A single-molecule photothermal agent with high photothermal conversion efficiency and its preparation method and application

Abstract

The present invention relates to the field of photothermal therapy technology, and in particular to a single-molecule photothermal agent with high photothermal conversion efficiency, and its preparation method and application. The preparation method comprises the following steps: step S1, synthesis of compound X1: under argon protection, 2,4-dimethylpyrrole is dissolved in dichloromethane, stirred under an ice bath, trifluoroacetic anhydride is added, stirred and warmed to room temperature, the reaction is quenched and then extracted, the organic phases are combined, dried, filtered, concentrated under reduced pressure and purified by silica gel column chromatography to obtain compound X1. The prepared single-molecule photothermal agent with high photothermal conversion efficiency has high water solubility, high photothermal conversion efficiency in aqueous solution, strong photostability, avoids the immune escape phenomenon that may be caused by the introduction of polymer materials, and solves the technical problem that existing photothermal agents are difficult to achieve high photothermal conversion efficiency in aqueous solution.

Description

Technical Field

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

[0002] Triple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer. Due to its insensitivity to traditional hormonal therapy and targeted therapies, effective treatment of TNBC remains a major challenge. Photothermal therapy (PTT), a selective, non-invasive cancer treatment, has attracted considerable attention due to its high spatiotemporal precision. By adjusting laser intensity, irradiation duration, and photothermal agent concentration, PTT can precisely control heat generation. After photothermal agents (PTAs) are delivered to tumor tissue, PTT utilizes external lasers to irradiate the lesion. The heat generated raises the temperature of the tumor site, triggering nonspecific damage to biomacromolecules and leading to cell death.

[0003] Currently, organic molecular photothermal agents are widely used in photothermal therapy due to their high photothermal conversion efficiency (PCE) and easily modifiable structures. However, few single-molecule PTAs have achieved high PCEs in aqueous solutions. This is primarily due to the fact that most organic PTAs require complex structural manipulation and modification to enhance their PCE, which in turn reduces their water solubility, leading to disordered aggregation in aqueous systems and, in turn, weakening their PCE. Therefore, these photothermal agents are often combined with amphiphilic polymers to enhance their solubility in aqueous solutions and facilitate their application in biological systems. However, long-term or repeated use of polymers such as PEGylated ones can induce the production of anti-PEG antibodies in the body, reducing drug efficacy and increasing the risk of adverse reactions. In contrast, single-molecule PTAs, because they do not contain polymers, can circumvent these antibody-mediated immune responses. This makes single-molecule PTAs more suitable for patients who are sensitive to polymers such as PEG or who already have anti-PEG antibodies.

[0004] Therefore, when designing photothermal therapy strategies, especially for models of diseases requiring long-term treatment or repeated dosing, the immune response properties of PTAs must be fully considered. The development of high-performance single-molecule photothermal agents offers a potential solution. However, achieving high PCEs for single-molecule PTAs in aqueous solutions remains a significant challenge, primarily due to the poor water solubility and aggregation of most purely organic photothermal materials, which compromises their functionality in biological systems. Summary of the Invention

[0005] In response to the problems raised in the background technology, the purpose of the present invention is to propose a single-molecule photothermal agent with high photothermal conversion efficiency. It 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. It solves 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, and to show 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:

[0009] 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:

[0010] .

[0011] A method for preparing a single-molecule photothermal agent with high photothermal conversion efficiency comprises the following steps:

[0012] 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 stirred and warmed to room temperature. 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: ;

[0013] Step S2, Synthesis of Compound X2: Under argon protection, 2,4-dimethylpyrrole and Compound X1 were dissolved in dichloromethane, stirred in an ice bath, phosphorus oxychloride was added, and the temperature was raised to room temperature with stirring. Boron trifluoride etherate complex and triethylamine were added, and stirring was continued. After extraction, the organic phases were combined, 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: ;

[0014] Step S3, Synthesis of Compound X3: Phenoxazine, 4-bromobenzaldehyde, palladium acetate, tri-tert-butylphosphine tetrafluoroborate, and cesium carbonate were dissolved in toluene, heated and stirred for reaction, and the reaction solution was cooled to room temperature. The toluene was removed under reduced pressure, and the residue was extracted with dichloromethane. The organic phases were combined, dried, concentrated, and purified by silica gel column chromatography to obtain Compound X3. The structural formula of Compound X3 is as follows: ;

[0015] 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.

[0016] Further, in step S1, the molar ratio of 2,4-dimethylpyrrole to trifluoroacetic anhydride is 1:(1-2); in step S2, the molar ratio of compound X1 to 2,4-dimethylpyrrole is 1:(1-2); in step S2, the molar ratio of boron trifluoride ether complex to triethylamine is 1.1:(1-2); in step S3, the molar ratio of phenoxazine to 4-bromobenzaldehyde is 1:(1-2); in step S3, the reaction temperature of the heating and stirring reaction is 90°C to 120°C, and the reaction time is 18h to 30h; in step S4, the molar ratio of compound X2 to compound X3 is 1:(4-6), and the reaction temperature under nitrogen protection is 60°C to 100°C; in step S4, the volume ratio of glacial acetic acid to piperidine is 1:(1-2.5).

[0017] The application of the single-molecule photothermal agent with high photothermal conversion efficiency in the preparation of triple-negative breast cancer anti-tumor drugs.

[0018] 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 photostability, avoids the immune escape phenomenon that may be caused by the introduction of polymer materials, and solves the technical problem that existing photothermal agents are difficult to achieve high photothermal conversion efficiency in aqueous solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a molecular structure diagram of the twisted state and the stretched state of the single-molecule photothermal agent with high photothermal conversion efficiency of the present invention.

[0020] Figure 2 This is a synthetic route for the single-molecule photothermal agent with high photothermal conversion efficiency of the present invention.

[0021] Figure 3 is the compound X1 of Example 1 in deuterated chloroform 1H NMR spectrum (500 MHz).

[0022] Figure 4 is the compound X1 of Example 1 in deuterated chloroform 13 C NMR spectrum (126 MHz).

[0023] Figure 5 is the compound X2 of Example 1 in deuterated chloroform 1 H NMR spectrum (500 MHz).

[0024] Figure 6 is the compound X2 of Example 1 in deuterated chloroform 13 C NMR spectrum (126 MHz).

[0025] Figure 7 is the compound X3 of Example 1 in deuterated chloroform 1 H NMR spectrum (500 MHz).

[0026] Figure 8 is the compound X3 of Example 1 in deuterated chloroform 13 C NMR spectrum (126 MHz).

[0027] Figure 9 The BDP1-PNZ of Example 1 in deuterated chloroform 1 H NMR spectrum (500 MHz).

[0028] Figure 10 The BDP1-PNZ of Example 1 in DMSO-d6 13 C NMR spectrum (126 MHz).

[0029] Figure 11 The UV-visible absorption and fluorescence spectra of BDP1-PNZ (10 μM concentration) in different solvents are shown in FIG. Figure 11 (A) is the UV-visible absorption spectrum. Figure 11 (B) is the fluorescence spectrum.

[0030] Figure 12 1 is the UV-visible absorption spectrum and fluorescence spectrum of BDP1-PNZ (concentration of 10 μM) in dimethyl sulfoxide in Example 1.

[0031] Figure 13 In the presence of BDP1-PNZ of Example 1, ABDA (used as 1 Absorption spectra of the O2 probe) at different irradiation times (0-6 minutes).

[0032] Figure 14This is the infrared thermal imaging image of BDP1-NEt2 of Control Example 1 and BDP1-PNZ of Example 1 under laser irradiation. Figure 14 (A) is an infrared thermal imaging image of an aqueous solution of BDP1-NEt2 of Control Example 1 and BDP1-PNZ (10 μM) of Example 1 under laser irradiation at different laser power densities of 808 / 716 nm; Figure 14 (B) shows the BDP1-NEt2 of the control example 1 and the BDP1-PNZ of the example 1 at different concentrations at 0.5W / cm 2 Infrared thermal imaging diagram under laser irradiation with laser power density.

[0033] Figure 15 is a temperature curve diagram of the BDP1-NEt2 aqueous solution of Control Example 1 and the BDP1-PNZ aqueous solution of Example 1 under laser irradiation at different laser power densities of 808 / 716 nm (concentration = 10 μM), Figure 15 (A) Temperature curve of BDP1-NEt2 of control example 1, Figure 15 (B) is a temperature curve diagram of BDP1-PNZ in Example 1.

[0034] Figure 16 The BDP1-NEt2 of the control example 1 and the BDP1-PNZ aqueous solution of the example 1 were irradiated with 808 / 716 nm laser (0.5 W / cm 2 ) temperature curve diagram, Figure 16 (A) is the temperature curve of BDP1-NEt2 in comparative example 1. Figure 16 (B) is a temperature curve diagram of BDP1-PNZ in Example 1.

[0035] Figure 17 is a temperature change diagram of BDP1-NEt2 of comparative example 1 and BDP1-PNZ of example 1, Figure 17 (A) is a temperature change curve of the aqueous solution of BDP1-NEt2 of Control Example 1 and BDP1-PNZ of Example 1 under laser irradiation of different laser power densities of 808 / 716 nm (concentration = 10 μM); Figure 17 (B) is the aqueous solution of BDP1-NEt2 of control example 1 and BDP1-PNZ of example 1 at different concentrations, irradiated by 808 / 716 nm laser (0.5 W / cm 2 ) (concentration gradient change, irradiation time: 600 seconds).

[0036] Figure 18 is a photothermal effect diagram of BDP1-PNZ of Example 1 and BDP1-NEt2 of Control Example 1, Figure 18(A) is the photothermal effect diagram of BDP1-PNZ in Example 1, Figure 18 (B) is the photothermal effect diagram of BDP1-NEt2 of control example 1, where η represents the photothermal conversion efficiency.

[0037] Figure 19 is a graph showing the relationship between the cooling time and the negative natural logarithm (-ln(θ)) corresponding to the temperature in the cooling stage of BDP1-PNZ of Example 1 and BDP1-NEt2 of Comparative Example 1, Figure 19 (A) is a graph showing the relationship between the cooling time and the negative natural logarithm (-ln(θ)) corresponding to the temperature of BDP1-PNZ in Example 1 during the cooling stage. Figure 19 (B) is a graph showing the relationship between the cooling time and the negative natural logarithm (-ln(θ)) corresponding to the temperature of BDP1-NEt2 in the cooling stage of Control Example 1.

[0038] Figure 20 is the infrared thermal image of BDP1-NEt2 of comparative example 1 and BDP1-PNZ of example 1, Figure 20 (A) is an infrared thermal image of BDP1-NEt2 of Control Example 1 at a concentration of 10 μM. Figure 20 (B) is an infrared thermal image of BDP1-PNZ of Example 1 at a concentration of 10 μM, which shows the infrared thermal image of BDP1-PNZ of Example 1 at a concentration of 10 μM. 2 ) Temperature changes after several heating / cooling cycles after 30 min of irradiation.

[0039] Figure 21 It uses 808 / 716nm laser (0.5W / cm 2 ) Comparative dissolution of BDP1-NEt2 (10 μM) of Control Example 1 and BDP1-PNZ (10 μM) of Example 1 in aqueous solution after irradiation for 30 minutes.

[0040] Figure 22 This is the absorption intensity change diagram of BDP1-PNZ (716nm) and BDP1-NEt2 (808nm) at different concentrations in aqueous solution. Figure 22 (A) is the absorption intensity change diagram of BDP1-PNZ. Figure 22 (B) The absorption intensity change diagram of BDP1-NEt2. When the concentration of BDP1-PNZ is greater than 61.33 μM, the absorption peak red-shifts (monomers transform into aggregates); a similar phenomenon occurs when the concentration of BDP1-NEt2 is greater than 17.86 μM.

[0041] Figure 23This figure shows the results of the CCK-8 assay for the dark cytotoxicity of BDP1-PNZ in Example 1. The figure shows the viability of 4T1 cells treated with different concentrations of BDP1-PNZ (0-50 μM) for 36 hours. The viability of cells in the untreated group is defined as 100%. The results are expressed as the mean ± standard deviation of five independent measurements.

[0042] Figure 24 The BDP1-NEt2 of control example 1 (0.5 W / cm 2 , 10 minutes) and BDP1-PNZ of Example 1 (0.5 W / cm 2 Comparison of 4T1 cell survival rates after laser irradiation (10 minutes). Figure 24 (A) is the BDP1-NEt2 of control example 1 with different concentrations (0.5 W / cm 2 Comparison of 4T1 cell survival rates after laser irradiation (10 minutes). Figure 24 (B) is the BDP1-PNZ of Example 1 with different concentrations (0.5 W / cm 2 Comparison of 4T1 cell survival rates after laser irradiation (10 minutes, 3 days). Non-irradiated and irradiated groups were set up for experiments. In the figure, IR (-) refers to the non-irradiated group, and IR (+) refers to the irradiated group.

[0043] Figure 25 This is a graph showing the photocytotoxicity test of BDP1-PNZ (10 μM) in Example 1, in which 4T1 cells were treated (or untreated) with BDP1-PNZ and then exposed to different laser powers (716 nm laser irradiation for 3 minutes). Error bars represent the standard deviation of each group (sample size n=5). Results are expressed as mean ± standard deviation (sample size n=3), and significant differences were analyzed (NS: not significantly different; ***P<0.001).

[0044] Figure 26 This is a staining experiment to evaluate the photothermal treatment effect of BDP1-PNZ. Figure 26 (A) is a microscopic image (bright field) of 4T1 cells cultured with BDP1-NEt2 (10 μM) from Control Example 1, after 808 nm laser irradiation and without laser irradiation; Figure 26 (B) is a microscopic image of 4T1 cells cultured with BDP1-NEt2 (10 μM) from Control Example 1, irradiated with 808 nm laser, and not irradiated with laser (stained with Calcein-AM / PI); Figure 26 (C) is a microscopic image (bright field) of 4T1 cells cultured with BDP1-PNZ (10 μM) of Example 1 and irradiated with 716 nm laser and without laser irradiation; Figure 26(D) in the middle is a microscopic image (Calcein-AM / PI staining) of 4T1 cells cultured with BDP1-PNZ (10 μM) in Example 1, irradiated with 716 nm laser, and not irradiated with laser.

[0045] Figure 27 This is a test chart of BDP1-PNZ's ability to produce reactive oxygen species at the cellular level. Figure 27 (A) Figure 27 (B) and Figure 27 (C) in the middle are the microscopic images of the control group, BDP1-PNZ light-protected group and BDP1-PNZ light-exposed group under bright field. Figure 27 (D) Figure 27 (E) and Figure 27 (F) in the middle are the microscopic images of the control group, BDP1-PNZ light-protected group, and BDP1-PNZ light-exposed group under the fluorescence field; Figure 27 (G) Figure 27 (H) and Figure 27 (I) in the figure are the microscopic images of the control group, BDP1-PNZ light-protected group and BDP1-PNZ light-exposed group under superimposed fields.

[0046] Figure 28 yes Figure 27 Quantitative analysis of fluorescence intensity in the middle, where the data are expressed as mean ± standard deviation (sample size n = 3), and significant difference analysis (NS: no significant difference; ***P < 0.001).

[0047] Figure 29 is the photoacoustic imaging image of the tumor at a specified time after BDP1-PNZ and ICG treatment in Example 1, Figure 29 (A) Figure 29 (B) Figure 29 (C) Figure 29 (D) Figure 29 (E) and Figure 29 (F) is the photoacoustic imaging of the tumor at 0h, 1h, 3h, 6h, 9h and 12h after treatment with BDP1-PNZ in Example 1. Figure 29 (a) in Figure 29 (b) Figure 29 (c) Figure 29 (d) Figure 29 (e) and Figure 29 (f) in the figure is the photoacoustic imaging of the tumor at 0h, 1h, 3h, 6h, 9h and 12h after ICG treatment, where only part of the tumor area is shown in the image.

[0048] Figure 30 Yes Figure 29 Quantitative analysis of photoacoustic intensity.

[0049] Figure 31 4T1 tumor mice were treated with 716 nm laser (0.5 W / cm 2 ) Infrared thermal imaging image after irradiation, Figure 31 (A) Figure 31 (B) Figure 31 (C) and Figure 31 (D) in the figure are infrared thermal images at 0h, 1min, 2min and 3min after injection of BDP1-PNZ in Example 1, Figure 31 (a) in Figure 31 (b) Figure 31 (c) and Figure 31 (d) in the figure are infrared thermal images at 0h, 1min, 2min and 3min after PBS injection.

[0050] Figure 32 This is a curve diagram of the in vivo temperature change of the tumor site during the laser irradiation time after the injection of BDP1-PNZ and PBS in Example 1.

[0051] Figure 33 This is a picture of the size and appearance of the tumor taken on the 14th day of the in vivo phototherapy trial.

[0052] Figure 34 This is the experimental analysis chart of the in vivo phototherapy experiment. Figure 34 (A) shows the changes in tumor volume over time in mice of different groups (mean ± standard deviation, sample size n=5). Figure 34 (B) shows the average weight of tumors in mice of different groups (mean ± standard deviation, sample size n=5). Figure 34 (C) shows the changes in body weight of mice in different groups during the treatment period.

[0053] Figure 35 These are hematoxylin and eosin (H&E)-stained images of heart, liver, spleen, lung, and kidney sections from 4T1 tumor-bearing mice in different treatment groups. Scale bars are 100 μm.

[0054] Figure 36 This is the blood analysis chart of mice in different treatment groups. Figure 36 (A) is an analysis of the hemolysis rate of mouse erythrocytes by the photothermal agent BDP1-PNZ at concentrations ranging from 1 to 100 μM (PBS is the negative control, and water is the positive control); Figure 36 (B) shows the liver function biochemical indicators of mice in different treatment groups (mean ± standard deviation, sample size n=5); Figure 36(C) shows the biochemical indices of renal function in mice in different treatment groups (mean ± standard deviation, sample size n=5). DETAILED DESCRIPTION

[0055] 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:

[0056] .

[0057] In response to the technical problems in the background technology, single-molecule PTAs need to meet the following key biomedical and photophysical properties: (1) good solubility in physiological environment; (2) excellent photostability under light to maintain photothermal performance; (3) high PCE in aqueous solution. Among many 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, resistance to photobleaching and environmental insensitivity. However, due to its inherent hydrophobicity and rigid structure, BODIPY and its analogs easily form a large number of ineffective aggregates and precipitates in aqueous solution, which greatly limits their practical application in photothermal therapy under physiological conditions. Therefore, it is urgent to develop new high-performance single-molecule BODIPY derivatives. In order to improve the PCE in aqueous solution, the focus is on improving the water solubility of the molecule, and at the same time, it is necessary to significantly enhance the intramolecular non-radiative transition ability (the key to improving PCE is to increase the non-radiative transition process). Dark electron transfer states are often accompanied by the enhancement of 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 transition). Dark electron transfer states are usually associated with 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.

[0058] The present invention introduces a phenoxazine (PNZ) group into BODIPY to construct a single-molecule photothermal agent (BDP1-PNZ) with high photothermal conversion efficiency, thereby realizing the PET process between PNZ and BODIPY. The central CF3 group and the adjacent methyl group in the BDP1-PNZ structure (induce the bending of the molecular main chain to promote non-radiative transitions), and the introduction of a bulky electron-donating group phenoxazine (PNZ), form a new type of single-molecule photothermal agent (BDP1-PNZ). The introduction of the PNZ group enhances the π-π stacking effect within the BDP1-PNZ molecule, causing its conformation to change 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, thereby significantly improving its solubility in aqueous solution. The conformational change also greatly enhances the non-radiative transition process to release heat. Figure 1The twisted and extended states of the BDP1-PNZ molecular structure are shown. In addition, the introduction of the PNZ electron-donating group induces photoinduced electron transfer (PET) during photoexcitation, significantly promoting the formation of low-lying dark electron transfer states in the excited state. The effective formation of this dark state is the basis for the enhanced photothermal capacity of BDP1-PNZ. The high efficiency of BDP1-PNZ is due to the introduction of the PNZ group, which can be summarized into three core factors: (1) The introduction of the PNZ group produces steric hindrance due to the bending of the fluorophore bracket, further aggravating the overall distortion of the molecule (the steric effect of the -CF3 group introduced in the middle position and the two adjacent methyl groups, which causes the BODIPY skeleton to show obvious bending in both the ground state and the excited state); (2) A significant transition from a twisted conformation to an extended conformation in the excited state. This conformational change and the related internal conversion process are crucial to 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 a low-lying dark electron transfer state. The efficient formation of these low-lying dark electron transfer states is the basis for the enhanced photothermal performance of BDP1-PNZ, and its formation process also enhances the photostability of the photothermal agent.

[0059] The single-molecule photothermal agent (BDP1-PNZ) of this invention exhibits high water solubility, virtually no fluorescence emission or singlet oxygen generation, and significantly improved PCE. The introduction of PNZ enhances π-π interactions within the BDP1-PNZ molecule, leading to the coexistence of twisted and extended conformations. Transitions between these conformations also promote nonradiative transitions. The high photothermal conversion efficiency stems from the extremely low energy of the PET state and the small energy gap between it and the S0 state (the singlet ground state of the molecule). According to the energy gap law, this condition enhances the rate of nonradiative transitions, resulting in two advantages: (a) shorter excited state duration leads to greater molecular stability; and (b) faster deexcitation (the rate at which an excited state returns from a high energy state to the ground state or other lower energy state) enhances photothermal efficiency. Because each "light absorption-heat release" cycle is shortened, the molecule can complete more cycles per unit time, thereby enhancing heating efficiency. The single-molecule photothermal agent with high photothermal conversion efficiency of the present invention has high water solubility, high photothermal conversion efficiency in aqueous solution, and strong photostability. It avoids the immune escape phenomenon that may be caused by the introduction of polymer materials, and still maintains high photothermal conversion efficiency due to the improved solubility and enhanced non-radiative transition brought by the PNZ group, thus solving the technical problem that existing photothermal agents are difficult to achieve high photothermal conversion efficiency in aqueous solution.

[0060] A method for preparing a single-molecule photothermal agent with high photothermal conversion efficiency comprises the following steps:

[0061] 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 stirred and warmed to room temperature. 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: ;

[0062] Step S2, Synthesis of Compound X2: Under argon protection, 2,4-dimethylpyrrole and Compound X1 were dissolved in dichloromethane, stirred in an ice bath, phosphorus oxychloride was added, and the temperature was raised to room temperature with stirring. Boron trifluoride etherate complex and triethylamine were added, and stirring was continued. After extraction, the organic phases were combined, 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: ;

[0063] Step S3, Synthesis of Compound X3: Phenoxazine, 4-bromobenzaldehyde, palladium acetate, tri-tert-butylphosphine tetrafluoroborate, and cesium carbonate were dissolved in toluene, heated and stirred for reaction, and the reaction solution was cooled to room temperature. The toluene was removed under reduced pressure, and the residue was extracted with dichloromethane. The organic phases were combined, dried, concentrated, and purified by silica gel column chromatography to obtain Compound X3. The structural formula of Compound X3 is as follows: ;

[0064] 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.

[0065] The synthetic route of BDP1-PNZ is as follows Figure 2 The method demonstrates the preparation of meso-trifluoromethyl BODIPY (Compound X2) and a phenoxazine derivative (Compound X3). BDP1-PNZ is then synthesized via a Knoevenagel condensation reaction of Compound X2 and Compound X3 using glacial acetic acid and piperidine as catalysts. The resulting single-molecule photothermal agent exhibits high water solubility, strong photostability, and high photothermal conversion efficiency in aqueous solution.

[0066] Further, 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 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, the reaction time is 18h to 30h, preferably the reaction temperature is 110°C, and the preferred reaction time is 24h; in the step S4, the molar ratio of compound X2 to compound X3 is 1: (4 to 6), preferably 1: 4; the reaction temperature of 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 to 2.5), preferably 1: 1.

[0067] The use of the single-molecule photothermal agent with high photothermal conversion efficiency in the preparation of an anti-tumor drug 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. It exhibits an extremely high molar extinction coefficient in the near-infrared region and can achieve dual-modal photoacoustic-photothermal imaging of tumors in vivo. It can be used to prepare an anti-tumor drug for triple-negative breast cancer and exhibits excellent anti-cancer ability in photothermal therapy, especially photothermal therapy for triple-negative breast cancer.

[0068] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. All reagents or instruments used without specifying the manufacturer are commercially available conventional products.

[0069] Raw materials: The raw materials in the following examples can all be obtained commercially, among which 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) were all purchased from Sigma-Aldrich.

[0070] Experimental instruments: Fluorescence spectra and UV-visible absorption spectra were recorded by Shimadzu RF-5301PC fluorescence spectrophotometer and Agilent 8453 spectrophotometer, respectively. 1 H and 13 C nuclear magnetic resonance (NMR) spectra were acquired with a Bruker 500 MHz NMR spectrometer. Thermal images and videos were recorded with a FLIR E60 thermal imaging camera.

[0071] Preparation of stock solutions for solution testing: Dissolve BDP1-NEt2 and BDP1-PNZ in DMSO to prepare the desired concentrations. For in vitro testing, prepare 10 mM stock solutions of BDP1-NEt2 and BDP1-PNZ in DMSO and further dilute to the desired concentration using a solution containing 0.1% Tween® 80.

[0072] Statistical analysis: Data were analyzed using one-way analysis of variance (ANOVA) using SPSS 21.0. Intergroup differences were assessed using Tukey's post hoc test. P < 0.05 was considered statistically significant.

[0073] Example 1

[0074] A method for preparing a single-molecule photothermal agent with high photothermal conversion efficiency is prepared by the following steps:

[0075] Step S1, Synthesis of Compound X1: Under argon protection, 2,4-dimethylpyrrole (1 mL, 9.71 mmol) was dissolved in dry dichloromethane (15 mL), and the mixture was stirred in an ice bath (0°C). Trifluoroacetic anhydride (1.5 mL, 10.68 mmol) was slowly added. The reaction solution was stirred at 0°C for 30 minutes, then warmed to room temperature and stirred for 3 hours. The reaction was quenched with saturated sodium bicarbonate solution, extracted with dichloromethane, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The organic phases were then purified by silica gel column chromatography (mobile phase gradient: n-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 is determined by 1 H NMR (hydrogen nuclear magnetic resonance) and 13 CNMR (carbon-13 nuclear magnetic resonance) spectroscopy characterization.

[0076] like Figure 3 and Figure 4 As shown:

[0077] 1 H NMR (500MHz, CDCl3) δ 9.25 (s, 1H), 5.97 (s, 1H), 2.36 (s, 3H), 2.32 (s, 3H); 13 CNMR (126MHz, CDCl3) δ 139.79, 120.83, 118.54, 116.25, 115.01, 113.95, 13.69, 13.45.

[0078] Step S2, Synthesis of Compound X2: Under argon, 2,4-dimethylpyrrole (250 μL, 2.43 mmol) and Compound X1 (300 mg, 1.62 mmol) were dissolved in dry dichloromethane (5 mL) and stirred in an ice bath (0°C). Phosphorus oxychloride (220 μL, 1.62 mmol) was added. The reaction mixture was warmed to room temperature and stirred for 4 hours, during which time the solution turned from colorless to red. Boron trifluoride etherate (0.98 mL, 7.89 mmol) and triethylamine (1 mL, 7.17 mmol) were then added, and stirring continued 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, v / v) to obtain Compound X2 (red solid, 31% yield). The structural formula of the prepared Compound X2 is as follows: The chemical structure of compound X2 is determined by 1 H NMR (hydrogen nuclear magnetic resonance) and 13 C NMR (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.

[0079] 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), and the mixture was heated at 110° C. with stirring for 24 hours. The reaction solution was cooled to room temperature, and the 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 determined by 1 H NMR (hydrogen nuclear magnetic resonance) and 13 C NMR (carbon-13 nuclear magnetic resonance) spectroscopy characterization, such as Figure 7 and Figure 8 As shown: 1 H NMR (500MHz, CDCl3) δ10.11 (s, 1H), 8.12 (d, J = 8.1Hz, 2H), 7.56 (d, J = 8.2Hz, 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 (126MHz, CDCl3) δ191.08, 145.18, 144.25, 136.04, 133.65, 132.41, 131.57, 123.45, 122.20,115.97, 113.59.

[0080] Step S4: Compound X2 (100 mg, 0.3125 mmol) and compound X3 (359 mg, 1.25 mmol) were dissolved in toluene (10 mL), glacial acetic acid (0.1 mL) and piperidine (0.1 mL) were added, and the mixture was heated at 80°C under nitrogen protection. The reaction was monitored by thin layer chromatography (TLC) until the main product spot turned green. The reaction was stopped and the solvent was removed under reduced pressure. The crude product was extracted with water and dichloromethane. The organic phase was dried over anhydrous sodium sulfate and concentrated, and then purified by silica gel column chromatography (mobile phase: n-hexane / dichloromethane = 4 / 1, volume ratio) to obtain a single-molecule photothermal agent BDP1-PNZ with high photothermal conversion efficiency (dark green powder, yield 51%). The chemical structure of BDP1-PNZ is shown in FIG. 1 H NMR (hydrogen nuclear magnetic resonance) and 13 C NMR (carbon-13 nuclear magnetic resonance) spectroscopy characterization, such as Figure 9 and Figure 10 As shown: 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). 13C 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:

[0081] .

[0082] Comparative Example 1

[0083] Before synthesizing the single-molecule photothermal agent (BDP1-PNZ) with high photothermal conversion efficiency of the present invention, the applicant researched 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 unsatisfactory photothermal conversion efficiency (PCE) under physiological conditions. The following tests will use BDP1-NEt2 for comparison with BDP1-PNZ.

[0084] 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):

[0085] Compound X2 (80 mg, 0.25 mmol) and 4-diethylaminobenzaldehyde (221 mg, 1.25 mmol) were dissolved in dry toluene (10 mL). Glacial acetic acid (0.1 mL) and piperidine (0.1 mL) were added to the solution. The mixture was heated at 80°C under a nitrogen atmosphere, and the reaction progress was monitored by TLC for 8 hours. The solvent was removed under reduced pressure, and the crude product was extracted with water and dichloromethane. The organic layer was dried over anhydrous Na2SO4, filtered, and the solvent was removed. The crude product was purified by silica gel column chromatography using n-hexane / dichloromethane (7 / 3, v / v) as the mobile phase to obtain BDP1-NEt2 (black powder, 51% yield). 1 H 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= 2.8Hz, 6H), 1.21 (t, J= 7.1 Hz, 12H). 13C 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. Molecular weight determination was performed by high-resolution mass spectrometry (HRMS) coupled with matrix-assisted laser desorption ionization (MALDI). HRMS (MALDI), calculated for (C36H41BF5N4 + ): m / z [M]+ : 635.334; found: m / z 635.332. The structural formula of the prepared BDP1-NEt2 is as follows:

[0086] .

[0087] 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:

[0088] 1. Photophysical properties test.

[0089] (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 tested.

[0090] like Figure 11 As shown in (A), BDP1-PNZ exhibits stable absorption spectra in solvents of different polarities, and its molar extinction coefficient at 716 nm in aqueous solution is 6.48×10 4 L·mol -1 cm -1 , indicating that it has strong light absorption ability. Figure 11 (B) and Figure 12 As shown in the figure, under excitation at the corresponding wavelength, BDP1-PNZ has almost no fluorescence emission, especially in aqueous solution, where its fluorescence quantum yield is less than 0.1%, indicating that the absorbed energy is almost completely released through non-radiative decay rather than fluorescence.

[0091] The fluorescence quantum yield was determined as follows:

[0092] Using indocyanine green (ICG, Φs = 0.132, ethanol, 298 K) as a reference, the relative fluorescence quantum yield was calculated using the following formula:

[0093] ;

[0094] Φ is the quantum yield, F is the integrated area of ​​the emission spectrum after correction, A is the absorbance at the excitation wavelength, λ ex is the excitation wavelength, the subscripts x and s represent the sample to be tested and the reference substance respectively, and n is the refractive index of the solvent. -6 -10 -7 mol / L), and the change in refractive index can be ignored.

[0095] (2) Test the generation of singlet oxygen by BDP1-PNZ in aqueous solution ( 1 O2) capabilities.

[0096] The singlet oxygen generation ability of BDP1-PNZ was detected by 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 BDP1-PNZ samples were added to cuvettes and then illuminated with a 716 nm laser (power density: 100 mW / cm 2 ) The samples were irradiated for different times (0-300 s), and the absorption spectra were recorded immediately after each irradiation.

[0097] like Figure 13 As shown in the figure, within 6 minutes of illumination, the characteristic absorption peak of 9,10-anthracenediyl-bis(methylene)dicarboxylic acid (ABDA) hardly changes, indicating that its singlet oxygen generation ability is extremely weak and can be ignored. It is known that organic phototherapeutic agents in the excited singlet state (S1) can release energy through three competing pathways: fluorescence radiation decay, non-radiative decay heat generation, and intersystem crossing (ISC) to the triplet state and possibly generate 1 O2 or other reactive oxygen species are used for photodynamic therapy. Combined with experimental data, it is speculated that BDP1-PNZ generates heat mainly through non-radiative transitions in aqueous solution.

[0098] (3) Test the photothermal performance of BDP1-PNZ.

[0099] Photothermal effect test method: Place a 10 μM compound aqueous solution (2.0 mL) in a quartz cuvette and irradiate it with laser light of different powers for 300 seconds (wavelengths as above), and record the temperature changes, such as Figure 14 (A) Figure 15 and Figure 17 As shown in (A);

[0100] Aqueous solutions (2.0 mL) of compounds with different concentrations (0, 10, 25 μM) were placed in a quartz cuvette and irradiated with 0.5 W / cm 2Laser (BDP1-PNZ: 716 nm; BDP1-NEt2: 808 nm) was irradiated for 300 s, and the temperature changes were recorded, e.g. Figure 14 (B) Figure 16 and Figure 17 As shown in (B).

[0101] In order to highlight the structural advantages of BDP1-PNZ, the heating curves of BDP1-PNZ of Example 1 and BDP1-NEt2 of Control Example 1 at different power densities and concentrations were compared. Figure 14 (A) and Figure 15 As shown in the figure, when the laser power density is increased from 0.1 W / cm 2 Increased to 1.0 W / cm 2 When , the temperature of BDP1-PNZ solution increased from 25℃ to 46℃ (△T = 21℃), while that of BDP1-NEt2 solution only increased from 25.2℃ to 31.2℃ (△T = 6℃). Figure 14 (B) and Figure 16 As shown in Figure 2, the temperature rise curve and infrared thermal imaging of BDP1-PNZ both show that its temperature change is directly related to its concentration. Figure 14 (B) and Figure 17 As shown in the figure, at a concentration of 25 μM, the final temperature of the BDP1-PNZ solution can reach above 55°C (△T≈34°C), while the BDP1-NEt2 solution with the same concentration only reaches 37.1°C (△T≈13°C), indicating that BDP1-PNZ with the introduction of PNZ groups has stronger photothermal conversion ability in aqueous solution.

[0102] Photothermal conversion efficiency (PCE) is a key parameter for evaluating the photothermal performance of molecules. The PCE (η) is calculated as follows: The temperature of the compound solution rises to a steady state (5 minutes) under continuous laser irradiation. The PCE (η) is calculated using the following formula:

[0103] ;

[0104] Where h is the heat transfer coefficient, A is the surface area of ​​the cuvette, and T max is the maximum steady-state temperature, T surr is the ambient temperature, Q dis is the heat dissipation generated by the solvent and the cuvette after absorbing light, I is the incident laser power, A 716 and A 808 is the absorbance of the sample at the laser wavelength (716 nm or 808 nm). The value of hA is calculated using the following formula:

[0105] ;

[0106] Where τ is the system heat transfer time constant, mD and c D are the mass and specific heat of the solution respectively. dis Calculated by the following formula:

[0107] ;

[0108] The experimental measurement of DMOS Q dis 0.0715W (716 nm laser), Q in water dis 0.179 W (716 nm laser) and 0.139 W (808 nm laser), T max(Solution) Refers to the highest temperature reached by the solution. Based on the above data and formula, calculate the photothermal conversion efficiency of each compound.

[0109] By comparing BDP1-PNZ and BDP1-NEt2 at 0.5W / cm 2 Heating curve under laser irradiation (such as Figure 18 As shown in the figure), combined with the relationship curve between the cooling time and the negative natural logarithm of the temperature (-ln(θ)) in the cooling stage (as shown in the figure), Figure 19 As shown in Figure 2), the PCE of BDP1-PNZ in aqueous solution is calculated to be 40.9%, which is significantly higher than the 9.2% of BDP1-NEt2. Figure 20 As shown in (B), BDP1-PNZ exhibits more stable temperature changes during three heating and cooling cycles. Figure 20 (A) and Figure 20 As shown in (B), its photothermal imaging also intuitively shows the cyclic stability, confirming that BDP1-PNZ has excellent photostability after structural modification.

[0110] (4) Test the water solubility of BDP1-PNZ.

[0111] The high photothermal conversion efficiency of BDP1-PNZ is closely related to its water solubility. Figure 21 As shown in Figure 2, the solubility of BDP1-PNZ is significantly improved compared to the hydrophobic and easily aggregated BDP1-NEt2. Figure 22 As shown in (A), the Lambert-Beer law is followed in a wide concentration range of 5-60 μM. Figure 22 As shown in (B), BDP1-NEt2 complies only in the range of 5-18 μM.

[0112] The above photophysical properties test results show that BDP1-PNZ is a potential photothermal agent with high photothermal conversion efficiency and strong and stable photothermal performance. It also verifies that the PNZ group plays an important role in improving the performance of single-molecule photothermal agents.

[0113] 2. In vitro photothermal performance test.

[0114] (1) Dark cytotoxicity evaluation of BDP1-PNZ.

[0115] Cell Culture: Human breast cancer 4T1 cells were cultured in RPMI 1640 medium (GIBCO) supplemented with 10% fetal bovine serum (HyClone), 100 U / mL penicillin, and 100 μg / mL streptomycin (HyClone). MCF10A cells were cultured in mammary epithelial cell basal medium (Lonza) supplemented with the Endothelial Cell Growth Medium BulletKit (Lonza). All cells were cultured in a humidified incubator at 37°C and 5% CO2.

[0116] Dark toxicity detection by CCK-8 method: Human breast cancer 4T1 cells (5000 per well) were seeded in 96-well plates and treated in 1640 complete culture medium at 37°C for 12 hours. Fresh culture medium with gradient concentrations (0-50μM) of BDP1-PNZ was then added to the well plate and the treatment continued for 36 hours. After removing the culture medium, the cells were carefully washed with PBS buffer. 100μL of 10-fold diluted CCK-8 solution (Cell Counting Kit-8, BIOMIKY) was added to each well and treated at 37°C and 5% CO2 for 20 minutes. After shaking for 5 minutes, the absorbance at 450nm was measured using a microplate reader (Multiskan Sky). Figure 23 As shown, after 4T1 cells were co-treated with different concentrations of BDP1-PNZ for 36 hours, the cell survival rate was still over 95% at a concentration of 50 μM, indicating good biocompatibility.

[0117] (2) Evaluation of the photothermal toxicity (photocytotoxicity) of BDP1-PNZ under laser irradiation.

[0118] CCK-8 assay for photothermal toxicity: 4T1 cells (5000 cells per well) were seeded into 96-well plates and treated in 1640 complete medium at 37°C for 12 hours. Fresh medium was then added with gradient concentrations of BDP1-PNZ (0-10 μM) and the treatment continued for 12 hours. In the experimental group (i.e., laser irradiation), cells were irradiated with a 716 nm laser (power density: 0.5 W / cm 2 ) for 10 minutes, followed by 12 hours in the dark (24 hours total). A control group (i.e., cells not irradiated with laser light) was treated in the dark for 24 hours under the same conditions. Subsequent steps were identical to those for the dark toxicity assay.

[0119] The experiment was conducted by setting up non-irradiated group and irradiated group, such as Figure 24As shown in (B), cells treated with different concentrations of BDP1-PNZ were irradiated with low power density laser (716 nm, 0.5 W / cm 2 ) After 10 minutes, the cell survival rate decreased significantly with increasing concentration, while the non-irradiated group treated in the dark showed no toxicity. As a control experiment, the PTT effect of BDP1-NEt2 in 4T1 cells was also evaluated. Figure 24 (A) In the figure, under the same irradiation conditions (BDP1-NEt2 was treated with 808nm laser irradiation), BDP1-NEt2 did not show significant phototoxicity, and its cell survival rate was similar to that of the non-irradiated group treated with light protection. This may be due to insufficient laser power density and short irradiation time, which failed to fully demonstrate the PTT effect of BDP1-NEt2.

[0120] In addition, power density dependence experiments of BDP1-PNZ in 4T1 cells were performed, as shown in Figure 25 As shown in the figure, when the BDP1-PNZ concentration was fixed at 10 μM, the cell survival rate decreased with increasing laser power. A PBS blank control group was also set up in the experiment, and no obvious cytotoxicity was observed under the same irradiation conditions.

[0121] (3) Evaluation of the photothermal therapeutic effect of BDP1-PNZ (staining experiment).

[0122] Staining experimental method: about 1×10 5 4T1 cells were seeded in glass-bottomed culture dishes and cultured overnight. The cells were treated with BDP1-NEt2 and BDP1-PNZ for 6 hours. BDP1-PNZ (using a 716 nm laser) and BDP1-NEt2 (using an 808 nm laser) were irradiated for 10 minutes and then cultured for 24 hours. The cells were stained with Calcein-AM and propidium iodide (PI) for 30 minutes, and fluorescence signals were recorded using confocal laser scanning microscopy (CLSM).

[0123] To directly evaluate the photothermal therapeutic effect of BDP1-PNZ, 4T1 cells were stained with Calcein-AM and propidium iodide (PI). Figure 26 As shown, two groups of cells were treated with the same concentration of BDP1-PNZ, with one group receiving laser irradiation. The results showed that only when BDP1-PNZ and laser irradiation were present simultaneously did the cells undergo extensive cell death and exhibit bright red fluorescence, confirming the phototoxicity findings. As a control, studies of the cellular PTT effect of BDP1-NEt2 revealed that, regardless of whether or not they were exposed to light, the cells emitted strong green fluorescence, with no significant difference between the groups, indicating that the cells were intact. These experiments confirm the excellent PTT performance of BDP1-PNZ at the cellular level and highlight its significant application potential.

[0124] (4) Testing the ability of BDP1-PNZ to produce reactive oxygen species (ROS) at the cellular level.

[0125] Intracellular reactive oxygen species (ROS) detection method: about 5×10 5 4T1 cells were seeded in glass-bottomed culture dishes and cultured at 37°C overnight. The cells were treated with 10 μM BDP1-PNZ for 6 hours and then treated with 2 μM ROS probe 2,7-dichlorofluorescein diacetate (DCFH-DA) for 30 minutes. After washing with PBS, the cells were illuminated with a 716 nm laser (power density: 0.5 W / cm 2 ) for 10 min, and the fluorescence signals were immediately recorded using a confocal laser scanning microscope (CLSM, excitation wavelength 488 nm, emission wavelength 500-550 nm).

[0126] Specifically, the 2,7-dichlorofluorescein diacetate (DCFH-DA) probe was used to detect the ability of BDP1-PNZ to produce reactive oxygen species (ROS) at the cellular level. Three groups were set up in the experiment: a PBS-cultured control group, a BDP1-PNZ light-protected group (no laser irradiation), and a BDP1-PNZ light-exposed group (laser irradiation). Figure 27 and Figure 28 As shown, the fluorescence signal intensities of the three groups were similar, indicating that BDP1-PNZ's ability to generate ROS after illumination is negligible. This result is consistent with the solution experiment, further confirming that the energy absorbed by BDP1-PNZ is mainly released as heat energy, achieving efficient energy focusing and significant therapeutic effects.

[0127] 3. In vivo photothermal performance test.

[0128] To further test the photoacoustic imaging (PAI) potential of BDP1-PNZ, a mouse tumor model was established: 5-6 week-old female Balb / c mice were housed under a normal light-dark cycle for 12 hours (with free access to food and water). A 4T1 tumor-bearing mouse model was established by subcutaneously injecting approximately 1×10^6 cells (100 μL suspension) into the left flank of male BALB / c mice. After seven days, the tumor volume grew to 70-100 mm. 3 , and the model was subsequently used for in vivo PAI experiments.

[0129] (1) In vivo photoacoustic imaging

[0130] When the tumor volume grows to 70-100 mm 3At 4 hr, 50 μL of BDP1-PNZ or ICG (0.5 mM in PBS containing 0.1% Tween 80, pH 7.4) was injected intratumorally. Photoacoustic images were acquired before and at different time points after injection (0, 1, 3, 6, 9, and 12 h) and the signal intensity was quantified. System parameters: frequency = 40 MHz, ultrasonic gain = 10 dB, photoacoustic gain = 40 dB. Since indocyanine green (ICG) is an organic dye currently widely used in in vivo PAI studies, it was selected as a control to evaluate the PAI ability of BDP1-PNZ. The mice were divided into two groups and injected intravenously with BDP1-PNZ and ICG, respectively, and then photoacoustic imaging was performed under 716 nm and 770 nm light excitation for more than 12 h. Figure 29 As shown, the PA signal (photoacoustic signal) of 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 a 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: the PA signal intensity of the ICG group reached a peak 3 hours after injection, while that of the BDP1-PNZ group reached the maximum value within about 1 hour, confirming the potential of BDP1-PNZ in rapid tumor diagnosis.

[0131] (2) In vivo photothermal imaging

[0132] The in vivo photothermal effect of BDP1-PNZ was evaluated by infrared thermal imaging. 4T1 tumor-bearing mice were injected with BDP1-PNZ and then irradiated with a 716 nm laser (0.5 W / cm 2 ) irradiated the tumor area, and the temperature changes were recorded every 1 minute. The PBS-injected group served as a control.

[0133] The body temperature of mice was monitored in real time using an infrared thermal imager. Figure 31 and Figure 32 As shown in the in vivo photothermal imaging, the temperature of the tumor area in the laser group (PBS+laser) increased by only △T≈1.5℃ within 3 minutes, indicating that 0.5W / cm 2 Continuous 716nm laser irradiation did not induce 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, which is high enough to ablate cancer cells, demonstrating the effective photothermal conversion ability of BDP1-PNZ in vivo.

[0134] (3) In vivo phototherapy trial

[0135] When the tumor volume reaches approximately 100 mm 3The 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 sample (0.5 mM) through the tail vein. One hour later, the mice were treated with 716 nm laser (0.5 W / cm 2 ) for 10 minutes. Tumor volume (length and width) and body weight were measured every two days after treatment, and tumors were photographed two weeks later.

[0136] like Figure 33 and Figure 34 As shown, treatment with PBS, PBS + laser, and BDP1-PNZ failed to inhibit tumor growth, with average tumor volume increasing approximately 8-9 times, indicating negligible efficacy in these groups. In contrast, the BDP1-PNZ + laser group demonstrated a strong tumor suppression effect, with tumors significantly suppressed and completely eliminated without recurrence. Furthermore, mice in all control and experimental groups showed a slow increase in body weight, indicating that treatment did not induce systemic toxic effects. These results fully demonstrate the excellent in vivo photothermal activity of BDP1-PNZ under light conditions.

[0137] (4) Histological and hematological analysis

[0138] Experimental methods: Blood samples were collected from mice on the 14th day of treatment for serum biochemistry and routine blood tests. Tumors and major organs (heart, liver, spleen, lungs, and kidneys) were taken, fixed with 4% formaldehyde overnight, and paraffin-embedded to prepare 5μm sections for observation. The body weights of all mice in the experimental and control groups did not change, indicating that these therapies did not induce systemic adverse reactions in mice. To evaluate the toxicity of BDP1-PNZ, the major organ tissues (heart, liver, spleen, lungs, and kidneys) of mice euthanized after treatment were tested. No pathological tissue damage or abnormalities (such as Figure 35 As shown in Figure 2, it was shown that BDP1-PNZ has good biosafety. Further, the blood of the treated mice was analyzed and compared with that of the control group. Figure 36 As shown in (A), BDP1-PNZ showed no hemolysis even at a concentration of 200 μM. Compared with the untreated group, the detailed biochemical indexes of the treated group (including liver and kidney function indicators) showed no significant differences, indicating that it had no significant hepatotoxicity ( Figure 36 (B)) and nephrotoxicity ( Figure 36 (C) After BDP1-PNZ combined with laser treatment, the mice's blood routine analysis parameters remained within normal ranges. These results suggest that the in vivo use of BDP1-PNZ does not pose a risk to the body.

[0139] In summary, the single-molecule photothermal agent BDP1-PNZ with high photothermal conversion efficiency of the present invention has 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 due to the introduction of the PNZ group, which can be summarized into three core factors: (1) The introduction of the PNZ group produces steric hindrance due to the bending of the fluorophore bracket, further aggravating the overall distortion of the molecule; (2) The significant transition from the twisted conformation to the extended conformation in the excited state. This conformational change and the related internal conversion process are crucial to 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 light excitation, thereby forming a low-lying dark electron transfer state. The efficient formation of these low-lying dark electron transfer states underlies the enhanced photothermal performance of BDP1-PNZ, and their formation process also enhances the photostability of the photothermal agent. By leveraging its exceptional energy release properties, BDP1-PNZ exhibits exceptional cell-killing efficiency. Importantly, BDP1-PNZ successfully overcomes the limitations of conventional organic small-molecule photothermal agents, which rely on amphiphilic polymer coatings. BDP1-PNZ can be directly applied as a single molecule to in vitro and in vivo systems, avoiding the potential immune escape associated with the introduction of polymers while maintaining high photothermal conversion efficiency due to the enhanced solubility and non-radiative transitions brought about by the PNZ group. Under the guidance of photothermal and photoacoustic imaging, BDP1-PNZ can precisely target tumors and achieve excellent tumor ablation effects via 718nm light irradiation. It can be applied to low-temperature photothermal therapy for triple-negative breast cancer while exhibiting good biocompatibility. BDP1-PNZ holds great promise as a versatile and highly effective photothermal therapeutic agent.

[0140] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by 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. The method for preparing a single-molecule photothermal agent with high photothermal conversion efficiency according to claim 1, wherein: The following steps are involved: 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 stirred and warmed to room temperature. 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 were dissolved in dichloromethane, stirred in an ice bath, phosphorus oxychloride was added, and the temperature was raised to room temperature with stirring. Boron trifluoride etherate complex and triethylamine were added, and stirring was continued. After extraction, the organic phases were combined, 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 were dissolved in toluene, heated and stirred for reaction, and the reaction solution was cooled to room temperature. The toluene was removed under reduced pressure, and the residue was extracted with dichloromethane. The organic phases were 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.

3. The method for preparing a single-molecule photothermal agent with high photothermal conversion efficiency according to claim 2, characterized in that: In step S1, the molar ratio of 2,4-dimethylpyrrole to trifluoroacetic anhydride is 1:(1-2).

4. The method for preparing a single-molecule photothermal agent with high photothermal conversion efficiency according to claim 2, characterized in that: In the step S2, the molar ratio of compound X1 to 2,4-dimethylpyrrole is 1:(1-2).

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

6. The method for preparing a single-molecule photothermal agent with high photothermal conversion efficiency according to claim 2, characterized in that: In step S3, the molar ratio of phenoxazine to 4-bromobenzaldehyde is 1:(1-2).

7. The method for preparing a single-molecule photothermal agent with high photothermal conversion efficiency according to claim 2, characterized in that: In 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.

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

9. The method for preparing a single-molecule photothermal agent with high photothermal conversion efficiency according to claim 2, characterized in that: In 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 claimed in claim 1 in the preparation of anti-triple-negative breast cancer drugs.

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