Photodynamic targeted antibacterial microneedle patch as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly discloses a photodynamic targeted antibacterial microneedle patch and a preparation method and application thereof.The antibacterial microneedle patch comprises a backing and needle tips, and the needle tips form an n * n array on the surface of the backing; and a photodynamic targeting gram-positive bacterium resisting photosensitizer is contained in the needle tip. The photosensitizer with a targeting group and pyridine cations is innovatively designed, is used for efficiently killing gram-positive bacteria and is loaded in the microneedle, and the antibacterial and photodynamic effects are achieved by generating active oxygen in a painless and minimally invasive local administration mode in combination with the photodynamic effect. According to the invention, gram-positive bacteria and biological membranes thereof can be effectively killed, and damage to normal tissues is avoided, so that the problem of poor antibacterial effect caused by low biological membrane penetration efficiency and poor drug permeability in existing photodynamic therapy is solved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and particularly relates to a photodynamic targeted antibacterial (Gram-positive bacteria) microneedle patch and its preparation method and application. Background Art

[0002] Chronic wound infection is a major complication of diabetes, often leading to delayed healing. These infections seriously affect the quality of life of millions of people and pose a significant burden on the global healthcare system. Different from the typical wound healing stage, the hyperglycemic levels experienced by diabetic patients promote the growth of bacteria in wound tissues. This exacerbates the dysregulation of key signaling molecules, including proteases, growth factors, and pro-inflammatory cytokines, further impairing tissue regeneration. In addition, excessive erosion within the wound tissue hinders basic processes such as collagen accumulation, new blood vessel formation, and the growth of hair follicles and granulation tissue. Therefore, there is an urgent need for new treatment strategies to effectively eliminate bacteria in the wound area, address the associated inflammatory response, and promote tissue regeneration, thereby accelerating the healing of diabetic wounds.

[0003] Traditional drug delivery systems have played an important role in the treatment of diabetic wound infections by providing an effective drug release method to accelerate wound healing and reduce the risk of infection. Among them, hydrogels, films, and scaffolds are the three most common types of delivery systems, which can provide a suitable moist environment to promote cell migration and tissue repair. However, there are still certain limitations in the drug delivery efficiency of these traditional systems. For example, although hydrogels can maintain good flexibility due to their high water content, the drug release process often lacks precise controllability; films may limit the local penetration depth of drugs due to their dense structure; although scaffolds have good mechanical support, their efficacy may be reduced due to insufficient synchronization between the degradation rate and drug release. In addition, these systems usually cannot achieve precise targeting and responsive release at the lesion site, resulting in low drug utilization.

[0004] Microneedles are patches composed of a micro-needle array with a length of 100 - 1000 μm. For example, PVP K30 and PVP K90 are used to prepare microneedle patches, and antibacterial agents, photosensitizers, etc. are encapsulated to achieve drug delivery through the stratum corneum, penetrate the biofilm structure in chronic wounds, improve the delivery efficiency, and enhance the antibacterial activity. At the same time, microneedles do not touch the dermal nerve endings and do not cause pain. The application scope of soluble microneedles includes but is not limited to blood sugar lowering, local anesthesia, anti-tumor treatment, immunization, and medical cosmetology, etc., and has broad research and application prospects.

[0005] The key to treating diabetic wounds is to remove bacteria and their biofilms from the infected wound area. However, the emergence of drug-resistant bacteria has rendered many antibiotics ineffective. Photodynamic therapy (PDT) has emerged as a promising strategy for treating bacterial infections. PDT uses photosensitizers (PSs) to generate reactive oxygen species (ROS) under light irradiation, and these ROS damage bacterial proteins, nucleic acids, and other cellular components, resulting in antibacterial and anti-biofilm effects. The efficacy of PDT highly depends on enhancing the interaction between PSs and bacteria. Therefore, it is highly necessary to develop a photodynamic targeted antibacterial microneedle patch that can effectively kill Gram-positive bacteria and their biofilms while avoiding damage to normal tissues, thereby overcoming the poor antibacterial effect caused by low biofilm penetration efficiency and poor drug permeability in existing photodynamic therapies. Summary of the Invention

[0006] In view of the above existing deficiencies, the present invention provides a photodynamic targeted antibacterial microneedle patch, its preparation method and application. The photosensitizer of the present invention is simultaneously provided with a targeting group phenoxyboronic acid and pyridinium cation, and the interaction between the two effectively kills Gram-positive bacteria and their biofilms while avoiding damage to normal tissues, thereby overcoming the poor antibacterial effect caused by low biofilm penetration efficiency and poor drug permeability in existing photodynamic therapies.

[0007] To achieve the above object, the present invention provides a targeted antibacterial photosensitizer, and the general formula of the targeted antibacterial photosensitizer is shown as the following formula TPI-BOB:

[0008]

[0009] In formula TPI-BOB, R 1 and R 2 each independently and arbitrarily selected from: hydrogen, methyl, methoxy, chlorine, bromine, iodine; n is arbitrarily selected from: 0, 2, 4, 6, 8.

[0010] According to one aspect of the present invention, the structural formula of the targeted antibacterial photosensitizer is any one of the structural formulas shown in the following formulas Ⅰ to Ⅹ:

[0011]

[0012]

[0013]

[0014] Based on the same inventive concept, the present invention also provides a preparation method of the above targeted antibacterial photosensitizer, including the following steps:

[0015] S1. Synthesis of compound TPI-Br:

[0016] Dissolve the compound shown in Formula XI, 4,7-dibromobenzo[c][1,2,5]thiadiazole, and base A in solvent A, then add [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium, and heat under nitrogen protection in an oil bath under vacuum at 80 - 120 °C for reaction. After the reaction is completed, cool to room temperature, dilute with water, and then carry out extraction, evaporation of the organic phase, and column chromatography separation to obtain compound TPI-Br; the synthetic route is as follows:

[0017]

[0018] S2. Synthesis of compound TPIP:

[0019] Dissolve TPI-Br, pyridine-4-boronic acid, base B, and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium in solvent B, heat to 80 - 120 °C for reaction under nitrogen protection. After the reaction is completed, cool to room temperature, dilute with water, and then carry out extraction, evaporation of the organic phase, and column chromatography separation to obtain compound TPIP; the synthetic route is as follows:

[0020]

[0021] S3. Synthesis of compound TPI-PN:

[0022] Dissolve TPIP and bromoamine alkane in solvent C, raise the temperature to 80 - 120 °C for reaction under stirring. After the reaction is completed, cool to room temperature, remove solvent C by vacuum evaporation, carry out HPLC separation, and freeze-dry to obtain compound TPI-PN; the synthetic route is as follows:

[0023]

[0024] S4. Synthesis of compound TPI-BOB:

[0025] Dissolve TPI-PN, 1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-5-carboxylic acid, and HATU in solvent D, then dropwise add N,N-diisopropylethylamine, and stir at room temperature for reaction. After the reaction is completed, dilute the reaction solution with water, carry out extraction, back-extract with water, evaporate to remove solvent D, and carry out HPLC separation to obtain compound TPI-BOB; the synthetic route is as follows:

[0026]

[0027] According to one aspect of the present invention, in step S1, the solvent A includes at least one of tetrahydrofuran, methanol, and 1,4-dioxane; the base A includes at least one of potassium carbonate and cesium carbonate; the molar volume concentration ratio of compound XI to solvent A is 1:3 - 4; the feeding molar ratio of compound XI to 4,7-dibromobenzo[c][1,2,5]thiadiazole is 1:1 - 2; the feeding molar ratio of compound XI to dichloropalladium [1,1'-bis(diphenylphosphino)ferrocene] is 1:0.01 - 0.02; the feeding molar ratio of compound XI to the base is 1:1 - 1.5; the reaction time is 6 - 8 h.

[0028] According to one aspect of the present invention, in step S2, the solvent B includes at least one of tetrahydrofuran, methanol, and 1,4-dioxane; the base B includes at least one of potassium carbonate and cesium carbonate; the molar volume concentration ratio of compound TPI-Br to solvent B is 1:2.5 - 4, the feeding molar ratio of compound TPI-Br to pyridine-4-boronic acid is 1:1 - 2, the feeding molar ratio of compound TPI-Br to dichloropalladium [1,1'-bis(diphenylphosphino)ferrocene] is 1:0.01 - 0.02, the feeding molar ratio of compound TPI-Br to the base B is 1:1 - 1.5; the reaction time is 15 - 20 h.

[0029] According to one aspect of the present invention, in step S3, the solvent C is CH 3 CN; the molar volume concentration ratio of compound TPIP to solvent C is 1:3 - 4; the feeding molar ratio of compound TPIP to bromoalkylamine is 1:1 - 2; the reaction time is 4 - 6 h.

[0030] According to one aspect of the present invention, in step S4, the solvent D is dimethylformamide; the molar volume concentration ratio of compound TPI-PN to solvent D is 1:3 - 4; the feeding molar ratio of compound TPI-PN to 1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-5-carboxylic acid is 1:1 - 2;

[0031] the feeding molar ratio of compound TPI-PN to HATU is 1:1 - 3; the feeding molar ratio of compound TPI-PN to N,N-diisopropylethylamine is 1:1 - 2; the reaction time is 4 - 6 h.

[0032] Based on the same inventive concept, the present invention also provides an antibacterial microneedle patch, including a needle tip and a backing supporting the needle tip, wherein the needle tip includes the above-mentioned targeted antibacterial photosensitizer wrapped and loaded or the targeted antibacterial photosensitizer prepared by the above-mentioned preparation method.

[0033] Based on the same inventive concept, the present invention also provides a preparation method of the above-mentioned antibacterial microneedle patch, including the following steps:

[0034] A1. Wrap and load 5 - 10 mg / mL of the targeted antibacterial photosensitizer with the matrix material of the microneedle tip at 2 - 5 g / mL to obtain the tip solution.

[0035] A2. Adjust the matrix material of the microneedle backing with a solvent to a concentration of 2 - 5 g / mL to obtain the backing solution.

[0036] A3. Add the tip solution into a mold, after vacuum drying, add the backing solution, and after drying and demolding, obtain the antibacterial microneedle patch.

[0037] Exemplarily, the matrix material of the microneedle tip is PVP K30 (commercially available).

[0038] Exemplarily, the matrix material of the microneedle backing is PVP K90 (commercially available).

[0039] According to one aspect of the present invention, it includes the following steps:

[0040] Based on the same inventive concept, the present invention also provides the application of any of the above - mentioned targeted antibacterial photosensitizers, the targeted antibacterial photosensitizers prepared by any of the above - mentioned preparation methods, any of the above - mentioned antibacterial microneedle patches, or the antibacterial microneedle patches prepared by any of the above - mentioned preparation methods in bacterial wash - free imaging, bacterial aggregation induction, bacterial near - infrared fluorescence imaging, or broad - spectrum antibacterial drugs.

[0041] The beneficial effects of the present invention:

[0042] (1) The targeted antibacterial photosensitizer TPI - BOB of the present invention realizes high - specific recognition and binding to Gram - positive bacteria through the phenoxyboronic acid (BOB) group. Specifically, the targeted antibacterial photosensitizer TPI - BOB of the present application can target the peptidoglycan rich on the bacterial surface through the phenoxyboronic acid (BOB) group. At the same time, the pyridinium cation on the targeted antibacterial photosensitizer TPI - BOB enhances the bacterial membrane affinity through electrostatic adsorption. In addition, the teichoic acid highly expressed by Gram - positive bacteria can provide a large number of binding sites, enabling the phenoxyboronic acid (BOB) to form stable reversible covalent bonds, improving the selectivity for specific bacteria. The pyridinium cation can not only enhance the bacterial membrane binding through electrostatic interaction, but also disrupt the bacterial membrane integrity through hydrophobic interaction, thereby promoting the photosensitizer to penetrate deep into the bacteria, making the BOB group easier to approach the binding sites on the bacterial membrane and further strengthening its targeted recognition ability. At the same time, the electronic effect of the phenoxyboronic acid (BOB) can regulate the polarity of the pyridinium cation, optimize its distribution in the aqueous solution and membrane environment, and improve the stability and antibacterial performance of the compound. This dual binding mode not only enhances the bacterial membrane penetration ability but also reduces the possibility of bacteria avoiding the drug action through a single drug - resistance mechanism, thus effectively reducing the generation of drug - resistant bacteria.

[0043] (2) Under light stimulation, the reactive oxygen species (ROS) generated by the targeted antibacterial photosensitizer TPI-BOB of the present invention can rapidly destroy the cell membrane, proteins, nucleic acids and other key biological macromolecules of bacteria, ultimately leading to bacterial death. Compared with the poor antibacterial effect caused by the short lifespan and limited diffusion range of ROS in traditional photodynamic therapy, the targeted antibacterial photosensitizer TPI-BOB of the present invention improves the local concentration of ROS on the bacterial membrane through its efficient targeting binding mechanism, thereby greatly enhancing the bactericidal efficiency. This mechanism not only enhances the antibacterial activity, but also realizes efficient targeted antibacterial and integrated diagnosis and treatment, providing a new strategy for antibacterial treatment.

[0044] (3) The targeted antibacterial microneedle patch provided by the present invention loads the targeted antibacterial photosensitizer TPI-BOB into the microneedle structure, and uses the microneedles to accurately deliver the drug to the infected tissue and biofilm site, ensuring the local enrichment of the photosensitizer, increasing the treatment concentration, and at the same time minimizing systemic exposure and potential side effects. Through the synergistic effect of photodynamic therapy and targeting, the patch significantly improves the antibacterial effect, can reduce the dosage of drugs, and reduce the risk of bacterial drug resistance, thereby achieving efficient antibacterial and biofilm clearance.

[0045] (4) The microneedles can physically penetrate the stratum corneum and penetrate the dense biofilm matrix, enabling the photosensitizer to be highly enriched at the infection site, thereby quickly inhibiting the deterioration of the infection and continuously releasing the drug to improve the treatment effect. Compared with traditional topical preparations, the photosensitizer is easily diluted or removed by body fluids, while the microneedles can provide continuous drug retention in the wound area, improving the stability of the treatment. At the same time, microneedle puncture can disrupt the dense biofilm structure, promote drug penetration, and enhance the role of reactive oxygen species (ROS) during photodynamic therapy, further improving the antibacterial performance. In addition, the unique design of the backing layer effectively prevents exogenous bacterial infection and improves the overall treatment safety.

[0046] (5) The soluble microneedles prepared by the vacuum casting method of the present invention have good mechanical properties, dissolution properties and high drug loading efficiency, can effectively penetrate the dense biofilm structure, and achieve high delivery efficiency. At the same time, the microneedles have good biocompatibility and will not cause adverse reactions such as skin inflammation, redness, bleeding, pain, etc. during use, further improving the safety and comfort of clinical applications. Description of the Drawings

[0047] Figure 1 It is the chemical synthesis route diagram of Compound I in Example 1 of the present invention;

[0048] Figure 2 It is the chemical synthesis route diagram of Compound TPI-IDT in Comparative Example 2 of the present invention;

[0049] Figure 3Optical property characterization diagrams of TPIP(I), TPI-IDT, and Compound I; among them, a is the normalized UV absorption spectra of TPIP(I), TPI-IDT, and Compound I; b is the

[0050] fluorescence emission spectra of TPIP(I), TPI-IDT, and Compound I; c is the fluorescence emission spectra of Compound I (10 μM) in THF / toluene mixed solutions with different ratios (Ex = 530 nm); d is the relative fluorescence intensity (I / I 0 ) diagrams of TPIP(I), TPI-IDT, and Compound I (10 μM) in THF / toluene with different ratios; e is the absorbance (A / A 0 ) of ABDA at 378 nm wavelength; f is the absorbance (I / I 0 ) of DCFH at 526 nm wavelength;

[0051] Figure 4 Results of the in vitro anti-MRSA activities of TPIP(I), TPI-IDT, and Compound I against bacteria; among them, a is the agar plate diagrams of MRSA cultured for 0.5 h under white light irradiation (60 mW / cm 2 ) or in the dark with different concentrations of the compound (TPIP(I) or TPI-IDT or Compound I); b is the counting and statistical analysis of the colonies on the agarose plate to quantify the survival rate of MRSA under the treatment condition of TPIP(I); c is the counting and statistical analysis of the colonies on the agarose plate to quantify the survival rate of MRSA under the treatment condition of TPI-IDT; d is the counting and statistical analysis of the colonies on the agarose plate to quantify the survival rate of MRSA under the treatment condition of Compound I; Note: The data are mean ± SD (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, ns: no significance;

[0052] Figure 5 Confocal characterization diagrams of the antibacterial abilities of TPIP(I), TPI-IDT, and Compound I;

[0053] Figure 6 Antibacterial mechanism diagrams of TPIP(I), TPI-IDT, and Compound I; among them, a is the morphological diagrams of the antibacterial activities of TPIP(I), TPI-IDT, and Compound I; b is the statistical diagram of the morphology of TPIP(I) antibacterial; c is the statistical diagram of the morphology of TPI-IDT antibacterial; d is the statistical diagram of the morphology of Compound I antibacterial;

[0054] Figure 7 Confocal diagrams of the anti-biofilm ability of Compound I;

[0055] Figure 8 It is a morphological characterization diagram of the needle tips in the antibacterial microneedle patch; among them, a is the morphological characterization diagram of the macro camera; b is the morphological characterization diagram of the fluorescence microscope; c is the morphological characterization diagram of the optical microscope;

[0056] Figure 9 It is a diagram for measuring the mechanical strength and biocompatibility of microneedles on mice and ex vivo porcine skin; among them, a is the diagram for measuring the mechanical strength and biocompatibility of microneedles on ex vivo porcine skin; b is the diagram for measuring the mechanical strength and biocompatibility of microneedles on live mice;

[0057] Figure 10 It is a diagram for measuring the mechanical strength of microneedles;

[0058] Figure 11 It is a diagram for characterizing the antibacterial activity of microneedles;

[0059] Figure 12 It is a confocal diagram for the anti-biofilm activity of microneedles;

[0060] Figure 13 It is a diagram for characterizing the blood compatibility of microneedles. Specific embodiments

[0061] To make the present invention easier to understand, the following further elaborates the present invention in combination with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope 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 belong to the scope of protection of the present invention. Unless otherwise defined, the professional terms used below have the same meaning as understood by those of ordinary skill in the art; unless otherwise specified, the raw materials and reagents involved herein can be purchased from the market or prepared by well-known methods.

[0062] It should be noted that the technical solution of this application has been simultaneously submitted to an English journal.

[0063] It should be noted that the abbreviations involved in this application are all common knowledge in the art, and some are as follows:

[0064] TPI is the abbreviation of "4-(benzo[c][1,2,5]thiadiazol-4-yl)-N,N-diphenylaniline";

[0065] HATU is the abbreviation of "2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate";

[0066] DMF is the abbreviation of "dimethylformamide";

[0067] THF is the abbreviation of "tetrahydrofuran";

[0068] MeOH is the abbreviation of "methanol";

[0069] TFA is the abbreviation of "trifluoroacetic acid";

[0070] EA is the abbreviation of "ethyl acetate".

[0071] Example 1

[0072] A preparation method of a targeted antibacterial photosensitizer (Compound I), and its synthetic route diagram is as Figure 1 shown, and specifically includes the following steps:

[0073] The first step: Synthesis of compound TPI-Br (I)

[0074] Dissolve (4-(diphenylamino)phenyl)boronic acid (867 mg, 3 mmol), 4,7-dibromobenzo[c][1,2,5]thiadiazole (1323 mg, 4.5 mmol) and K 2 CO 3 (300 mg) in THF (10 mL) and MeOH (20 mL), and stir to dissolve it. Then add [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (30 mg), then immediately evacuate, and heat in an oil bath at 90 °C under nitrogen protection for 6 h. Cool to room temperature, dilute with water, and then extract with dichloromethane. Dilute with water and then extract with dichloromethane. Evaporate the organic layer, and separate the product by silica gel column chromatography (petroleum ether:DCM = 100:1 - 5:1) to obtain TPI-Br (I) as an orange-red powder (1044 mg, yield: 76%). 1 1H NMR (400 MHz, CDCl 3 ) δ 7.91 (d, J = 7.6 Hz, 1H), 7.83 (d, J = 8.8 Hz, 2H), 7.56 (d, J = 7.6 Hz, 1H), 7.37 - 7.29 (m, 4H), 7.25 - 7.17 (m, 6H), 7.11 (dd, J = 11.5, 4.2 Hz, 2H). 13 13C NMR (101 MHz, CDCl 3 ) δ 153.96, 153.15, 148.44, 147.33, 133.55, 132.39, 129.92, 129.83, 129.43, 127.34, 125.05, 123.53, 122.63, 112.19.

[0075] The second step: Synthesis of compound TPIP (I)

[0076] Pyridine-4-boronic acid (131 mg, 1.5 mmol), TPI-Br (Ⅰ) (203.2 mg, 1 mmol), and K 2 CO 3 (300 mg) were dissolved in THF (5 mL). After stirring to dissolve, [1,1'-bis(diphenylphosphino)ferrocene] palladium(II) dichloride (30 mg) was added, and then immediately evacuated. It was heated in an oil bath at 90 °C under nitrogen protection for 12 h. The organic layer was evaporated, and the product was separated by silica gel column chromatography (DCM:MeOH = 1:0 - 500:1). Red powdery TPIP(Ⅰ) (260 mg, yield: 57%) was obtained. 1 H NMR (500 MHz, CDCl 3 ) δ 8.79 (d, J = 5.1 Hz, 2H), 7.94 (d, J = 5.1 Hz, 2H), 7.90 (d, J = 8.3 Hz, 2H), 7.87 - 7.75 (m, 2H), 7.31 (t, J = 7.7 Hz, 4H), 7.22 (t, J = 8.7 Hz, 6H), 7.10 (t, J = 7.4 Hz, 2H). 13 C NMR (126 MHz, CDCl 3 ) δ 154.03, 153.60, 150.17, 148.49, 147.32, 144.70, 134.65, 130.13, 130.10, 129.46, 129.17, 128.98, 126.87, 125.10, 123.58, 123.50, 122.56.

[0077] Step 3: Synthesis of compound TPI-PN(Ⅰ)

[0078] 2-Bromoethan-1-amine (264.89 mg, 1.5 mmol) and TPIP(Ⅰ) (456 mg, 1 mmol) were dissolved in CH 3 CN (15 mL), and stirred at 90 °C. The solution was cooled to room temperature, and the solvent was removed by vacuum evaporation. Separated by HPLC (solvent A: water containing 0.1% TFA; solvent B: CH 3 CN), and then lyophilized to obtain purple solid TPI-PN(Ⅰ)

[0079] (343 mg, yield: 62%). 11H NMR (500 MHz, DMSO-d6) δ 9.08 (dd, J = 102.2, 6.6 Hz, 4H), 8.52 (d, J = 7.6 Hz, 1H), 8.11 (d, J = 7.6 Hz, 1H), 8.06 (d, J = 8.7 Hz, 2H), 7.39 (t, J = 7.8 Hz, 4H), 7.13 (dd, J = 18.2, 8.3 Hz, 8H), 4.92 (t, J = 5.2 Hz, 3H), 3.61 (t, J = 5.3 Hz, 3H). 13 13C NMR (126 MHz, DMSO-d6) δ 153.71, 153.13, 152.67, 148.92, 148.88, 147.02, 145.72, 136.95, 132.83, 131.15, 130.28, 129.40, 127.23, 126.89, 125.46, 124.56, 121.88, 57.88, 55.37. HRMS (EI): calculated for C 31 H 26 BrN 5 S [M - Br] + : 500.1904; found: 500.2321.

[0080] Step 4: Synthesis of Compound I

[0081] Dissolve TPI-PN (I) (116 mg, 0.2 mmol), 1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-5-carboxylic acid (53.38 mg, 0.3 mmol) and HATU (80 mg, 0.2 mmol) in DMF (10 mL), then add dropwise N,N-diisopropylethylamine (0.72 mg, 0.005 mmol), and stir at room temperature for 12 h. Dilute the reaction solution with water, extract with EA, and then extract with water once more. After evaporating the solvent, separate by HPLC (solvent A: water containing 0.1% TFA; solvent B: CH 3 CN), and then lyophilize to obtain TPI-BOB (I) as a red powder

[0082] (65 mg, yield: 44%). 11H NMR (600 MHz, DMSO-d6) δ 9.29 (s, 1H), 9.11 (d, J = 7.0 Hz, 2H), 8.86 (d, J = 7.0 Hz, 2H), 8.75 (t, J = 5.9 Hz, 1H), 8.44 (d, J = 7.6 Hz, 1H), 8.02 (dd, J = 20.8, 8.2 Hz, 3H), 7.75–7.69 (m, 2H), 7.65 (d, J = 7.7 Hz, 1H), 7.37–7.31 (m, 4H), 7.13–7.03 (m, 8H), 4.97 (s, 2H), 4.78–4.73 (m, 2H), 3.88 (q, J = 5.4 Hz, 2H). 13 13C NMR (126 MHz, DMSO-d6) δ 152.58, 151.35, 148.32, 146.50, 144.49, 136.18, 132.23, 130.54, 129.70, 128.88, 126.64, 126.26, 124.88, 124.02, 123.97, 121.37, 60.09, 31.10, 30.65, 28.42, 28.35, 25.46, 21.99, 13.88. HRMS (EI): calculated for C 39 H 31 BN 5 O 3 S + [M - Br] + : 660.2236; found: 660.22192.

[0083] Example 2

[0084] A preparation method of a targeted antibacterial photosensitizer (Compound II), specifically including the following steps:

[0085] The synthesis steps of Compound II refer to Example 1, with the difference that: (4-(diphenylamino)phenyl)boronic acid in the first step is replaced with an equimolar amount of (4-(di-p-tolylamino)phenyl)boronic acid, and other steps and parameters are the same as in Example 1. HRMS (EI): calculated for C 41 H 35 BN 5 O 3 S + [M - Br] + : 688.2548; found: 688.2556.

[0086] Example 3

[0087] A preparation method of a targeted antibacterial photosensitizer (Compound III), specifically including the following steps:

[0088] The synthesis steps of Compound III refer to Example 1, with the difference that: (4-(diphenylamino)phenyl)boronic acid in Step 1 is replaced with an equimolar amount of (4-(bis(4-methoxyphenyl)amino)phenyl)boronic acid, and other steps and parameters are the same as those in Example 1. HRMS(EI): calculated for C 41 H 35 BN 5 O 5 S + [M - Br] + : 720.2446; found: 720.2453.

[0089] Example 4

[0090] A preparation method of a targeted antibacterial photosensitizer (Compound IV) specifically includes the following steps:

[0091] The synthesis steps of Compound IV refer to Example 1, with the difference that: (4-(diphenylamino)phenyl)boronic acid in Step 1 is replaced with an equimolar amount of (4-(bis(4-bromophenyl)amino)phenyl)boronic acid, and other steps and parameters are the same as those in Example 1. HRMS(EI): calculated for C 39 H 29 BBr 2 N 5 O 3 S + [M - Br] + : 816.0445; found: 816.0449.

[0092] Example 5

[0093] A preparation method of a targeted antibacterial photosensitizer (Compound V) specifically includes the following steps:

[0094] The synthesis steps of Compound V refer to Example 1, with the difference that: (4-(diphenylamino)phenyl)boronic acid in Step 1 is replaced with an equimolar amount of (4-(bis(4-iodophenyl)amino)phenyl)boronic acid, and other steps and parameters are the same as those in Example 1. HRMS(EI): calculated for C 39 H 29 B I2 N 5 O 3 S + [M - Br] + : 912.0168; found: 912.0177.

[0095] Example 6

[0096] A preparation method of a targeted antibacterial photosensitizer (Compound VI), specifically including the following steps:

[0097] For the synthesis steps of Compound VI, refer to Example 1, with the difference that: replace (4-(diphenylamino)phenyl)boronic acid in Step 1 with an equimolar amount of (4-(bis(4-chlorophenyl)amino)phenyl)boronic acid, and other steps and parameters are the same as in Example 1. HRMS(EI): calculated for C 39 H 29 BCl 2 N 5 O 3 S + [M - Br] + : 728.1456; found: 728.1488.

[0098] Example 7

[0099] A preparation method of a targeted antibacterial photosensitizer (Compound VII), specifically including the following steps:

[0100] For the synthesis steps of Compound VII, refer to Example 1, with the difference that: replace 2-bromoethan-1-amine in Step 3 with an equimolar amount of 4-bromobutan-1-amine, and other steps and parameters are the same as in Example 1. HRMS(EI): calculated for C 41 H 35 BN 5 O 3 S + [M - Br] + : 688.2548; found: 688.2566.

[0101] Example 8

[0102] A preparation method of a targeted antibacterial photosensitizer (Compound VIII), specifically including the following steps:

[0103] For the synthesis steps of Compound VIII, refer to Example 1, with the difference that: replace 2-bromoethan-1-amine in Step 3 with an equimolar amount of 6-bromohexan-1-amine, and other steps and parameters are the same as in Example 1. HRMS(EI): calculated for C 43 H 39 BN 5 O 3 S + [M - Br] + : 716.2861; found: 716.2873.

[0104] Example 9

[0105] A preparation method of a targeted antibacterial photosensitizer (Compound IX), specifically including the following steps:

[0106] The synthesis steps of Compound IX refer to Example 1, with the difference that: 2-bromoethan-1-amine in Step 3 is replaced with an equimolar amount of 8-bromo-1-octylamine, and the other steps and parameters are the same as those in Example 1. HRMS (EI): calculated for C 45 H 43 BN 5 O 3 S + [M - Br] + : 744.3174; found: 744.3189.

[0107] Example 10

[0108] A preparation method of a targeted antibacterial photosensitizer (Compound X), specifically including the following steps:

[0109] The synthesis steps of Compound X refer to Example 1, with the difference that: 2-bromoethan-1-amine in Step 3 is replaced with an equimolar amount of 10-bromo-1-decylamine, and the other steps and parameters are the same as those in Example 1. HRMS (EI): calculated for C 47 H 47 BN 5 O 3 S + [M - Br] + : 772.3487; found: 772.3499.

[0110] Example 11

[0111] A preparation method of an antibacterial microneedle patch, including the following steps:

[0112] (1) Prepare the tip solution, and the tip solution contains raw materials with the following concentrations: the microneedle matrix material (PVP K30) is 2 - 5 g / mL, and the encapsulated and loaded targeted antibacterial photosensitizer (any one of Compounds I - X) is 5 - 10 mg / mL, and the balance is the solvent. It should be noted that the microneedles with the loaded Compound I concentrations of 0, 0.125, 0.25, and 0.5 mM are named Blank DMN, DMN@BOB - 1, DMN@BOB - 2, and DMN@BOB - 3 respectively.

[0113] (2) Prepare the backing solution, and the backing layer contains raw materials with the following concentrations: the microneedle matrix material (PVP K90) is 2 - 5 g / mL, and the balance is the solvent.

[0114] (3) Add the needle tip solution to the mold, under the conditions of a pressure of -0.05 to -0.09 MPa and a temperature of 20 to 30 °C, the vacuum drying time is 5 to 15 min, then add the backing solution, and demold after drying to obtain the above antibacterial microneedle patch.

[0115] Comparative Example 1

[0116] A preparation method of a compound TPIP (Ⅰ) specifically includes the following steps:

[0117] The first step: Synthesis of the compound TPI-Br (Ⅰ)

[0118] Dissolve (4-(diphenylamino)phenyl)boronic acid (867 mg, 3 mmol), 4,7-dibromobenzo[c][1,2,5]thiadiazole (1323 mg, 4.5 mmol) and K 2 CO 3 (300 mg) in THF (10 mL) and MeOH (2 mL), stir to dissolve it. Then add [1,1'-bis(diphenylphosphino)ferrocene] palladium(II) dichloride (30 mg), then immediately evacuate, and heat in an oil bath at 90 °C for 6 h under nitrogen protection. Cool to room temperature, dilute with water, and then extract with dichloromethane. Dilute with water and then extract with dichloromethane. Evaporate the organic layer, and separate the product by silica gel column chromatography (petroleum ether:DCM = 100:1 - 5:1). The obtained TPI-Br (Ⅰ) is an orange-red powder (1044 mg, yield: 76%). 1 H NMR (400 MHz, CDCl 3 ) δ7.91 (d, J = 7.6 Hz, 1H), 7.83 (d, J = 8.8 Hz, 2H), 7.56 (d, J = 7.6 Hz, 1H), 7.37 - 7.29 (m, 4H), 7.25 - 7.17 (m, 6H), 7.11 (dd, J = 11.5, 4.2 Hz, 2H). 13 C NMR (101 MHz, CDCl 3 ) δ153.96, 153.15, 148.44, 147.33, 133.55, 132.39, 129.92, 129.83, 129.43, 127.34, 125.05, 123.53, 122.63, 112.19.

[0119] The second step: Synthesis of the compound TPIP (Ⅰ)

[0120] Dissolve pyridine-4-boronic acid (131 mg, 1.5 mmol), TPI-Br (Ⅰ) (203.2 mg, 1 mmol), and K 2 CO 3(300 mg) was dissolved in THF (5 mL). After stirring to dissolve, palladium(II) dichloride [1,1'-bis(diphenylphosphino)ferrocene] (30 mg) was added, and then immediately evacuated. It was heated in an oil bath at 90 °C for 12 h under nitrogen protection. The organic layer was evaporated, and the product was separated by silica gel column chromatography (DCM:MeOH = 1:0 - 500:1). Red powdery TPIP (I) (260 mg, yield: 57%) was obtained. 1 H NMR (500 MHz, CDCl 3 ) δ 8.79 (d, J = 5.1 Hz, 2H), 7.94 (d, J = 5.1 Hz, 2H), 7.90 (d, J = 8.3 Hz, 2H), 7.87 - 7.75 (m, 2H), 7.31 (t, J = 7.7 Hz, 4H), 7.22 (t, J = 8.7 Hz, 6H), 7.10 (t, J = 7.4 Hz, 2H). 13 C NMR (126 MHz, CDCl 3 ) δ 154.03, 153.60, 150.17, 148.49, 147.32, 144.70, 134.65, 130.13, 130.10, 129.46, 129.17, 128.98, 126.87, 125.10, 123.58, 123.50, 122.56.

[0121] Comparative Example 2

[0122] A preparation method of a compound TPI-IDT, and its synthetic route is as Figure 2 shown, and specifically includes the following steps:

[0123] The first step: Synthesis of compound TPI-Br (I)

[0124] (4-(Diphenylamino)phenyl)boronic acid (867 mg, 3 mmol), 4,7-dibromobenzo[c][1,2,5]thiadiazole (1323 mg, 4.5 mmol) and K 2 CO 3 (300 mg) were dissolved in THF (10 mL) and MeOH (2 mL), and stirred to dissolve. Then palladium(II) dichloride [1,1'-bis(diphenylphosphino)ferrocene] (30 mg) was added, and then immediately evacuated. It was heated in an oil bath at 90 °C for 6 h under nitrogen protection. It was cooled to room temperature, diluted with water, and then extracted with dichloromethane. Diluted with water and then extracted with dichloromethane. The organic layer was evaporated, and the product was separated by silica gel column chromatography (petroleum ether:DCM = 100:1 - 5:1). The obtained TPI-Br (I) was an orange-red powder (1044 mg, yield: 76%). 1 H NMR (400 MHz, CDCl3 )δ 7.91 (d, J = 7.6 Hz, 1H), 7.83 (d, J = 8.8 Hz, 2H), 7.56 (d, J = 7.6 Hz, 1H), 7.37 - 7.29 (m, 4H), 7.25 - 7.17 (m, 6H), 7.11 (dd, J = 11.5, 4.2 Hz, 2H). 13 C NMR (101 MHz, CDCl 3 )δ 153.96, 153.15, 148.44, 147.33, 133.55, 132.39, 129.92, 129.83, 129.43, 127.34, 125.05, 123.53, 122.63, 112.19.

[0125] Step 2: Synthesis of compound TPIP (Ⅰ)

[0126] Dissolve pyridine - 4 - boronic acid (131 mg, 1.5 mmol), TPI - Br (Ⅰ) (203.2 mg, 1 mmol), and K 2 CO 3 (300 mg) in THF (5 mL). After stirring to dissolve, add [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (30 mg), then immediately evacuate the air, and heat in an oil bath at 90 °C under nitrogen protection for 12 h. Evaporate the organic layer and separate the product using silica gel column chromatography (DCM:MeOH = 1:0 - 500:1). Obtain red powdered TPIP (Ⅰ) (260 mg, yield: 57%). 1 H NMR (500 MHz, CDCl 3 )δ 8.79 (d, J = 5.1 Hz, 2H), 7.94 (d, J = 5.1 Hz, 2H), 7.90 (d, J = 8.3 Hz, 2H), 7.87 - 7.75 (m, 2H), 7.31 (t, J = 7.7 Hz, 4H), 7.22 (t, J = 8.7 Hz, 6H), 7.10 (t, J = 7.4 Hz, 2H). 13 C NMR (126 MHz, CDCl 3 )δ 154.03, 153.60, 150.17, 148.49, 147.32, 144.70, 134.65, 130.13, 130.10, 129.46, 129.17, 128.98, 126.87, 125.10, 123.58, 123.50, 122.56.

[0127] Step 3: Synthesis of compound TPI - IDT

[0128] 1-Iodooctane (144.078 mg, 0.6 mmol) and TPIP (Ⅰ) (182.63 mg, 0.4 mmol) were dissolved in CH 3 CN (15 mL), and the mixture was stirred at 90 °C. The solution was cooled to room temperature, and the solvent was removed by vacuum evaporation. The organic layer was evaporated, and the product was separated by silica gel column chromatography

[0129] (DCM:MeOH = 1:0 - 100:1). The obtained TPI-IDT was a purple powder

[0130] (125.4 mg, yield: 45%). 1 H NMR (500 MHz, DMSO-d6) δ 9.04 (s, 2H), 8.69 (s, 2H), 8.29 (d, J = 5.8 Hz, 1H), 7.82 (dd, J = 17.8, 7.6 Hz, 3H), 7.24–6.80 (m, 12H), 4.45 (s, 2H), 1.78 (s, 2H), 1.08 (d, J = 39.9 Hz, 11H), 0.64 (d, J = 6.5 Hz, 3H). 13 C NMR (126 MHz, DMSO-d6) δ 153.26, 152.68, 152.10, 148.45, 146.58, 145.19, 136.47, 132.32, 130.63, 129.76, 128.94, 126.73, 126.31, 124.98, 124.09, 124.05, 121.42, 58.54, 40.43, 15.15. HRMS (EI): calculated for C 37 H 37 IN 4 S [M-I] + : 569.2733; found: 569.2746.

[0131] Performance detection and result analysis:

[0132] Verification of the AIE effect of TPIP (Ⅰ), TPI-IDT and Compound Ⅰ:

[0133] TPIP (Ⅰ), TPI-IDT and Compound Ⅰ were dissolved in DMSO to prepare a stock solution with a concentration of 10 mM. The stock solution was diluted to deionized water to obtain a diluted solution with a concentration of 20 μM, and it was scanned under a UV-visible spectrophotometer to obtain its absorption spectrum, and the maximum absorption wavelength was 530 nm ( Figure 3a). Dilute the mother liquor and mix it with toluene and tetrahydrofuran mixed solvents with different volume ratios (the volume contents of toluene are 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% respectively), and the concentrations of TPIP (Ⅰ), TPI-IDT and Compound Ⅰ are all 10 μM. Measure the fluorescence spectra of each sample at the maximum absorption wavelength (530 nm) under a fluorescence spectrophotometer ( Figure 3 b), and record the maximum emission wavelength and the maximum fluorescence intensity. As Figure 3 shown in c-d, with the increase of the proportion of toluene, the fluorescence intensities of the three compounds all increase to varying degrees. Among them, Compound Ⅰ shows aggregation-induced emission, and the fluorescence intensity increases significantly, reaching 160 times, showing the highest AIE property.

[0134] Verification of the production of reactive oxygen species by TPIP (Ⅰ), TPI-IDT and Compound Ⅰ:

[0135] ABDA is used to detect 1 O 2 production. There is Compound I (10 μM) in 1 mL of deionized water. After adding 10 μL of ABDA (10 mM), irradiate it under white light (60 mW / cm 2 ) for different times

[0136] (0 s, 20 s, 40 s, 60 s, 80 s, 100 s, 120 s, 140 s, 160 s), monitor its ultraviolet-visible spectrum, and the results are as Figure 3 shown in e. As can be seen from Figure 3 e, singlet oxygen generated by TPIP (I), TPI-IDT and Compound I (10 μM) and the positive control RB (10 μM) under white light irradiation (60 mW / cm 2 ) at different time points, the ultraviolet absorption rates of the three compounds at the wavelength of 378 nm all decrease, indicating the production of 1 O 2 . Among them, Compound I shows the most significant reduction efficiency, showing its strong 1 O 2 production rate, providing a good basis for the increase of its antibacterial activity.

[0137] DCFH is used to monitor the production of total reactive oxygen species. Convert DCFH to DCFH-DA under alkaline conditions, and mix DCFH-DA (50 μM) with Compound I (10 μM) into 1 mL of deionized water. The mixed solution is irradiated under white light (60 mW / cm 2 ) for different times (0 s, 10 s, 20 s, 30 s, 40 s, 50 s), and monitor its fluorescence spectrum (Ex = 485 nm). The increase in the fluorescence intensity at 526 nm can be used to monitor the total production amount of ROS, and the results are asFigure 3 as shown in f. From Figure 3 f, it can be seen that the ROS generated by TPIP(I), TPI-IDT, compound I (10 μM), and the positive control RB (10 μM) under white light irradiation (60 mW / cm 2 ) at different time points. The significant increase in fluorescence intensity of the three compounds at 526 nm indicates the generation of total reactive oxygen species. In the presence of compound I, total reactive oxygen species are rapidly generated, exceeding the positive control compound RB, showing a strong total ROS generation efficiency.

[0138] Verification of the in vitro antibacterial ability of TPIP(Ⅰ), TPI-IDT, and compound Ⅰ-Ⅹ:

[0139] Staphylococcus aureus (S. aureus) and methicillin-resistant Staphylococcus aureus (MRSA), as representatives of Gram-positive bacteria, were used to evaluate the antibacterial activities of TPIP(Ⅰ), TPI-IDT, and compound Ⅰ-Ⅹ. All these bacteria were cultured overnight on agar plates, and then single colonies were picked for culture. The isolated single colonies were cultured overnight under shaking (37 °C, 180 rpm), and the next day, the OD was adjusted to 600 1 and then used for bacterial experiments.

[0140] The in vitro antibacterial activities of compound Ⅰ-Ⅹ were evaluated by the plate coating technique. The bacteria were cultured overnight under shaking (180 rpm, 37 °C), diluted to OD 600 of 1, and then diluted 10 3 times with PBS. Different concentrations of TPIP(Ⅰ), TPI-IDT, and compound Ⅰ-Ⅹ were added to 1 mL of the bacterial suspension to obtain concentration gradients of 0 μM, 0.5 μM, 2 μM, 8 μM, 16 μM, and 32 μM. Then the mixture was cultured at 37 °C for 30 min, and then irradiated with white light (60 mW / cm 2 ) for 30 min or not irradiated. 100 μL of the bacterial solution was spread on an agar plate and cultured overnight under shaking (180 rpm, 37 °C). The bacterial survival rate can be calculated by comparing the number of colonies on the plate, and the results are as shown in Figure 4 and Table 1. From Figure 4It can be seen that the MICs of Compound Ⅰ against S. aureus and MRSA under white light irradiation (light irradiation group) are 8 μM and 16 μM respectively, showing significant antibacterial activity. In contrast, TPIP(Ⅰ) and TPI-IDT require 32 μM and 16 μM respectively to completely inhibit the growth of S. aureus. For MRSA, TPI-IDT and TPIP(Ⅰ) require a concentration of 32 μM or higher to achieve a similar inhibitory level. All these experimental results indicate that the synergistic effect produced after the addition of the two targeting molecules plays a significant role in enhancing the antibacterial effect.

[0141] Table 1 In vitro antibacterial experimental results of Compounds Ⅰ-Ⅹ under white light irradiation (light irradiation group)

[0142]

[0143] Bacterial viability staining experiments of TPIP(Ⅰ), TPI-IDT and Compound Ⅰ:

[0144] The bacteria were cultured overnight with shaking (180 rpm, 37 °C) and diluted to an OD 600 of 1. 1 mL of a bacterial solution containing 10 μM of the compound (TPIP(Ⅰ), TPI-IDT and Compound Ⅰ), and the mixture was co-cultured at 37 °C for 1 hour, and then irradiated with white light (60 mW / cm 2 ) or treated in the dark for 30 min. Then, it was co-stained with Hoechst 33342 and YO-PRO-1 for 15 min. The bacteria were collected by centrifugation (3000 rpm, 5 min), rinsed three times with PBS, and then mixed with glycerol for preparation of slides and observed under a microscope, and the results are as Figure 5 shown. Among them, Hoechst 33342 can stain all bacteria, while YO-PRO-1 can only stain dead bacteria. Therefore, a higher proportion of green fluorescence indicates a larger number of dead bacteria. From Figure 5 this, it can be seen that for the light irradiation group, the green fluorescence of YO-PRO-1 in Compound Ⅰ almost completely overlaps with the blue fluorescence of Hoechst 33342, indicating good bactericidal effect. For the non-light irradiation group, the green fluorescence does not match well with the blue fluorescence, indicating that the in vitro killing ability of Compound Ⅰ against bacteria comes from the production of ROS under light irradiation.

[0145] Mechanism study of TPIP(Ⅰ), TPI-IDT and Compound Ⅰ against MRSA:

[0146] The bacteria were cultured overnight with shaking (180 rpm, 37 °C) and diluted to an OD 600was 1. A bacterial solution containing 10 μM of the compound (TPIP(I), TPI-IDT, and compound I) in 1 mL was co-cultured at 37 °C for 1 hour, and then co-stained with Hoechst 33342 for 15 min. The bacteria were collected by centrifugation (3000 rpm, 5 min), rinsed three times with PBS, and then mixed with glycerol for slide preparation and observed under a microscope. The results are as Figure 6 shown. As can be seen from Figure 6 , the red fluorescence of compound I was closely co-localized with the blue fluorescence produced by the cytoplasmic dye Hoechst 33342, indicating that compound I penetrated into the bacteria and exerted antibacterial effects. In contrast, the overlap between the red fluorescence of TPIP(I) and TPI-IDT and the blue fluorescence of Hoechst 33342 was limited, indicating their poor binding ability to bacteria, which was related to their low antibacterial activity. The experimental results showed that the compound modified with phenoxyboronic acid (BOB) molecules and pyridinium cations had selective targeting and strong antibacterial activity against Gram-positive bacteria. The cell wall of Gram-positive bacteria contains abundant peptidoglycan, providing a large number of binding sites for compound I.

[0147] The bacteria were cultured overnight by shaking (180 rpm, 37 °C) and diluted to an OD 600 of 1. A bacterial solution containing 10 μM of compound 1 in 1 mL was co-cultured at 37 °C for 1 hour, and then irradiated with or without white light (60 mW / cm 2 ) for 30 min. The bacteria were collected by centrifugation (3000 rpm, 5 min), rinsed three times with PBS, and then the bacterial samples were suspended in 1 mL of 2.5% glutaraldehyde,

[0148] dehydrated, sputter-coated with gold, and then observed under an electron microscope. Scanning electron microscopy analysis of the bacteria after light irradiation showed that S. aureus and MRSA treated with PBS maintained intact cell walls and clear morphology. In contrast, the bacteria treated with TPIP(I), TPI-IDT, and compound I showed varying degrees of damage, including surface fusion and release of intracellular contents. In the light-irradiated group of compound I, the bacteria showed obvious outer membrane rupture and content release.

[0149] Evaluation experiment of the in vitro antibiofilm activity of compound I:

[0150] (1) Biofilm growth inhibition experiment

[0151] The MRSA in the logarithmic growth phase was diluted to 3×10 6CFU / mL and add it to a confocal dish. Divide it into a control group, a non-irradiation group and an irradiation group, and add 20 μL of PBS, 20 μL of Compound I (1 mM) and 20 μL of Compound I (1 mM) respectively. Then incubate them in the dark for 2 h, and subsequently place the irradiation group under white light for 20 min. After all confocal dishes are incubated at 37 °C for 24 h, 48 h, and 72 h, remove the culture medium and carefully wash it three times with PBS, then add Hoechst 33342 and YO-PRO-1 for co-incubation for 15 min for staining. Subsequently, observe and image the biofilm under a laser confocal microscope, and the results are as Figure 7 shown.

[0152] (2) Mature biofilm disruption experiment

[0153] Dilute MRSA in the logarithmic growth phase to 3×10 6 CFU / mL and add it to a confocal dish. After culturing at 37 °C for 24 h, 48 h, and 72 h to form mature biofilms of different degrees, divide them into a control group, a non-irradiation group and an irradiation group, and add 20 μL of PBS, 20 μL of Compound I (1 mM) and 20 μL of Compound I (1 mM) respectively. Then incubate them in the dark for 2 h, and subsequently place the irradiation group under white light for 20 min. After removing the culture medium from all confocal dishes and carefully washing it three times with PBS, add Hoechst 33342 and YO-PRO-1 for co-incubation for 15 min for staining. Subsequently, observe and image the biofilm under a laser confocal microscope, and the results are as Figure 7 shown.

[0154] As Figure 7 can be seen, the biofilms in the control group all showed blue fluorescence, indicating a large number of live bacteria in the biofilm. As time increased, the thickness of the biofilm also increased significantly, showing a dense biofilm structure. After adding Compound I and light treatment, the structure of the MRSA biofilm showed more pores, the mature biofilm structure was destroyed, and only a few bacteria remained in the form of colonies. At the same time, the biomass and roughness coefficient were significantly reduced.

[0155] Study on the in vitro anti-biofilm mechanism of Compound I:

[0156] Dilute MRSA in the logarithmic growth phase to 3×10 6 CFU / mL and add it to a confocal dish. After culturing at 37 °C for 72 h to form a mature biofilm, remove the culture medium from all confocal dishes and carefully wash it three times with PBS, then add Compound I for co-incubation for 1 h for staining. Observe by layer scanning under a confocal microscope.

[0157] Dilute MRSA in the logarithmic growth phase to 3×10 6 CFU / mL, and add it to the cell culture slide on the confocal dish. It was divided into a control group, a non-irradiation group, and an irradiation group, and 20 μL of PBS, 20 μL of Compound I (1 mM), and 20 μL of Compound I (1 mM) were added respectively. Then they were all placed in the dark for incubation for 2 h, and subsequently the irradiation group was placed under white light for irradiation for 20 min. Then it was rinsed three times with PBS, and then the bacterial sample was suspended with 1 mL of 2.5% glutaraldehyde, dehydrated, sputter-coated with gold, and then observed under an electron scanning microscope.

[0158] MRSA biofilms were stained with Compound I, and red fluorescence was observed, indicating that the probe binds to the bacteria within the biofilm. The results confirmed that Compound I can penetrate the biofilm barrier and bind to the resident bacteria, thus achieving the effect of eliminating the biofilm. The SEM images further showed that the MRSA biofilm treated with PBS maintained a dense structure and a complete surface, while the biofilm in the Compound I photo-treatment group was significantly damaged, and only some bacterial colonies remained. All these illustrate the good in vitro anti-biofilm activity of Compound I.

[0159] Physical evaluation of the micro-needle tip solution DMN@BOB-3 in the antibacterial microneedle patch:

[0160] (1) Investigation of micro-needle morphology

[0161] After the micro-needles were prepared, a macro camera and a fluorescence microscope were used for observation respectively, as specifically shown in Figure 8 the figure. As shown in Figure 8 a-b, the blank microneedle patch had 144 needle tips arranged in an orderly manner, the distance between each needle tip was about 700 μm, and the needle tip length was about 750 μm. As shown in Figure 8 c, the red fluorescence of Compound I was detected when observing DMN@BOB-3 through an optical microscope, confirming that Compound I was successfully loaded onto the micro-needle tips.

[0162] (2) Investigation of micro-needle mechanical properties

[0163] The mechanical strength of DMN@BOB-3 and Blank DMN can be measured by an MTS biomechanical testing machine. Place the micro-needles on the turntable of the MTS biomechanical testing machine with the needle tips facing up. Use the sensor to move downward at a uniform speed of 10 μm / s, and record the stress intensity and displacement in real time to obtain the stress-displacement curve, and the results are as shown in Figure 10 the figure.

[0164] To verify the penetration ability of the micro-needles, press the micro-needles into the ex vivo skin of pigs and live mice for 2 min, and then remove them. Take photos of the ex vivo pig skin and live mouse skin over time, and the results are asFigure 9 as shown

[0165] As can be seen from Figure 9 it, the force-displacement curve shows that there is no significant difference in the mechanical strength of the microneedles before and after loading Compound I. The mechanical strengths of the blank microneedles (Blank DMN) and the microneedles loaded with the compound (DMN@BOB-3) are 0.55 N and 0.42 N respectively, which are sufficient to insert into the biological membrane and skin. In addition, uniform and complete microneedle imprints are left on the isolated pig skin, and the tips of the microneedles dissolve as expected. In live mice, the microneedle imprints remain intact on the skin after the microneedles are removed, and the skin completely recovers within 8 minutes, indicating that the microneedles have good skin compatibility and adaptability.

[0166] (3) Investigation of the drug release ability of microneedles

[0167] To determine the in vitro drug release ability of the microneedles, the tips of the needles were scraped off and dissolved in 1 mL of PBS. 10 μL of the solution was sampled at different times, and the OD 530 value was measured using a micro UV instrument, and then 10 μL of PBS was added. The ratio of the measured OD 530 value to the theoretical OD 530 value at complete release was calculated to obtain the drug release rate. Using PBS to simulate the in vivo environment, the microneedles showed rapid release, and 73.8% of Compound I was released within the first hour, with a cumulative release of 90% within 36 hours. The rapid release of the drug provided sufficient drug concentration for the rapid inhibition of acute infections, helped to efficiently inhibit bacterial activity, and promoted wound healing.

[0168] (4) Investigation of the ROS generation ability of microneedles

[0169] ABDA was used to detect 1 O 2 generation. The tips of the needles of DMN@BOB-3 were in 1 mL of water, and after mixing, the supernatant was centrifuged (3000 rpm, 10 minutes) and used for subsequent experiments. After adding 10 μL of ABDA (10 mM), it was irradiated under white light (60 mW / cm 2 ) for different times, and its ultraviolet-visible spectrum was measured. The decrease in the ultraviolet absorption at a wavelength of 378 nm can be used to detect 1 O 2 generation. The experimental results showed that 1 O 2 generation efficiency was slightly lower than that of Compound I used alone, but it was still sufficient to combat biofilm infections.

[0170] In vitro antibacterial activity evaluation of the microneedles in the antibacterial microneedle patch:

[0171] The in vitro antibacterial activity of DMNs (Blank DMN, DMN@BOB-1, DMN@BOB-2, DMN@BOB-3) was evaluated using the flat coating technique. The bacteria were cultured overnight with shaking (180 rpm, 37 °C) and diluted to an OD 600 of 1, and then diluted 10 3 times with PBS. The microtip leachate was centrifuged (3000 rpm, 10 min), and the supernatant was taken for subsequent experiments. The bacterial solution was mixed with the supernatant of the leachate at a ratio of 1:1, cultured at 37 °C for 30 min, and then irradiated with or without white light (60 mW / cm 2 ) for 30 min. 100 μL of the bacterial solution was spread on an agar plate and cultured with shaking (180 rpm, 37 °C) overnight. The bacterial survival rate could be calculated by comparing the number of colonies on the plate, and the results are as shown in Figure 11 . As can be seen from Figure 11 , for S. aureus and MRSA, the Blank DMN group had almost no antibacterial activity, while the drug-loaded microneedles all showed significant antibacterial activity, and the inhibition rate of DMN@BOB-3 in the light treatment group against S. aureus and MRSA reached 99%.

[0172] Evaluation of the in vitro antibiofilm activity of microneedles:

[0173] (1) Biofilm growth inhibition experiment

[0174] MRSA in the logarithmic growth phase was diluted to 3×10 6 CFU / mL and added to a confocal dish. It was divided into a control group, a non-irradiation group, and an irradiation group, and Blank DMN microneedle patches, DMN@BOB-1 drug-loaded microneedle patches, DMN@BOB-2 drug-loaded microneedle patches, and DMN@BOB-3 drug-loaded microneedle patches were added respectively, and then all were incubated in the dark for 2 h, and then the irradiation group was placed under white light for 20 min. After all confocal dishes were incubated at 37 °C for 24 h, 48 h, and 72 h, the culture medium was removed and carefully washed three times with PBS, and then Hoechst 33342 and YO-PRO-1 were added for co-incubation for 15 min for staining. Subsequently, the biofilm was observed and imaged under a laser confocal microscope, and the results are as shown in Figure 11 .

[0175] (2) Mature biofilm disruption experiment

[0176] MRSA in the logarithmic growth phase was diluted to 3×10 6CFU / mL and added it to a confocal dish. After culturing at 37 °C for 24 h, 48 h, and 72 h, different degrees of mature biofilms were formed, which were divided into a control group, a non-irradiation group, and an irradiation group, and Blank DMN microneedle patches and DMN@BOB-3 drug-loaded microneedle patches were added respectively. Then all were placed in the dark for incubation for 2 h, and subsequently the irradiation group was placed under white light for irradiation for 20 min. After removing the culture medium from all confocal dishes and carefully washing three times with PBS, Hoechst33342 and YO-PRO-1 were added for co-incubation for 15 min for staining. Subsequently, the biofilms were observed and imaged under a laser confocal microscope, and the results are as Figure 12 shown. As Figure 12 can be seen, the biofilms treated with PBS showed little change after irradiation, maintaining a complete and dense structure. In contrast, the biofilms treated with DMN@BOB-3 solution and irradiation in the early stage of incubation showed a loose structure, increased surface voids, an elevated roughness coefficient, and a significant reduction in biomass, indicating immature and intact biofilms. Even after 72 h, they still showed strong growth inhibition, indicating that DMN@BOB-3 has persistent biofilm growth inhibition activity. After incubating the biofilms for 24 h, 48 h, and 72 h, the DMN@BOB-3 light treatment groups with different treatments showed obvious biofilm structure damage, and most of the resident bacteria were killed, indicating its strong activity against mature biofilms.

[0177] Hemolysis experiment of microneedles:

[0178] Blood (1.5 mL) from healthy mice was donated. First, the collected red blood cells were centrifuged at 1500 rpm for 5 min, the supernatant was aspirated and resuspended in PBS, and then washed three times until the supernatant was clear. Then the solution was resuspended in PBS (21 mL) to prepare a 2% red blood cell solution. Compounds I (0 μM, 0.5 μM, 1 μM, 2 μM, 4 μM, 8 μM, 16 μM, 32 μM, 64 μM, 128 μM) and DMN tips dissolved in PBS (Blank DMN, DMN@BOB-1, DMN@BOB-2, DMN@BOB-3) were added to the same volume of 2% red blood cell solution. After incubation at 37 °C for 2 h, the supernatant was centrifuged at 1500 rpm for 5 min and then transferred to a 96-well plate. The OD 540 value was measured using a Multiskan FC microplate spectrophotometer (Thermo), and its hemolysis rate was calculated by the following formula and a blood compatibility characterization graph was plotted based on the hemolysis rate. Specifically as Figure 13 shown. In this work, PBS and deionized water were used as negative control and positive control respectively.

[0179] The hemolysis percentage is calculated according to the following formula:

[0180]

[0181] It can be seen from Figure 13 that even at the highest concentration, the hemolysis rate is low, and for DMN@BOB-3 with drug loading, the hemolysis rate is only 0.86%, showing excellent biocompatibility.

[0182] Microneedles for in vivo anti-biofilm infection:

[0183] All animal experimental procedures were approved by the Animal Protection and Use Committee of Central South University (No.: 2021-XMSB-0147) and complied with all relevant ethical regulations. A diabetic mouse model was established by intraperitoneal injection of 150 mg / kg STZ. One week later, the blood glucose of the mice stabilized above 17 mmol / L, confirming the successful establishment of the diabetic model. After anesthesia, the hair on the back of the mice was removed, and circular wounds with a size of 8 mm were punched out the next day. At the same time, MRSA bacterial solution (10 μL, OD600 = 1) was dropped on the wound surface of the mice for one day. 35 mice were randomly divided into 7 groups: (1) blank DMN (G1); (2) blank DMN + light (G2); (3) Ce6 (G3); (4) Ce6 + light (G4); (5) vancomycin (G5); (6) DMN@BOB-3 (G6); (7) DMN@BOB-3 + light (G7). The wounds of the mice were photographed, measured, and weighed on days 0, 3, 7, 11, and 14. On day 14, the wound tissues of the mice were collected, immersed in 2 mL PBS, the tissue fluid was cut, and diluted 10 times for agar plate coating. The bacterial survival rate can be calculated by comparing the number of colonies on the plates.

[0184] The results showed that all groups had a tendency of wound healing, and the wound closure could be clearly observed in groups G5 and G7 on day 3. By day 14, the wounds of the mice in groups G7 and G5 were almost completely healed, and the wound closure rate exceeded 95%. In addition, on day 14, the wound tissues of the mice were homogenized, and the number of bacterial colonies was measured by the plate counting method. The negative control group treated with PBS showed a high number of bacterial colonies on all plates, while groups G3 and G4 showed a decrease in the number of colonies. In contrast, groups G7 and G5 had almost no bacterial colonies, showing significant in vivo anti-biofilm activity.

[0185] As described above, it is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claimed rights.

Claims

1. A targeted antibacterial photosensitizer for Gram-positive bacteria, characterized in that: The general formula of the targeted antibacterial photosensitizer is shown in the following formula TPI-BOB: In the formula TPI-BOB, R1 and R2 are each arbitrarily selected from the group consisting of hydrogen, methyl, methoxy, chlorine, bromine, and iodine; and n is arbitrarily selected from the group consisting of 0, 2, 4, 6, and 8.

2. The targeted antibacterial photosensitizer according to claim 1, characterized in that: The structural formula of the targeted antibacterial photosensitizer is any one of the structural formulas shown in the following formulas I to X:

3. The method for preparing the targeted antibacterial photosensitizer according to claim 1, characterized in that: The following steps are involved: S1. Synthesis of compound TPI-Br: The compound shown in the following formula Ⅺ, 4,7-dibromobenzo[c][1,2,5]thiadiazole and base A are dissolved in solvent A, and [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride is added, and the mixture is heated to 80-120°C in an oil bath vacuum under nitrogen protection for reaction. After the reaction, the mixture is cooled to room temperature, diluted with water, extracted, evaporated the organic phase, and separated by chromatography to obtain compound TPI-Br. The synthetic route is as follows: S2. Synthesis of compound TPIP: TPI-Br, pyridine-4-boric acid, base B and [1,1'-bis(diphenylphosphino)ferrocene] palladium dichloride are dissolved in solvent B, heated to 80-120°C under nitrogen protection for reaction, cooled to room temperature after the reaction is completed, diluted with water, extracted, evaporated the organic phase, and separated by chromatography to obtain compound TPIP; the synthetic route is as follows: S3. Synthesis of compound TPI-PN: Dissolve TPIP and bromoamine in solvent C, raise the temperature to 80-120°C for reaction while stirring, cool to room temperature after the reaction, remove solvent C by vacuum evaporation, separate by HPLC, and freeze-dry to obtain compound TPI-PN; the synthesis route is as follows: S4. Synthesis of compound TPI-BOB: TPI-PN, 1-hydroxy-1,3-dihydrobenzo[c][1,2]oxyborane-5-carboxylic acid and HATU were dissolved in solvent D, and N,N-diisopropylethylamine was added dropwise, and the mixture was stirred at room temperature for reaction. After the reaction, the reaction solution was diluted with water, extracted, back-extracted with water, evaporated to remove solvent D, and separated by HPLC to obtain compound TPI-BOB. The synthesis route is as follows:

4. The method for preparing the targeted antibacterial photosensitizer according to claim 3, characterized in that: In step S1, the solvent A includes at least one of tetrahydrofuran, methanol and 1,4-dioxane; the base A includes at least one of potassium carbonate and cesium carbonate; the molar volume concentration ratio of compound XI to solvent A is 1:3-4; the feeding molar ratio of compound XI to 4,7-dibromobenzo[c][1,2,5]thiadiazole is 1:1-2; the feeding molar ratio of compound XI to [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride is 1:0.01-0.02; the feeding molar ratio of compound XI to base is 1:1-1.5; and the reaction time is 6-8h.

5. The method for preparing the targeted antibacterial photosensitizer according to claim 3, characterized in that: In step S2, the solvent B includes at least one of tetrahydrofuran, methanol and 1,4-dioxane; the base B includes at least one of potassium carbonate and cesium carbonate; the molar volume concentration ratio of the compound TPI-Br to the solvent B is 1:2.5-4, the feeding molar ratio of the compound TPI-Br to pyridine-4-boric acid is 1:1-2, the feeding molar ratio of the compound TPI-Br to [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride is 1:0.01-0.02, and the feeding molar ratio of the compound TPI-Br to the base B is 1:1-1.5; the reaction time is 15-20h.

6. The method for preparing the targeted antibacterial photosensitizer according to claim 3, characterized in that: In step S3, the solvent C is CH3CN; the molar volume concentration ratio of the compound TPIP to the solvent C is 1:3-4; the feeding molar ratio of the compound TPIP to the bromamine is 1:1-2; and the reaction time is 4-6h.

7. The method for preparing the targeted antibacterial photosensitizer according to claim 3, characterized in that: In step S4, the solvent D is dimethylformamide; the molar volume concentration ratio of the compound TPI-PN to the solvent D is 1:3-4; the molar ratio of the compound TPI-PN to 1-hydroxy-1,3-dihydrobenzo[c][1,2]oxyborane-5-carboxylic acid is 1:1-2; the molar ratio of the compound TPI-PN to HATU is 1:1-3; the molar ratio of the compound TPI-PN to N,N-diisopropylethylamine is 1:1-2; and the reaction time is 4-6h.

8. An antibacterial microneedle patch, comprising a needle tip and a backing supporting the needle tip, characterized in that: The needle tip includes a targeted antibacterial photosensitizer loaded with the targeted antibacterial photosensitizer according to any one of claims 1 to 2 or a targeted antibacterial photosensitizer prepared by the preparation method according to any one of claims 3 to 7.

9. The method for preparing the antibacterial microneedle patch according to claim 8, comprising the following steps: A1. Wrap the 2-5 g / mL matrix material of the microneedle tip with 5-10 mg / mL of targeted antibacterial photosensitizer to obtain a needle tip solution; A2, adding a solvent to the matrix material of the microneedle backing to a concentration of 2-5 g / mL to obtain a backing solution; A3, adding the needle tip solution into the mold, adding the backing solution after vacuum drying, and obtaining the antibacterial microneedle patch after drying and demoulding.

10. Use of the targeted antibacterial photosensitizer according to any one of claims 1 to 2, the targeted antibacterial photosensitizer prepared by the preparation method according to any one of claims 3 to 7, the antibacterial microneedle patch according to claim 8, or the antibacterial microneedle patch prepared by the preparation method according to claim 9 in bacterial wash-free imaging, bacterial aggregation induction, bacterial near-infrared fluorescence imaging or broad-spectrum antibacterial drugs.

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