Monomolecular photothermal agent with high photothermal conversion efficiency as well as preparation method and application of monomolecular photothermal agent

Abstract

The invention relates to the technical field of photo-thermal therapy, in particular to a monomolecular photo-thermal agent with high photo-thermal conversion efficiency and a preparation method and application of the monomolecular photo-thermal agent. The preparation method comprises the following steps: S1, synthesis of a compound X1: under the protection of argon, dissolving 2, 4-dimethyl pyrrole in dichloromethane, stirring in an ice bath, adding trifluoroacetic anhydride, heating to room temperature after stirring, extracting after quenching reaction, combining organic phases, drying, filtering, concentrating under reduced pressure and purifying by silica gel column chromatography to obtain the compound X1. The prepared monomolecular photothermal agent with the high photothermal conversion efficiency is high in water solubility, has the high photothermal conversion efficiency in an aqueous solution and is high in light stability, the immune escape phenomenon possibly caused by introduction of a high polymer material is avoided, and the technical problem that an existing photothermal agent is difficult to achieve the high photothermal conversion efficiency in the aqueous solution is solved.

Description

Technical Field

[0001] The present invention relates to the technical field of photothermal therapy, and particularly relates to a single-molecule photothermal agent with high photothermal conversion efficiency, a preparation method thereof, and an application thereof. Background Art

[0002] Triple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer. Due to its insensitivity to traditional hormone therapies and targeted therapies, the effective treatment of TNBC remains a major challenge. As a selective and non-invasive cancer treatment method, photothermal therapy (PTT) has attracted much attention due to its high spatio-temporal precision characteristics. By adjusting the laser intensity, irradiation time, and concentration of the photothermal agent, PTT can precisely control heat generation. After the photothermal agent (PTAs) is delivered to the tumor tissue, PTT uses external laser irradiation on the lesion, and the generated heat can increase the temperature of the tumor site, causing non-specific damage to biological macromolecules and leading to cell death.

[0003] Currently, organic molecular photothermal agents have been widely used in photothermal therapy due to their high photothermal conversion efficiency (PCE) and easily modified structures. However, there are few single-molecule PTAs that achieve high PCE in aqueous solutions. The main reason is that most organic PTAs need to be modified through complex structural regulation to improve PCE, but this will simultaneously reduce their water solubility, resulting in disordered aggregation in the water system, which instead weakens the PCE. Therefore, it is often necessary to assemble such photothermal agents with amphiphilic polymer materials to improve their solubility in aqueous solutions, so as to better apply them to biological systems. However, long-term or repeated use of polymer materials such as polyethylene glycolylation (PEGylated) may induce the production of anti-PEG antibodies in the body, reducing the drug effectiveness and increasing the risk of adverse reactions. In contrast, single-molecule photothermal agents do not contain polymer materials and can avoid such antibody-mediated immune responses. This makes single-molecule PTAs more suitable for patient groups that are sensitive to polymers such as PEG or already have anti-PEG antibodies.

[0004] Therefore, when designing photothermal therapy strategies, especially in models of diseases that require long-term treatment or repeated drug administration, the immune response characteristics of PTAs must be fully considered. Developing high-performance single-molecule photothermal agents provides a potential solution for this. However, achieving high PCE of single-molecule PTAs in aqueous solutions still faces major challenges, mainly because most pure organic photothermal materials have poor water solubility and are prone to aggregation, affecting their functions in biological systems. Summary of the Invention

[0005] In response to the problems raised by the background technology, the purpose of the present invention is to propose a single-molecule photothermal agent with high photothermal conversion efficiency, which has high water solubility, high photothermal conversion efficiency in aqueous solution, strong photostability, and avoids the immune escape phenomenon that may be caused by the introduction of polymer materials, thereby solving the technical problem that existing photothermal agents are difficult to achieve high photothermal conversion efficiency in aqueous solution.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned single-molecule photothermal agent with high photothermal conversion efficiency. The prepared single-molecule photothermal agent has the advantages of high water solubility, strong photostability and high photothermal conversion efficiency in aqueous solution.

[0007] Another object of the present invention is to propose the use of the above-mentioned single-molecule photothermal agent with high photothermal conversion efficiency in the preparation of anti-tumor drugs for triple-negative breast cancer, and to use it to prepare anti-tumor drugs for triple-negative breast cancer, which exhibits excellent anti-cancer ability in photothermal therapy, especially photothermal therapy for triple-negative breast cancer.

[0008] The above technical objectives of the present invention are achieved through the following technical solutions: A single-molecule photothermal agent with high photothermal conversion efficiency, the structural formula of the single-molecule photothermal agent with high photothermal conversion efficiency is as follows: .

[0009] A method for preparing a single-molecule photothermal agent with high photothermal conversion efficiency comprises the following steps: Step S1, synthesis of compound X1: Under argon protection, 2,4-dimethylpyrrole was dissolved in dichloromethane, stirred in an ice bath, trifluoroacetic anhydride was added, and the mixture was heated to room temperature after stirring. The reaction was quenched and then extracted. The organic phases were combined, dried, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain compound X1. The structural formula of compound X1 is as follows: ; Step S2, synthesis of compound X2: Under argon protection, 2,4-dimethylpyrrole and compound X1 are dissolved in dichloromethane, stirred in an ice bath, phosphorus oxychloride is added, the temperature is raised to room temperature and stirred, boron trifluoride ether complex and triethylamine are added, stirring is continued, and the organic phases are combined after extraction, dried, filtered, concentrated under reduced pressure and purified by silica gel column chromatography to obtain compound X2, the structural formula of compound X2 is as follows: ; Step S3, synthesis of compound X3: phenoxazine, 4-bromobenzaldehyde, palladium acetate, tri-tert-butylphosphine tetrafluoroborate and cesium carbonate are dissolved in toluene, heated and stirred for reaction, the reaction solution is cooled to room temperature, toluene is removed under reduced pressure, the residue is extracted with dichloromethane, the organic phases are combined, dried, concentrated and purified by silica gel column chromatography to obtain compound X3, the structural formula of compound X3 is as follows: ; Step S4: Dissolve compound X2 and compound X3 in toluene, add glacial acetic acid and piperidine, heat under nitrogen protection, monitor the reaction by thin-layer chromatography, remove the solvent under reduced pressure, extract the crude product, and after drying, concentrating and purifying by silica gel column chromatography for the organic phase, the single-molecule photothermal agent with high photothermal conversion efficiency is obtained.

[0010] Further explanation: In the said step S1, the molar ratio of 2,4-dimethylpyrrole to trifluoroacetic anhydride is 1:(1-2); in the said step S2, the molar ratio of compound X1 to 2,4-dimethylpyrrole is 1:(1-2); in the said step S2, the molar ratio of boron trifluoride diethyl ether complex to triethylamine is 1.1:(1-2); in the said step S3, the molar ratio of phenoxazine to 4-bromobenzaldehyde is 1:(1-2); in the said step S3, the reaction temperature for the heating and stirring reaction is 90°C - 120°C, and the reaction time is 18h - 30h; in the said step S4, the molar ratio of compound X2 to compound X3 is 1:(4-6), and the reaction temperature for heating under nitrogen protection is 60°C - 100°C; in the said step S4, the volume ratio of glacial acetic acid to piperidine is 1:(1-2.5).

[0011] Application of the said single-molecule photothermal agent with high photothermal conversion efficiency in the preparation of anti-tumor drugs for triple-negative breast cancer.

[0012] Compared with the prior art, the embodiments of the present invention have the following beneficial effects: The above-mentioned single-molecule photothermal agent with high photothermal conversion efficiency has high water solubility, high photothermal conversion efficiency in aqueous solution, strong light stability, avoids the immune escape phenomenon that may be caused by introducing polymer materials, and solves the technical problem that it is difficult for existing photothermal agents to achieve high photothermal conversion efficiency in aqueous solution. Description of the Drawings

[0013] Figure 1 It is the molecular structure diagram of the twisted state and extended state of the single-molecule photothermal agent with high photothermal conversion efficiency of the present invention.

[0014] Figure 2 It is the synthesis route diagram of the single-molecule photothermal agent with high photothermal conversion efficiency of the present invention.

[0015] Figure 3 It is of compound X1 in deuterated chloroform in Example 1 1 1H NMR spectrum (500 MHz).

[0016] Figure 4 It is of compound X1 in deuterated chloroform in Example 1 13 13C NMR spectrum (126 MHz).

[0017] Figure 51H NMR spectrum (500 MHz) of Compound X2 of Example 1 in deuterated chloroform. 1 1H NMR spectrum (500 MHz) of Compound X2 of Example 1 in deuterated chloroform.

[0018] Figure 6 13C NMR spectrum (126 MHz) of Compound X2 of Example 1 in deuterated chloroform. 13 13C NMR spectrum (126 MHz) of Compound X2 of Example 1 in deuterated chloroform.

[0019] Figure 7 1H NMR spectrum (500 MHz) of Compound X3 of Example 1 in deuterated chloroform. 1 1H NMR spectrum (500 MHz) of Compound X3 of Example 1 in deuterated chloroform.

[0020] Figure 8 13C NMR spectrum (126 MHz) of Compound X3 of Example 1 in deuterated chloroform. 13 13C NMR spectrum (126 MHz) of Compound X3 of Example 1 in deuterated chloroform.

[0021] Figure 9 1H NMR spectrum (500 MHz) of BDP1-PNZ of Example 1 in deuterated chloroform. 1 1H NMR spectrum (500 MHz) of BDP1-PNZ of Example 1 in deuterated chloroform.

[0022] Figure 10 13C NMR spectrum (126 MHz) of BDP1-PNZ of Example 1 in DMSO-d6. 13 13C NMR spectrum (126 MHz) of BDP1-PNZ of Example 1 in DMSO-d6.

[0023] Figure 11 UV-Vis absorption spectra and fluorescence spectra of BDP1-PNZ (concentration 10 μM) of Example 1 in different solvents, Figure 11 where (A) in is the UV-Vis absorption spectrum, Figure 11 and (B) in is the fluorescence spectrum.

[0024] Figure 12 UV-Vis absorption spectrum and fluorescence spectrum of BDP1-PNZ (concentration 10 μM) of Example 1 in dimethyl sulfoxide.

[0025] Figure 13 Absorption spectra of ABDA (used as 1 O2 probe) at different irradiation times (0 - 6 minutes) in the presence of BDP1-PNZ of Example 1.

[0026] Figure 14 Infrared thermal imaging diagrams of BDP1-NEt2 of Comparative Example 1 and BDP1-PNZ of Example 1 under laser irradiation, Figure 14 where (A) in is the infrared thermal imaging diagram of BDP1-NEt2 of Comparative Example 1 and BDP1-PNZ (10 μM) aqueous solution under laser irradiation with different laser power densities at 808 / 716 nm; Figure 14In (B), the infrared thermal imaging diagrams of BDP1-NEt2 in Comparative Example 1 with different concentrations and BDP1-PNZ in Example 1 under laser irradiation with a laser power density of 0.5 W / cm 2 are shown.

[0027] Figure 15 is the temperature curve diagram of the aqueous solutions of BDP1-NEt2 in Comparative Example 1 and BDP1-PNZ in Example 1 under laser irradiation with different laser power densities at 808 / 716 nm (concentration = 10 μM). Figure 15 In (A), the temperature curve diagram of BDP1-NEt2 in Comparative Example 1. Figure 15 In (B), the temperature curve diagram of BDP1-PNZ in Example 1.

[0028] Figure 16 is the temperature curve diagram of the aqueous solutions of BDP1-NEt2 in Comparative Example 1 and BDP1-PNZ in Example 1 at different concentrations under laser irradiation at 808 / 716 nm (0.5 W / cm 2 ). Figure 16 In (A), the temperature curve diagram of BDP1-NEt2 in Comparative Example 1. Figure 16 In (B), the temperature curve diagram of BDP1-PNZ in Example 1.

[0029] Figure 17 is the temperature change diagram of BDP1-NEt2 in Comparative Example 1 and BDP1-PNZ in Example 1. Figure 17 In (A), it is the temperature change curve diagram of the aqueous solutions of BDP1-NEt2 in Comparative Example 1 and BDP1-PNZ in Example 1 under laser irradiation with different laser power densities at 808 / 716 nm (concentration = 10 μM); Figure 17 In (B), it is the temperature change diagram of the aqueous solutions of BDP1-NEt2 in Comparative Example 1 and BDP1-PNZ in Example 1 at different concentrations under laser irradiation at 808 / 716 nm (0.5 W / cm 2 ). (The concentration gradient changes, irradiation time: 600 seconds).

[0030] Figure 18 is the photothermal effect diagram of BDP1-PNZ in Example 1 and BDP1-NEt2 in Comparative Example 1. Figure 18 In (A), it is the photothermal effect diagram of BDP1-PNZ in Example 1. Figure 18 In (B), it is the photothermal effect diagram of BDP1-NEt2 in Comparative Example 1, where η represents the photothermal conversion efficiency.

[0031] Figure 19It is a graph showing the relationship between the cooling time corresponding to the temperature in the cooling stage of BDP1-PNZ in Example 1 and BDP1-NEt2 in Comparative Example 1 and the negative natural logarithm (-ln(θ)). Figure 19 In (A) of [Figure], it is a graph showing the relationship between the cooling time corresponding to the temperature in the cooling stage of BDP1-PNZ in Example 1 and the negative natural logarithm (-ln(θ)). Figure 19 In (B) of [Figure], it is a graph showing the relationship between the cooling time corresponding to the temperature in the cooling stage of BDP1-NEt2 in Comparative Example 1 and the negative natural logarithm (-ln(θ)).

[0032] Figure 20 It is the infrared thermal images of BDP1-NEt2 in Comparative Example 1 and BDP1-PNZ in Example 1. Figure 20 In (A) of [Figure], it is the infrared thermal image of BDP1-NEt2 in Comparative Example 1 with a concentration of 10 μM. Figure 20 In (B) of [Figure], it is the infrared thermal image of BDP1-PNZ in Example 1 with a concentration of 10 μM, which shows the temperature changes of several heating / cooling cycles after irradiation with a laser of 808 / 716 nm (0.5 W / cm 2 ) for 30 minutes.

[0033] Figure 21 It is a comparison graph of the dissolution of BDP1-NEt2 (10 μM) in Comparative Example 1 and BDP1-PNZ (10 μM) in Example 1 in aqueous solution after irradiation with a laser of 808 / 716 nm (0.5 W / cm 2 ) for 30 minutes.

[0034] Figure 22 It is a graph showing the change in absorption intensity of BDP1-PNZ (716 nm) and BDP1-NEt2 (808 nm) at different concentrations in aqueous solution. Figure 22 In (A) of [Figure], it is a graph showing the change in absorption intensity of BDP1-PNZ. Figure 22 In (B) of [Figure], it is a graph showing the change in absorption intensity of BDP1-NEt2. When the concentration of BDP1-PNZ > 61.33 μM, the absorption peak redshifts (transition from monomer to aggregate); a similar phenomenon occurs when the concentration of BDP1-NEt2 > 17.86 μM.

[0035] Figure 23 It is a graph showing the detection results of the dark cytotoxicity of BDP1-PNZ in Example 1 detected by the CCK-8 method. The survival rate of 4T1 cells treated with different concentrations of BDP1-PNZ (0 - 50 μM) for 36 hours. The survival rate of the untreated group of cells was defined as 100%. The results are expressed as the mean ± standard deviation of five independent measurements.

[0036] Figure 24is a comparison chart of the survival rates of 4T1 cells after laser irradiation with BDP1-NEt2 (0.5 W / cm 2 , 10 minutes) of Comparative Example 1 at different concentrations and BDP1-PNZ (0.5 W / cm 2 , 10 minutes) of Example 1. Figure 24 In (A) of , it is a comparison chart of the survival rates of 4T1 cells after laser irradiation with BDP1-NEt2 (0.5 W / cm 2 , 10 minutes) of Comparative Example 1 at different concentrations. Figure 24 In (B) of , it is a comparison chart of the survival rates of 4T1 cells after laser irradiation with BDP1-PNZ (0.5 W / cm 2 , 10 minutes) of Example 1 at different concentrations. Experiments were conducted by setting up an unirradiated group and an irradiated group. In the figure, IR(-) refers to the unirradiated group, and IR(+) refers to the irradiated group.

[0037] Figure 25 is a test chart of the photocytotoxicity of BDP1-PNZ (10 μM) of Example 1. Among them, after 4T1 cells were treated (or not treated) with BDP1-PNZ, they were exposed to different laser powers (irradiated with 716 nm laser for 3 minutes). The error bars represent the standard deviation of each group (sample size n = 5), and the results are expressed as mean ± standard deviation (sample size n = 3). Analysis of significant differences (N.S.: no significant difference; ***P < 0.001).

[0038] Figure 26 is a chart for evaluating the photothermal therapy effect of BDP1-PNZ through a staining experiment. Figure 26 In (A) of , it is a microscopic image (under bright field) of 4T1 cells cultured with BDP1-NEt2 (10 μM) of Comparative Example 1 after 808 nm laser irradiation and without laser irradiation. Figure 26 In (B) of , it is a microscopic image (stained with Calcein-AM / PI) of 4T1 cells cultured with BDP1-NEt2 (10 μM) of Comparative Example 1 after 808 nm laser irradiation and without laser irradiation. Figure 26 In (C) of , it is a microscopic image (under bright field) of 4T1 cells cultured with BDP1-PNZ (10 μM) of Example 1 after 716 nm laser irradiation and without laser irradiation. Figure 26 In (D) of , it is a microscopic image (stained with Calcein-AM / PI) of 4T1 cells cultured with BDP1-PNZ (10 μM) of Example 1 after 716 nm laser irradiation and without laser irradiation.

[0039] Figure 27 is a detection chart of the ability of BDP1-PNZ to produce reactive oxygen species at the cellular level. Figure 27in (A), Figure 27 in (B), and Figure 27 in (C) are the microscopic images of the control group, the BDP1-PNZ dark-light group, and the BDP1-PNZ light-exposed group under bright field, respectively; Figure 27 in (D), Figure 27 in (E), and Figure 27 in (F) are the microscopic images of the control group, the BDP1-PNZ dark-light group, and the BDP1-PNZ light-exposed group under fluorescence field, respectively; Figure 27 in (G), Figure 27 in (H), and Figure 27 in (I) are the microscopic images of the control group, the BDP1-PNZ dark-light group, and the BDP1-PNZ light-exposed group under superposition field, respectively.

[0040] Figure 28 is Figure 27 the quantitative analysis chart of the fluorescence intensity in, where the data are expressed as mean ± standard deviation (sample size n = 3), and the significant difference analysis (N.S.: no significant difference; ***P < 0.001).

[0041] Figure 29 is the photoacoustic imaging chart of the tumor at the specified time after the BDP1-PNZ and ICG treatments in Example 1, Figure 29 in (A), Figure 29 in (B), Figure 29 in (C), Figure 29 in (D), Figure 29 in (E), and Figure 29 in (F) are the photoacoustic imaging charts of the tumor at 0 h, 1 h, 3 h, 6 h, 9 h, and 12 h after the BDP1-PNZ treatment in Example 1, Figure 29 in (a), Figure 29 in (b), Figure 29 in (c), Figure 29 in (d), Figure 29 in (e), and Figure 29 in (f) are the photoacoustic imaging charts of the tumor at 0 h, 1 h, 3 h, 6 h, 9 h, and 12 h after the ICG treatment, where only part of the tumor area is shown in the images.

[0042] Figure 30 is for Figure 29 the quantitative analysis chart of the photoacoustic intensity in.

[0043] Figure 31 is the infrared thermal imaging chart of 4T1 tumor-bearing mice after being irradiated with 716 nm laser (0.5 W / cm 2 ) Figure 31 in (A), Figure 31 in (B),Figure 31 in (C) and Figure 31 in (D) are infrared thermal imaging diagrams at 0 h, 1 min, 2 min, and 3 min after injection of BDP1-PNZ in Example 1, Figure 31 in (a), Figure 31 in (b), Figure 31 in (c) and Figure 31 in (d) are infrared thermal imaging diagrams at 0 h, 1 min, 2 min, and 3 min after injection of PBS.

[0044] Figure 32 is a graph of the in-vivo temperature change at the tumor site during the laser irradiation time after injection of BDP1-PNZ in Example 1 and injection of PBS.

[0045] Figure 33 is a graph of the tumor size and appearance when taking pictures of the tumor on the 14th day in the in-vivo phototherapy experiment.

[0046] Figure 34 is an analysis diagram of the in-vivo phototherapy experiment, Figure 34 in (A) is a graph of the change in tumor volume over time for mice in different groups (mean ± standard deviation, sample size n = 5), Figure 34 in (B) is a graph of the average weight of tumors of mice in different groups (mean ± standard deviation, sample size n = 5), Figure 34 in (C) is a graph of the change in body weight of mice in different groups during the treatment period.

[0047] Figure 35 are hematoxylin and eosin (H&E) staining images of sections of the heart, liver, spleen, lungs, and kidneys of 4T1 tumor-bearing mice in different treatment groups, with a scale of 100 microns.

[0048] Figure 36 is an analysis diagram of the blood of mice in different treatment groups, Figure 36 in (A) is an analysis diagram of the hemolysis rate of the photothermal agent BDP1-PNZ at concentrations of 1 to 100 μM on mouse red blood cells (PBS as the negative control and water as the positive control); Figure 36 in (B) is a graph of the biochemical indexes of liver function of mice in different treatment groups (mean ± standard deviation, sample size n = 5); Figure 36 in (C) is a graph of the biochemical indexes of kidney function of mice in different treatment groups (mean ± standard deviation, sample size n = 5). Detailed implementation mode

[0049] A single-molecule photothermal agent with high photothermal conversion efficiency, and the structural formula of the single-molecule photothermal agent with high photothermal conversion efficiency is as follows: .

[0050] To address the technical problems in the background art, single-molecule PTAs need to meet the following key biomedical and photophysical properties: (1) good solubility in physiological environments; (2) excellent photostability under light irradiation to maintain photothermal performance; (3) high PCE in aqueous solutions. Among numerous organic dyes, boron dipyrromethene (BODIPY) has been widely used in light-mediated technologies (such as photocontrolled release, photoacoustic imaging, and phototherapy) due to its high molar extinction coefficient, anti-photobleaching property, and environmental insensitivity. However, due to their inherent hydrophobicity and rigid structures, BODIPY and its analogs are prone to forming a large number of ineffective aggregates and precipitates in aqueous solutions, greatly limiting their practical applications in photothermal therapy under physiological conditions. Therefore, there is an urgent need to develop novel high-performance single-molecule BODIPY derivatives. To improve the PCE in aqueous solutions, the key is to improve the molecular water solubility, and at the same time, the ability of intramolecular non-radiative transitions needs to be significantly enhanced (the key to improving PCE lies in increasing the non-radiative transition process). Dark electron transfer states are often accompanied by enhanced non-radiative processes. These states are usually restricted by symmetry or selection rules, making it difficult for electrons to return to the ground state through luminescence (radiative transitions). Dark electron transfer states are usually related to non-radiative transitions (such as internal conversion and intersystem crossing), which release energy in the form of heat rather than photons. Therefore, effectively inducing the formation of dark electron transfer states in photothermal agents can significantly contribute to the enhancement of photoacoustic signals. The photoinduced electron transfer (PET) effect can greatly promote the formation of dark electron transfer states.

[0051] In the present invention, a phenoxazine (PNZ) group is introduced into BODIPY to construct a single-molecule photothermal agent (BDP1-PNZ) with high photothermal conversion efficiency of the present invention, realizing the PET process between PNZ and BODIPY. The central CF3 group and adjacent methyl groups in the BDP1-PNZ structure (inducing the bending of the molecular backbone to promote non-radiative transitions), and a large-volume electron-donating group phenoxazine (PNZ) is introduced to form a novel single-molecule photothermal agent (BDP1-PNZ). The introduction of the PNZ group enhances the π-π stacking interaction within the BDP1-PNZ molecule, changing its conformation from an extended state to a twisted state. This twisted three-dimensional conformation can limit molecular aggregation, thereby reducing the precipitation tendency of BDP1-PNZ, and then significantly improving its solubility in aqueous solutions. Also, due to the conformational change, the non-radiative transition process is greatly enhanced to release heat. Figure 1Shows the twisted and extended states of the BDP1-PNZ molecular structure. In addition, the introduction of the PNZ electron-donating group induces photoinduced electron transfer (PET) during photoexcitation, significantly promoting the formation of the lower-lying dark electron transfer state in the excited state, and the effective formation of such dark states is the basis for the enhanced photothermal ability of BDP1-PNZ. The high efficiency of BDP1-PNZ stems from the introduction of the PNZ group, which can be specifically attributed to three core factors: (1) The introduction of the PNZ group generates steric hindrance due to the bending of the fluorophore scaffold, further exacerbating the overall molecular twist (the steric effect generated by the -CF3 group introduced in the middle position and two adjacent methyl groups, which results in an obvious bend of the BODIPY skeleton in both the ground state and the excited state); (2) The significant transition from the twisted conformation to the extended conformation in the excited state, and this conformational change and the related internal conversion process are crucial for the photothermal efficiency of BDP1-PNZ; (3) The PNZ group, as a bulky electron-donating group, can activate the photoinduced electron transfer (PET) ability of BDP1-PNZ under photoexcitation, thereby forming the lower-lying dark electron transfer state. The effective formation of these lower-lying dark electron transfer states is the basis for the enhanced photothermal performance of BDP1-PNZ, and the formation process also enhances the photostability of the photothermal agent.

[0052] The single-molecule photothermal agent (BDP1-PNZ) of the present invention has high water solubility, and almost no fluorescence emission or singlet oxygen generation, and the PCE is greatly improved. The introduction of PNZ enhances the π-π interaction within the BDP1-PNZ molecule, resulting in the coexistence of twisted and extended conformations, and the conformational transformation also promotes the generation of non-radiative transitions. The high photothermal conversion efficiency stems from the extremely low energy of this PET state and the small energy gap between it and the S0 state (the S0 state refers to the singlet state of the electronic ground state of the molecule). According to the Energy Gap Law, this condition strengthens the non-radiative transition rate, which brings two advantages: (a) The shorter the excited state duration, the higher the molecular stability; (b) The accelerated de-excitation rate (the de-excitation rate refers to the rate at which the excited state returns from the high-energy state to the ground state or other low-energy states) can improve the photothermal efficiency because each "light absorption - heat release" cycle period is shortened, and the molecule can complete more cycles per unit time, thereby enhancing the heating efficiency. The single-molecule photothermal agent of the present invention with high photothermal conversion efficiency has high water solubility, high photothermal conversion efficiency in aqueous solution, and strong photostability. It not only avoids the immune escape phenomenon that may be caused by the introduction of polymer materials, but also maintains high photothermal conversion efficiency due to the improved solubility and enhanced non-radiative transition brought by the PNZ group, solving the technical problem that it is difficult for existing photothermal agents to achieve high photothermal conversion efficiency in aqueous solution.

[0053] A preparation method of a single-molecule photothermal agent with high photothermal conversion efficiency, comprising the following steps: Step S1, synthesis of compound X1: Under argon protection, 2,4-dimethylpyrrole was dissolved in dichloromethane, stirred in an ice bath, trifluoroacetic anhydride was added, and the mixture was heated to room temperature after stirring. The reaction was quenched and then extracted. The organic phases were combined, dried, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain compound X1. The structural formula of compound X1 is as follows: ; Step S2, synthesis of compound X2: Under argon protection, 2,4-dimethylpyrrole and compound X1 are dissolved in dichloromethane, stirred in an ice bath, phosphorus oxychloride is added, the temperature is raised to room temperature and stirred, boron trifluoride ether complex and triethylamine are added, stirring is continued, and the organic phases are combined after extraction, dried, filtered, concentrated under reduced pressure and purified by silica gel column chromatography to obtain compound X2, the structural formula of compound X2 is as follows: ; Step S3, synthesis of compound X3: phenoxazine, 4-bromobenzaldehyde, palladium acetate, tri-tert-butylphosphine tetrafluoroborate and cesium carbonate are dissolved in toluene, heated and stirred for reaction, the reaction solution is cooled to room temperature, toluene is removed under reduced pressure, the residue is extracted with dichloromethane, the organic phases are combined, dried, concentrated and purified by silica gel column chromatography to obtain compound X3, the structural formula of compound X3 is as follows: ; Step S4, dissolving compound X2 and compound X3 in toluene, adding glacial acetic acid and piperidine, heating under nitrogen protection, monitoring the reaction by thin layer chromatography, removing the solvent under reduced pressure, extracting the crude product, and drying, concentrating and purifying the organic phase by silica gel column chromatography to obtain the single-molecule photothermal agent with high photothermal conversion efficiency.

[0054] The synthetic route of BDP1-PNZ is as follows Figure 2 As shown: meso-trifluoromethyl BODIPY (compound X2) and phenoxazine derivatives (compound X3) were prepared, and then BDP1-PNZ was synthesized by Knoevenagel condensation reaction of compound X2 and compound X3 using glacial acetic acid and piperidine as catalysts. The single-molecule photothermal agent prepared by this preparation method has the advantages of high water solubility, strong photostability and high photothermal conversion efficiency in aqueous solution.

[0055] Further illustration, in the step S1, the molar ratio of 2,4-dimethylpyrrole to trifluoroacetic anhydride is 1:(1-2), preferably 1:1; in the step S2, the molar ratio of compound X1 to 2,4-dimethylpyrrole is 1:(1-2), preferably 1:1.5; in the step S2, the molar ratio of boron trifluoride diethyl ether complex to triethylamine is 1.1:(1-2), preferably 1.1:1; in the step S3, the molar ratio of phenoxazine to 4-bromobenzaldehyde is 1:(1-2), preferably 1:1.1; in the step S3, the reaction temperature of the heating and stirring reaction is 90°C to 120°C, and the reaction time is 18 h to 30 h. The preferred reaction temperature is 110°C, and the preferred reaction time is 24 h; in the step S4, the molar ratio of compound X2 to compound X3 is 1:(4-6), preferably 1:4; the reaction temperature for heating under nitrogen protection is 60°C to 100°C, preferably 80°C; in the step S4, the volume ratio of glacial acetic acid to piperidine is 1:(1-2.5), preferably 1:1.

[0056] Application of the single-molecule photothermal agent with high photothermal conversion efficiency in the preparation of anti-tumor drugs for triple-negative breast cancer. The single-molecule photothermal agent with high photothermal conversion efficiency of the present invention has the advantages of high water solubility, strong photostability and high photothermal conversion efficiency in aqueous solution, exhibits extremely high molar extinction coefficients in the near-infrared region, can realize dual-modal photoacoustic-photothermal imaging of in vivo tumors, and is used in the preparation of anti-tumor drugs for triple-negative breast cancer, showing excellent anti-cancer ability in photothermal therapy, especially in photothermal therapy for triple-negative breast cancer.

[0057] For those not specifying specific technologies or conditions in the examples, they shall be carried out according to the technologies or conditions described in the literature in this field or according to the product specifications. For the reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0058] Raw materials: The raw materials in the following examples can all be obtained commercially. Among them, 2,4-dimethylpyrrole, trifluoroacetic anhydride, boron trifluoride diethyl ether complex (BF3·OEt2), phenoxazine, 4-bromobenzaldehyde, 9,10-anthracenediyl-bis(methylene)dicarboxylic acid (ABDA), indocyanine green (ICG), and 2,7-dichlorofluorescein diacetate (DCFH-DA) are all purchased from Sigma-Aldrich.

[0059] Experimental instruments: Fluorescence spectra and ultraviolet-visible absorption spectra are respectively recorded by a Shimadzu RF-5301PC fluorescence spectrophotometer and an Agilent 8453 spectrophotometer. 1 H and 13 1H and 13C nuclear magnetic resonance (NMR) spectra are collected by a Bruker 500 MHz nuclear magnetic resonance spectrometer. Thermal imaging and videos are recorded by a FLIR E60 infrared thermal imager.

[0060] Preparation of stock solution for solution testing: Dissolve BDP1-NEt2 and BDP1-PNZ used for solution testing in DMSO to prepare a test solution with the required concentration. In in vitro testing, BDP1-NEt2 and BDP1-PNZ are dissolved in DMSO to prepare a 10 mM stock solution, and further diluted to the required concentration with a solution containing 0.1% Tween® 80.

[0061] Statistical analysis: The data was analyzed by one-way ANOVA using SPSS 21.0, and the significance of differences between groups was evaluated by Tukey's post hoc test. P < 0.05 was considered a statistically significant difference.

[0062] Example 1 A preparation method of a single-molecule photothermal agent with high photothermal conversion efficiency is prepared by the following steps: Step S1, synthesis of compound X1: Under argon protection, dissolve 2,4-dimethylpyrrole (1 mL, 9.71 mmol) in dry dichloromethane (15 mL), stir in an ice bath (0 °C), slowly add trifluoroacetic anhydride (1.5 mL, 10.68 mmol), stir the reaction solution at 0 °C for 30 minutes and then raise the temperature to room temperature, continue stirring for 3 hours, quench the reaction with saturated sodium bicarbonate solution, extract with dichloromethane, combine the organic phases, dry over anhydrous sodium sulfate, filter, concentrate under reduced pressure, and purify by silica gel column chromatography (mobile phase gradient: hexane / ethyl acetate = 20 / 1, volume ratio) to obtain compound X1 (white solid, yield 81%). The structural formula of the obtained compound X1 is as follows: . The chemical structure of compound X1 was characterized by 1 1H NMR (proton nuclear magnetic resonance) and 13 13C NMR (carbon-13 nuclear magnetic resonance) spectra.

[0063] As Figure 3 and Figure 4 shown: 1 1H NMR (500 MHz, CDCl3) δ 9.25 (s, 1H), 5.97 (s, 1H), 2.36 (s, 3H), 2.32 (s, 3H); 13 13C NMR (126 MHz, CDCl3) δ 139.79, 120.83, 118.54, 116.25, 115.01, 113.95, 13.69, 13.45.

[0064] Step S2, synthesis of compound X2: Under argon protection, 2,4-dimethylpyrrole (250 μL, 2.43 mmol) and compound X1 (300 mg, 1.62 mmol) were dissolved in dry dichloromethane (5 mL), stirred in an ice bath (0 ° C), and phosphorus oxychloride (220 μL, 1.62 mmol) was added. The reaction solution was warmed to room temperature and stirred for 4 hours, and the solution changed from colorless to red. Subsequently, boron trifluoride ether complex (0.98 mL, 7.89 mmol) and triethylamine (1 mL, 7.17 mmol) were added and continued to stir for 10 minutes. After extraction with dichloromethane, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (mobile phase: n-hexane / ethyl acetate = 20 / 1, volume ratio) to obtain compound X2 (red solid, yield 31%). The structural formula of the prepared compound X2 is as follows: The chemical structure of compound X2 is 1 H NMR (hydrogen nuclear magnetic resonance) and 13 CNMR (Carbon-13 Nuclear Magnetic Resonance) spectroscopy characterization, such as Figure 5 and Figure 6 As shown: 1 H NMR (500 MHz, CDCl3) δ6.15 (s, 2H), 2.54 (s, 6H), 2.30 (d, J = 3.2 Hz, 6H); 13 C NMR (126MHz, CDCl3) δ158.97, 143.27, 131.42,124.54, 123.57, 121.38, 15.99, 15.15.

[0065] Step S3, synthesis of compound X3: Phenoxazine (549.0 mg, 3 mmol), 4-bromobenzaldehyde (617 mg, 3.38 mmol), palladium acetate (56 mg, 0.25 mmol), tri-tert-butylphosphine tetrafluoroborate (217 mg, 0.75 mmol) and cesium carbonate (2.44 g, 14.6 mmol) were dissolved in toluene (50 mL), heated and stirred at 110° C. for 24 hours, the reaction solution was cooled to room temperature, toluene was removed under reduced pressure, the residue was extracted with dichloromethane, the organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography (mobile phase: n-hexane / dichloromethane = 10 / 1, volume ratio) to obtain compound X3 (orange crystals, yield 45%). The structural formula of the prepared compound X3 is as follows: The chemical structure of compound X3 is shown by 1 H NMR (hydrogen nuclear magnetic resonance) and 13 C NMR (carbon-13 nuclear magnetic resonance) spectroscopy characterization, such as Figure 7 andFigure 8 as shown below 1 H NMR (500 MHz, CDCl3) δ 10.11 (s, 1H), 8.12 (d, J = 8.1 Hz, 2H), 7.56 (d, J = 8.2 Hz, 2H), 6.78 - 6.68 (m, 4H), 6.62 (t, J = 7.1 Hz, 2H), 5.97 (d, J = 7.5 Hz, 2H). 13 C NMR (126 MHz, CDCl3) δ 191.08, 145.18, 144.25, 136.04, 133.65, 132.41, 131.57, 123.45, 122.20, 115.97, 113.59.

[0066] Step S4: Dissolve compound X2 (100 mg, 0.3125 mmol) and compound X3 (359 mg, 1.25 mmol) in toluene (10 mL), add glacial acetic acid (0.1 mL) and piperidine (0.1 mL), heat at 80 °C under nitrogen protection, monitor the reaction by thin layer chromatography (TLC) until the main product spot turns green, then stop the reaction, remove the solvent under reduced pressure, extract the crude product with water and dichloromethane, dry the organic phase over anhydrous sodium sulfate, concentrate, and purify by silica gel column chromatography (mobile phase: n - hexane / dichloromethane = 4 / 1, v / v) to obtain the single - molecule photothermal agent BDP1 - PNZ with high photothermal conversion efficiency (dark green powder, yield 51%). The chemical structure of BDP1 - PNZ was characterized by 1 1H NMR (proton nuclear magnetic resonance) and 13 13C NMR (carbon - 13 nuclear magnetic resonance) spectra, as Figure 9 and Figure 10 shown below 1 H NMR (500 MHz, CDCl3) δ 7.84 (d, J = 8.4 Hz, 4H), 7.79 (d, J = 16.3 Hz, 2H), 7.43–7.36 (m, 6H), 6.88 (s, 2H), 6.67 (dtd, J = 9.2, 7.8, 1.6 Hz, 8H), 6.60 (td, J = 7.6, 1.7 Hz, 4H), 5.99 (dd, J = 7.9, 1.5 Hz, 4H), 2.41 (s, 6H). 1313C NMR (126 MHz, CDCl3) δ 154.57, 144.15, 142.11, 140.23, 137.32, 136.51, 134.21, 131.47, 130.40, 123.44, 121.72, 121.19, 120.25, 115.72, 113.44, 53.56, 31.74, 22.81, 16.11, 14.26. The structural formula of the prepared BDP1-PNZ is as follows: 。

[0067] Comparative Example 1 Before synthesizing the single-molecule photothermal agent (BDP1-PNZ) with high photothermal conversion efficiency of the present invention, the applicant studied and synthesized a photoacoustic imaging agent (BDP1-NEt2). BDP1-NEt2 has poor solubility in aqueous solution and is prone to ineffective aggregation and precipitation, resulting in an unsatisfactory photothermal conversion efficiency (PCE) under physiological conditions. The following partial tests will compare BDP1-NEt2 with BDP1-PNZ.

[0068] The preparation method of BDP1-NEt2 is as follows (the preparation method of compound X2 adopts the preparation method of compound X2 in Example 1): Dissolve compound X2 (80 mg, 0.25 mmol) and 4-diethylaminobenzaldehyde (221 mg, 1.25 mmol) in dry toluene (10 mL). Add glacial acetic acid (0.1 mL) and piperidine (0.1 mL) to the above solution. Heat the mixture at 80 °C under a nitrogen atmosphere and monitor the progress of the reaction by TLC. The reaction time is 8 hours. Remove the solvent under reduced pressure, and extract the crude product with water and dichloromethane. The organic layer is dried over anhydrous Na2SO4, filtered, and the solvent is removed. The crude product is purified by silica gel column chromatography using hexane / dichloromethane (7 / 3, v / v) as the mobile phase to obtain BDP1-NEt2 (black powder, yield 51%). 1 1H NMR (500 MHz, CDCl3) δ 7.53 (t, J = 13.3 Hz, 6H), 7.24 (d, J = 16.2 Hz, 2H), 6.77 (s, 2H), 6.67 (d, J = 8.9 Hz, 4H), 3.42 (q, J = 7.0 Hz, 8H), 2.33 (d, J = 2.8 Hz, 6H), 1.21 (t, J = 7.1 Hz, 12H). 1313C NMR (126 MHz, DMSO) δ 153.52, 149.05, 139.70, 138.42, 132.47, 129.8, 123.15, 120.99, 112.44, 111.72, 43.96, 15.32, 12.52. High-resolution mass spectrometry (HRMS) combined with matrix-assisted laser desorption ionization (MALDI) was used for molecular weight determination. HRMS (MALDI), calcd for (C36H41BF5N4 + ): m / z [M]+ : 635.334; found: m / z 635.332. The structural formula of the prepared BDP1-NEt2 is as follows: 。

[0069] The single-molecule photothermal agent BDP1-PNZ with high photothermal conversion efficiency prepared in Example 1 was subjected to photophysical property tests, in vitro photothermal performance tests, and in vivo photothermal performance tests. The test process and test results are as follows: 1. Photophysical property tests.

[0070] (1) The absorption and fluorescence spectra of BDP1-PNZ in acetone, acetonitrile (ACN), dichloromethane (DCM), dioxane, dimethyl sulfoxide (DMSO), methanol, tetrahydrofuran (THF), and toluene were measured.

[0071] As Figure 11 shown in (A) below, BDP1-PNZ showed stable absorption spectra in different polar solvents, and its molar extinction coefficient at 716 nm in aqueous solution was 6.48×10 4 L·mol -1 ·cm -1 , indicating its strong light absorption ability. As Figure 11 shown in (B) below and Figure 12 , BDP1-PNZ showed almost no fluorescence emission under excitation at the corresponding wavelengths. Especially in aqueous solution, its fluorescence quantum yield was less than 0.1%, indicating that the absorbed energy was almost completely dissipated through non-radiative decay rather than fluorescence emission.

[0072] The method for measuring the fluorescence quantum yield is as follows: Using indocyanine green (ICG, Φs = 0.132, ethanol, 298 K) as a reference, the relative fluorescence quantum yield was calculated by the following formula: ; Φ is the quantum yield, F is the integrated area of the corrected emission spectrum, A is the absorbance at the excitation wavelength, λex is the excitation wavelength, where the subscripts x and s represent the sample to be measured and the reference respectively, and n is the refractive index of the solvent. When the solution concentration is low (10 -6 -10 -7 mol / L), the change in refractive index can be ignored.

[0073] (2) Test the ability of BDP1-PNZ to generate singlet oxygen ( 1 O2) in aqueous solution.

[0074] The singlet oxygen generation ability of BDP1-PNZ was detected using the singlet oxygen scavenger 9,10-anthracenediyl-bis(methylene)dicarboxylic acid (ABDA) as an indicator. Briefly, the absorbance of ABDA at 380 nm in aqueous solution was adjusted to approximately 1.0. 10 μM of the BDP1-PNZ sample was added to the cuvette, and then the sample was irradiated with a 716 nm laser (power density: 100 mW / cm 2 ) for different times (0 - 300 s), and the absorption spectrum was recorded immediately after each irradiation.

[0075] As Figure 13 shown, within 6 minutes of light irradiation, the characteristic absorption peak of 9,10-anthracenediyl-bis(methylene)dicarboxylic acid (ABDA) hardly changed, indicating that its singlet oxygen generation ability is extremely weak and can be ignored. It is known that an organic phototherapeutic agent in the excited singlet state (S1) can release energy through three competing pathways: fluorescence radiative decay, non-radiative decay to generate heat, and intersystem crossing (ISC) to the triplet state and possibly generate 1 O2 or other reactive oxygen species for photodynamic therapy. Combining the experimental data, it is speculated that BDP1-PNZ mainly generates heat through non-radiative transitions in aqueous solution.

[0076] (3) Test the photothermal performance of BDP1-PNZ.

[0077] Photothermal effect test method: Place 10 μM of the compound aqueous solution (2.0 mL) in a quartz cuvette and irradiate it with a laser of different powers for 300 s (the wavelength is the same as above), and record the temperature change, as shown in (A) of Figure 14 and Figure 15 and Figure 17 ; Place 2.0 mL of the compound aqueous solution with different concentrations (0, 10, 25 μM) in a quartz cuvette and irradiate it with a 0.5 W / cm 2 laser (BDP1-PNZ: 716 nm; BDP1-NEt2: 808 nm) for 300 s, and record the temperature change, as shown in (B) of Figure 14 and Figure 16 and Figure 17 .

[0078] To highlight the structural advantages of BDP1-PNZ, the heating curves of BDP1-PNZ in Example 1 and BDP1-NEt2 in Comparative Example 1 were compared at different power densities and concentrations. As Figure 14 shown in (A) of Figure 15 and 2 shown in 2 , when the laser power density increased from 0.1 W / cm Figure 14 to 1.0 W / cm Figure 16 , the temperature of the BDP1-PNZ solution increased from 25 °C to 46 °C (ΔT = 21 °C), while the temperature of the BDP1-NEt2 solution only increased from 25.2 °C to 31.2 °C (ΔT = 6 °C). In addition, as Figure 14 shown in (B) of Figure 17 and

[0079] shown in , both the heating curve and infrared thermal imaging of BDP1-PNZ showed that its temperature change was directly related to the concentration. As shown in (B) of max and surr shown in dis , at a concentration of 25 μM, the final temperature of the BDP1-PNZ solution could reach above 55 °C (ΔT ≈ 34 °C), while the BDP1-NEt2 solution at the same concentration only reached 37.1 °C (ΔT ≈ 13 °C), indicating that BDP1-PNZ with the introduced PNZ group had stronger photothermal conversion ability in aqueous solution. 716 and 808 were the absorbances of the sample at the laser wavelength (716 nm or 808 nm). The value of hA was calculated by the following formula: ; where τ was the system heat transfer time constant, m D and c D were the mass and specific heat capacity of the solution, respectively. Q dis was calculated by the following formula: ; The experimentally measured Q dis in DMOS was 0.0715 W (laser at 716 nm), and Q in waterdis are 0.179 W (laser of 716 nm) and 0.139 W (laser of 808 nm), T max(Solution) refers to the highest temperature reached by the solution. Based on the above data and formula, the photothermal conversion efficiency of each compound is calculated.

[0080] By comparing the heating curves of BDP1-PNZ and BDP1-NEt2 under 0.5 W / cm 2 laser irradiation (as Figure 18 shown), and combining with the relationship curve between the cooling time and the negative natural logarithm of temperature (-ln(θ)) in the cooling stage (as Figure 19 shown), the PCE of BDP1-PNZ in aqueous solution is calculated to be 40.9%, which is significantly higher than 9.2% of BDP1-NEt2. It is worth noting that, as Figure 20 shown in (B) of Figure 20 , BDP1-PNZ shows more stable temperature changes during three heating and cooling cycles, as Figure 20 shown in (A) of

[0081] (4) Test the water solubility of BDP1-PNZ.

[0082] The high photothermal conversion efficiency of BDP1-PNZ is inseparable from its water solubility. As Figure 21 shown, compared with hydrophobic and easily aggregated BDP1-NEt2, the solubility of BDP1-PNZ is significantly improved, and as Figure 22 shown in (A) of Figure 22 , it conforms to the Lambert-Beer law in the wide concentration range of 5 - 60 μM, while as

[0083] shown in (B) of

[0084] BDP1-NEt2 only conforms in the range of 5 - 18 μM.

[0085] (1) Evaluation of the dark cytotoxicity of BDP1-PNZ.

[0086] Cell culture: Human breast cancer 4T1 cells were cultured in RPMI 1640 medium (GIBCO) containing 10% fetal bovine serum (HyClone), 100 U / mL penicillin, and 100 μg / mL streptomycin (HyClone). MCF 10A cells were cultured in mammary epithelial cell basal medium (Lonza) supplemented with endothelial cell growth medium BulletKit (Lonza). All cells were cultured in a humidified incubator at 37 °C and 5% CO2.

[0087] Detection of dark toxicity by CCK-8 method: Human breast cancer 4T1 cells (5000 cells per well) were seeded in 96-well plates and treated at 37 °C in complete 1640 medium for 12 hours. Subsequently, fresh medium containing BDP1-PNZ at gradient concentrations (0 - 50 μM) was added to the wells, and the treatment was continued for 36 hours. After removing the medium, the cells were carefully washed with PBS buffer. 100 μL of CCK-8 solution diluted 10-fold (Cell Counting Kit-8, BIOMIKY) was added to each well, and the cells were treated at 37 °C and 5% CO2 for 20 minutes. After shaking for 5 minutes, the absorbance at 450 nm was measured using a microplate reader (Multiskan Sky). As Figure 23 shown, after co-treating 4T1 cells with different concentrations of BDP1-PNZ for 36 hours, the cell viability was still over 95% at a concentration of 50 μM, indicating good biocompatibility.

[0088] (2) Evaluation of the photothermal toxicity (photocytotoxicity) of BDP1-PNZ under laser irradiation.

[0089] Detection of photothermal toxicity by CCK-8 method: 4T1 cells cultured in cell culture (5000 cells per well) were seeded in 96-well plates and treated at 37 °C in complete 1640 medium for 12 hours. Subsequently, fresh medium containing BDP1-PNZ at gradient concentrations (0 - 10 μM) was added, and the treatment was continued for 12 hours. The cells in the experimental group (i.e., laser irradiated) were irradiated with a 716 nm laser (power density: 0.5 W / cm 2 ) for 10 minutes at room temperature, and then treated in the dark for 12 hours (total 24 hours). The cells in the control group (i.e., not laser irradiated) were treated in the dark for 24 hours under the same conditions. The subsequent steps were the same as those for the dark toxicity detection.

[0090] By setting up an experiment with an unirradiated group and an irradiated group, as Figure 24 shown in (B) of 2)After 10 minutes, as the concentration increased, the cell viability decreased significantly, while the unirradiated group treated with light avoidance showed no toxicity. As a control experiment, the PTT effect of BDP1-NEt2 in Example 1 was also evaluated in 4T1 cells. As shown in Figure 24 (A) below, under the same irradiation conditions (laser irradiation at 808 nm for the BDP1-NEt2-treated group), BDP1-NEt2 did not show significant phototoxicity, and its cell viability was similar to that of the unirradiated group treated with light avoidance. This may be due to insufficient laser power density and short irradiation time, which failed to fully demonstrate the PTT effect of BDP1-NEt2.

[0091] In addition, a power density-dependent experiment of BDP1-PNZ in 4T1 cells was also carried out. As shown in Figure 25 below, when the concentration of BDP1-PNZ was fixed at 10 μM, the cell viability decreased as the laser power increased. A PBS blank control group was also set up in the experiment, and no obvious cytotoxicity was observed under the same irradiation conditions.

[0092] (3) Evaluation of the photothermal therapy effect of BDP1-PNZ (staining experiment).

[0093] Staining experiment method: Approximately 1×10 5 4T1 cells were seeded in a bottom glass culture dish and cultured overnight. The cells were treated with BDP1-NEt2 and BDP1-PNZ for 6 hours, irradiated with BDP1-PNZ (using a 716 nm laser) and BDP1-NEt2 (using an 808 nm laser) for 10 minutes, and then cultured for 24 hours. Stained with Calcein-AM and propidium iodide (PI) for 30 minutes, and the fluorescence signal was recorded by a laser scanning confocal microscope (CLSM).

[0094] To visually evaluate the photothermal therapy effect of BDP1-PNZ, 4T1 cells were stained with Calcein-AM and propidium iodide (PI). As shown in Figure 26 below, both groups of cells were treated with the same concentration of BDP1-PNZ, and one group received laser irradiation. The results showed that: only when BDP1-PNZ and laser irradiation were present simultaneously, the cells showed extensive death and bright red fluorescence, verifying the results of the phototoxicity experiment. As a control, the study on the cell PTT effect of BDP1-NEt2 showed that: whether irradiated or not, the cells emitted strong green fluorescence and there was no significant difference between groups, indicating that the cells were not damaged. These experiments confirmed the excellent PTT performance of BDP1-PNZ at the cell level, demonstrating its significant application potential.

[0095] (4) Test of the ability of BDP1-PNZ to produce reactive oxygen species (ROS) at the cell level.

[0096] Intracellular reactive oxygen species (ROS) detection method: Approximately 5×10 5 4T1 cells were seeded in a bottom glass culture dish and cultured overnight at 37 °C. After the cells were treated with 10 μM BDP1-PNZ for 6 hours, 2 μM ROS probe 2,7-dichlorofluorescein diacetate (DCFH-DA) was added and treated for 30 minutes. After washing with PBS, it was irradiated with a 716 nm laser (power density: 0.5 W / cm 2 ) for 10 minutes, and the fluorescence signal was immediately recorded by a laser scanning confocal microscope (CLSM, excitation wavelength 488 nm, emission wavelength 500 - 550 nm).

[0097] Specifically, the 2,7-dichlorofluorescein diacetate (DCFH-DA) probe was used to detect the ability of BDP1-PNZ to generate reactive oxygen species (ROS) at the cellular level. Three groups were set up in the experiment: a control group cultured with PBS, a BDP1-PNZ dark group (without laser irradiation), and a BDP1-PNZ light group (with laser irradiation). As Figure 27 shown Figure 28 by the results, the fluorescence signal intensities of the three groups were similar, indicating that the ROS generation ability of BDP1-PNZ after light irradiation was negligible. This result was consistent with the solution experiment, further confirming that the energy absorbed by BDP1-PNZ was mainly released in the form of heat energy, achieving efficient energy focusing and significant therapeutic effects.

[0098] 3. In vivo photothermal performance test.

[0099] To further detect the photoacoustic imaging (PAI) potential of BDP1-PNZ, a mouse tumor model was established: Female Balb / c mice at 5 - 6 weeks old were raised for 12 hours under normal light-dark cycles (with free access to food and water). A 4T1 tumor-bearing mouse model was constructed by subcutaneously injecting approximately 1×10^6 cells (100 μL suspension) into the left ventral side of male BALB / c mice. Seven days later, the tumor volume grew to 70 - 100 mm 3 , and then this model was used for in vivo PAI experiments.

[0100] (1) In vivo photoacoustic imaging detection When the tumor volume grew to 70 - 100 mm 3At that time, 50 μL of BDP1-PNZ or ICG (0.5 mM, PBS containing 0.1% Tween 80, pH 7.4) was injected into the tumor. Photoacoustic images were acquired and the signal intensity was quantified at different time points before and after injection (0, 1, 3, 6, 9, 12 hours). System parameters: frequency = 40 MHz, ultrasonic gain = 10 dB, photoacoustic gain = 40 dB. Since indocyanine green (ICG) is an organic dye widely used in current in vivo PAI research, it was selected as a control to evaluate the PAI ability of BDP1-PNZ. The mice were divided into two groups and injected with BDP1-PNZ and ICG intravenously, respectively, and then photoacoustic imaging was performed under 716 nm and 770 nm light excitation for more than 12 hours. As Figure 29 shown, the PA signal (photoacoustic signal) of the mice injected with BDP1-PNZ was significantly stronger than that of the ICG group, indicating that BDP1-PNZ still exhibited a better PA effect (photoacoustic effect) even in the complex tumor microenvironment. Figure 30 The normalized PA intensity (photoacoustic signal intensity) at different time points (0, 1, 3, 6, 9, and 12) was calculated as follows: the PA signal intensity of the ICG group reached the peak at 3 hours after injection, while that of the BDP1-PNZ group reached the maximum within about 1 hour, confirming the potential of BDP1-PNZ in rapid tumor diagnosis.

[0101] (2) In vivo photothermal imaging The in vivo photothermal effect of BDP1-PNZ was evaluated by an infrared thermal imager. BDP1-PNZ was injected into 4T1 tumor-bearing mice. One hour later, the tumor area was irradiated with a 716 nm laser (0.5 W / cm 2 ), and the temperature change was recorded every 1 minute. The PBS injection group was used as a control.

[0102] The body temperature change of the mice was monitored in real time using an infrared thermal imager. As Figure 31 and Figure 32 shown, the in vivo photothermal imaging showed that the temperature of the tumor area in the laser group (PBS + laser) only increased by ΔT≈1.5 °C within 3 minutes, indicating that continuous irradiation with a 716 nm laser at 0.5 W / cm 2 would not cause tissue hyperthermia (a necessary condition for in vivo phototherapy). In contrast, the tumor temperature in the BDP1-PNZ + laser group rapidly increased from 37.1 °C to 50.1 °C within 3 minutes, and this temperature was sufficient to ablate cancer cells, demonstrating the effective in vivo photothermal conversion ability of BDP1-PNZ.

[0103] (3) In vivo phototherapy experiment When the tumor volume reached approximately 100 mm 3At that time, the mice were randomly divided into four groups: (1) PBS control group (Control); (2) PBS control group + laser (Control + Laser); (3) BDP1 - PNZ; (4) BDP1 - PNZ + laser (BDP1 - PNZ + Laser). Each group was injected with 50 μL of the sample (0.5 mM) via the tail vein. One hour later, it was irradiated with 716 nm laser (0.5 W / cm 2 ) for 10 minutes. After treatment, the tumor volume (length, width) and body weight were measured every two days. After 2 weeks, the tumors of the mice were excised and photographed.

[0104] As Figure 33 and Figure 34 shown, the treatments in the PBS group, PBS + laser group, and BDP1 - PNZ group failed to inhibit tumor growth, and the average tumor volume increased by about 8 - 9 times, indicating that the efficacy of these groups was negligible; while the BDP1 - PNZ + laser group showed good antitumor effects, the tumors were significantly inhibited and completely eliminated without recurrence. In addition, the body weights of all control group and experimental group mice increased slowly, indicating that the treatment did not cause systemic toxic effects. The above results fully demonstrated the excellent in - vivo photothermal activity of BDP1 - PNZ under light irradiation conditions.

[0105] (4) Histological and hematological analysis Test method: On the 14th day of treatment, blood samples of the mice were collected for serum biochemical and blood routine tests. The tumors and major organs (heart, liver, spleen, lung, kidney) were taken, fixed in 4% formaldehyde overnight, embedded in paraffin, and 5 - μm sections were prepared for observation. The body weights of all mice in the experimental group and the control group did not change, indicating that these therapies did not cause systemic adverse reactions in mice. To evaluate the toxicity of BDP1 - PNZ, the major organ tissues (heart, liver, spleen, lung, and kidney) of the euthanized mice after treatment were examined. No pathological tissue damage or abnormalities were observed in all groups (as Figure 35 shown), indicating that BDP1 - PNZ had good biosafety. Further, the blood of the treated mice was analyzed and compared with the control group. As shown in (A) of Figure 36 , even when the concentration was as high as 200 μM, BDP1 - PNZ still showed no hemolysis phenomenon. Compared with the untreated group, the detailed biochemical index results of the treated group (including liver and kidney function indexes) did not show significant differences, indicating that it had no significant hepatotoxicity ( Figure 36 (B)) and nephrotoxicity ( Figure 36 (C)). After the combined treatment of BDP1 - PNZ and laser, the blood routine analysis indexes of the mice were still within the normal range. These results indicated that the in - vivo application of BDP1 - PNZ would not pose a risk to the body.

[0106] In summary, the single-molecule photothermal agent BDP1-PNZ with high photothermal conversion efficiency of the present invention has a high photothermal conversion efficiency in aqueous solution. BDP1-PNZ exhibits an extremely high molar extinction coefficient in the near-infrared region. The fluorescence emission and singlet oxygen generation ability of BDP1-PNZ are extremely weak and almost negligible, so that the absorbed energy is mainly released through non-radiative transitions (mainly in the form of heat energy), thereby significantly improving its photothermal conversion efficiency at the single-molecule level in aqueous solution. The high efficiency of BDP1-PNZ is attributed to the introduction of the PNZ group, which can be summarized into three core factors: (1) The introduction of the PNZ group generates steric hindrance due to the bending of the fluorophore scaffold, further exacerbating the overall molecular distortion; (2) A significant conformational change from the twisted conformation to the extended conformation in the excited state, and this conformational change and related internal conversion processes are crucial for the photothermal efficiency of BDP1-PNZ; (3) The PNZ group, as a large-volume electron-donating group, can activate the photoinduced electron transfer (PET) ability of BDP1-PNZ under photoexcitation, and then form a low-lying dark electron transfer state. The effective formation of these low-lying dark electron transfer states is the basis for the enhanced photothermal performance of BDP1-PNZ, and the formation process also enhances the photostability of the photothermal agent. By exerting the excellent characteristics of energy release, BDP1-PNZ exhibits excellent cell killing efficiency. Particularly importantly, BDP1-PNZ successfully overcomes the limitation of traditional organic small-molecule photothermal agents relying on amphiphilic polymer material coating. BDP1-PNZ can be directly applied as a single molecule in in vitro and in vivo systems, which not only avoids the immune escape phenomenon that may be caused by the introduction of polymer materials, but also maintains a high photothermal conversion efficiency due to the improved solubility and enhanced non-radiative transitions brought by the PNZ group. Under the guidance of photothermal and photoacoustic imaging, BDP1-PNZ can accurately locate tumors and achieve excellent tumor ablation effects through 718 nm light irradiation, and can be applied to the cryogenic photothermal treatment of triple-negative breast cancer, while showing good biocompatibility. BDP1-PNZ has good application prospects as a multifunctional and highly efficient photothermal therapeutic agent.

[0107] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several deformations and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A single-molecule photothermal agent with high photothermal conversion efficiency, characterized in that, The structural formula of the single-molecule photothermal agent with high photothermal conversion efficiency is as follows: 。 2. A preparation method of a single-molecule photothermal agent with high photothermal conversion efficiency, characterized in that, It includes the following steps: Step S1, Synthesis of Compound X1: Under argon protection, dissolve 2,4-dimethylpyrrole in dichloromethane, stir in an ice bath, add trifluoroacetic anhydride, stir and then raise the temperature to room temperature. After quenching the reaction, extract, combine the organic phases, dry, filter, concentrate under reduced pressure and purify by silica gel column chromatography to obtain Compound X1. The structural formula of Compound X1 is as follows: ; Step S2: Synthesis of compound X2: Under argon protection, 2,4-dimethylpyrrole and compound X1 are dissolved in dichloromethane, stirred in an ice bath, phosphorus oxychloride is added, the temperature is raised to room temperature and stirred, boron trifluoride ether complex and triethylamine are added, stirring is continued, the organic phases are combined after extraction, and after drying, filtration, concentration under reduced pressure and purification by silica gel column chromatography, compound X2 is obtained. The structural formula of the compound X2 is as follows: ; Step S3, synthesis of compound X3: phenoxazine, 4-bromobenzaldehyde, palladium acetate, tri-tert-butylphosphine tetrafluoroborate and cesium carbonate are dissolved in toluene, heated and stirred for reaction, the reaction solution is cooled to room temperature, toluene is removed under reduced pressure, the residue is extracted with dichloromethane, the organic phases are combined, dried, concentrated and purified by silica gel column chromatography to obtain compound X3, the structural formula of compound X3 is as follows: ; Step S4: Dissolve compound X2 and compound X3 in toluene, add glacial acetic acid and piperidine, heat under nitrogen protection, monitor the reaction by thin-layer chromatography, remove the solvent under reduced pressure, extract the crude product, and after drying, concentrating and purifying by silica gel column chromatography of the organic phase, obtain the single-molecule photothermal agent with high photothermal conversion efficiency.

3. The preparation method of the single-molecule photothermal agent with high photothermal conversion efficiency according to claim 2, wherein, In the said step S1, the molar ratio of 2,4-dimethylpyrrole to trifluoroacetic anhydride is 1:(1-2).

4. The preparation method of the single-molecule photothermal agent with high photothermal conversion efficiency according to claim 2, wherein, In the said step S2, the molar ratio of compound X1 to 2,4-dimethylpyrrole is 1:(1-2).

5. The preparation method of the single-molecule photothermal agent with high photothermal conversion efficiency according to claim 2, characterized in that, In the said step S2, the molar ratio of boron trifluoride diethyl ether complex to triethylamine is 1.1:(1-2).

6. The preparation method of the single-molecule photothermal agent with high photothermal conversion efficiency according to claim 2, wherein In the said step S3, the molar ratio of phenoxazine to 4-bromobenzaldehyde is 1:(1-2).

7. The preparation method of the single-molecule photothermal agent with high photothermal conversion efficiency according to claim 2, characterized in that, In the said step S3, the reaction temperature of the heating and stirring reaction is 90°C - 120°C, and the reaction time is 18h - 30h.

8. The preparation method of the single-molecule photothermal agent with high photothermal conversion efficiency according to claim 2, characterized in that, In the said step S4, the molar ratio of compound X2 to compound X3 is 1:(4-6), and the reaction temperature of heating under nitrogen protection is 60°C - 100°C.

9. The preparation method of the single-molecule photothermal agent with high photothermal conversion efficiency according to claim 2, characterized in that, In the said step S4, the volume ratio of glacial acetic acid to piperidine is 1:(1-2.5).

10. Use of the single-molecule photothermal agent with high photothermal conversion efficiency as described in claim 1 in the preparation of an anti-tumor drug for triple-negative breast cancer.

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