotary evaporation, and then perform chromatography purification using a solvent prepared by mixing petroleum ether and dichloromethane in a ratio of 19:1, and vacuum drying to obtain 2,7-dibromo-9,9-bis-(6-bromohexyl)fluorene.

[0028] Further, the preparation method of 5-bromo-1-(6-bromohexyl)-2-(5-bromothiophen-2-yl)-1H-benzo[d]imidazole is specifically as follows:

[0029] C1: 5-bromo-2-(5-bromothien-2-yl)-1H-benzo[d]imidazole and K 2 CO 3 Dissolved in DMF, reacted at 95°C for 2h under argon atmosphere; 5-bromo-2-(5-bromothiophen-2-yl)-1H-benzo[d]imidazole and K 2 CO 3 The molar ratio of K is 1.08:1.62; and 2 CO 3 The dosage ratio of DMF is 1.62mmol:7mL;

[0030] C2: Add 1,6-dibromohexane to the product and react at 95℃ for 4h; 1,6-dibromohexane and K 2 CO 3 The molar ratio is 10.8:1.62;

[0031] C3: After cooling to room temperature, deionized water was added and extracted with dichloromethane to extract the organic layer;

[0032] C4: The organic layer was treated with anhydrous MgSO 4 Drying, filtering and rotary evaporation;

[0033] C5: The material after rotary evaporation was subjected to chromatography using a solvent prepared with petroleum ether:ethyl acetate in a ratio of 10:1 to obtain 5-bromo-1-(6-bromohexyl)-2-(5-bromothiophen-2-yl)-1H-benzo[d]imidazole.

[0034] Further, the preparation method of 5-bromo-2-(5-bromothiophen-2-yl)-1H-benzo[d]imidazole is specifically as follows:

[0035] D1: Add 4-bromo-1,2-phenylenediamine, sodium 5-bromothiophene-2-sulfonate, DMF and activated carbon into a reaction bottle, raise the temperature to reflux, and maintain the reflux temperature for 3.5 hours; the amount ratio of 4-bromo-1,2-phenylenediamine, sodium 5-bromothiophene-2-sulfonate and DMF is 6.96mmol:7.02mmol:17mL;

[0036] D2: Filter the solution before cooling to obtain a filtrate, pour the filtrate into ice water and stir to obtain an emulsion;

[0037] D3: After adding saturated salt water to the emulsion, filter it with suction, take the filter cake, wash it with water and then dry it;

[0038] D4: The dried solid was purified by chromatography using a solvent prepared with petroleum ether: ethyl acetate in a ratio of 3:1. The purified product was decolorized by recrystallization using ethanol to obtain 5-bromo-2-(5-bromothiophen-2-yl)-1H-benzo[d]imidazole.

[0039] Further, the preparation method of sodium 5-bromothiophene-2-sulfonate is specifically as follows:

[0040] E1: Dissolve 5-bromothiophene-2-carboxaldehyde in ethanol;

[0041] E2: Add sodium bisulfite solution to the 5-bromothiophene-2-carboxaldehyde-ethanol solution and stir the reaction at room temperature for 2.5 hours;

[0042] E3: The product is filtered to obtain a filter cake, which is washed with anhydrous ethanol and then dried to obtain sodium 5-bromothiophene-2-sulfonate.

[0043] The present invention also provides a photodynamic antibacterial material prepared by the above-mentioned preparation method.

[0044] The present invention also provides a use of the above-mentioned photodynamic antibacterial material in inhibiting the activity of Gram-positive bacteria and Gram-negative bacteria.

[0045] The beneficial effects of the present invention are:

[0046] The present invention obtains a polymer PFTIM by polymerizing benzimidazole and fluorene, and then uses PFTIM as a repeating unit for conjugated polymerization to obtain a conjugated polyelectrolyte (+)-PFTIM. The conjugated polyelectrolyte (+)-PFTIM can excite fluorescence in the visible light range, can inhibit the activity of both Gram-negative bacteria and Gram-positive bacteria, and can effectively inhibit the growth of biofilms of strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The figure is a comparison of the antibacterial results of different concentrations of antibacterial material aqueous solutions on MRSA;

[0048] Figure 2 The figure is a comparison of the antibacterial results of aqueous solutions of antibacterial materials with different concentrations on MDR-PA;

[0049] Figure 3 The crystal violet staining results and Biomass statistical results in Example 7 are shown;

[0050] Figure 4 This is the result of biofilm staining of bacteria incubated with antibacterial materials. DETAILED DESCRIPTION

[0051] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0052] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially. The sources of the reagents used in the examples are shown in Table 1 below;

[0053] Table 1

[0054] name Type / Model company Solid culture medium LB Agar Cool Laibo Liquid culture medium LB Broth Cool Laibo Crystal violet dye Crystal violet Cologne Chemical Dead cell stain PI (Propidium Iodide) Solebao Composite dyes Merge Solebao

[0055] Example 1 Synthesis of 2,7-dibromo-9,9-bis-(6-bromohexyl)fluorene

[0056] Under argon atmosphere, 2,7-dibromofluorene (2.5 g, 7.7 mmol), 1,6-dibromohexane (38 g, 156 mmol) and tetrabutylammonium bromide (0.25 g, 0.78 mmol) were added to a 100 mL three-necked flask in sequence. After complete dissolution, KOH (50%, 8 mL) was added, the temperature was raised to 75 ° C, and stirred for 1.5 h. The mixture was cooled to room temperature, poured into 100 mL of deionized water, extracted with dichloromethane, and the combined organic layer was washed with water 3 times, and then washed with anhydrous MgSO 4 After the solvent and excess 1,6-dibromohexane were removed by rotary evaporation, the product was purified by silica gel column chromatography using petroleum ether and dichloromethane (19:1) as solvent and vacuum dried to obtain 2.3688 g of white 2,7-dibromo-9,9-bis-(6-bromohexyl)fluorene solid with a yield of 59.1%.

[0057] Example 2 Synthesis of 2,7-bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-9,9-bis-(6-bromohexyl)fluorene

[0058] Take 2,7-dibromo-9,9-bis-(6-bromohexyl)fluorene (1.4g, 4.54mmol), bis-pinacol (2.075g, 27.2mmol), potassium acetate (1.335g, 27.2mmol) prepared in Example 1 and add them to a 50mL two-necked flask, add DMF (10mL) to dissolve, and evacuate and ventilate three times. Stir for 20min, fully dissolve, add catalyst Pd (dppf) Cl2 (0.111g, 0.274mmol) from the mouth of the flask connected to the condenser, evacuate and ventilate 3 times, heat to 85°C and react for 12h. After the reaction is completed, pour the reaction product in the flask into 20mL water, extract with ethyl acetate, add an appropriate amount of NaCl to emulsify, wash the organic phase with NaCl solution three times, dry with anhydrous MgSO4, filter, rotary evaporate, and dry. Ethyl acetate and petroleum ether (20:1) were used as solvents, purified by silica gel column chromatography, and vacuum dried to obtain 1.1361 g of a white solid with a yield of 61.93%. The obtained white solid was detected by nuclear magnetic resonance hydrogen spectrum, and the detection results were: 1HNMR (400MHz, Chloroform-d) δ7.81 (d, J = 8.1 Hz, 2H), 7.72 (d, J = 7.4 Hz, 4H), 3.25 (t, J = 6.9 Hz, 4H), 2.05-1.97 (m, 4H), 1.62 (dt, J = 14.5, 7.0 Hz, 4H), 1.39 (s, 24H), 1.19-1.10 (m, 4H), 1.04 (p, J = 7.2 Hz, 4H), 0.60-0.49 (m, 4H). According to the 1HNMR results, the structural formula of the white solid is 2,7-bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-9,9-bis-(6-bromohexyl)fluorene, and the reaction formula is as follows:

[0059]

[0060] Example 3 Synthesis of Sodium 5-Bromothiophene-2-Sulfonate

[0061] 5-bromothiophene-2-carboxaldehyde (4.775 g, 25 mmol) was added to a 100 mL round-bottom flask, and 12.5 mL of ethanol was added. The mixture was stirred thoroughly to dissolve. Then, sodium bisulfite was dissolved in 6 mL of distilled water. The prepared 4 mol / L sodium bisulfite solution was added to the ethanol solution of 2-bromo-5-formaldehyde thiophene. When the solution changed from brown to white, the sodium bisulfite solution was stopped and continued to be added dropwise. The mixture was stirred at room temperature for 2.5 h. After the reaction was completed, the mixture was filtered, and the filter cake was rinsed with ice anhydrous ethanol (12.5 mL). The mixture was dried under vacuum to obtain 5.9691 g of white 5-bromothiophene-2-sodium sulfonate solid, with a yield of 80.90%.

[0062] Example 4 Synthesis of 5-bromo-2-(5-bromothiophen-2-yl)-1H-benzo[d]imidazole

[0063] Take the sodium 5-bromothiophene-2-sulfonate prepared in Example 3, add a spoonful of activated carbon, 4-bromo-1,2-phenylenediamine (1.0415g, 6.96mmol), sodium 5-bromothiophene-2-sulfonate (1.6612g, 7.02mmol) and DMF (17mL) to a 50mL three-necked flask, quickly heat to reflux, and react at this temperature for 3.5h. After the reaction is completed, filter while hot to remove insoluble matter, pour the filtrate into 350mL of ice water, stir, and immediately produce a yellow emulsion, add saturated salt water to precipitate it. Filter by suction, wash with distilled water several times, and put it in a drying oven to dry. Use petroleum ether and ethyl acetate (3:1) as solvents and purify by silica gel column chromatography. The product was decolorized by recrystallization with ethanol and dried in vacuo to give 0.8324 g of yellow solid 5-bromo-2-(5-bromothiophen-2-yl)-1H-benzo[d]imidazole with a yield of 33.97%.

[0064] Example 5 Synthesis of 5-bromo-1-(6-bromohexyl)-2-(5-bromothiophen-2-yl)-1H-benzo[d]imidazole

[0065] Take 5-bromo-2-(5-bromothiophen-2-yl)-1H-benzo[d]imidazole (0.3867 g, 1.08 mmol) prepared in Example 4 and K 2 CO 3 (0.2230 g, 1.62 mmol) was dissolved in DMF (7 mL), reacted at 95 ° C for 2 h under argon protection, and then 1,6-dibromohexane (3.75 g, 10.8 mmol) was added and reacted at 95 ° C for 4 h. After cooling to room temperature, an appropriate amount of deionized water was added, and the mixture was extracted with dichloromethane for 3 times. The organic layers were combined and washed with anhydrous Na 2 SO 4Dry, filter, and evaporate. Column chromatography was performed using petroleum ether: ethyl acetate = 10:1 to obtain 0.6263 g of 5-bromo-1-(6-bromohexyl)-2-(5-bromothiophen-2-yl)-1H benzo[d]imidazole, with a yield of 55.64%; H NMR: 1H NMR (400 MHz, Chloroform-d) δ 7.90 (d, J = 1.6 Hz, 1H), 7.39 (dd, J = 8.6, 1.8 Hz, 1 H), 7.29–7.26 (m, 1H), 7.22 (d, J = 8.6 Hz, 1H), 7.15 (d, J = 4.0 Hz, 1H), 4.34–4.29 (m, 2H), 3.39 (t, J = 6.6 Hz, 2H), 1.85 (dq, J = 14.4, 7.2, 6.7 Hz, 4H), 1.45 (dh, J = 28.8, 7.5, 6.6 Hz, 4H). The reaction formula is as follows:

[0066]

[0067] Example 6 Synthesis of photodynamic antibacterial material

[0068] 2,7-bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-9,9-bis-(6-bromohexyl)fluorene (0.1606 g, 0.25 mmol) prepared in Example 2, 5-bromo-1-(6-bromohexyl)-2-(5-bromothiophen-2-yl)-1H-benzo[d]imidazole (0.1303 g, 0.25 mmol) prepared in Example 5, toluene (10 mL), K 2 CO 3 (2M, 1.5mL), then add 2 drops of Aq336 (methyl trioctyl ammonium chloride) (0.1ml). Vacuum and ventilate 3 times, stir for 20min under argon atmosphere and then deoxygenate, then add Pd(OAc)2 (0.003g, 0.013mmol) and tricyclohexylphosphine (0.006g, 0.021mmol), ventilate and ventilate 4 times. React at 90℃ for 9h, drop methanol (200mL) into a cotton filter tube, let stand and precipitate, filter, and dry in a vacuum oven to obtain 0.0875g of yellow PFTIM solid with a yield of 39.70%. The reaction formula is as follows:

[0069]

[0070] Weigh 20 mg of the neutral polymer PFTIM into a reaction tube, add THF (3 mL) and trimethylamine aqueous solution (1 mL) in sequence, and react at 60°C for 48 h. After the reaction stops, rinse the reaction tube with methanol, collect the solvent, rotary evaporate, and dry to obtain 0.0225 g of a yellow conjugated polyelectrolyte (+)-PFTIM solid with a yield of 93.66%.

[0071] Example 7 Verification of the antibacterial ability of photodynamic antibacterial materials

[0072] The conjugated polyelectrolyte (+)-PFTIM prepared in Example 6 was used to prepare 5 μM, 10 μM and 20 μM aqueous solutions of antibacterial materials, respectively.

[0073] MRSA (drug-resistant Staphylococcus aureus) and MDR-PA (drug-resistant Pseudomonas aeruginosa) were selected as target bacteria for antibacterial test verification. Specifically:

[0074] The target strains were activated three times with LB solid medium, and the activated strains were cultured in LB liquid medium. Since the optimal growth temperatures of the two bacteria were different, they were incubated with different concentrations of antibacterial material aqueous solutions at 37°C (MRSA) and 25°C (MDR-PA) for 24 hours, and a blank control group was set up. After the culture was completed, 50uL of gradient dilution was transferred with a pipette, and the appropriate concentration was selected to ensure that the colonies on the plate were uniform and independent of each other. In this experiment, the cultured bacterial solution was diluted 1×10 6 After the incubation is completed, 100uL of bacterial solution is transferred with a pipette and spread on the plate, and the light group (Light) and the light-shielding group (Dark) are set. After 12 hours of incubation, the antibacterial ability and photodynamic antibacterial activity are evaluated according to the number of colonies. In addition, the plate is photographed with a scanning electron microscope to observe the morphology of the bacteria and determine whether cell lysis occurs. The results are shown in Figure 1 and Figure 2 As shown, Figure 1 A is a comparison of MRSA colonies incubated with aqueous solutions of antibacterial materials of different concentrations under light and light-shielding conditions. Figure 1 B is the scanning electron micrograph of MRSA incubated with 20uM aqueous solution of antibacterial material under light and light shielding conditions; Figure 2 A is a comparison of the colonies of MDR-PA incubated with aqueous solutions of antibacterial materials of different concentrations under light and shade conditions. Figure 2 B is the scanning electron micrograph of MDR-PA incubated with 20uM aqueous solution of antibacterial material under light and light-shielding conditions;

[0075] Depend on Figure 1 and Figure 2 It can be seen that the use of different concentrations of antibacterial material aqueous solutions can inhibit the activity of both Gram-positive and Gram-negative bacteria. Among them, at a concentration of 5μM, the inhibitory effect is weak; at a concentration of 10μM, the inhibitory effect is obvious; at a concentration of 20μM, both Gram-positive and Gram-negative bacteria can hardly survive. Using a 20uM antibacterial material aqueous solution under white light irradiation conditions, both bacteria can be completely killed. In the absence of light, the cell morphology remains intact.

[0076] A biofilm inhibition test was also set up to verify the ability to inhibit biofilm formation. The biofilm inhibition test was specifically as follows: the bacteria were taken out of the -80°C refrigerator and revived on LB solid culture medium (repeated three times), and the revived bacteria were picked out and inoculated into LB liquid culture medium for amplification and culture to the logarithmic growth phase.

[0077] The amplified bacterial solution was diluted 100 times and inoculated into a 24-well plate, with 100uL of bacterial solution in each well. 100uL of aqueous solution of antibacterial materials at different concentrations was added to the positive wells, and an equal amount of PBS buffer solution was added to the negative wells. The well plate was placed in a constant temperature incubator at 37°C and incubated for 24h.

[0078] After the culture was completed, the culture medium was aspirated and washed three times with PBS to remove impurities and floating bacteria. 200uL of methanol was added to each well for fixation. After 15 minutes, the methanol was aspirated and dried.

[0079] Add 200uL of 0.1% crystal violet to each well and stain for 20 minutes, then let it dry.

[0080] The absorbance at 595 nm was measured using an ELISA instrument, and the biomass (%) of each well was measured with the negative plate as 100%. Figure 3 As shown, Figure 3 A is the crystal violet staining result of 20 μM antibacterial material aqueous solution (PS) and blank group (Blank) and negative group (PBS) for incubation of MRSA. Figure 3 B is the crystal violet staining result of 20 μM aqueous solution of antibacterial material and blank group and negative group used to incubate MDR-PA; Figure 3 C is a comparison of MRSA biomass incubated with different concentrations of antibacterial material aqueous solutions shaded or not; Figure 3 D is a comparison of the biomass of MDR-PA incubated with or without light shielding in aqueous solutions of antibacterial materials of different concentrations.

[0081] Depend on Figure 3 A and Figure 3 B shows that after incubating the bacteria with a 20 μM aqueous solution of antibacterial material under light conditions, the crystal violet staining became significantly lighter, while there was no significant change in staining under dark conditions.

[0082] Depend on Figure 3 C and Figure 3 D shows that as the concentration of the antibacterial material aqueous solution increases, the bacterial biomass in the positive wells decreases. In summary, the antibacterial material prepared in Example 6 can effectively inhibit the activity of Gram-negative bacteria and Gram-positive bacteria, and the higher the concentration of the antibacterial material, the stronger the ability to inhibit the activity of bacteria.

[0083] To further verify the effect of antibacterial materials on biofilms, PI (propidium iodide) and Merge dye were used to stain the bacterial biofilms incubated with antibacterial materials, and the biofilm staining results were observed.

[0084] Specifically, the cell slide was immersed in hydrofluoric acid (AR, ≥40%) for five seconds, then washed with pure water and 75% ethanol and sterilized by high-pressure steam. After being completely dried, it was placed in a 24-well polystyrene microwell culture plate. 300uL of LB culture medium was added to each well, and 30uL of bacterial solution in the logarithmic growth phase was inoculated. The plate was incubated at 37°C for 36h; then the culture medium was aspirated.

[0085] 40uL of antibacterial material was incubated with the biofilm and then treated with light and darkness. The commercially available dye propidium iodide (PI) and the composite dye Merage were used for staining and then the growth status of the two biofilms was observed under a laser confocal microscope. The staining results are shown in Figure 4 As shown, Ps is the experimental group, Figure 4 It can be seen that after light treatment, the conjugated polyelectrolyte (+)-PFTIM prepared in Example 6 has an excellent inhibitory effect on the cell membranes of MRSA and MDR-PA.

Claims

1. A method for preparing a photodynamic antibacterial material with fluorene and benzimidazole as the skeleton, characterized in that: The steps include: S1: 2,7-bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-9,9-bis-(6-bromohexyl)fluorene, 5-bromo-1-(6-bromohexyl)-2-(5-bromothiophen-2-yl)-1H-benzo[d]imidazole, toluene and 2M K2CO3 were mixed, and methyltrioctylammonium chloride was added dropwise, and the mixture was stirred for 20 min under argon atmosphere; S2: Pd(OAc)2 and tricyclohexylphosphine were added continuously, and the reaction was carried out at 90°C for 9 hours under an argon atmosphere, and then a neutral polymer PFTIM was obtained by extraction and filtration using methanol. The structure of the neutral polymer PFTIM is as follows: S3: Use THF and trimethylamine aqueous solution to conjugate polymerize PFTIM to obtain the photodynamic antibacterial material, and the structure of the photodynamic antibacterial material is:

2. The method for preparing the photodynamic antibacterial material with fluorene and benzimidazole as the skeleton according to claim 1, characterized in that: The amount ratio of 2,7-bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)-9,9-bis-(6-bromohexyl)fluorene, 5-bromo-1-(6-bromohexyl)-2-(5-bromothiophene-2-yl)-1H-benzo[d]imidazole, toluene and K2CO3 is 0.25mmol:0.25mmol:10mL:1.5mL; The dosage ratio of the methyl trioctyl ammonium chloride to toluene is 0.1 mL: 10 mL; The usage ratio of toluene, Pd(OAc)2 and tricyclohexylphosphine is 10 mL: 0.013 mmol: 0.021 mmol.

3. The method for preparing the photodynamic antibacterial material with fluorene and benzimidazole as the skeleton according to claim 2, characterized in that: The preparation method of the 2,7-bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)-9,9-bis-(6-bromohexyl)fluorene is specifically as follows: A1: Add 2,7-dibromo-9,9-bis-(6-bromohexyl)fluorene, bis-pinacol and potassium acetate into a reaction bottle, add DMF to dissolve, stir for 20 minutes, and fully dissolve; the amount ratio of 2,7-dibromo-9,9-bis-(6-bromohexyl)fluorene, bis-pinacol, potassium acetate and DMF is 4.54mmol:27.2mmol:27.2mmol:10mL; A2: Pd(dppf)Cl2 was added to the mixed solution, and the mixture was reacted at 85°C for 12 hours under an argon environment; the molar ratio of Pd(dppf)Cl2 to 2,7-dibromo-9,9-bis-(6-bromohexyl)fluorene was 0.274:4.54; A3: After the reaction is completed, the product is extracted with ethyl acetate, washed and dried to obtain 2,7-bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)-9,9-bis-(6-bromohexyl)fluorene.

4. The method for preparing the photodynamic antibacterial material with fluorene and benzimidazole as the skeleton according to claim 3, characterized in that: The step A3 is specifically as follows: A31: Add the reaction product of A2 to water and mix, then add ethyl acetate and NaCl; A32: The organic phase was washed three times with NaCl solution, dried over anhydrous MgSO4, filtered, rotary evaporated, and vacuum dried in sequence; A33: Using ethyl acetate:petroleum ether as a solvent of 20:1, purify the dried solid by silica gel column chromatography, and then dry in vacuo to obtain 2,7-bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)-9,9-bis-(6-bromohexyl)fluorene.

5. The method for preparing the photodynamic antibacterial material with fluorene and benzimidazole as the skeleton according to claim 3, characterized in that: The preparation method of the 2,7-dibromo-9,9-bis-(6-bromohexyl)fluorene is specifically as follows: B1: Under argon atmosphere, add 2,7-dibromofluorene, 1,6-dibromohexane and tetrabutylammonium bromide in a molar ratio of 7.7:156:0.78 into the reaction bottle in sequence and wait for complete dissolution; B2: Add 50% KOH and stir at 75°C for 1.5h to wait for the reaction to complete; the ratio of KOH to 2,7-dibromofluorene is 8mL:7.7mmol; B3: After the reaction is completed, deionized water is added and extracted with dichloromethane. The organic layer is washed with water three times and then dried with anhydrous MgSO4; B4: Perform rotary evaporation, and then perform chromatography purification using a solvent prepared by mixing petroleum ether and dichloromethane in a ratio of 19:1, and vacuum drying to obtain 2,7-dibromo-9,9-bis-(6-bromohexyl)fluorene.

6. The method for preparing the photodynamic antibacterial material with fluorene and benzimidazole as the skeleton according to claim 1, characterized in that: The preparation method of the 5-bromo-1-(6-bromohexyl)-2-(5-bromothiophene-2-yl)-1H-benzo[d]imidazole is specifically as follows: C1: 5-bromo-2-(5-bromothiophen-2-yl)-1H-benzo[d]imidazole and K2CO3 were dissolved in DMF and reacted at 95°C for 2h under argon environment; the molar ratio of 5-bromo-2-(5-bromothiophen-2-yl)-1H-benzo[d]imidazole and K2CO3 was 1.08:1.62; and the amount ratio of K2CO3 to DMF was 1.62mmol:7mL; C2: Add 1,6-dibromohexane to the product and react at 95°C for 4 hours; the molar ratio of 1,6-dibromohexane to K2CO3 is 10.8:1.62; C3: After cooling to room temperature, deionized water was added and extracted with dichloromethane to extract the organic layer; C4: The organic layer was dried over anhydrous MgSO4, filtered and rotary evaporated; C5: The material after rotary evaporation was subjected to chromatography using a solvent prepared with petroleum ether:ethyl acetate in a ratio of 10:1 to obtain 5-bromo-1-(6-bromohexyl)-2-(5-bromothiophen-2-yl)-1H-benzo[d]imidazole.

7. The method for preparing the photodynamic antibacterial material with fluorene and benzimidazole as the skeleton according to claim 6, characterized in that: The preparation method of the 5-bromo-2-(5-bromothiophene-2-yl)-1H-benzo[d]imidazole is specifically as follows: D1: Add 4-bromo-1,2-phenylenediamine, sodium 5-bromothiophene-2-sulfonate, DMF and activated carbon into a reaction bottle, heat to reflux, and maintain the reflux temperature for 3.5 hours; the amount ratio of 4-bromo-1,2-phenylenediamine, sodium 5-bromothiophene-2-sulfonate and DMF is 6.96mmol:7.02mmol:17mL; D2: Filter the solution before cooling to obtain a filtrate, pour the filtrate into ice water and stir to obtain an emulsion; D3: After adding saturated salt water to the emulsion, filter it with suction, take the filter cake, wash it with water and then dry it; D4: The dried solid was purified by chromatography using a solvent prepared with petroleum ether: ethyl acetate in a ratio of 3:

1. The purified product was decolorized by recrystallization using ethanol to obtain 5-bromo-2-(5-bromothiophen-2-yl)-1H-benzo[d]imidazole.

8. The method for preparing the photodynamic antibacterial material with fluorene and benzimidazole as the skeleton according to claim 7, characterized in that: The preparation method of the sodium 5-bromothiophene-2-sulfonate is specifically as follows: E1: Dissolve 5-bromothiophene-2-carboxaldehyde in ethanol; E2: Add sodium bisulfite solution to the 5-bromothiophene-2-carboxaldehyde-ethanol solution and stir the reaction at room temperature for 2.5 hours; E3: The product is filtered to obtain a filter cake, which is washed with anhydrous ethanol and then dried to obtain sodium 5-bromothiophene-2-sulfonate.

9. A photodynamic antibacterial material prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the photodynamic antibacterial material according to claim 9 in inhibiting the activity of Gram-positive bacteria and Gram-negative bacteria.

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