Photosensitizer with active oxygen conversion and membrane targeting dual functions and preparation method thereof

Abstract

The invention discloses a photosensitizer with active oxygen conversion and membrane targeting dual functions. The photosensitizer is prepared from the following raw materials: a BBR / NO complex and an embedding carrier, the BBR / NO complex is prepared from the following raw materials: natural AIEgens BBR and an NO donor; the preparation raw materials of the embedding carrier comprise an embedding cavity and a cell membrane targeting positioning peptide; the cell membrane targeted positioning peptide is a transduction activation peptide. The photosensitizer disclosed by the invention can be used for quickly and efficiently converting the generated ROS into active nitrogen (RNS) and efficiently killing common pathogenic bacteria in food.

Description

Technical Field

[0001] The present invention relates to the technical field of novel non-thermal sterilization, and in particular to a photosensitizer with dual functions of active oxygen conversion and membrane targeting and a preparation method thereof. Background Art

[0002] Foodborne diseases caused by pathogenic bacteria are common all over the world, causing huge losses and economic burdens. In the past 25 years, the incidence of global foodborne pathogenic bacteria infection has increased significantly, and the health status of one-quarter of infected people is not optimistic (Ahmed, AM, & Shimamoto, T. Isolation and molecular characterization of Salmonella enterica, Escherichia coli O157: H7 and Shigella spp. from meat and dairy products in Egypt. International Journal of Food Microbiology, 2014, 168, 57-62). Therefore, the development of new and efficient sterilization technology to combat foodborne pathogens has always been highly concerned by researchers, manufacturers and consumers, and is of great significance to food safety and human health.

[0003] Compared with traditional thermal sterilization technology, non-thermal sterilization can maintain the natural color, aroma, taste and other qualities of food to a greater extent and reduce the negative impact on food. Photodynamic inactivation (PDI), as a new non-thermal sterilization technology, has been widely regarded as an effective method to kill microorganisms and ensure food quality and safety. Due to its advantages such as green and safe, non-toxic and residue-free, and will not induce bacteria to develop drug resistance, it has gradually become a research hotspot in the food field in recent years. The three elements of the PDI system are light, oxygen and photosensitizer. The principle is that under the irradiation of a light source of a specific wavelength, the photosensitizer absorbs energy to produce a transition, and transfers the energy to oxygen molecules, converting O2 into reactive oxygen species (ROS) with high oxidizing ability. ROS destroys cell microstructures, DNA, lipids, proteins, etc., leading to bacterial cell death.

[0004] Bertoloni et al. first reported the inactivation effect of PDI on microorganisms. The study pointed out that exogenous hematoporphyrin showed inactivation effect on Gram-positive bacteria and yeast at a wavelength of 380nm and 250W light (Bertoloni, G., Salvato, B., & Dallacqua, M. Hematoporphyrin-sensitized photoinactivation of Streptococcus faecalis. Photochemistry & Photobiology, 1984, 39, 811-816). Current research shows that PDI has a broad-spectrum bactericidal function against most pathogens. Wu et al. studied the inactivation efficiency of PDI mediated by the natural photosensitizer curcumin on V. parahaemolyticus. When the curcumin concentration was 10 and 20μM, under 470nm LED irradiation (3.6J / cm 2 ), the cell number of V. parahaemolyticus was reduced to an undetectable level (Wu, J., Mou, HJ, Xue, CH, Leung, AW, Xu, CS, & Tang, QJ (2016). Photodynamic effect of curcuminon Vibrio parahaemolyticus. Photodiagnosis and Photodynamic Therapy, 2016, 15, 34-39). However, most natural photosensitizers have poor water solubility, are easily decomposed by light, have unstable physicochemical properties, and traditional photosensitizers tend to form aggregates under high concentration conditions, resulting in a photoquenching effect, thereby weakening the generation of ROS.

[0005] Berberine (BBR) is a natural aggregation-induced emission substance (AIEgens) with a torsional intramolecular charge transfer (TICT) effect. Under white light (10 mW / cm2), it can kill 90% of Gram-positive Staphylococcus aureus and 49% of Gram-negative Escherichia coli (Lee, MMS, Zheng, L., Yu, B., Xu, W., Kwok, RTK, Lam, JWY, Xu, F., Wang, D., & Tang. BZ (2019). A highly efficient and AIE-active theranostic agent from natural herbs. Materials Chemistry Frontiers, 3, 1454–1461). Therefore, the PDI system developed based on the natural AIE photosensitizer BBR can produce a large amount of ROS in the aggregated state to kill bacterial cells, without being limited by the light quenching effect caused by aggregation. However, its bactericidal efficiency cannot reach the effective bactericidal standard (>3Log CFU / mL). Therefore, the bactericidal efficiency of the BBR-mediated PDI system needs to be further improved. In addition, BBR is a water-soluble substance. In order to realize its AIE properties, it is necessary to aggregate BBR molecules in organic reagents to limit intramolecular vibration. However, organic reagents are cytotoxic and are not suitable for the food industry. In order to avoid the use of organic reagents, CN110755610A discloses an antibacterial hydrogel with aggregation-induced luminescence properties and a preparation method thereof. The hydrogel is prepared by blending BBR with an amino acid derivative Fmoc-Phe-OH, and the nanofiber network structure and electrostatic interaction formed by self-assembly of the amino acid derivatives are used to limit the intramolecular movement of berberine, further realizing the aggregation-induced luminescence of berberine, and preparing a hydrogel with photodynamic antibacterial effect; however, in this invention, the ROS generated by BBR has a short half-life (3μs) and a small action radius (100nm), which greatly limits the bactericidal efficiency of PDI in practical applications (Zhang, A., Wu, H., Chen, X., Chen, Z., Pan, Y., Qu, W., Hao, H., Chen, D., & Xie, S. (2023). Targeting and arginine-driven synergizing photodynamic therapy with nutritional immunotherapy nanosystems for combating MRSA biofilms. Science Advances, 2023, 9, 9116).

[0006] In summary, after extensive searching by the applicant, it is found that there is at least one problem in this field: the traditional PDI technology cannot provide persistent oxidation of biomacromolecules in cells and tissues from the source, and the photosensitizer cannot act efficiently on the target cells due to its poor stability. Therefore, it is necessary to develop or improve a photosensitizer with dual functions of active oxygen conversion and membrane targeting and a preparation method thereof. Summary of the invention

[0007] Based on this, in order to solve the problem that the traditional PDI technology cannot provide persistent oxidation of biomacromolecules in cells and tissues from the source, and the photosensitizer cannot act on the target cells efficiently due to its poor stability, the present invention provides a photosensitizer with dual functions of active oxygen conversion and membrane targeting and a preparation method thereof. The specific technical scheme is as follows:

[0008] A photosensitizer with dual functions of active oxygen conversion and membrane targeting, wherein the raw materials for its preparation include a BBR / NO complex and an embedding carrier;

[0009] The raw materials for preparing the BBR / NO complex include natural AIEgens BBR and NO donor;

[0010] The raw materials for preparing the embedding carrier include an embedding cavity and a cell membrane targeting peptide;

[0011] The cell membrane targeting peptide is a transduction activation peptide.

[0012] Further, the NO donor includes natural amino acids;

[0013] The natural amino acids include at least one of asparagine, glutamine, histidine, lysine, and L-arginine.

[0014] Furthermore, the embedding cavity comprises at least one of β-cyclodextrin, cucurbituril and carboxymethyl β-cyclodextrin.

[0015] Furthermore, the preparation method of the BBR / NO complex comprises the following steps:

[0016] 250-350 μM of the natural AIEgens BBR and 50-1500 μM of the NO donor are mixed, and then stirred at 45-55° C. in the dark for 0.5-1.5 h, and then cooled to room temperature, and then placed at 3-5° C. for 10-15 h, and then filtered using a 0.4-0.5 μm filter membrane, and then dried to obtain the BBR / NO complex;

[0017] The stoichiometric ratio of the natural AIEgens BBR and the NO donor is 1-2:1-4.

[0018] Furthermore, the method for preparing the embedding carrier comprises the following steps:

[0019] Adding 50-150 μM of the cell membrane targeting peptide to an EDC / NHS conjugate solution for activation treatment, then adding 50-500 μM of the embedding cavity, then performing an oscillation reaction, and then placing it in a dialysis membrane for flowing water dialysis treatment to obtain the embedding carrier;

[0020] The stoichiometric ratio of the cell membrane targeting peptide and the embedding cavity is 1:1-4;

[0021] The molecular weight of the dialysis membrane is 1800-2200D;

[0022] The duration of the running water dialysis treatment is 22 to 28 hours.

[0023] Furthermore, the activation treatment conditions are a rotation speed of 100 to 300 rpm, a temperature of 30 to 50° C., and a reaction time of 1 to 3 hours;

[0024] The conditions for the shaking reaction are a rotation speed of 100 to 300 rpm, a temperature of 30 to 50° C., and a reaction time of 3 to 5 hours.

[0025] Furthermore, the preparation method of the EDC / NHS conjugate solution comprises the following steps:

[0026] 0.05-0.15M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.01-0.03M N-hydroxysuccinimide are dissolved in 0.05-0.15M 2-morpholineethanesulfonic acid buffer, and then the pH value of the conjugate solution is adjusted to 5-6 to obtain the EDC / NHS conjugate solution.

[0027] The technical solution also provides a method for preparing a photosensitizer having dual functions of active oxygen conversion and membrane targeting, which comprises the following steps:

[0028] The BBR / NO complex and the embedding carrier are stirred at 40-60° C. in the dark for 1-3 hours, then cooled to room temperature, then placed at 3-5° C. for 10-15 hours, and then placed in a dialysis membrane for flowing water dialysis to obtain the photosensitizer.

[0029] Further, the stoichiometry of the BBR / NO complex and the embedding carrier is 1-3:1-3;

[0030] The molecular weight of the dialysis membrane is 1500-2500D;

[0031] The duration of the running water dialysis treatment is 20 to 30 hours.

[0032] Furthermore, the technical solution also provides an application of a photosensitizer for killing methicillin-resistant Staphylococcus aureus, with a sterilization rate of >99%.

[0033] The above technical solution uses a photosensitizer obtained by embedding a BBR / L-Arg complex prepared in a ratio of 1:2 in a CMCD / TAT cavity, which can quickly and efficiently convert the generated ROS into reactive nitrogen (RNS), thereby effectively killing common pathogenic bacteria in food. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is the NO production rate of the BBR / NO complex;

[0035] Figure 2 It is the ITC titration cycle diagram of BBR and L-Arg;

[0036] Figure 3 It is the complete isotherm of the interaction between BBR and L-Arg;

[0037] Figure 4 is the thermodynamic parameter for the binding of BBR and L-Arg;

[0038] Figure 5 is the NO yield of BBR and L-Arg in a simple mixed and non-covalently bound state;

[0039] Figure 6 is the ROS production rate of BBR / Arg / CMCD / TAT photosensitizer;

[0040] Figure 7 is the NO production rate of BBR / Arg / CMCD / TAT photosensitizer;

[0041] Figure 8 is the ONOO- yield of the BBR / Arg / CMCD / TAT photosensitizer;

[0042] Fig. 9 It is the bactericidal efficiency of PDI mediated by BBR / Arg / CMCD / TAT photosensitizer against MRSA. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with its embodiments. It should be understood that the specific implementation methods described herein are only used to explain the present invention and do not limit the protection scope of the present invention.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0045] A photosensitizer with dual functions of active oxygen conversion and membrane targeting in one embodiment of the present invention, wherein the raw materials for its preparation include a BBR / NO complex and an embedding carrier;

[0046] The raw materials for preparing the BBR / NO complex include natural AIEgens BBR and NO donor;

[0047] The raw materials for preparing the embedding carrier include an embedding cavity and a cell membrane targeting peptide;

[0048] The cell membrane targeting peptide is a transduction activation peptide.

[0049] In one embodiment, the NO donor comprises a natural amino acid;

[0050] The natural amino acids include at least one of asparagine, glutamine, histidine, lysine, and L-arginine.

[0051] In one embodiment, the embedding cavity comprises at least one of β-cyclodextrin, cucurbituril and carboxymethyl β-cyclodextrin.

[0052] In one embodiment, the preparation method of the BBR / NO complex comprises the following steps:

[0053] 250-350 μM of the natural AIEgens BBR and 50-1500 μM of the NO donor are mixed, and then stirred at 45-55° C. in the dark for 0.5-1.5 h, and then cooled to room temperature, and then placed at 3-5° C. for 10-15 h, and then filtered using a 0.4-0.5 μm filter membrane, and then dried to obtain the BBR / NO complex;

[0054] The stoichiometric ratio of the natural AIEgens BBR and the NO donor is 1-2:1-4.

[0055] Preferably, the method for preparing the BBR / NO complex comprises the following steps:

[0056] 250-350 μM of the natural AIEgens BBR and 100-1200 μM of the NO donor are mixed, and then stirred at 45-55° C. in the dark for 0.5-1.5 h, and then cooled to room temperature, and then placed at 3-5° C. for 10-15 h, and then filtered using a 0.4-0.5 μm filter membrane, and then dried to obtain the BBR / NO complex;

[0057] The stoichiometric ratio of the natural AIEgens BBR and the NO donor is 1-2:1-4.

[0058] Further preferably, the preparation method of the BBR / NO complex comprises the following steps:

[0059] 250-350 μM of the natural AIEgens BBR and 300-900 μM of the NO donor are mixed, and then stirred at 45-55° C. in the dark for 0.5-1.5 h, and then cooled to room temperature, and then placed at 3-5° C. for 10-15 h, and then filtered using a 0.4-0.5 μm filter membrane, and then dried to obtain the BBR / NO complex;

[0060] The stoichiometric ratio of the natural AIEgens BBR and the NO donor is 1-2:1-4.

[0061] More preferably, the method for preparing the BBR / NO complex comprises the following steps:

[0062] 250-350 μM of the natural AIEgens BBR and 300-600 μM of the NO donor are mixed, and then stirred at 45-55° C. in the dark for 0.5-1.5 h, and then cooled to room temperature, and then placed at 3-5° C. for 10-15 h, and then filtered using a 0.4-0.5 μm filter membrane, and then dried to obtain the BBR / NO complex;

[0063] The stoichiometric ratio of the natural AIEgens BBR and the NO donor is 1-2:1-4.

[0064] In one embodiment, the method for preparing the embedding carrier comprises the following steps:

[0065] Adding 50-150 μM of the cell membrane targeting peptide to an EDC / NHS conjugate solution for activation treatment, then adding 50-500 μM of the embedding cavity, then performing an oscillation reaction, and then placing it in a dialysis membrane for flowing water dialysis treatment to obtain the embedding carrier;

[0066] The stoichiometric ratio of the cell membrane targeting peptide and the embedding cavity is 1:1-4;

[0067] The molecular weight of the dialysis membrane is 1800-2200D;

[0068] The duration of the running water dialysis treatment is 22 to 28 hours.

[0069] In one embodiment, the activation treatment conditions are a rotation speed of 100 to 300 rpm, a temperature of 30 to 50° C., and a reaction time of 1 to 3 h;

[0070] The conditions for the shaking reaction are a rotation speed of 100 to 300 rpm, a temperature of 30 to 50° C., and a reaction time of 3 to 5 hours.

[0071] In one embodiment, the method for preparing the EDC / NHS conjugate solution comprises the following steps:

[0072] 0.05-0.15M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.01-0.03M N-hydroxysuccinimide are dissolved in 0.05-0.15M 2-morpholineethanesulfonic acid buffer, and then the pH value of the conjugate solution is adjusted to 5-6 to obtain the EDC / NHS conjugate solution.

[0073] In one embodiment, the present technical solution provides a method for preparing a photosensitizer having dual functions of active oxygen conversion and membrane targeting, which comprises the following steps:

[0074] The BBR / NO complex and the embedding carrier are stirred at 40-60° C. in the dark for 1-3 hours, then cooled to room temperature, then placed at 3-5° C. for 10-15 hours, and then placed in a dialysis membrane for flowing water dialysis to obtain the photosensitizer.

[0075] In one embodiment, the stoichiometry of the BBR / NO complex and the embedding carrier is 1-3:1-3;

[0076] The molecular weight of the dialysis membrane is 1500-2500D;

[0077] The time of the running water dialysis treatment is 20 to 30 hours.

[0078] In one embodiment, the technical solution provides an application of a photosensitizer for killing methicillin-resistant Staphylococcus aureus, with a sterilization rate of >99%.

[0079] The embodiments of the present invention will be described in detail below with reference to specific examples.

[0080] The bacterial species used in the following Example 27: Methicillin-resistant Staphylococcus aureus (ATCC 43300) was purchased from the China Industrial Microbiological Culture Collection Center and was used as a representative methicillin-resistant Staphylococcus aureus.

[0081] The reagents used in the following examples are described as follows:

[0082] Berberine (BBR) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; asparagine (Asn), glutamine (Gln), histidine (His), lysine (Lys) and L-arginine (L-Arg) were purchased from Shanghai Shenggong Biotechnology Co., Ltd.; transduction activating peptide (TAT) was purchased from Anhui Zhuanpeptide Biological Co., Ltd.; toluidine blue O (TBO), β-cyclodextrin (CD), carboxymethyl β-cyclodextrin (CMCD), cucurbit[7]uril (CB), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and 2-morpholineethanesulfonic acid buffer (MES) were purchased from Shanghai Merrill Chemical Technology Co., Ltd. All chemical reagents were of analytical grade.

[0083] Instruments used: IT-09A5 heating magnetic stirrer from Shanghai Nenggong Industrial Co., Ltd.; TGL16M desktop high-speed refrigerated centrifuge from Shanghai Wancheng Medical Instrument Co., Ltd.; 9272 water-proof constant temperature incubator and LED combination light box from Shanghai Yiheng Technology Co., Ltd.

[0084] Embodiment 1:

[0085] Preparation of BBR / NO complex: 300 μM natural AIEgens BBR (berberine) and 300 μM asparagine (Asn) were mixed in a 1:1 stoichiometric ratio, then stirred at 50 °C in the dark for 1 h, then cooled to room temperature, then placed at 4 °C for 12 h, then filtered using a 0.45 μm filter membrane, and then dried to obtain the BBR / Asn complex.

[0086] Embodiment 2:

[0087] Preparation of BBR / NO complex: 300 μM natural AIEgens BBR (berberine) and 300 μM glutamine (Gln) were mixed in a 1:1 stoichiometric ratio, then stirred at 50 °C in the dark for 1 h, then cooled to room temperature, then placed at 4 °C for 12 h, then filtered using a 0.45 μm filter membrane, and then dried to obtain the BBR / Gln complex.

[0088] Embodiment 3:

[0089] Preparation of BBR / NO complex: 300 μM natural AIEgens BBR (berberine) and 300 μM histidine (His) were mixed in a 1:1 stoichiometric ratio, then stirred at 50°C in the dark for 1 h, then cooled to room temperature, then placed at 4°C for 12 h, then filtered using a 0.45 μm filter membrane, and then dried to obtain the BBR / His complex.

[0090] Embodiment 4:

[0091] Preparation of BBR / NO complex: 300 μM natural AIEgens BBR (berberine) and 300 μM lysine (Lys) were mixed in a 1:1 stoichiometric ratio, then stirred at 50 °C in the dark for 1 h, then cooled to room temperature, then placed at 4 °C for 12 h, then filtered using a 0.45 μm filter membrane, and then dried to obtain the BBR / Lys complex.

[0092] Embodiment 5:

[0093] Preparation of BBR / NO complex: 300 μM natural AIEgens BBR (berberine) and 300 μM L-arginine (L-Arg) were mixed in a 1:1 stoichiometric ratio, then stirred at 50 °C in the dark for 1 h, then cooled to room temperature, then placed at 4 °C for 12 h, then filtered using a 0.45 μm filter membrane, and then dried to obtain the BBR / Arg-3 complex.

[0094] The number of binding sites of the BBR / Arg complex obtained in Example 5 was determined:

[0095] Isothermal titration calorimetry (ITC) was used to determine the Gibbs free energy and number of binding sites of the interaction between BBR and L-Arg. 80 μL of BBR solution (300 μM) was added to the burette, 240 μL of L-Arg solution (1200 μM) was added to the sample cell, and the experimental parameters were set as follows: temperature 25 °C, reference energy 10.0 μcal / s, oscillation rate 750 rpm, initial delay 60 s, injection interval 150 s, and injection duration 4.0 s.

[0096] Embodiment 6:

[0097] Preparation of BBR / NO complex: 300 μM natural AIEgens BBR (berberine) and 100 μM L-arginine (L-Arg) were mixed in a stoichiometric ratio of 3:1, then stirred at 50°C in the dark for 1 h, then cooled to room temperature, then placed at 4°C for 12 h, then filtered using a 0.45 μm filter membrane, and then dried to obtain the BBR / Arg-1 complex.

[0098] Embodiment 7:

[0099] Preparation of BBR / NO complex: 300 μM natural AIEgens BBR (berberine) and 300 μM L-arginine (L-Arg) were mixed in a 1:1 stoichiometric ratio, then stirred at 50 °C in the dark for 1 h, then cooled to room temperature, then placed at 4 °C for 12 h, then filtered using a 0.45 μm filter membrane, and then dried to obtain the BBR / Arg-3 complex.

[0100] Embodiment 8:

[0101] Preparation of BBR / NO complex: 300 μM natural AIEgens BBR (berberine) and 600 μM L-arginine (L-Arg) were mixed in a stoichiometric ratio of 1:2, then stirred at 50°C in the dark for 1 h, then cooled to room temperature, then placed at 4°C for 12 h, then filtered using a 0.45 μm filter membrane, and then dried to obtain the BBR / Arg-6 complex.

[0102] Embodiment 9:

[0103] Preparation of BBR / NO complex: 300 μM natural AIEgens BBR (berberine) and 900 μM L-arginine (L-Arg) were mixed in a stoichiometric ratio of 1:3, then stirred at 50 °C in the dark for 1 h, then cooled to room temperature, then placed at 4 °C for 12 h, then filtered using a 0.45 μm filter membrane, and then dried to obtain the BBR / Arg-9 complex.

[0104] Embodiment 10:

[0105] Preparation of BBR / NO complex: 300 μM natural AIEgens BBR (berberine) and 1200 μM L-arginine (L-Arg) were mixed in a stoichiometric ratio of 1:4, then stirred at 50°C in the dark for 1 h, then cooled to room temperature, then placed at 4°C for 12 h, then filtered using a 0.45 μm filter membrane, and then dried to obtain the BBR / Arg-12 complex.

[0106] Embodiment 11:

[0107] Preparation of embedding carrier: 0.1M EDC and 0.02M NHS were dissolved in 0.1M MES buffer, and the pH value of the conjugate solution was adjusted to 5.4 to prepare EDC / NHS conjugate solution. 100μM TAT was activated in the conjugate solution for 2h (200rpm, 40℃). Subsequently, 100μM CD was added to the conjugate solution at a 1:1 stoichiometric ratio, and the reaction was shaken at 40℃ and 200rpm for 4h. The above reaction product was placed in a dialysis membrane with a molecular weight of 2000D and dialyzed in running water for 24h to obtain CD / TAT-1.

[0108] Embodiment 12:

[0109] Preparation of embedding carrier: 0.1M EDC and 0.02M NHS were dissolved in 0.1M MES buffer, and the pH value of the conjugate solution was adjusted to 5.4 to prepare EDC / NHS conjugate solution. 100μM TAT was activated in the conjugate solution for 2h (200rpm, 40℃). Subsequently, 200μM CD was added to the conjugate solution at a stoichiometric ratio of 1:2, and the reaction was shaken at 40℃ and 200rpm for 4h. The above reaction product was placed in a dialysis membrane with a molecular weight of 2000D and dialyzed in running water for 24h to obtain CD / TAT-2.

[0110] Embodiment 13:

[0111] Preparation of embedding carrier: 0.1M EDC and 0.02M NHS were dissolved in 0.1M MES buffer, and the pH value of the conjugate solution was adjusted to 5.4 to prepare EDC / NHS conjugate solution. 100μM TAT was activated in the conjugate solution for 2h (200rpm, 40°C). Subsequently, 300μM CD was added to the conjugate solution at a stoichiometric ratio of 1:3, and the reaction was shaken at 40°C and 200rpm for 4h. The above reaction product was placed in a dialysis membrane with a molecular weight of 2000D and dialyzed in running water for 24h to obtain CD / TAT-3.

[0112] Embodiment 14:

[0113] Preparation of embedding carrier: 0.1M EDC and 0.02M NHS were dissolved in 0.1M MES buffer, and the pH value of the conjugate solution was adjusted to 5.4 to prepare EDC / NHS conjugate solution. 100μM TAT was activated in the conjugate solution for 2h (200rpm, 40°C). Subsequently, 400μM CD was added to the conjugate solution at a stoichiometric ratio of 1:4, and the reaction was shaken at 40°C and 200rpm for 4h. The above reaction product was placed in a dialysis membrane with a molecular weight of 2000D and dialyzed in running water for 24h to obtain CD / TAT-4.

[0114] Embodiment 15:

[0115] Preparation of embedding carrier: 0.1M EDC and 0.02M NHS were dissolved in 0.1M MES buffer, and the pH of the conjugate solution was adjusted to 5.4 to prepare EDC / NHS conjugate solution. 100μM TAT was activated in the conjugate solution for 2h (200rpm, 40°C). Subsequently, 100μM CB was added to the conjugate solution at a 1:1 stoichiometric ratio, and the reaction was shaken at 40°C and 200rpm for 4h. The above reaction product was placed in a dialysis membrane with a molecular weight of 2000D and dialyzed in running water for 24h to obtain CB / TAT-1.

[0116] Embodiment 16:

[0117] Preparation of embedding carrier: 0.1M EDC and 0.02M NHS were dissolved in 0.1M MES buffer, and the pH of the conjugate solution was adjusted to 5.4 to prepare EDC / NHS conjugate solution. 100μM TAT was activated in the conjugate solution for 2h (200rpm, 40°C). Subsequently, 200μM CB was added to the conjugate solution at a stoichiometric ratio of 1:2, and the reaction was shaken at 40°C and 200rpm for 4h. The above reaction product was placed in a dialysis membrane with a molecular weight of 2000D and dialyzed in running water for 24h to obtain CB / TAT-2.

[0118] Embodiment 17:

[0119] Preparation of embedding carrier: 0.1M EDC and 0.02M NHS were dissolved in 0.1M MES buffer, and the pH of the conjugate solution was adjusted to 5.4 to prepare EDC / NHS conjugate solution. 100μM TAT was activated in the conjugate solution for 2h (200rpm, 40°C). Subsequently, 300μM CB was added to the conjugate solution at a stoichiometric ratio of 1:3, and the reaction was shaken at 40°C and 200rpm for 4h. The above reaction product was placed in a dialysis membrane with a molecular weight of 2000D and dialyzed in running water for 24h to obtain CB / TAT-3.

[0120] Embodiment 18:

[0121] Preparation of embedding carrier: 0.1M EDC and 0.02M NHS were dissolved in 0.1M MES buffer, and the pH of the conjugate solution was adjusted to 5.4 to prepare EDC / NHS conjugate solution. 100μM TAT was activated in the conjugate solution for 2h (200rpm, 40°C). Subsequently, 400μM CB was added to the conjugate solution at a stoichiometric ratio of 1:4, and the reaction was shaken at 40°C and 200rpm for 4h. The above reaction product was placed in a dialysis membrane with a molecular weight of 2000D and dialyzed in running water for 24h to obtain CB / TAT-4.

[0122] Embodiment 19:

[0123] Preparation of embedding carrier: 0.1M EDC and 0.02M NHS were dissolved in 0.1M MES buffer, and the pH value of the conjugate solution was adjusted to 5.4 to prepare EDC / NHS conjugate solution. 100μM TAT was activated in the conjugate solution for 2h (200rpm, 40℃). Subsequently, 100μM CMCD was added to the conjugate solution at a 1:1 stoichiometric ratio, and the reaction was shaken at 40℃ and 200rpm for 4h. The above reaction product was placed in a dialysis membrane with a molecular weight of 2000D and dialyzed in running water for 24h to obtain CMCD / TAT-1.

[0124] Embodiment 20:

[0125] Preparation of embedding carrier: 0.1M EDC and 0.02M NHS were dissolved in 0.1M MES buffer, and the pH value of the conjugate solution was adjusted to 5.4 to prepare EDC / NHS conjugate solution. 100μM TAT was activated in the conjugate solution for 2h (200rpm, 40℃). Subsequently, 200μM CMCD was added to the conjugate solution at a stoichiometric ratio of 1:2, and the reaction was shaken at 40℃ and 200rpm for 4h. The above reaction product was placed in a dialysis membrane with a molecular weight of 2000D and dialyzed in running water for 24h to obtain CMCD / TAT-2.

[0126] Embodiment 21:

[0127] Preparation of embedding carrier: 0.1M EDC and 0.02M NHS were dissolved in 0.1M MES buffer, and the pH value of the conjugate solution was adjusted to 5.4 to prepare EDC / NHS conjugate solution. 100μM TAT was activated in the conjugate solution for 2h (200rpm, 40℃). Subsequently, 300μM CMCD was added to the conjugate solution at a stoichiometric ratio of 1:3, and the reaction was shaken at 40℃ and 200rpm for 4h. The above reaction product was placed in a dialysis membrane with a molecular weight of 2000D and dialyzed in running water for 24h to obtain CMCD / TAT-3.

[0128] Embodiment 22:

[0129] Preparation of embedding carrier: 0.1M EDC and 0.02M NHS were dissolved in 0.1M MES buffer, and the pH value of the conjugate solution was adjusted to 5.4 to prepare EDC / NHS conjugate solution. 100μM TAT was activated in the conjugate solution for 2h (200rpm, 40°C). Subsequently, 400μM CMCD was added to the conjugate solution at a stoichiometric ratio of 1:4, and the reaction was shaken at 40°C and 200rpm for 4h. The above reaction product was placed in a dialysis membrane with a molecular weight of 2000D and dialyzed in running water for 24h to obtain CMCD / TAT-4.

[0130] The embedding carriers obtained in Examples 11 to 22 were tested for coupling efficiency:

[0131] The embedding carriers prepared in Examples 11 to 22 were mixed with 5 mL of TBO (500 μM, pH 10.0) and shaken at 25° C. and 25 rpm for 2 h. Excess TBO was rinsed with deionized water, and then the dye was desorbed in 0.5 mL of 50% acetic acid solution and shaken (25 rpm) for 2 h. The absorbance of the desorbed solution at OD631 nm was measured, and the covalent coupling efficiency of the embedding carrier was calculated. The results are shown in Table 1.

[0132] Embodiment 23:

[0133] Preparation of photosensitizer: With the stoichiometric ratio of CMCD and BBR as 1:1, 10 mL of the embedding carrier CMCD / TAT-3 and 10 mL of the BBR / Arg-1 complex were vigorously stirred at 50 °C in the dark for 2 h. After cooling to room temperature, they were allowed to stand at 4 °C for 12 h. Then, they were placed in a dialysis membrane with a molecular weight of 2000D and dialyzed in flowing water for 24 h to obtain the photosensitizer BBR / Arg / CMCD / TAT-1.

[0134] Embodiment 24:

[0135] Preparation of photosensitizer: With the stoichiometric ratio of CMCD and BBR as 1:1, 10 mL of the embedding carrier CMCD / TAT-3 and 10 mL of the BBR / Arg-3 complex were vigorously stirred at 50°C in the dark for 2 h. After cooling to room temperature, they were allowed to stand at 4°C for 12 h. Then, they were placed in a dialysis membrane with a molecular weight of 2000D and dialyzed in flowing water for 24 h to obtain the photosensitizer BBR / Arg / CMCD / TAT-3.

[0136] Embodiment 25:

[0137] Preparation of photosensitizer: With the stoichiometric ratio of CMCD and BBR as 1:1, 10 mL of the embedding carrier CMCD / TAT-3 and 10 mL of the BBR / Arg-6 complex were vigorously stirred at 50 °C in the dark for 2 h. After cooling to room temperature, they were allowed to stand at 4 °C for 12 h. Then, they were placed in a dialysis membrane with a molecular weight of 2000D and dialyzed in flowing water for 24 h to obtain the photosensitizer BBR / Arg / CMCD / TAT-6.

[0138] Embodiment 26:

[0139] Preparation of photosensitizer: With the stoichiometric ratio of CMCD and BBR as 1:1, 10 mL of the embedding carrier CMCD / TAT-3 and 10 mL of the BBR / Arg-9 complex were vigorously stirred at 50 °C in the dark for 2 h. After cooling to room temperature, they were allowed to stand at 4 °C for 12 h. Then, they were placed in a dialysis membrane with a molecular weight of 2000D and dialyzed in flowing water for 24 h to obtain the photosensitizer BBR / Arg / CMCD / TAT-9.

[0140] Embodiment 27:

[0141] Preparation of photosensitizer: With the stoichiometric ratio of CMCD and BBR as 1:1, 10 mL of the embedding carrier CMCD / TAT-3 and 10 mL of the BBR / Arg-12 complex were vigorously stirred at 50 °C in the dark for 2 h. After cooling to room temperature, they were allowed to stand at 4 °C for 12 h. Then, they were placed in a dialysis membrane with a molecular weight of 2000D and dialyzed in flowing water for 24 h to obtain the photosensitizer BBR / Arg / CMCD / TAT-12.

[0142] The photosensitizers obtained in Examples 23 to 27 were used to measure the reactive oxygen species (ROS) production rate of the photosensitizer complex:

[0143] Add 970 μL of methicillin-resistant S. aureus (ATCC 43300) bacterial solution (10 6 Log CFU / mL), 10 μL ROS probe DCFH-DA and 20 μL photosensitizer, incubate in dark for 30 min, place the bacterial solution in a light-proof light box of the PDI device, adjust the height between the light source (20 W) and the bacterial solution to 5.0 cm, and irradiate with an energy of 28.05 mW / cm 2 The illumination time was 15 min. The fluorescence intensity was then measured at an excitation wavelength of 488 nm and an emission wavelength of 535 nm.

[0144] The photosensitizers obtained in Examples 23 to 27 were subjected to determination of the yield of active nitrogen (RNS):

[0145] The NO production rate was determined using the nitric oxide (NO) probe DAF-FM. 975 μL of methicillin-resistant S. aureus (ATCC 43300) bacterial solution (10 6 Log CFU / mL), 5μL DAF-FM probe and 20μL photosensitizer, incubate in dark for 30min, place the bacterial solution in a light-proof light box of the PDI device, and adjust the height between the light source (20W) and the bacterial solution to 5.0cm, corresponding to an irradiation energy of 28.05mW / cm 2 The illumination time was 15 min. The fluorescence intensity was then measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm.

[0146] The ONOO- yield was determined using the peroxynitrite anion (ONOO-) probe rhodamine 123 (DHR123). 880 μL of methicillin-resistant S. aureus (ATCC 43300) bacterial solution (10 6 Log CFU / mL), 100μL DHR123 probe and 20μL photosensitizer complex, incubate in dark for 30min, then place the bacterial solution in a light-proof light box of the PDI device, and adjust the height between the light source (20W) and the bacterial solution to 5.0cm, corresponding to an irradiation energy of 28.05mW / cm 2 The light treatment time was 15 min. Then the absorbance in the range of 350-800 nm was scanned by UV.

[0147] The photosensitizer obtained in Examples 23 to 27 was used for photodynamic sterilization:

[0148] White LED (20 W) was purchased from Shenzhen, China (GeTian Optoelectronics Co., Ltd., China). The LED tube was surrounded by a photo-taking light box to prevent external light from entering. The distance between the LED light source and the bacterial suspension was adjusted to 5.0 cm, corresponding to an irradiation energy of 28.05 mW / cm 2 , calculate the dose obtained for each sample using the following formula:

[0149] E = Pt; where E = dose (energy density), in J / cm 2 , p = irradiance (power density), unit is W / cm 2 , t = time, in seconds.

[0150] Methicillin-resistant S. aureus was used as a representative pathogen. After the strain was activated, the bacterial concentration was adjusted to 8Log CFU / mL with 0.85% sterile saline. 180μL of bacterial suspension was mixed evenly with 20μL of photosensitizer complex, incubated in the dark for 30 minutes, and then the bacterial solution was placed in a light-proof light box of the PDI device and treated with PDI for 10 minutes. 100μL of the bacterial suspension treated with PDI was diluted and spread, and the number of colonies was calculated after incubation at 37°C for 24 hours. Three parallel samples were made for each treatment, and each dilution was repeated 3 times.

[0151] Data analysis and processing:

[0152] The experimental data were processed and analyzed using OriginPro 9.1 and SPSS 17.0 software packages (SPSS Inc., Chicago, USA). The least significant difference (LSD) method was used to compare the significance of the data (p = 0.05).

[0153] Table 1:

[0154]

[0155] In Examples 11 to 22, CD, CB and CMCD were used as embedding cavities, respectively, and covalently coupled with the transduction activation peptide TAT at a ratio of 1:1, 2:1, 3:1 and 4:1, respectively, to prepare CD / TAT-1, CD / TAT-2, CD / TAT-3, CD / TAT-4, CB / TAT-1, CB / TAT-2, CB / TAT-3, CB / TAT-4, CMCD / TAT-1, CMCD / TAT-2, CMCD / TAT-3, CMCD / TAT-4 embedding carriers. The coupling efficiency of the embedding cavity and TAT was calculated by detecting the change in the absorbance of toluidine blue O (TBO) before and after binding with TAT. As can be seen from Table 1, the coupling efficiency of the prepared CD / TAT and CB / TAT embedding carriers is low, and there is no significant difference in the coupling efficiency of the embedding carriers prepared with different stoichiometric ratios. The coupling efficiency of the CMCD / TAT embedding carrier prepared by CMCD and TAT is relatively high, with the coupling efficiency of CMCD / TAT-1 being 33.43%. As the CMCD content increases, the coupling efficiencies of CMCD / TAT-2 and CMCD / TAT-3 increase to 54.11% and 87.25%, respectively. However, when the CMCD content increases further, the coupling efficiency of CMCD / TAT-4 (88.95%) does not increase significantly compared to CMCD / TAT-3. This is because the CMCD molecule contains a large number of carboxyl groups, which can be covalently coupled with the amino groups on the TAT molecule through amide reaction. The above results show that CMCD is the most suitable embedding cavity material for coupling with TAT, and the CMCD / TAT embedding carrier prepared with a stoichiometric ratio of 3:1 has the best coupling efficiency.

[0156] from Figure 1 As can be seen from the figure, in Examples 1 to 5 of the present invention, BBR is used as an AIE photosensitizer, and is hydrogen bonded with natural amino acids Asn, Gln, His, Lys and L-Arg to prepare BBR / Asn, BBR / Gln, BBR / His, BBR / Lys and BBR / Arg complexes, and their NO yields are detected ( Figure 1). Although Asn, Gln, His, Lys and L-Arg are all AA with high nitrogen content (number of N atoms ≥ 2), only the BBR / Arg complex prepared by non-covalent binding of BBR and L-Arg showed significant NO production ability. This is because the L-Arg molecule contains a unique guanidine structure. The nitrogen atom in the guanidine group is oxidized by ROS produced by BBR under the catalysis of nitric oxide synthase, converting ROS into NO. The above results show that compared with other natural amino acids, the unique guanidine group in the L-Arg molecule makes it a superior NO donor with efficient ROS conversion ability.

[0157] Examples 5 to 10 prepared a series of BBR / Arg complexes with different L-Arg addition amounts, wherein the BBR concentration was 300 μM and the L-Arg concentrations were 100 μM, 300 μM, 600 μM, 900 μM and 1200 μM, respectively. The ITC titration cycle diagram obtained by titrating L-Arg with BBR is shown in FIG. Figure 2 As shown, the area below each injection peak is equal to the heat released by the injection. When this integrated heat is plotted against the molar ratio of the ligand added to the sample cell, the complete isotherm of the interaction is obtained ( Figure 3 ). The molar ratio corresponding to the midpoint of the logarithmic region of the enthalpy change ΔH is the number of binding sites between the BBR molecule and the L-Arg molecule. Figure 3 It can be seen that the number of binding sites between BBR and L-Arg is 0.518, which means that each BBR molecule can bind to two L-Arg molecules. Further, from the thermodynamic parameters of the binding between BBR and L-Arg, it can be seen that ( Figure 4 ), the Gibbs free energy of interaction between the two, ΔG, is less than 0, which proves that the reaction between BBR and L-Arg can proceed spontaneously at room temperature.

[0158] from Figure 5 As can be seen, the NO production of the mixture of BBR and L-Arg and the BBR / Arg complex at the same concentration was detected by using a simple mixture of BBR and L-Arg as a control ( Figure 5 ). Although the NO yield of BBR and L-Arg increases with the increase of L-Arg concentration after simple mixing, the NO yield of the BBR / Arg complex bound by hydrogen bonding is significantly higher than that of the mixture of BBR and L-Arg at the same L-Arg concentration. When the L-Arg concentration is 600μM, the difference in NO yield reaches the maximum value, at which time the NO yield of the BBR / Arg complex is 3.18 times that of the mixture of BBR and L-Arg. The above results show that hydrogen bonding can significantly improve the NO yield of the BBR / Arg complex, and when the stoichiometric ratio of BBR and L-Arg is 1:2, the NO yield benefit can be maximized.

[0159] Examples 23 to 27 load the BBR / Arg complex with the best NO generation ability into the CMCD / TAT carrier with the highest coupling efficiency (stoichiometric ratio of 3:1) to prepare the photosensitizer BBR / Arg / CMCD / TAT with both active oxygen conversion and membrane targeting functions. Generally speaking, natural photosensitizers will be excited to produce a large amount of ROS when irradiated with light of a specific wavelength. In the BBR / Arg / CMCD / TAT photosensitizer prepared by the present invention, BBR is a natural AIE photosensitizer, which will be excited to produce ROS after being treated with PDI. Subsequently, these ROS further react with L-Arg and are converted into RNS (NO, ONOO-, etc.) with stronger oxidation ability and longer lifespan. Therefore, the ROS yield of the photosensitizer complex and its various components was first determined. From Figure 6 It can be seen that L-Arg and BBR alone hardly generate ROS. However, when BBR is loaded into the CMCD / TAT carrier, due to the restricted intramolecular motion, the AIE effect is produced. 2 ), a large amount of ROS was generated. As the L-Arg content increased from 100μM to 1200μM, the ROS content generated by the photosensitizer complex gradually decreased. It is worth noting that the ROS yield of the BBR / Arg-6 complex also decreased compared to that of BBR alone. These results mean that L-Arg consumes the ROS generated by the photosensitizer BBR / Arg / CMCD / TAT.

[0160] from Figure 7 It can be seen that L-Arg, BBR and BBR / CMCD / TAT alone will not produce NO, while the NO yield of the BBR / Arg complex is significantly increased. This means that L-Arg and BBR are indispensable for the production of NO. In the BBR / Arg / CMCD / TAT photosensitizer, with the increase of L-Arg content, the NO yield gradually increased and reached the maximum value when the L-Arg concentration was 600μM. Thereafter, as L-Arg further increased to 1200μM, the NO yield of the photosensitizer complex did not change significantly. The above results show that the BBR / Arg / CMCD / TAT photosensitizer prepared by the present invention can effectively convert ROS into NO, and when the concentrations of BBR and L-Arg are 300μM and 600μM respectively, the NO yield reaches the highest value, that is, the optimal stoichiometric ratio of BBR and L-Arg is 1:2. This result is consistent with the above Figure 3 and Figure 5 The results are consistent.

[0161] Figure 8 is the ONOO- yield of the BBR / Arg / CMCD / TAT photosensitizer complex, from Figure 8 It can be seen that the trend of ONOO- produced by the photosensitizer is consistent with that of NO, that is, when the ratio of BBR and L-Arg is 1:2, the yield of ONOO- reaches the maximum. In general, under white light irradiation, the BBR / Arg / CMCD / TAT photosensitizer complex can efficiently convert the ROS produced by the reaction into RNS with stronger oxidative ability and longer life, thereby improving the bactericidal efficiency of the PDI system.

[0162] Fig. 9 The inactivation of MRSA by the BBR / Arg / CMCD / TAT photosensitizer complex with different L-Arg addition amounts after white light irradiation is demonstrated. The negative control group was a bacterial suspension without any treatment, and its initial inoculation amount was 8Log CFU / mL. After exposure to light, L-Arg and BBR alone did not show obvious bactericidal ability compared to the control group. Similarly, the BBR / Arg / CMCD / TAT photosensitizer did not reduce the number of MRSA cells when not exposed to light. As the illumination time increased from 5min (26.04J / cm 2 ) was extended to 15min (78.12J / cm 2) , the number of MRSA cells treated with BBR / Arg / CMCD / TAT-1 decreased from 5.59Log CFU / mL to 2.68Log CFU / mL (>99.9%). When the concentration of L-Arg was increased to 600μM, BBR / Arg / CMCD / TAT-6 was treated with PDI for 15min (78.12J / cm 2 ) reduced the number of MRSA cells to an uncountable level (>8Log), and its bactericidal efficiency was significantly increased by >99.9% compared with BBR / CMCD / TAT without L-Arg (3.67Log CFU / mL). This is because L-Arg successfully converted ROS into RNS (NO, ONOO-) with stronger oxidative ability and longer life, achieving persistent oxidation of biological macromolecules in cells. As L-Arg was further increased to 900μM and 1200μM, the bactericidal efficiency of BBR / Arg / CMCD / TAT-9 and BBR / Arg / CMCD / TAT-12 was not significantly different from that of BBR / Arg / CMCD / TAT-6. This indicates that the optimal combination ratio of BBR (300μM) and L-Arg (600μM) is 1:2. The BBR / Arg / CMCD / TAT photosensitizer prepared under this condition can convert ROS into NO with maximum efficiency and kill bacterial cells efficiently.

[0163] In addition, compared with BBR / Arg-6 (4.21Log CFU / mL) not embedded in the CMCD / TAT carrier, the bactericidal efficiency of BBR / Arg / CMCD / TAT photosensitizer was significantly improved by >99.99%. On the one hand, the hydrophobic inner cavity of CMCD provides a non-polar microenvironment for BBR and restricts the intramolecular movement of BBR, thereby greatly improving the ROS yield of the BBR / Arg / CMCD / TAT photosensitizer complex ( Figure 5 ), promoting the generation of NO; ​​on the other hand, the transduction activation peptide TAT gives the BBR / Arg / CMCD / TAT photosensitizer complex cell membrane targeting ability, enhances the interaction between the photosensitizer and bacterial cells, and promotes the generated NO to act on the target cells more quickly and efficiently, thereby significantly improving the PDI bactericidal efficiency. In general, the BBR / Arg / CMCD / TAT photosensitizer prepared based on the natural AIE photosensitizer BBR of the present invention has both cell membrane targeting ability and ROS conversion ability, and when the stoichiometric ratio of BBR and L-Arg is 1:2, under the excitation of a white LED light source, ROS can be converted into RNS with maximum efficiency, achieving efficient killing of foodborne pathogens (>8Log).

[0164] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0165] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A photosensitizer with dual functions of active oxygen conversion and membrane targeting, characterized in that: The preparation raw materials include BBR / NO complex and embedding carrier; The raw materials for preparing the BBR / NO complex include natural AIEgens berberine (BBR) and NO donor; The raw materials for preparing the embedding carrier include an embedding cavity and a cell membrane targeting peptide; The cell membrane targeting peptide is a transduction activation peptide.

2. The photosensitizer according to claim 1, characterized in that The NO donor includes natural amino acids; The natural amino acids include at least one of asparagine, glutamine, histidine, lysine, and L-arginine, preferably L-arginine.

3. The photosensitizer according to claim 1 or 2, characterized in that The embedding cavity comprises at least one of β-cyclodextrin, cucurbituril and carboxymethyl β-cyclodextrin, preferably carboxymethyl β-cyclodextrin.

4. The photosensitizer according to claim 3, characterized in that The preparation method of the BBR / NO complex comprises the following steps: 250-350 μM of the natural AIEgens BBR and 50-1500 μM of the NO donor are mixed, and then stirred at 45-55° C. in the dark for 0.5-1.5 h, and then cooled to room temperature, and then placed at 3-5° C. for 10-15 h, and then filtered using a 0.4-0.5 μm filter membrane, and then dried to obtain the BBR / NO complex; The stoichiometric ratio of the natural AIEgens BBR and the NO donor is 1-2:1-4.

5. The photosensitizer according to claim 3, characterized in that The method for preparing the embedding carrier comprises the following steps: Adding 50-150 μM of the cell membrane targeting peptide to an EDC / NHS conjugate solution for activation treatment, then adding 50-500 μM of the embedding cavity, then performing an oscillation reaction, and then placing it in a dialysis membrane for flowing water dialysis treatment to obtain the embedding carrier; The stoichiometric ratio of the cell membrane targeting peptide to the embedding cavity is 1:1-4, preferably 1:3, and more preferably the embedding cavity is carboxymethyl β-cyclodextrin; The molecular weight of the dialysis membrane is 1800-2200D; The duration of the running water dialysis treatment is 22 to 28 hours.

6. The photosensitizer according to claim 5, characterized in that The activation treatment conditions are as follows: a rotation speed of 100 to 300 rpm, a temperature of 30 to 50° C., and a reaction time of 1 to 3 hours; The conditions for the shaking reaction are a rotation speed of 100 to 300 rpm, a temperature of 30 to 50° C., and a reaction time of 3 to 5 hours.

7. The photosensitizer according to claim 5, characterized in that The preparation method of the EDC / NHS conjugate solution comprises the following steps: 0.05-0.15M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.01-0.03M N-hydroxysuccinimide are dissolved in 0.05-0.15M 2-morpholineethanesulfonic acid buffer, and then the pH value of the conjugate solution is adjusted to 5-6 to obtain the EDC / NHS conjugate solution.

8. A method for preparing a photosensitizer having dual functions of active oxygen conversion and membrane targeting, characterized in that: The preparation method is used to prepare the photosensitizer according to any one of claims 1 to 7, and comprises the following steps: The BBR / NO complex and the embedding carrier are stirred at 40-60° C. in the dark for 1-3 hours, then cooled to room temperature, then placed at 3-5° C. for 10-15 hours, and then placed in a dialysis membrane for flowing water dialysis to obtain the BBR / NO complex.

9. The preparation method according to claim 8, characterized in that: The stoichiometric ratio of the BBR / NO complex and the embedding carrier is 1-3:1-3, to obtain the photosensitizer; The molecular weight of the dialysis membrane is 1500-2500D; The time of the running water dialysis treatment is 20 to 30 hours.

10. An application of a photosensitizer, characterized in that: The photosensitizer according to any one of claims 1 to 7 or the photosensitizer prepared by the preparation method according to any one of claims 8 to 9 is used in killing methicillin-resistant Staphylococcus aureus.

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