Preparation method and application of boric acid-based photosensitive molecule and gallium ion compound thereof

Abstract

The invention discloses a boric acid-based photosensitive molecule and a preparation method and application of a gallium ion compound of the boric acid-based photosensitive molecule, and belongs to the technical field of photodynamic antibiosis. The invention provides preparation and application of a compound TPB-Ga formed by TPB and gallium ions as an antibacterial agent, a compound with a structure as shown in a formula TTCPy, 4-(bromomethyl) phenylboronic acid and an organic solvent are mixed, the mixture is heated under a protective atmosphere for a reaction, the mixture is rapidly stirred into a gallium nitrate aqueous solution and ultrapure water mixture, TPB-Ga compound nanoparticles are obtained, and the TPB-Ga compound nanoparticles are used as the antibacterial agent. The method comprises the following steps: dissolving TPB-Ga in ultrapure water to obtain a TPB-Ga compound solution, preserving at 4 DEG C for later use, and finding that the compound has efficient reactive oxygen species (ROS) generation capability, good photodynamic sterilization effect and good biocompatibility through the antibacterial rate and the cell survival rate.

Description

Technical Field

[0001] The present invention relates to the technical field of photodynamic antibacterial technology, and particularly to a preparation method and application of a borate-based antibacterial photosensitizer and its gallium ion complex. Background Art

[0002] Antibiotic therapy is one of the main means for treating bacterial infectious diseases at present. However, the long-term and excessive use of antibiotics has promoted bacteria to develop drug resistance and even evolved into superbugs. Traditional antibiotics not only show poor efficacy in the treatment of bacterial infections, but also force pathogens to develop antibiotic resistance through selection pressure, thus accelerating the evolution process of drug-resistant strains.

[0003] Antimicrobial Photodynamic Therapy (aPDT) is an emerging non-invasive antibacterial strategy. Its core mechanism is that through the photochemical reaction of a photosensitizer under specific wavelength light irradiation, reactive oxygen species (ROS) with strong oxidizing properties are generated, and efficient sterilization is achieved by ROS destroying the lipid, protein and DNA of the bacterial cell membrane. Compared with traditional antibacterial therapies, aPDT has the advantages of simple operation, controllable treatment process, repeatable administration, etc. More importantly, its non-antibiotic-dependent mechanism of action fundamentally avoids the generation of bacterial drug resistance.

[0004] In recent years, photosensitizers based on organic molecules have become the most attractive class of materials in the field of antimicrobial photodynamic therapy due to their molecular design flexibility, biocompatibility and controllability. However, traditional antimicrobial photodynamic therapy research mainly focuses on type II photosensitizers, and their efficacy for hypoxic bacterial infection lesions is limited. Summary of the Invention

[0005] In order to solve the above problems, the present invention proposes a kind of.

[0006] In view of this, the present invention provides a preparation method and antibacterial application of a borate-based photosensitive small molecule and its gallium ion complex. The photosensitizer provided by the present invention has aggregation-induced emission (AIE) properties, has high reactive oxygen species (ROS) generation ability, good photodynamic bactericidal effect and good biocompatibility.

[0007] In order to achieve the above invention purpose, the present invention provides the following technical solutions: An antibacterial photosensitizer, comprising a borate-based photosensitive molecule and / or a borate-based photosensitive molecule-gallium ion complex, and the borate-based photosensitive molecule has the following structure: 。

[0008] The present invention also provides a method for preparing a borate-based photosensitizer molecule, comprising the following steps: Mix a compound having the structure shown by formula TTCPy, 4-(bromomethyl)phenylboronic acid and an organic solvent, and heat for reaction under a protective atmosphere to obtain the borate-based photosensitizer molecule (TPB); the structure of formula TTCPy is: 。

[0009] Preferably, the organic solvent is toluene, and the molar ratio of the compound having the structure of formula TTCPy to 4-(bromomethyl)phenylboronic acid is 1:1.1-1.2.

[0010] Preferably, the heating reaction is carried out at the reflux temperature of toluene at 110 °C for 5-8 h.

[0011] The present invention also provides a method for preparing a borate-based photosensitive molecule-gallium ion complex, comprising the following steps: (1) Drop an aqueous solution of gallium nitrate into ultrapure water, and stir rapidly to obtain a gallium nitrate solution for standby; (2) Prepare an organic solution of the borate-based photosensitizer molecule (TPB) as claimed in claim 1, drop the organic solution of the borate-based photosensitizer molecule into the gallium nitrate solution, stir, and then successively carry out ultrasonic treatment, centrifugation, washing again, and centrifugation again to obtain a precipitate; (3) Lyophilize the precipitate to obtain borate-based photosensitive molecule-gallium ion (TPB-Ga) complex nanoparticles. Dissolve the borate-based photosensitive molecule-gallium ion (TPB-Ga) complex nanoparticles in ultrapure water to obtain a TPB-Ga complex solution, and store it at 4 °C for standby.

[0012] Preferably, in step (1), the concentration of the aqueous solution of gallium nitrate is 1 mg / mL, and the volume is 1 mL; the volume of ultrapure water is 8 mL; Preferably, in step (2), the organic solution of the borate-based photosensitizer molecule is a dimethyl sulfoxide solution of the borate-based photosensitizer molecule at 1 mg / mL.

[0013] Preferably, in step (2), the stirring time is 20-40 min, the ultrasonic time is 5-10 min; the centrifugal force for centrifugation is 15000-18000 × g, the centrifugation time is 20-40 min, and the centrifugation temperature is 4 °C.

[0014] The present invention also discloses the application of the borate-based photosensitive molecule (TPB) and / or the borate-based photosensitive molecule-gallium ion (TPB-Ga) complex in the preparation of antibacterial agents and photodynamic antibacterial drugs.

[0015] The beneficial effects of the preparation method and application of the borate-based photosensitive molecule and its gallium ion complex of the present invention are as follows: (1) The antibacterial photosensitizer provided by the present invention has a borate structure in its molecular structure and is positively charged, while the surface of bacteria has polysaccharides that can coordinate with borate and is negatively charged. Due to electrostatic interaction, the antibacterial photosensitizer with the structure shown by TPB can effectively bind to bacteria, laying a foundation for efficient sterilization. At the same time, the antibacterial photosensitizer TPB provided by the present invention has an obvious aggregation-induced emission (AIE) effect, so it also has high reactive oxygen species generation ability in aqueous media and will not reduce the sterilization effect due to aggregation quenching of ROS generation. In addition, TPB has a donor-Π-acceptor conjugated structure, which is beneficial for photodynamic sterilization in the near-infrared light region window.

[0016] (2) Under light irradiation conditions (460 nm - 660 nm), TPB can not only generate singlet oxygen (1O2), but also efficiently generate superoxide anion (•O2−) through type I photodynamic reaction. Low concentration of TPB (≤ 10 μM) combined with light irradiation can achieve an inhibition rate of not less than 98% against Staphylococcus aureus, and shows excellent biocompatibility (cell survival rate is greater than 85% and hemolysis rate is less than 5%).

[0017] (3) TPB-Ga provided by the present invention shows an inhibition rate of not less than 80% against drug-resistant strains (methicillin-resistant Staphylococcus aureus, multi-drug resistant Escherichia coli) under the preferred light irradiation conditions through the dual mechanisms of photodynamic killing and Ga³⁺ metabolic interference, and the antibacterial efficiency is higher than that of single components. The present invention provides an important theoretical basis and practical scheme for the development of a new broad-spectrum antibacterial strategy independent of antibiotics. Description of the Drawings

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention, and those of ordinary skill in the art can also obtain other drawings without creative efforts.

[0019] Figure 1 It is the ultraviolet-visible absorption spectrum of TPB. In the figure, the abscissa is the wavelength; the ordinate is the absorbance; Figure 2 It is the fluorescence spectrum of TPB; in the figure, the abscissa is the wavelength; the ordinate is the fluorescence intensity; Figure 3(A) Fluorescence spectra of TPB at different ratios of DMSO / toluene (Wavelength: wavelength; Intensity: fluorescence intensity); (B) Fluorescence intensity at 725 nm (Abscissa: volume fraction of toluene; Ordinate: fluorescence intensity at 725 nm, I0 refers to the fluorescence intensity at 725 nm when the volume fraction of toluene is 0), indicating that TPB has aggregation-induced emission; Figure 4 For the generation of 1 O2 by TPB; (A) Absorption spectra after TPB reacts with ABDA after illumination for a certain time (Abscissa: wavelength; Ordinate: absorbance); (B) Change in absorbance at 378 nm (A / A0 refers to the ratio of the absorbance at 378 nm at a certain illumination time to the absorbance at 0 moment); Figure 5 For the generation of • O2 − by TPB. (A) Fluorescence spectra after TPB reacts with DHR 123 (Abscissa: wavelength; Ordinate: fluorescence intensity); (B) Change in relative fluorescence intensity at 526 nm (Abscissa: illumination time; Ordinate: I / I0 refers to the ratio of the fluorescence intensity at 526 nm at a certain illumination time to the fluorescence intensity at 0 moment); Figure 6 For the generation of 1 O2 by TPB-Ga. (A) Absorption spectra after TPB-Ga reacts with ABDA after illumination for a certain time (Abscissa: wavelength; Ordinate: absorbance); (B) Change in absorbance at 378 nm (A / A0 refers to the ratio of the absorbance at 378 nm at a certain illumination time to the absorbance at 0 moment); Figure 7 For the generation of • O2 − by TPB-Ga. (A) (A) Fluorescence spectra after TPB-Ga reacts with DHR 123 (Abscissa: wavelength; Ordinate: fluorescence intensity); (B) Change in relative fluorescence intensity at 526 nm (Abscissa: illumination time; Ordinate: I / I0 refers to the ratio of the fluorescence intensity at 526 nm at a certain illumination time to the fluorescence intensity at 0 moment); Figure 8 For the photodynamic bactericidal effect of TPB on Staphylococcus aureus. Under illumination conditions, an obvious antibacterial effect can be observed when the concentration of TPB is 0.5 μM. When the concentration of TPB reaches 2 μM, the average inhibition rate against Staphylococcus aureus is as high as 99.85%, showing a significant concentration dependence; Figure 9The bactericidal effect of TPB-Ga against multidrug-resistant Staphylococcus aureus. Under light illumination, the average inhibition rate of TPB-Ga against multidrug-resistant Staphylococcus aureus was as high as 93.80%, which was superior to that of the Control group (10.54%), the TPB group (32.21%), and the Ga 3+ group (43.01%); Figure 10 The photodynamic bactericidal effect of TPB-Ga against Escherichia coli. Under light illumination, TPB-Ga had better antibacterial activity against Escherichia coli compared with the control group and pure TPB and Ga 3+ , and the inhibition rate was as high as 85.57%; Figure 11 The bactericidal effect of TPB-Ga against multidrug-resistant Escherichia coli. Under light illumination, TPB-Ga had better antibacterial activity against multidrug-resistant Escherichia coli compared with the control group and pure TPB and Ga 3+ , and the inhibition rate reached 80.55%; Figure 12 shows the cell viability of TPB under light (Laser) and dark (Dark) conditions (the concentration of TPB-Ga on the abscissa and the viability of Raw 264.7 cells on the ordinate); Figure 13 shows the cell viability of TPB-Ga under light (Laser) and dark (Dark) conditions (the concentration of TPB-Ga on the abscissa and the viability of Raw 264.7 cells on the ordinate). Detailed implementation manners

[0020] To enable those skilled in the art of the present technology to better understand the solution of the present invention, the technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0021] Unless otherwise specifically stated, all kinds of raw materials, reagents, instruments, and equipment used in this application can be obtained through market purchase or can be prepared by existing methods.

[0022] The terms "preferred", "preferably", "more preferably", etc. in the present invention refer to the embodiments of the present invention that can provide certain beneficial effects in certain cases. However, in the same or other cases, other embodiments may also be preferred. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not available, nor is it intended to exclude other embodiments from the scope of the present invention.

[0023] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0024] Example 1

[0025] A preparation method of a borate-based photosensitizer molecule (TPB) includes the following steps: Under the protection of an argon atmosphere, add compound TTCPy (100 mg, 0.22 mmol) and 4-(bromomethyl)phenylboronic acid (56 mg, 0.26 mmol) into a eggplant-shaped flask, as well as anhydrous toluene (8 mL); heat the reaction system to reflux and stir for 7 hours. After the reaction is completed, remove the solvent by a rotary evaporator. The obtained crude product is purified by silica gel column chromatography (methylene chloride: methanol = 20:1~10:1) to obtain 42 mg of a purple solid product with a yield of 52%. The structural characterization data of the product are as follows: 1 H NMR (400 MHz, DMSO- d 6) δ 9.24 – 9.13 (m, 2H), 9.07 (dt, J = 4.7, 2.3Hz, 1H), 8.35 (d, J = 6.5 Hz, 2H), 8.15 (s, 2H), 8.04 (d, J = 4.2 Hz, 1H), 7.85(d, J = 7.8 Hz, 2H), 7.76 – 7.66 (m, 3H), 7.49 (d, J = 7.3 Hz, 2H), 7.37 (td, J =7.8, 2.1 Hz, 4H), 7.14 (dd, J = 19.7, 6.6 Hz, 6H), 6.97 (d, J = 8.7 Hz, 2H), 5.84(s, 2H). 1313C NMR (101 MHz, DMSO) δ 155.08, 150.47, 149.49, 146.67, 145.07, 144.65, 142.85, 136.55, 135.31, 135.17, 130.31, 128.13, 128.00, 125.71, 125.40, 124.89, 123.18, 121.77, 117.07, 99.31, 62.76, 49.06, 40.60, 40.39, 40.18, 39.97, 39.76, 39.56, 39.35. HRMS (ESI): m / z calcd. For [C 37 H 29 BN3O2S] + ,[M] ＋ 590.2068, found 590.20532。

[0026] According to the above characterization data, the structural formula of the obtained borate-based photosensitizer molecule (TPB) is: .

[0027] Example 2

[0028] A preparation method of a borate-based photosensitive molecule-gallium ion (TPB-Ga) complex, comprising the following steps: (1) Take 1 mL of gallium nitrate solution with a Ga3+ concentration of 1 mg / mL and drop it into 8 mL of ultrapure water, stir rapidly to obtain a gallium nitrate solution for standby; (2) Then take 1 mL of dimethyl sulfoxide and prepare a DMSO organic solution of TPB with a concentration of 1 mg / mL. The borate-based photosensitizer molecule (TPB) is prepared into a borate-based photosensitizer molecule organic solution. Drop the DMSO organic solution of TPB into the gallium nitrate solution in step (1), stir for 30 min and then sonicate for 3 min; then centrifuge at 17000 × g and 4 °C for 30 min, wash with 5 mL again, and centrifuge again to obtain a precipitate; (3) Lyophilize the precipitate to obtain borate-based photosensitive molecule-gallium ion (TPB-Ga) complex nanoparticles. Dissolve the borate-based photosensitive molecule-gallium ion (TPB-Ga) complex nanoparticles in ultrapure water to obtain a TPB-Ga complex solution with a concentration of 1 mg / mL, and store it at 4 °C for standby.

[0029] Example 3

[0030] Photodynamic bactericidal performance test of borate-based photosensitive molecule (TPB): Take 5 μL of a 3 mM TPB DMSO organic solution and dilute it to 3 mL with pure water to prepare a TPB solution with a final concentration of 5 μM for testing the absorption spectrum and fluorescence spectrum. All spectral measurements were carried out at room temperature using a 1 cm pathlength quartz cuvette for measurement.

[0031] (1) Absorption spectrum of TPB: The UV-visible absorption spectrum was measured using a UV-1800 PC ultraviolet-visible spectrophotometer produced by the United States, and the scanning range was 200 - 800 nm. The results obtained are as Figure 1 shown. From Figure 1 it can be seen that TPB shows a maximum absorption wavelength of 530 nm in the UV-visible absorption spectrum.

[0032] (2) Fluorescence spectrum of TPB: The fluorescence spectrum was measured using a Hitachi FL-4700 fluorescence spectrophotometer produced by Japan. The excitation wavelength was set at 550 nm, the emission spectrum was scanned and the fluorescence intensity was recorded. The results obtained are as Figure 2 shown. Figure 2 It shows that TPB has strong fluorescence emission characteristics in the near-infrared region, and its maximum emission wavelength (λ em ) is 820 nm. This characteristic indicates that TPB can be used for photodynamic therapy and imaging of bacteria with near-infrared light with a large penetration depth.

[0033] (3) Test of the AIE property of TPB: Toluene is a poor solvent for TPB, and DMSO is a good solvent for TPB. Accurately pipette 50 μL of a 3 mM TPB DMSO stock solution and add it to DMSO / toluene mixed solvents with different volume ratios (the volume fractions of toluene are 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% and 0%) respectively to prepare a series of TPB test solutions with a final concentration of 50 μM. The AIE characteristics were studied using a Hitachi FL-4700 fluorescence spectrophotometer. The excitation wavelength was set at 550 nm, the scanning range was 560 - 800 nm, and the emission spectrum and the maximum fluorescence intensity were recorded. The results obtained are as Figure 3 shown. Figure 3 It shows that when the volume fraction of the hydrophobic poor solvent toluene exceeds 70%, the fluorescence intensity of TPB shows a significant increasing trend, accompanied by a blue shift of the fluorescence emission peak. This experimental result indicates that TPB has typical aggregation-induced emission (AIE) characteristics.

[0034] (4) TPB generates 1O2 capacity: 1 mM TPB DMSO organic solution was mixed with 50 μM ABDA solution to prepare a TPB test solution with a final concentration of 5 μM. Detection was performed using a UV-visible spectrophotometer. 1 The absorption spectrum was collected in the range of 300-450nm to monitor the O2 generation. During the experiment, the light (660nm, 300w) was irradiated at fixed time intervals (0, 30, 60, 90, 120s) and the absorption spectrum was measured once, focusing on the intensity change of the ABDA characteristic absorption peak at 378nm. The TPB was evaluated by analyzing the change curve of ABDA absorbance at 378nm over time. 1 O2 generation efficiency, the results are as follows Figure 4 shown. Figure 4 It shows that TPB can be efficiently produced after photoexcitation 1 O2 resulted in rapid photodegradation of ABDA, which reached a plateau at 120 s.

[0035] (5) TPB generation • O2 − Ability: 1 mM TPB DMSO organic solution was mixed with 2 μM DHR 123 solution to prepare a TPB test solution with a final concentration of 2 μM. Detection was performed using a fluorescence spectrophotometer. • O2 − The excitation wavelength was set to 488 nm, and the emission spectrum was collected in the range of 500 ~ 675 nm. During the experiment, the light (660 nm, 300 W) was applied at fixed time intervals (0, 30, 60, 90, 120, 150, 180 s), and the fluorescence spectrum was measured once, and the characteristic fluorescence intensity at 526 nm was used as the fluorescence intensity. • O2 − The quantitative index generated is calculated by analyzing the change curve of fluorescence intensity at 526 nm over time. • O2 − The generation rate constant is as follows: Figure 5 shown. Figure 5 The results showed that the fluorescence intensity of DHR 123 was positively correlated with the time of TPB exposure, indicating that TPB can be rapidly generated. • O2 − .

[0036] Example 4

[0037] Photodynamic bactericidal performance test of borate-based photosensitive molecules-gallium ions (TPB-Ga) complex A TPB-Ga complex solution with a concentration of 1 mg / mL was used as the test solution.

[0038] (1) Ability of TPB-Ga to generate 1O2: A solution with a final concentration of 5 μg / mL was prepared by mixing a TPB-Ga complex with a concentration of 1 mg / mL and an ABDA solution with a concentration of 50 μM, and then tested according to the TPB experimental method. The results are as Figure 6 shown. Figure 6 It shows that TPB-Ga can efficiently generate 1 O2 after photoexcitation, leading to rapid photodegradation of ABDA.

[0039] (2) Ability of TPB-Ga to generate • O2 − : A solution with a final concentration of 2 μg / mL was prepared by mixing a TPB-Ga stock solution with a concentration of 1 mg / mL and a DHR123 solution with a concentration of 2 μM, and then tested according to the TPB experimental method. The results are as Figure 7 shown. Figure 7 It shows that the fluorescence intensity of DHR 123 is positively correlated with the irradiation time of TPB-Ga, and TP-Ga can rapidly generate • O2 − .

[0040] Example 5

[0041] Test on the photodynamic killing ability of PB against Staphylococcus aureus. The specific steps are as follows: Staphylococcus aureus was streaked on an LB agar plate and cultured in a constant temperature incubator at 37 °C for 18 - 24 hours. A single colony was picked using a sterile inoculation loop and inoculated into 5 mL of LB liquid medium. The inoculated medium was placed in a constant temperature shaker at 37 °C and shaken at a speed of 240 rpm for 12 - 16 hours. An appropriate amount of the bacterial solution was centrifuged at 7000 × g for 5 min (room temperature), and the supernatant was discarded. An equal volume of PBS buffer was added, and the bacterial cells were gently resuspended; the centrifugation-resuspension step was repeated twice to ensure thorough washing of the bacterial cells; finally, the bacterial cells were resuspended with an appropriate amount of PBS buffer, and the concentration of the bacterial solution was adjusted to 1 × 10 7 CFU / mL (Colony-Forming Unit per Milliliter) for standby. All operations were carried out in a biosafety cabinet of Class II to ensure aseptic operation. All waste generated during the experiment was autoclaved at 121 °C for 30 min to ensure biosafety.

[0042] Take 475 μL of a solution with a concentration of 1 × 10 7A suspension of Staphylococcus aureus (S. aureus) containing 100 CFU / mL of TPB solution was mixed with 25 μL of different concentrations of TPB solution (0, 10, 20, 40, 100, and 200 μM) to achieve a predetermined final TPB concentration gradient (0, 0.5, 1, 2, 5, and 10 μM). The mixture was incubated in a 37°C incubator in the dark for 1 hour. The incubated mixture was illuminated using a 300 W 660 nm laser array (660 nm, 300 W, 0 and 5 min). A negative control group (a TPB-free suspension protected from light) was also established. After illumination, the bacterial suspension from each experimental group was serially diluted in sterile PBS buffer. 100 μL of the diluted bacterial suspension was evenly spread on pre-prepared LB agar plates, with three replicates for each concentration. The plated plates were incubated in a 37°C incubator for 16 to 24 hours. After the culture was completed, Image J software was used to count the colonies and calculate the CFU. The results were as follows: Figure 8 shown. Figure 8 The results showed that the light-exposed group exhibited significant differences in antibacterial activity compared to the dark-protected group. Under light conditions, a significant antibacterial effect was observed at a TPB concentration of 0.5 μM, with an average inhibition rate against Staphylococcus aureus reaching 97.70%. When the TPB concentration reached 2 μM, the average inhibition rate against Staphylococcus aureus reached 99.85%, showing a significant concentration-dependent effect.

[0043] Example 6

[0044] Test of the photodynamic killing ability of TPB-Ga on microorganisms (1) Test of photodynamic killing ability of methicillin-resistant Staphylococcus aureus. The specific steps are as follows: Methicillin-resistant Staphylococcus aureus was streaked onto LB agar plates, and bacteria culture was performed according to the method in Example 5.

[0045] Take 425 μL of the solution with a concentration of 1 × 10 7 CFU / mL of methicillin-resistant Staphylococcus aureus suspension was mixed with 75 μL TPB and Ga 3+ TPB-Ga solution, TPB solution, and Ga(NO)3, all with a content of 0.5 mg / mL, were mixed. The mixed system was placed in a 37°C incubator in the dark for 1 hour. A 660 nm laser array with a power of 300 W was used to illuminate the incubated bacterial-drug mixed system (660 nm, 300 W, 0 and 10 min). A negative control group (containing only physiological saline without TPB-Ga, TPB, and Ga) was also set up. 3+And the bacterial suspension treated with light avoidance). After light treatment, the bacterial suspensions of each experimental group were serially diluted with sterile PBS buffer. Take 100 μL of the diluted bacterial suspension and evenly spread it on the surface of a pre-prepared LB agar plate. Set three parallel samples for each concentration. Place the spread plates in an incubator at 37 °C for 16 - 24 hours. After the incubation, use Image J software to count the colonies and calculate the CFU. The results are as Figure 9 shown. Figure 9 The results showed that there were significant differences in antibacterial activity between the light-exposed group and the light-avoided group. Under light conditions, the average inhibition rate of TPB-Ga against methicillin-resistant Staphylococcus aureus was as high as 93.80%, which was better than that of the Control group (10.54%), the TPB group (32.21%), and the Ga 3+ group (43.01%).

[0046] (II) Test on the photodynamic killing ability of TPB-Ga against Escherichia coli. The specific steps are as follows: Inoculate Escherichia coli on an LB agar plate and culture the bacteria in a similar method as in Example 5.

[0047] Then, according to the method in (I) of Example 6, replace the bacterial suspension with an Escherichia coli bacterial suspension. Test the photodynamic killing ability of TPB-Ga against Escherichia coli. The results are as Figure 10 shown. Figure 10 It was shown that TPB-Ga had better antibacterial activity against Escherichia coli compared with the control group, the pure TPB group, and the Ga 3+ group, and the inhibition rate was as high as 85.57%.

[0048] (III) Test on the photodynamic killing ability of TPB-Ga against multidrug-resistant Escherichia coli. The specific steps are as follows: Inoculate multidrug-resistant Escherichia coli on an LB agar plate and culture the bacteria in a similar method as in Example 5.

[0049] Then, according to the method in (II) of Example 6, replace the bacterial suspension with a multidrug-resistant Escherichia coli bacterial suspension. Test the photodynamic killing ability of TPB-Ga against multidrug-resistant Escherichia coli. The results are as Figure 11 shown. Figure 11 It was shown that under light conditions, TPB-Ga had better antibacterial activity against multidrug-resistant Escherichia coli compared with the control group, the pure TPB, and Ga 3+ , and the inhibition rate reached 80.55%.

[0050] Example 7

[0051] Toxicity test The MTT method was used to evaluate the phototoxicity and dark toxicity of TPB on macrophages RAW264.7. The specific steps are as follows: 7.1 Seed the cells in a 96-well plate at a concentration of 1 × 10 4 cells / well, add 100 μL of cell suspension to each well, and incubate in a 37 °C, 5% CO2 incubator for 24 hours. Aspirate the medium in the wells, and add 100 μL of complete medium containing different concentrations of TPB (0 - 150 μM) respectively. Incubate at 37 °C in the dark for 30 min, and then irradiate with a 660 nm laser row lamp with a power of 300 W for 10 min. After irradiation, place the cells back into the incubator and continue to culture for 24 hours. After 24 hours, add 10 μL of MTT solution (5 mg / mL, prepared with PBS) to each well and incubate at 37 °C for 4 hours. After 4 hours, aspirate the supernatant, add 100 μL of DMSO to each well and shake gently at low speed for 10 min to fully dissolve the formazan crystals. Measure the absorbance (OD value) of each well at a wavelength of 570 nm using an enzyme-linked immunosorbent assay reader. Set control groups: normal cell control group (without drugs), blank control group (containing only medium). The results are as Figure 12 shown Figure 12 The results show that within the effective antibacterial concentration range of TPB (0.5 - 10 μM), whether under light or dark conditions, TPB did not show obvious cytotoxicity to Raw 264.7 cells (cell survival rate > 95%). Even at a high concentration of 150 μM, the Raw 264.7 cells still maintained a survival rate of 51.41%, indicating that TPB has good safety.

[0052] The MTT method was used to evaluate the phototoxicity and dark toxicity of TPB-Ga on macrophages RAW264.7. The specific steps were similar to those in 7.1 above. The concentrations of TPB-Ga were 0, 1, 5, 10, 20, 30, 40, 50, 100, 150 μg / mL. The results obtained are as Figure 13 shown. Figure 13 The results show that the cytotoxicity of TPB-Ga is concentration-dependent. Under light conditions, when the concentration of TPB-Ga was 100 μg / mL, the survival rate of Raw 264.7 cells was 51.94%; when the concentration increased to 150 μg / mL, the cell survival rate decreased to less than 50%, indicating that TPB-Ga has relatively high safety when its concentration is lower than 100 μg / mL.

[0053] As can be seen from the above embodiments, the type I photosensitizer TPB provided by the present invention not only has good bacterial targeting and enrichment ability, but also exhibits the AIE effect and excellent photodynamic performance. TPB shows significant antibacterial activity against Staphylococcus aureus. The TPB-Ga complex exhibits more excellent antibacterial performance than the monomer. More importantly, TPB-Ga can compensate for the performance defects of TPB in killing Gram-negative bacteria and drug-resistant bacteria, further broadening the antibacterial spectrum. The antibacterial photosensitizer provided by the present invention has a simple synthesis step and is easy to operate, and has broad application prospects in photodynamic antibacterial chemotherapy.

[0054] References to "embodiments" in this specification mean that the particular features, structures, or characteristics described in connection with the embodiments can be included in at least one embodiment of the invention. The phrase appears in various places in the specification and does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment mutually exclusive of other embodiments. Those skilled in the art will explicitly and implicitly understand that the embodiments described herein can be combined with other embodiments.

[0055] Finally, it should be noted that: the embodiments disclosed in the present invention are only the preferred embodiments of the present invention, and are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. An antibacterial photosensitizer, characterized in that, Comprising a boronic acid-based photosensitive molecule and / or a boronic acid-based photosensitive molecule-gallium ion complex, and the boronic acid-based photosensitive molecule has the structure shown below: 。 2. A preparation method of a borate-based photosensitizer molecule, characterized in that, Comprising the following steps: Mixing a compound having the structure shown by formula TTCPy, 4-(bromomethyl)phenylboronic acid and an organic solvent, and heating and reacting under a protective atmosphere to obtain the boronic acid-based photosensitizer molecule; the structure of formula TTCPy is: 。 3. The preparation method of the borate-based photosensitizer molecule according to claim 2, wherein The organic solvent is toluene, and the molar ratio of the compound having the structure of formula TTCPy to 4-(bromomethyl)phenylboronic acid is 1:1.1-1.

2.

4. The preparation method of the borate-based photosensitizer molecule according to claim 2 or 3, characterized in that, The heating reaction is: heating and reacting under the condition that the reflux temperature of toluene is 110 °C, and the reaction time is 5-8 h.

5. A method for preparing a borate-based photosensitive molecule-gallium ion complex, characterized in that, Comprising the following steps: (1) Dropwise adding an aqueous solution of gallium nitrate into ultrapure water, and rapidly stirring to obtain a gallium nitrate solution for standby; (2) Formulating the boronic acid-based photosensitizer molecule described in claim 1 into an organic solution of the boronic acid-based photosensitizer molecule, dropwise adding the organic solution of the boronic acid-based photosensitizer molecule into the gallium nitrate solution, stirring, and then successively performing ultrasonic treatment, centrifugation, re-washing, and re-centrifugation to obtain a precipitate; (3) Freeze-drying the precipitate to obtain a boronic acid-based photosensitive molecule-gallium ion complex.

6. The preparation method of the borate-based photosensitive molecule-gallium ion complex according to claim 5, characterized in that, In step (1), the concentration of the aqueous solution of gallium nitrate is 1 mg / mL, and the volume is 1 mL; the volume of ultrapure water is 8 mL.

7. The preparation method of the borate-based photosensitive molecule-gallium ion complex according to claim 5, wherein In step (2), the organic solution of the boronic acid-based photosensitizer molecule is a 1 mg / mL dimethyl sulfoxide solution of the boronic acid-based photosensitizer molecule.

8. The preparation method of the borate-based photosensitive molecule-gallium ion complex according to claim 5, wherein, In step (2), the stirring time is 20-40 min, the ultrasonic time is 5-10 min; the centrifugal force for centrifugation is 15000-18000 × g, the centrifugation time is 20-40 min, and the centrifugation temperature is 4 °C.

9. Use of the boronic acid-based photosensitive molecule and / or the boronic acid-based photosensitive molecule-gallium ion complex according to claim 1 in the preparation of a photodynamic antibacterial drug.

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