Preparation and application of water-stable organic free radical supramolecular crystal

Abstract

The invention discloses preparation and application of a water-stable organic free radical supramolecular crystal, and relates to the technical field of photo-thermal antibacterial agents. The photo-thermal antibacterial agent comprises closed dodecane borane anions (B12H12 < 2->) and a fluorescent dye N, N '-dimethyl bipyridine-thiazole [5, 4-d] thiazole (MPT < 2 + >). In the photo-thermal antibacterial agent disclosed by the invention, the supramolecular crystal (MB12-1) is constructed through a chaotropic effect between B12H12 < 2-> and MPT < 2 + > molecules. And then, carrying out photoinduced electron transfer between the MB12-1 and the TEOA to obtain the MB12-2 containing the organic free radical MPT. +. The introduction of the chaotropic effect not only facilitates the synthesis of the supramolecular crystal, but also can cooperate with the long-distance cation-pi interaction between MPT < 2 + > molecules to maintain the stability of the supramolecular crystal. N2 adsorption test analysis proves that the MB12-2 has no obvious pores inside, and molecules are tightly arranged. Therefore, the half-life period of MB12-2 in water can be as long as 20 days. The stability of free radicals is the basis of photo-thermal conversion, and MB12-2 shows high photo-thermal conversion capacity under irradiation of 660 nm laser, so that MB12-2 can be used as a photo-thermal antibacterial agent to promote healing of bacterial infected wounds.

Description

Technical Field

[0001] The present invention relates to the technical field of photothermal antibacterial materials, and in particular to the preparation and application of a water-stable organic free radical supramolecular crystal. Background Art

[0002] In recent years, photothermal therapy (PTT) has attracted widespread attention in broad-spectrum anti-infective treatment due to its advantages such as low toxicity, minimal invasiveness, and no drug resistance. PTT can convert light energy into heat energy (≥50 °C) through photothermal agents, thereby destroying the bacterial cell membrane and causing bacterial death. At present, with the development of science and technology, various organic molecules and inorganic materials are being developed as photothermal agents for photothermal therapy, such as porphyrin, anthocyanin, polydopamine, gold nanoparticles, graphene oxide, etc. In addition, organic free radicals have become promising photothermal agents in photothermal therapy due to their unique optical properties. However, most organic free radicals have problems such as complex synthesis and easy photobleaching under light irradiation. Therefore, it is very important to design and develop more effective organic free radical photothermal agents to meet the growing demand for PTT.

[0003] The chaotropic effect, which includes but is different from hydrogen bonding and electrostatic interactions, is a new type of non-covalent interaction that can provide a strong driving force for the self-assembly of supramolecular complexes. 12 H 12 2- ) is a highly water-soluble super-chaotropic anion. B 12 H 12 2- As a three-dimensional analog of benzene, it is also a highly stable cluster molecule. Based on these characteristics, using B 12 H 12 2- Supramolecular complexes prepared by the chaotropic effect between hydrophobic neutral or positively charged molecules have been applied in many fields such as photothermal or photodynamic therapy, catalysis, and selective adsorption.

[0004] Therefore, through B 12 H 12 2- Designing a supramolecular crystal containing water-stable organic free radicals for photothermal antibacterial application based on the chaotropic effect between hydrophobic neutral or positively charged molecules is an urgent problem to be solved in the present invention. Summary of the invention

[0005] The present invention is achieved by 12 H 12 2- With positively charged molecules (MPT 2+) is used to design a supramolecular crystal containing water-stable organic free radicals. The invention aims to solve the problems of the existing organic free radical photothermal agents in the background technology, such as the complex synthesis and the easy bleaching under light.

[0006] In order to achieve the above technical objectives, the present invention mainly adopts the following technical solutions: In the first aspect, the present invention prepares a supramolecular crystal containing water-stable organic free radicals that can be used for photothermal antibacterial treatment. The supramolecular crystal is composed of a closed dodecaborane anion (B 12 H 12 2- ) and the fluorescent dye N, N'-dimethyldipyridyl-thiazolyl[5,4-d]thiazolyl(MPT 2+ )form.

[0007] Closed dodecaborane anion (B 12 H 12 2- ) has a regular icosahedral structure and carries two permanent negative charges. 12 H 12 2- As a highly water-soluble super-chaotropic anion, it can self-assemble with hydrophobic neutral or positively charged molecules through the chaotropic effect to prepare photothermal agents for antibacterial purposes.

[0008] Here, through B 12 H 12 2- and MPT 2+ A novel photothermal antibacterial agent as described above was designed based on the intermolecular chaotropic effect, which has excellent water stability and efficient photothermal conversion performance.

[0009] First, use B 12 H 12 2- and MPT 2+ The chaotropic effect between molecules constructs supramolecular crystal MB 12 -1, and then reduce MB via photoinduced electron transfer (PET) 12 -1 forms MPT in supramolecular crystals ·+ Free radicals, thereby obtaining supramolecular crystals MB containing free radicals 12 -2. The chaotropic effect is not only beneficial to the synthesis of supramolecular crystals, but also can cooperate with MPT through steric effect and spin density effect. 2+ Intermolecular long-range cation-π interactions enable free radical MPT ·+ It has long-term water stability. In addition, MB 12 -2 has significant photothermal effect and photothermal stability under 660 nm laser irradiation, and exhibits strong antibacterial activity both in vitro and in vivo.

[0010] In a preferred embodiment of the present invention, the photothermal antibacterial agent, B 12 H 12 2- and MPT 2+ The molar ratio is 1:1.

[0011] In a second aspect, the present invention provides a method for preparing the photothermal antibacterial agent as described in the first aspect, comprising: 12 H 12 2- and MPT 2+ The aqueous solution of MB is mixed to obtain 12 -1. Then restore MB through light 12 -1 obtained supramolecular crystal MB with water stability and high-efficiency photothermal antibacterial activity 12 -2.

[0012] In a third aspect, the present invention provides a use of the photothermal antibacterial agent as described in the first aspect for inhibiting bacteria in vitro.

[0013] The bacteria are Escherichia coli and Staphylococcus aureus.

[0014] In a fourth aspect, the present invention provides a method for inhibiting bacteria on mammalian skin using the photothermal antibacterial agent as described in the first aspect, wherein an effective dose of the photothermal antibacterial agent is applied to the mammalian skin.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. The ultraviolet-visible light absorption spectrum (UV-Vis) experimental results of the photothermal antibacterial agent of the present invention show that MB 12 -2 exhibits long-term water stability.

[0016] 2. In the photothermal antibacterial agent of the present invention, MPT 2+ The potential cytotoxicity and skin irritation of B 12 H 12 2- The side effects of photothermal antibacterial agents are significantly reduced by complexation.

[0017] 3. In the photothermal antibacterial agent of the present invention, MPT 2+ By 12 H 12 2- The chaotropic effect of the two molecules led to the self-assembly of supramolecular crystals (MB) containing water-stable organic free radicals at a molar ratio of 1:1. 12 -2), this supramolecular crystal showed better performance than MPT alone. 2+Higher stability and photothermal conversion efficiency. Single crystal structure analysis (SXRD) shows that B 12 H 12 2- and MPT 2+ The strong chaotropic effect between molecules leads to MPT 2+ The molecules form long-range cation-π interactions, which effectively enhances MPT ·+ Electron paramagnetic resonance (EPR) spectroscopy shows that MPT ·+ At the same time, N2 adsorption-desorption test showed that MB 12 -2 has almost no solvent accessible volume. In addition, MB 12 -2 has a half-life of up to 20 days in water. Therefore, MB with excellent water stability 12 -2 exhibits efficient photothermal conversion under 660 nm laser irradiation and can be further used for photothermal antibacterial. Its high antibacterial rate can effectively promote wound healing.

[0018] 4. The present invention provides a simple method for designing supramolecular crystals with long-term water-stable organic free radicals, and also expands the application of free radical-doped crystal materials in photothermal therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 B in photothermal antibacterial agent 12 H 12 2- and MPT 2+ Supramolecular self-assembly process diagram and MB 12 -2 preparation flow chart; Figure 2 For B 12 H 12 2- and MPT 2+ Structural analysis of self-assembled supramolecular crystals, where a) and b) are B 12 H 12 2- and MPT 2+ X-ray structure of B 12 H 12 2- and MPT 2+ The chaotropic effect between (the value is in Å); d) is B 12 H 12 2- and MPT 2+ Simulated electrostatic potential (ESP) of B 12 H 12 2- Cluster-supported MPT2+ Cation-π interactions between molecules (labeled values ​​are in Å, represented by dashed black lines).

[0020] Figure 3 MB 12 -1 and MB 12 -2 characterization detection diagram, where a) is MPT 2+ and UV-vis spectra of excess TEOA with different irradiation times; b) MB 12 UV-Vis spectra of -1 and excess TEOA with different irradiation times. The inset shows MB 12 -1 to MB 12 -2 photos of color changes; c) MPT 2+ 、MB 12 -1 and MB 12 -2 EPR spectrum; d) and e) are MB 12 -1 and MB 12 -2 transmission electron microscope (TEM) image; f) is MB 12 -1 and MB 12 -2 powder x-ray diffraction (PXRD) spectrum.

[0021] Figure 4 MB 12 -1 and MB 12 -2 N2 adsorption-desorption experiment.

[0022] Figure 5 MB 12 -2 Water stability test graph and photothermal conversion performance test graph, where a) is MB 12 -2 is the picture of the color change under different environmental conditions; b) is the picture of MB at room temperature 12 -2 in water over time; c) is the UV-Vis spectrum of MB at room temperature 12 -2In water, the absorption peak decay curve at 637 nm wavelength changes with time. The curve fitting adopts the exponential fitting method; d) is at a power of 1.20 W cm -2 MB with different concentrations under 660 nm laser irradiation 12 -2 Infrared thermal image of aqueous dispersion and e) temperature rise curve; f) at 1.20 W cm -2 MB under 660 nm laser irradiation 12 -2 aqueous dispersion (200 μg mL -1 ) Temperature changes with power density; g) is at 1.20 W cm -2 MB under 660 nm laser irradiation 12-2 Photothermal conversion efficiency calculation process and heating and cooling diagram; h) is at 1.20 W cm -2 MB under 660 nm laser irradiation 12 -2 heating and cooling cycle curve diagram.

[0023] Figure 6 MB 12 -2 In vitro antibacterial test, a) and b), c) and d) were performed under laser irradiation at a concentration of 200 μg mL -1 MB 12 -2 against Escherichia coli and Staphylococcus aureus (660 nm, 1.2 W cm -2 , n = 3, **p < 0.01, ***p < 0.001, Student's t-test).

[0024] Figure 7 MB 12 -1 and MB 12 -2 cytotoxicity assay.

[0025] Figure 8 MB 12 -1 and MB 12 -2 In vivo anti-infection test, a) from day 0 to day 11, using 200 μg mL -1 a) is a photo of the wound healing process after different sample treatments; b) is a schematic diagram of the wound contraction comparison on the 3rd day and the 11th day; c) is a photo of the colony of Staphylococcus aureus at the wound infection site on the 9th day after different treatments and d) the corresponding colony data; e) is the difference in relative wound area between different treatment groups (n=3, ***p < 0.001, NS means not significant, Student's t-test); f) is the H&E histological image of the wound tissue of different treatment groups on the 11th day. (Where L+ indicates 660 nm laser irradiation, L- indicates no 660 nm laser irradiation). DETAILED DESCRIPTION

[0026] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

[0027] It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence, nor do they play a substantial limiting role on the subsequent technical features. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices. Example

[0028] MB 12 -1 preparation.

[0029] MB 12 -1's assembly process involves only two additives, MPT and Cs2B 12 H 12 , both are water soluble. 2+ With B 12 H 12 2- The aqueous solution is mixed to obtain solid powder MB 12 -1. Example

[0030] Photothermal antibacterial agent MB 12 -2.

[0031] Excess TEOA was added to the dispersed MB 12 -1 aqueous solution, MB was reduced by photoreduction 12 -1, solid powder MB can be obtained 12 -2.

[0032] Test example 1. Experimental part 1. Materials Closed dodecaborane anion (B 12 H 12 2- ) and the fluorescent dye N, N'-dimethyldipyridyl-thiazolyl[5, 4-d]thiazolyl(MPT 2+) was prepared according to the methods in the literature (Angew. Chem. Int. Ed. 2019, 58, 15526-15531; New J. Chem. 2022, 46, 5470-5473). Dithioformamide was purchased from Shanghai McLean Biochemical Co., Ltd. (Shanghai, China); pyridine-4-carboxaldehyde and other original reagents (analytical grade) were purchased from Beijing Eno Chemical Technology Co., Ltd. (Shanghai, China); yeast extract, trypsin and agar were obtained from BioFrox; live / dead bacterial staining kit (i.e., propidium iodide (PI) red dye and Calcine-AM green dye) was purchased from Shanghai Yuanye Biological Co., Ltd., China; ultrapure water (18.25 MΩ, Eco-S15UVF; HHitech, Shanghai, China) was used for the preparation of all aqueous solutions.

[0033] 2. Instruments The UV-vis spectra were measured using an SP-756P UV-vis spectrometer. Scanning electron microscopy (SEM) images were obtained on a Hitachi S-4800 field emission SEM (FE-SEM) instrument. Transmission electron microscopy (TEM) images were obtained using a Hitachi H-7000FA high-resolution transmission electron microscope with an operating voltage of 200 kV. Powder X-ray diffraction (PXRD) patterns of the prepared products were recorded using an X-ray diffractometer (Bruker D8 advance) with a scanning speed of 4° min -1 ; N2 adsorption-desorption experiments of LZU-111 were carried out on a Micromeritics ASAP 2020 surface area and porosity analyzer; fluorescence images were obtained under ambient conditions using an inverted scanning microscope (Olympus, FV1000).

[0034] 3. In vitro antibacterial test Drug-resistant Gram-negative Escherichia coli (DR -E. coli ) and Gram-positive Staphylococcus aureus (DR- S. aureus ) is a bacterial model to evaluate MB 12 -1 and MB 12 -2 antibacterial performance. Take different concentrations of antibacterial samples (MB 12- 1 and MB 12 -2) were mixed with diluted bacterial solution (1 × 10 6 CFU mL -1 ) were mixed in a 1.5 mL centrifuge tube. The mixed solutions of substances and bacteria with different concentrations were divided into a laser irradiation group and a non-irradiation group. The laser irradiation group was irradiated at 1.2 W cm -2The cells were irradiated with a 660 nm laser for 5 min, 100 μL of the mixed solution was spread on the surface of LB solid culture medium, and cultured in a 37°C constant temperature incubator for 15 h. The bacterial PBS solution without laser irradiation was used as the control group. Finally, the total number of viable bacteria was determined by dilution method and plate count method.

[0035] 4. Conduct in vitro cytotoxicity assessment MTT test to evaluate MB 12 MDA-MB-231 cells were diluted in fresh complete medium and seeded in 96-well plates (1.5 × 10 cells per well) containing DMEM (200 μL). 4 After incubation (37 °C, 5% CO2) for 24 h, different concentrations of MB were used. 12 -1 and MB 12 -2 treated cells. Then 20 μL of MTT dye solution (5 mg / mL) was added to each well. -1 ) for 24 h. Subsequently, the MTT medium was removed and 150 μL of dimethyl sulfoxide was added to each well. The OD value at 450 nm was measured using a microplate reader (BIO-RAD 550). Cell viability was calculated as follows: Viability = (OD treated / OD control )×100%, where OD treated From drug-treated cells, OD control from cells that had not been treated in any way.

[0036] 5. In vivo antibacterial test The mouse model of Staphylococcus aureus infection wound was used to investigate the 12 -2 in vivo therapeutic effect on bacterially infected wounds. All animal experiments were performed in accordance with the procedures and regulations of Wuhan University of Science and Technology. The average weight of mice used in the experiment was about 20 g. After the mice were anesthetized, the backs were depilated, and the full-thickness skin was removed by biopsy puncture to form a circular wound. Immediately, 30 μL of Staphylococcus aureus (1.0 × 10 8 CFU mL -1 ) to establish a full-thickness Staphylococcus aureus infection wound model. The dressing was fixed with a medical elastic bandage. The wound area and body weight of the mice were observed and recorded every 2 days until the 11th day. All mice were divided into 5 groups: control group, MB before and after laser irradiation, and control group. 12 -1Solution group, MB before and after laser irradiation 12-2 solution group, with 3 replicates in each group. After the wound healing experiment, the mice were killed, and the wound and surrounding tissues were stained with H&E. The tissues collected 2 days later were homogenized with physiological saline (100 μL). The diluted aliquots of the homogenate were spread on LB medium and cultured in a constant temperature incubator at 37 °C. The colonies were counted after 15 h of culture.

[0037] 6. X-ray crystallography Single crystal X-ray diffraction measurements were performed on a Bruker D8 Venture diffractometer equipped with a PHOTON-100 CMOS detector. All calculations were performed using the SHELXTL-97 program package, and the structures were solved using the direct method and refined using full matrix least squares fitting on F2. Crystallographic data reports for supramolecular crystals were exported using Olex2 software.

[0038] 7. Quantum computing The electrostatic potential diagrams were modified by Multiwfn software and visualized by VMD software. All DFT calculations were performed at the B3LYP / 6-31G*+ level using Gaussian 09 software.

[0039] 8. Statistical analysis The collected quantitative data are expressed as mean ± standard deviation. Statistical significance was analyzed by Student's t-test. Statistical significance was inferred at p < 0.05.

[0040] 2. Results and Analysis 1. B 12 H 12 2- and MPT 2+ The crystal structure of Slowly diffuse the aqueous solution of MPT into Cs2B 12 H 12 Supramolecular crystals (MB 12 -1). Single crystal X-ray diffraction (SXRD) results revealed that B 12 H 12 2- With MPT 2+ The single crystal structure confirms that MB 12 -1 per MPT 2+ With 6 Bs 12 H 12 2- Interaction, each B 12 H 12 2- With 6 MPT 2+ Interaction. 12 H12 2- and MPT 2+ Self-assembled into MB at a molar ratio of 1:1 12 -1. Crystal data and structure refinement such as Figure 2 As shown. In the crystal arrangement, B 12 H 12 2- Evenly and symmetrically distributed in MPT 2+ The pyridine groups at both ends further revealed that B 12 H 12 2- With MPT 2+ The chaotropic effect between the two bonds contains BH…HC (the distance of dihydrogen bonds is 2.20 to 2.66 Å), and the distance of BH…N bonds is 2.68 and 2.99 Å ( Figure 2 c) and negatively charged B 12 H 12 2- With positively charged MPT 2+ The electrostatic interaction between Figure 2 d) Interestingly, in B 12 H 12 2- Surrounded by MPT, 2+ The positively charged N ions interact with MPT via cation-π interactions. 2+ The π electron cloud interaction (3.79 Å), such as Figure 2 e. MPT stacking via cation-π interaction 2+ The network can be further connected with B 12 H 12 2- The clusters are electrostatically bonded, making B 12 H 12 2- The interactions within the supramolecular crystal are maximized. All these weak intermolecular interactions make B 12 H 12 2- With MPT 2+ Tightly combined to form an orderly and stable MB 12 -1.

[0041] 2. MB 12 Preparation and characterization of -2 Because B 12 H 12 2- With MPT 2+ To solve the chaotropic effect between MPT and Cs2B, we only need to 12 H 12MB can be quickly obtained by simply mixing the aqueous solutions of the two additives. 12 -1( Figure 1 a) Subsequently, MB is photoreduced 12 MB containing free radicals was prepared 12 -2. Electron paramagnetic resonance (EPR) spectroscopy confirmed that MB 12 -2 MPT ·+ The generation of Figure 3 e) UV-Vis spectroscopy for MPT detection 2+ and MB 12 -1 Absorption changes during reduction. Figure 3 As shown in c and d, during the irradiation process, MPT 2+ Convert to MPT ·+ , absorbs at 466 nm. 12 -2, MPT ·+ The absorption peak of MB is at 637 nm. In addition, we further studied MB by transmission electron microscopy (TEM). 12 -1 and MB 12 -2 morphological features ( Figure 3 g).

[0042] As mentioned above, during PET processing, MB 12 -1 and MB 12 -2 has obvious differences in EPR and UV-Vis spectra, but no significant changes in PXRD patterns ( Figure 3 h), which indicates that the supramolecular crystals did not undergo obvious photolysis before and after UV irradiation and were able to maintain structural stability. 12 -1 and MB 12 -2 was subjected to N2 adsorption-desorption test ( Figure 4 ), the results showed that the internal porosity of the crystal was not significant and there was almost no volume accessible to the solvent, which further verified the high stability of the supramolecular crystal.

[0043] 3. MB 12 -2 Stability Thanks to MB 12 -2 crystal structure and MB 12 -1, we speculate that under the synergistic effect of the chaotropic effect and the cation-π interaction, B 12 H 12 2- Cluster and MPT ·+ The networks interpenetrate each other to form radical-doped supramolecular crystals with densely packed structures and no obvious pores, which will make MPT ·+ Has better stability.

[0044] Therefore, to evaluate the MPT·+ To ensure the stability of MB 12 -2 stored under ambient conditions (open air and laboratory light). Encouragingly, under ambient conditions, MB 12 -2 has been virtually unaffected for over a week ( Figure 5 a), indicating MB 12 -2 has good environmental stability. In addition, MB 12 -2 is quite stable in water. Figure 5 As shown in b and c, the MB in water was analyzed by UV-Vis spectroscopy. 12 -2 monitor and calculate MB 12 The half-life of MB in water is about 20.60 days. 12 The densely packed structure of -2 effectively avoids the diffusion of O2 and H2O, making MPT ·+ It is not easily bleached in air at room temperature and has excellent water stability.

[0045] 4. MB 12 -2 photothermal conversion Thanks to MB 12 -2 has a new absorption at 637 nm and excellent stability, we will MB 12 -2 is used as a photothermal material to explore its photothermal properties. Figure 5 As shown in e, under the influence of 660 nm laser (1.2 W cm -2 ) , as MB in aqueous solution 12 -2 With the increase of concentration, the temperature of the solution rises. When the concentration reaches 200 μg mL -1 The temperature can quickly reach 61°C, and the FLIR thermal imager visually verifies this ( Figure 5 d). At the same time, at the same concentration (200 μg mL -1 ) conditions, MB under different power density irradiation 12 -2 heating curve is as follows Figure 5 f. According to MB 12 -2 cooling curve, and calculated its photothermal conversion efficiency (η) to be 35.80% ( Figure 5 g). In addition, after 5 cycles of irradiation, MB 12 -2 has no significant decrease in the photothermal performance ( Figure 5 h), which indicates that MB 12 -2 has satisfactory photothermal performance and photothermal stability under 660 nm laser irradiation.

[0046] 5. In vitro antibacterial test Thanks to MB 12-2 has excellent photothermal activity, so it has a great influence on MB 12 The in vitro antibacterial ability of -2 was explored. Escherichia coli and Staphylococcus aureus were selected as models. Figure 6 ad shows different concentrations of MB 12 The antibacterial efficiency of the -2 solution after different treatments was significantly higher than that of the experimental group without laser irradiation at 660 nm (1.2 W cm -2 ) laser irradiation, the survival rates of both bacteria increased with MB 12 -2 concentration decreased significantly. When the concentration was 200 μg mL -1 When the bacterial mortality rate was as high as 97%, it showed that MB 12 -2 has strong photothermal antibacterial ability.

[0047] 6. In vivo anti-infection test Use MB 12 -1 and MB 12 -2 (200 μg mL -1 ) After 24 h of culture, the cell survival rate remained above 90% ( Figure 7 ). The results show that MB 12 -2 has good biocompatibility and low cytotoxicity.

[0048] Based on the above antimicrobial activity and cytotoxicity evaluation, MB 12 -2 solution (200 μg mL -1 ) Further wound healing experiments were performed to evaluate its photothermal antibacterial application in vivo. The back of the mouse was scratched and Staphylococcus aureus (1x10 8 CFU mL -1 ) to prepare the infection model. We set up 5 experimental groups, namely control group, MB 12 -1 laser (L+) and no laser (L-) irradiation groups, and MB 12 -2 laser and no laser irradiation groups, in which MB 12 -1 and MB 12 -2 concentration was 200 μgmL -1 .like Figure 8 As shown in a, the wound healing process of mice in different treatment groups was monitored by photography. The corresponding wound contraction schematic diagram and wound contraction calculation are shown in Figure 8 b and e. For all experimental groups, the wound size gradually decreased over time. The wounds in the first four groups healed slowly, and large wounds and obvious bacterial infection were still visible on the 11th day. In contrast, MB 12 On the 5th day, the wound surface treated with MB-2 (L+) showed obvious scar healing. 12The wound surface of MB-2 (L+) was the least open (3.2%), and the wound surface was smooth with some new epidermal and dermal tissues. 12 The wound healing effect of the -2 (L+) group was the best. In addition, we extracted the wound tissues of mice in each group on the second day after different treatments, and detected the bacteria in the wound site using the agar plate method to evaluate its bactericidal effect. Figure 8 c and d show that the control group and MB 12 -1 (L+) / (L-) group and MB 12 Many colonies were observed on the agar plates of the -2 (L-) group, while MB 12 -2 (L+) treatment did not result in colonies. This is consistent with wound healing and confirms that MB 12 -2 has excellent antibacterial properties at 660 nm (1.2 W cm -2 ) Laser irradiation can perform photothermal antibacterial treatment, thereby promoting the healing of bacterially infected wounds.

[0049] In addition, to further verify the above results, we evaluated the details of wound healing by histological changes in skin tissue. After the treatment, the rats were killed at the scheduled time and the tissues of each group were collected. These skin tissues were fixed with formalin, embedded in paraffin, sliced ​​with a microtome, and stained with hematoxylin and eosin (H&E) ( Figure 8 f) H&E staining results showed that on day 11, MB 12 -2 (L+) group had the smallest scar width; meanwhile, H&E images showed that the scar width of the control group and MB 12 -1 (L-) / (L+) group and MB 12 Many inflammatory signals (blood cells and neutrophils) appeared in the skin tissue of the -2(L-) group, while MB 12 -2 (L+) treatment resulted in fewer inflammatory cells and the detection of regenerated dermal tissue, such as hair follicles and other appendages, which confirmed that MB 12 -2 is a promising in vivo photothermal antibacterial agent.

[0050] In summary, the present invention prepares supramolecular crystals (MBs) with long-term water-stable organic free radicals and high photothermal conversion efficiency based on the chaotropic effect. 12 -2), can be used for photothermal therapy. 12 -1 crystal structure analysis shows that B 12 H 12 2- With MPT 2 + The chaotropic effect between them is not only the main non-covalent interaction of supramolecular self-assembly, but also the main factor affecting MPT 2+Therefore, under the synergistic effect of multiple non-covalent interactions, the tightly packed supramolecular crystals can maintain structural stability under light irradiation, thereby generating free radical MPT with a half-life of at least 20 days in water. ·+ It is worth noting that MB dispersed in aqueous solution 12 -2 exhibited efficient photothermal conversion performance under 660 nm laser irradiation. Both in vivo and in vitro experiments showed that MB 12 -2 has excellent photothermal antibacterial properties. The present invention provides an effective and simple method for designing water-stable organic free radicals using the chaotropic effect, and provides a new idea for the design of new photothermal antibacterial agents.

[0051] The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within the protection scope of the present application.

Claims

1. A photothermal antibacterial agent, characterized in that: The photothermal antibacterial agent is composed of a closed dodecaborane anion (B 12 H 12 2- ) and the fluorescent dye N, N'-dimethyldipyridyl-thiazolyl[5, 4-d]thiazolyl(MPT 2+ ) is formed through the chaotropic effect.

2. The photothermal antibacterial agent according to claim 1, characterized in that: In the photothermal antibacterial agent, B 12 H 12 2- and MPT 2+ Mix in a 1:1 molar ratio.

3. The method for preparing the photothermal antibacterial agent according to claim 2, characterized in that: The B 12 H 12 2- and MPT 2+ The aqueous solution is mixed evenly to obtain supramolecular crystal MB 12 -1. Then restore MB through light 12 -1 obtained supramolecular crystal MB with water stability and photothermal antibacterial activity 12 -2.

4. Use of the photothermal antibacterial agent according to any one of claims 1 to 3 for inhibiting bacteria in vitro.

5. The use according to claim 3, characterized in that: The bacteria are Escherichia coli or Staphylococcus aureus.

6. The method for inhibiting bacteria on mammalian skin using a photothermal antibacterial agent according to any one of claims 1 to 4, characterized in that: An effective dose of photothermal antibacterial agent is applied to the mammalian skin.

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