Tumor diagnosis and treatment agent with targeting function and preparation method thereof

Abstract

The invention provides a tumor diagnosis and treatment agent with a targeting function and a preparation method thereof, and belongs to the field of biomedicine. According to the method, triphenylamine is taken as a starting unit, a pi conjugated system is expanded through a benzothiadiazole group, pyridinium salt is introduced as a strong electron withdrawing unit, alkyl chains with different lengths are connected into molecules, and finally, diagnosis and treatment integrated molecules with fluorescence imaging and tumor targeting functions are synthesized. The fluorescent targeted diagnosis and treatment agent not only has good tumor cell plasma membrane targeting accuracy, but also has excellent aggregation-induced emission performance which is obviously higher than that of a traditional commercial photosensitizer, so that accurate targeted localization of tumor cells is realized. Meanwhile, the fluorescent targeting diagnosis and treatment agent TBPPM has excellent singlet oxygen generation capability, and further shows relatively strong tumor cell killing capability. Therefore, the prepared fluorescent targeted diagnosis and treatment agent provides a new material for accurate positioning and treatment of tumors under fluorescence guidance, and also provides a new thought for development of targeted diagnosis and treatment agents for breast cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a tumor diagnostic and therapeutic agent with targeted function and its preparation method. Background Technology

[0002] Malignant tumors are a major public health issue and one of the leading causes of death worldwide. Over the past four decades, the lack of precise diagnostic methods and effective treatments has prevented significant improvement in cancer mortality rates. Current mainstream treatment options include surgical resection and drug therapy, but these still face numerous challenges such as drug resistance and adverse reactions. Meanwhile, individual variability in immunotherapy leads to delays in evaluating treatment efficacy. These factors collectively limit the improvement of cancer treatment outcomes. Therefore, there is an urgent need for highly sensitive monitoring methods and innovative treatment strategies to improve clinical efficacy while reducing side effects.

[0003] Currently, near-infrared fluorescence imaging (NIR) is increasingly becoming an indispensable diagnostic tool due to its advantages such as low cost, non-invasiveness, high sensitivity, and real-time imaging. However, traditional organic fluorescent probes such as rhodamine, fluorescein, and coumarin are prone to generating strong non-specific background signals in organic environments. More importantly, when a large number of traditional fluorophores with aromatic and planar chemical structures accumulate at the target site, false negative results often occur due to fluorescence attenuation or quenching. To address this problem of "aggregation leading to quenching," Academician Tang Benzhong's team proposed the concept of aggregation-induced emission (AIE) in 2001, utilizing the mechanism of significantly enhanced fluorescence due to the restricted rotation of molecules and the vibration of group motion within the aggregated state. Compared to traditional phosphors, AIE luminescent agents possess excellent photophysical properties, such as bright fluorescence in the aggregated or solid state, large Stokes shift, good photostability, and long retention time in live samples. In addition, AIE photosensitizers can also enhance the generation efficiency of reactive oxygen species (ROS) in the aggregated state by increasing the intersystem traversal rate. Therefore, bioprobes based on AIE luminescent agents have been widely used in the construction of integrated diagnosis and treatment systems for tumors.

[0004] As a method of treating cancer and other diseases through light modulation, photodynamic therapy (PDT) combines photosensitizers, light, and oxygen accumulated at the disease site to generate toxic reactive oxygen species (ROS), thereby killing cancer cells and destroying diseased tissue. Its mechanism of action is as follows: under light irradiation, the photosensitizer is excited to a singlet excited state, subsequently producing fluorescence via radiation or transitioning to a triplet state via intersystemic crossover (ISC), and then reacting with surrounding oxygen or substrates to generate ROS. As a highly toxic substance, ROS can cause diseased tissue death by inducing apoptosis or necrosis, damaging blood vessels, and stimulating immune responses. Further research shows that ROS has an extremely short half-life and a limited diffusion distance (<20 nm) in biological systems, making it difficult to migrate long distances to the target site before rapidly degrading and becoming ineffective. Due to its limited diffusion capacity and lifespan, the effective action of ROS is strictly confined to the vicinity of its generation site, giving photodynamic therapy a high degree of spatial controllability. Therefore, to ensure therapeutic efficacy, the generation process of ROS must be precisely controlled within the target area to cause lethal oxidative damage locally. Meanwhile, the short radius of action and transient nature of ROS help to strictly limit its destructive effects to cancer cells or microorganisms exposed to specific light, thereby minimizing damage to adjacent normal tissues and significantly improving the selectivity and accuracy of treatment. Based on these characteristics, PDT has been widely regarded as a highly efficient and non-invasive disease treatment strategy.

[0005] The cell membrane, a crucial interface composed of a phospholipid bilayer, not only separates the intracellular and extracellular environments, serving as the primary barrier for substance exchange, but also forms the basis for maintaining cell morphological integrity and functional stability, making it a highly promising therapeutic target. Specific targeting tumor therapeutic agents designed for the tumor cell membrane can efficiently generate reactive oxygen species (ROS) locally on the membrane under light exposure, directly oxidizing cholesterol and unsaturated phospholipids in the membrane structure, disrupting membrane permeability, fluidity, and structural integrity, ultimately leading to rapid cell death. This membrane-targeting strategy can be considered to not only directly disrupt the survival barrier of tumor cells but also significantly reduce off-target toxicity to normal cells due to the specificity of its action site, providing new possibilities for developing highly effective and low-toxicity tumor treatments. Summary of the Invention

[0006] This specification aims to provide a tumor therapeutic agent with targeting function and its preparation method. The fluorescent targeted therapeutic agent TBPPM prepared using this method not only exhibits excellent precision in targeting tumor cell plasma membranes, but also demonstrates significantly superior aggregation-induced emission performance compared to traditional commercial photosensitizers, thereby enabling precise targeting of tumor cells. Simultaneously, the prepared fluorescent targeted therapeutic agent possesses excellent singlet oxygen generation capability, exhibiting strong tumor cell killing efficacy. It can be considered that the newly prepared fluorescent targeted therapeutic agent TBPPM provides a novel material for the precise localization and treatment of tumor cells under fluorescence image guidance, and also offers new insights for the development of targeted diagnostic and therapeutic platforms for breast cancer.

[0007] Experimental steps

[0008] A tumor therapeutic agent with targeting function and its preparation method are characterized by the fluorescent targeted therapeutic agent having high targeting and killing ability of breast cancer cells. The specific synthesis method includes the following steps:

[0009] (1) Synthesis of Intermediate I: 1.5–2.5 g of 4,7-dibromo-2,1,3-benzothiadiazole, 10–50 mg of potassium carbonate, and 40–80 mg of tetra(triphenylphosphine)palladium were dissolved sequentially in 20–40 mL of tetrahydrofuran solution. Then, 1.1–1.7 g of triphenylamine 4-borate was added to the solution, and the reaction mixture was heated at 60–120 °C for 8–16 h. After the reaction was complete and the mixture was cooled to room temperature, the reaction solution was extracted with ethyl acetate and washed with distilled water. Finally, the product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1–15:1, v / v) to obtain 1.1–1.7 g of Intermediate I.

[0010] (2) Synthesis of TBP: Under nitrogen protection, 1.1–1.7 g of intermediate I, 350–750 mg of pyridine 4-borate, 1.2–1.8 g of potassium carbonate, and 80–160 mg of dichlorobis(1,1'-bis(diphenylphosphine)ferrocene)palladium were successively added to a mixed solvent of toluene and methanol in 20–40 mL, and reacted at 100–150 °C for 8–16 h. After the reaction was completed, the reaction solution was extracted with ethyl acetate, washed with distilled water, dried over anhydrous sodium sulfate, and dried under reduced pressure. Finally, the obtained product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1–15:1, v / v) to obtain 0.6–1.2 g of TBP.

[0011] (3) Synthesis of TBP-1: At 70–100 °C, 0.6–1.2 g of TBP solution was slowly added dropwise to 20–30 mL of acetonitrile solution containing 1.6–2.1 g of 1,2-bis(2-iodoethoxy)ethane, and the mixture was stirred at this temperature for 8–16 h. Subsequently, the reaction solution was cooled to room temperature, and the solvent was removed by concentration under reduced pressure. Finally, the solution was purified by silica gel column chromatography (dichloromethane:methanol = 15:1–25:1, v / v) to obtain 0.6–1.2 g of TBP-1.

[0012] (4) Synthesis of TBP-2: 0.6–1.2 g of compound TBP-1 was dissolved in 5–10 mL of acetonitrile, and 1.8–2.6 g of triethylamine was added to the reaction solution. The mixture was stirred at 80–160 °C for 8–16 h. After the reaction was completed and cooled to room temperature, 0.6–1.2 g of TBP-2 was obtained by concentration under reduced pressure.

[0013] (5) Synthesis of TBPPM: 0.6–1.2 g of compound TBP-2 was dissolved in a mixture of 10–20 mL of acetone and methanol, and 83.5–167 g of potassium hexafluorophosphate was slowly added to the reaction solution. The reaction mixture was then stirred at 80–160 °C for 10–15 h. After the reaction was complete, the mixture was cooled to room temperature and concentrated under reduced pressure to obtain 0.6–1.2 g of the tumor cell membrane-targeting photosensitive probe TBPPM.

[0014] Furthermore, in step (1), the concentration of 4,7-dibromo-2,1,3-benzothiadiazole is 8–26.67 g / mL, and the mass ratio of potassium carbonate, tetra(triphenylphosphine)palladium, and triphenylamine 4-borate to 4,7-dibromo-2,1,3-benzothiadiazole is 0.004:0.016:0.44:1–0.033:0.053:1.133:1.

[0015] Furthermore, in step (2), the volume ratio of toluene to methanol is 1:1 to 1:5, and the concentration of intermediate I is 11.76 to 36.36 g / mL.

[0016] Furthermore, in step (2), the mass ratio of pyridine 4-borate, potassium carbonate, and palladium dichlorobis(1,1'-bis(diphenylphosphino)ferrocene) to intermediate I is 0.21: 0.71: 0.005:1 to 0.69: 1.64: 0.15:1.

[0017] Furthermore, in step (3), the concentration of TBP is 16.67 to 50.0 g / mL, and the mass ratio of TBP to 1,2-bis(2-iodoethoxy)ethane is 1.3:1 to 3.5:1.

[0018] Furthermore, in step (4), the concentration of TBP-1 is 4.17 to 10.67 g / mL, and the mass ratio of TBP-1 to triethylamine is 1.5:1 to 4.4:1.

[0019] Furthermore, in step (5), the volume ratio of acetone to methanol is 1:1 to 1:5, the concentration of TBP-2 is 8.4 to 33.4 g / mL, and the mass ratio of potassium hexafluorophosphate to TBP-2 is 69.6:1 to 278.5:1.

[0020] Furthermore, the fluorescent targeted therapeutic agent TBPPM not only has good precision in targeting tumor cell plasma membranes, but also has superior aggregation-induced emission performance that is significantly higher than that of traditional commercial photosensitizers, thereby achieving precise targeting of tumor cells.

[0021] Furthermore, the fluorescent targeted therapeutic agent TBPPM has excellent singlet oxygen generation capability, thereby exhibiting strong tumor cell killing ability.

[0022] Furthermore, the fluorescent targeted therapeutic agent TBPPM provides a new material for the precise localization and treatment of tumor cells under fluorescence imaging guidance, and also provides new ideas for the development of a targeted diagnosis and treatment platform for breast cancer.

[0023] A tumor therapeutic agent with targeting function and its preparation method are characterized by the fluorescent targeted therapeutic agent having high targeting and killing ability of breast cancer cells. The specific synthesis method includes the following steps: Synthesis of Intermediate I: 1.5–2.5 g of 4,7-dibromo-2,1,3-benzothiadiazole, 10–50 mg of potassium carbonate, and 40–80 mg of tetra(triphenylphosphine)palladium were sequentially dissolved in 20–40 mL of tetrahydrofuran solution. Next, 1.1–1.7 g of triphenylamine 4-borate was added to the solution, and the reaction mixture was heated at 60–120 °C for 8–16 h. After the reaction was complete and the mixture was cooled to room temperature, the reaction solution was extracted with ethyl acetate and washed with distilled water. Finally, the product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1–15:1, v / v) to obtain 1.1–1.7 g of Intermediate I.

[0024] (2) Synthesis of TBP: Under nitrogen protection, 1.1–1.7 g of intermediate I, 350–750 mg of pyridine 4-borate, 1.2–1.8 g of potassium carbonate, and 80–160 mg of dichlorobis(1,1'-bis(diphenylphosphine)ferrocene)palladium were successively added to a mixed solvent of toluene and methanol in 20–40 mL, and reacted at 100–150 °C for 8–16 h. After the reaction was completed, the reaction solution was extracted with ethyl acetate, washed with distilled water, dried over anhydrous sodium sulfate, and dried under reduced pressure. Finally, the obtained product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1–15:1, v / v) to obtain 0.6–1.2 g of TBP.

[0025] (3) Synthesis of TBP-1: At 70–100 °C, 0.6–1.2 g of TBP solution was slowly added dropwise to 20–30 mL of acetonitrile solution containing 1.6–2.1 g of 1,2-bis(2-iodoethoxy)ethane, and the mixture was stirred at this temperature for 8–16 h. Subsequently, the reaction solution was cooled to room temperature, and the solvent was removed by concentration under reduced pressure. Finally, the solution was purified by silica gel column chromatography (dichloromethane:methanol = 15:1–25:1, v / v) to obtain 0.6–1.2 g of TBP-1.

[0026] (4) Synthesis of TBP-2: 0.6–1.2 g of compound TBP-1 was dissolved in 5–10 mL of acetonitrile, and 1.8–2.6 g of triethylamine was added to the reaction solution. The mixture was stirred at 80–160 °C for 8–16 h. After the reaction was completed and cooled to room temperature, 0.6–1.2 g of TBP-2 was obtained by concentration under reduced pressure.

[0027] (5) Synthesis of TBPPM: 0.6–1.2 g of compound TBP-2 was dissolved in a mixture of 10–20 mL of acetone and methanol, and 83.5–167 g of potassium hexafluorophosphate was slowly added to the reaction solution. The reaction mixture was then stirred at 80–160 °C for 10–15 h. After the reaction was complete, the mixture was cooled to room temperature and concentrated under reduced pressure to obtain 0.6–1.2 g of the tumor cell membrane-targeting photosensitive probe TBPPM.

[0028] Furthermore, in step (1), the concentration of 4,7-dibromo-2,1,3-benzothiadiazole is 8–26.67 g / mL, and the mass ratio of potassium carbonate, tetra(triphenylphosphine)palladium, and triphenylamine 4-borate to 4,7-dibromo-2,1,3-benzothiadiazole is 0.004:0.016:0.44:1–0.033:0.053:1.133:1.

[0029] Furthermore, in step (2), the volume ratio of toluene to methanol is 1:1 to 1:5, and the concentration of intermediate I is 11.76 to 36.36 g / mL.

[0030] Furthermore, in step (2), the mass ratio of pyridine 4-borate, potassium carbonate, and palladium dichlorobis(1,1'-bis(diphenylphosphino)ferrocene) to intermediate I is 0.21: 0.71: 0.005:1 to 0.69: 1.64: 0.15:1.

[0031] Furthermore, in step (3), the concentration of TBP is 16.67 to 50.0 g / mL, and the mass ratio of TBP to 1,2-bis(2-iodoethoxy)ethane is 1.3:1 to 3.5:1.

[0032] Furthermore, in step (4), the concentration of TBP-1 is 4.17 to 10.67 g / mL, and the mass ratio of TBP-1 to triethylamine is 1.5:1 to 4.4:1.

[0033] Furthermore, in step (5), the volume ratio of acetone to methanol is 1:1 to 1:5, the concentration of TBP-2 is 8.4 to 33.4 g / mL, and the mass ratio of potassium hexafluorophosphate to TBP-2 is 69.6:1 to 278.5:1.

[0034] Furthermore, the fluorescent targeted therapeutic agent TBPPM not only has good precision in targeting tumor cell plasma membranes, but also has superior aggregation-induced emission performance that is significantly higher than that of traditional commercial photosensitizers, thereby achieving precise targeting of tumor cells.

[0035] Furthermore, the fluorescent targeted therapeutic agent TBPPM has excellent singlet oxygen generation capability, thereby exhibiting strong tumor cell killing ability.

[0036] Furthermore, the fluorescent targeted therapeutic agent TBPPM provides a new material for the precise localization and treatment of tumor cells under fluorescence imaging guidance, and also provides new ideas for the development of a targeted diagnosis and treatment platform for breast cancer. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in the embodiments or prior art of this specification, the drawings used in the description of the embodiments or prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this specification. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0039] Figure 1The fluorescent targeted therapeutic agent TBPPM of this invention 1 H NMR spectrum; Figure 2 The fluorescent targeted therapeutic agent TBPPM of this invention 13 C NMR spectrum; Figure 3 The fluorescence spectrum of the fluorescent targeted therapeutic agent TBPPM of the present invention in DMSO / toluene mixed solutions with different toluene contents is shown below. Figure 4 This is a molecular orbital energy level diagram of the fluorescent targeted therapeutic agent TBPPM of this invention; Figure 5 The graph shows the change rate of absorbance of ABDA with irradiation time in the presence of the fluorescent targeted therapeutic agent TBPPM of the present invention, where A0 and A are the absorbance of ABDA at 378 nm before and after irradiation. Figure 6 This is the ultraviolet absorption spectrum of the photodegradation of ABDA by the fluorescent targeted therapeutic agent TBPPM of this invention; Figure 7 The relative change (I / I0-1) of the fluorescence intensity of HPF mixed with the fluorescent targeted therapeutic agent TBPPM of the present invention at 515 nm; Figure 8 The relative change (I / I0-1) in fluorescence intensity at 525 nm of the H2DCF-DA mixed solution mixed with the fluorescent targeted therapeutic agent TBPPM of the present invention. Figure 9 The activity of NIH-3T3 cells co-incubated with different concentrations of the fluorescent targeted therapeutic agent TBPPM of the present invention under dark conditions; Figure 10 Blood biochemistry and routine blood parameters of mice on days 0, 1, 7, and 21 after tail vein injection of TBPPM; Figure 11 H&E histological staining of major organ sections from mice injected with TBPPM via tail vein for 1, 7, and 21 days and normal mice; Figure 12 This is a fluorescence confocal imaging image of NIH-3T3 and 4T1 cells after incubation of the fluorescent targeted therapeutic agent TBPPM of the present invention with NIH-3T3 and 4T1 cells for different times; Figure 13 This is a laser confocal image of 4T1 cells after co-incubation with the fluorescent targeted therapeutic agents TBPPM (5 μmol / L) and Dio (5 μmol / L) of this invention, followed by co-staining at 37°C for 15 min; Mito Tracker Green and Dio: green channels; TBPPM and RBPMI: red channels; Figure 14The activity of 4T1 cells co-incubated with different concentrations of the fluorescent targeted therapeutic agent TBPPM of the present invention under light conditions; Figure 15 ROS levels in 4T1 cells were measured. The concentration of TBPPM was 5 μmol / L, the concentration of DCFH-DA was 10 μmol / L, and the power of white light was 100 mW / cm². 2 ; Figure 16 The effect of different light exposure times on the survival rate of 4T1 cells by the fluorescent targeted therapeutic agent TBPPM of this invention; Figure 17 These are fluorescence images of mouse tumors at different time points after intratumoral injection of the fluorescent targeted therapeutic agent TBPPM of the present invention. Figure 18 This image shows the tumor in mice after treatment with the fluorescent targeted therapeutic agent TBPPM of this invention. Detailed Implementation

[0040] To enable those skilled in the art to better understand the technical solutions in this specification, the technical solutions in the embodiments of this specification will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this specification, and not all embodiments. Based on the embodiments in this specification, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this specification.

[0041] Example 1: (1) Synthesis of Intermediate I: 1.7 g of 4,7-dibromo-2,1,3-benzothiadiazole, 20 mg of potassium carbonate, and 70 mg of tetra(triphenylphosphine)palladium were dissolved sequentially in 30 mL of tetrahydrofuran solution. Then, 1.3 g of triphenylamine 4-borate was added to the solution, and the reaction mixture was heated at 70 °C for 13 h. After the reaction was complete and the mixture was cooled to room temperature, the reaction solution was extracted with ethyl acetate and washed with distilled water. Finally, the crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 10:1, v / v) to obtain 1.7 g of Intermediate I; (2) Synthesis of TBP: Under nitrogen protection, 1.5 g of intermediate I, 650 mg of pyridine 4-borate, 1.4 g of potassium carbonate and 110 mg of dichlorobis(1,1'-bis(diphenylphosphine)ferrocene)palladium were added sequentially to a mixed solvent of toluene and methanol, and reacted at 100 °C for 10 h. After the reaction was completed, the reaction solution was extracted with ethyl acetate, washed with distilled water, dried over anhydrous sodium sulfate and vacuum dried. Finally, the crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 10:1, v / v) to obtain 1.1 g of TBP; (3) Synthesis of TBP-1: At 75 °C, 0.9 g of TBP solution was slowly added dropwise to 22 mL of acetonitrile solution containing 1.7 g of 1,2-bis(2-iodoethoxy)ethane, and the mixture was stirred at this temperature for 14 h. Subsequently, the reaction solution was cooled to room temperature, and the solvent was removed by concentration under reduced pressure. Finally, the solution was purified by silica gel column chromatography (dichloromethane:methanol = 20:1, v / v) to obtain 1.1 g of TBP-1; (4) Synthesis of TBP-2: 0.8 g of compound TBP-1 was dissolved in 7 mL of acetonitrile, and 1.9 g of triethylamine was added to the reaction solution. The mixture was stirred at 100 °C for 12 h. After the reaction was completed and cooled to room temperature, 1.1 g of TBP-2 was obtained by concentration under reduced pressure. (5) Synthesis of TBPPM: 0.9 g of compound TBP-2 was dissolved in a mixture of acetone and methanol in 15 mL, and 83.5 g of potassium hexafluorophosphate was slowly added to the reaction mixture. Then, the reaction mixture was stirred at 100 °C for 12 h. After the reaction was completed, the mixture was cooled to room temperature and concentrated under reduced pressure to obtain 0.8 g of the fluorescent targeted therapeutic agent TBPPM.

[0042] To verify the successful preparation of the fluorescent targeted therapeutic agent TBPPM, we used proton nuclear magnetic resonance spectroscopy (NMR spectroscopy). Figure 1 ) and carbon spectrum ( Figure 2 Structural characterization revealed that TBPPM, a fluorescent targeted therapeutic agent with good solubility in DMSO, had a maximum absorption of 491 nm, but no fluorescence. Figure 3 With increasing toluene volume fraction, the fluorescence intensity of the therapeutic agent TBPPM significantly increased. When the toluene content reached 99%, the fluorescence emission intensity of the therapeutic agent TBPPM increased 37-fold, indicating that TBPPM has good AIE performance. Density function theory (DFT) calculations at the TD-PBE0 / TZVP level showed that… Figure 4The HOMO and LUMO orbital distributions of TBPPM are concentrated on the TBP structure, indicating that the prepared therapeutic agent TBPPM has good AIE photophysical properties. Furthermore, the maximum emission wavelength of the therapeutic agent TBPPM in DMSO was calculated to be 624.69 nm, consistent with our actual test results, further demonstrating that TBPPM has good photophysical properties in the dispersed state.

[0043] Compared to its ROS, singlet oxygen ( 1 O2 exhibits higher selectivity and stronger oxidizing effect. Therefore, we used 9,10-anthracitedicarboxylo-bis(methylene)-dimalonic acid (ABDA) to test the efficacy of the therapeutic agent TBPPM under light irradiation. 1 O2 generation capacity, results as follows Figure 5 As shown. With prolonged illumination time, the indicator ABDA mixture mixed with the diagnostic agent TBPPM under illumination conditions (100 mW / cm²) showed [results]. 2 The characteristic absorption peak decreases rapidly. Meanwhile, we used the commercial photosensitizer Bengal rose red (RB) as a control to compare oxygen production capacity. As shown in Figure 6, RB in water... 1 The O2 generation efficiency is 75%, while the oxygen production rate of TBPPM is 232%, indicating that TBPPM has superior performance compared to commercial photosensitizers. 1 O2 generation capacity. Furthermore, we used different ROS indicators: HPF and H2DCF-DA, to evaluate the types of ROS generated by TBPPM under illumination. Figure 7 and Figure 8 As shown, the total ROS generation rate of TBPPM is higher than that of commercial RB, and ROS is generated at a rate of 1 O2 is the main component.

[0044] Next, we used a CCK-8 assay kit to investigate the biosafety of the preparation of the therapeutic agent TBPPM. For example... Figure 9 As shown, even when co-incubated with 10 μmol / L TBPPM, the survival rate of NIH-3T3 fibroblasts under light-protected conditions remained around 80%, indicating that the prepared therapeutic agent TBPPM had no significant cytotoxicity. Simultaneously, blood biochemistry and routine blood tests in mice after tail vein injection of the therapeutic agent TBPPM showed that both biochemical and routine indicators were within the normal range. Figure 10 This indicates that therapeutic concentrations of TBPPM have no significant toxicity or side effects in mice. Furthermore, H&E staining of organ tissue sections showed no significant differences between day 1, 7, and 21 after TBPPM injection and day 0. Figure 11Furthermore, the absence of obvious histopathological abnormalities or lesions indicates that TBPPM does not cause significant biological damage to mice. Therefore, our prepared therapeutic agent TBPPM exhibits good biocompatibility.

[0045] To verify the targeting of the therapeutic agent TBPPM to breast cancer tumor cells 4T1, we co-incubated the agent with 4T1 cells or normal cells (NIH-3T3) for 5 seconds to 30 minutes, and observed the uptake using confocal microscopy. Figure 12 As shown, 4T1 cells exhibit stronger red fluorescence than NIH-3T3 cells, indicating that 4T1 cells take up more TBPPM than NIH-3T3 cells. Further investigation into the localization region of TBPPM in the fluorescence image of 4T1 cells revealed that TBPPM illuminates the circular cell membrane and provides good contrast with the cell background. Furthermore, to further demonstrate the cell membrane localization of TBPPM, we performed co-staining using the commercial fluorescent probe Dio's Cell Plasma Membrane Staining Kit. The results in the fused image show (…). Figure 13 The Pearson correlation coefficient between the two is over 90%, indicating that TBPPM has high cell membrane targeting specificity.

[0046] The results of the cytotoxicity experiment showed that ( Figure 14 The survival rate of 4T1 cells incubated with TBPPM was only 7%, indicating that TBPPM showed a good therapeutic effect on 4T1 cells. Next, we assessed the level of ROS production by TBPPM in 4T1 cells by detecting the DCF fluorescence generated by the ROS indicator H2DCF-DA. Figure 15 As shown, the fluorescence signal in the cells gradually increased with prolonged exposure time, further demonstrating that TBPPM can effectively generate ROS. Next, we further verified the PDT killing effect of TBPPM on tumor cells using the Calcein-AM / PI live / dead cell double staining kit, and the results are as follows. Figure 16 As shown, after co-incubation with TBPPM and exposure to white light for 10 minutes, almost all 4T1 cells died, consistent with the results of CCK8, indicating that TBPPM is very effective in photodynamic destruction of cancer cells.

[0047] Benefiting from the excellent targeting and therapeutic properties of TBPPM in cell experiments, we further validated its diagnostic and therapeutic functions in tumor-bearing mice. Figure 17As shown, subcutaneously injected TBPPM near the tumor slowly diffused into the tumor area within 4 hours, and the fluorescence intensity within the tumor gradually increased over time. Even after 24 hours, red fluorescence was still clearly visible within the tumor, while other areas showed only weak fluorescence signals, indicating that TBPPM has excellent tumor imaging performance. Treatment results showed that tumor growth in the TBPPM + light irradiation group was significantly inhibited compared to the control group. Figure 18 This indicates that TBPPM has a good therapeutic effect on tumors.

Claims

1. A tumor diagnostic and therapeutic agent with targeted function and its preparation method, characterized in that, The method includes the following steps: (1) Synthesis of Intermediate I: 1.5–2.5 g of 4,7-dibromo-2,1,3-benzothiadiazole, 10–50 mg of potassium carbonate, and 40–80 mg of tetra(triphenylphosphine)palladium were dissolved sequentially in 20–40 mL of tetrahydrofuran solution. Then, 1.1–1.7 g of triphenylamine 4-boronate was added to the solution, and the reaction solution was heated at 60–120 °C for 8–16 h. After the reaction was complete and the mixture was cooled to room temperature, the reaction solution was extracted with ethyl acetate and washed with distilled water. Finally, the crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1–15:1, v / v) to obtain 1.1–1.7 g of Intermediate I. Intermediate I; (2) Synthesis of TBP: Under nitrogen protection, 1.1–1.7 g of intermediate I, 350–750 mg of pyridine 4-borate, 1.2–1.8 g of potassium carbonate, and 80–160 mg of dichlorobis(1,1'-bis(diphenylphosphine)ferrocene)palladium were successively added to a mixed solvent of toluene and methanol in 20–40 mL, and reacted at 100–150 °C for 8–16 h. After the reaction was completed, the reaction solution was extracted with ethyl acetate, washed with distilled water, dried over anhydrous sodium sulfate, and dried under reduced pressure. Finally, the crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1–15:1, v / v) to obtain 0.6–1.2 g of TBP. TBP; (3) Synthesis of TBP-1: At 70–100 °C, 0.6–1.2 g of TBP solution was slowly added dropwise to 20–30 mL of acetonitrile solution containing 1.6–2.1 g of 1,2-bis(2-iodoethoxy)ethane, and the mixture was stirred at this temperature for 8–16 h. The reaction mixture was then cooled to room temperature, and the solvent was removed by concentration under reduced pressure. Finally, the solution was purified by silica gel column chromatography (dichloromethane:methanol = 15:1–25:1, v / v) to obtain 0.6–1.2 g of TBP-1. TBP-1; (4) Synthesis of TBP-2: 0.6–1.2 g of compound TBP-1 was dissolved in 5–10 mL of acetonitrile. 1.8–2.6 g of triethylamine was added to the reaction mixture, and the mixture was stirred at 80–160 °C for 8–16 h. After the reaction was complete and cooled to room temperature, 0.6–1.2 g of TBP-2 was obtained by concentration under reduced pressure. TBP-2; (5) Synthesis of TBPPM: 0.6–1.2 g of compound TBP-2 was dissolved in a mixture of 10–20 mL of acetone and methanol, and 83.5–167 g of potassium hexafluorophosphate was slowly added to the reaction mixture. The reaction mixture was then stirred at 80–160 °C for 10–15 h. After the reaction was complete, the mixture was cooled to room temperature and concentrated under reduced pressure to obtain 0.6–1.2 g of the tumor cell membrane-targeting photosensitizing probe TBPPM. TBPPM.

2. The tumor diagnostic and therapeutic agent with targeted function and its preparation method as described in claim 1, characterized in that: In step (1), the concentration of 4,7-dibromo-2,1,3-benzothiadiazole is 8–26.67 g / mL, and the mass ratio of potassium carbonate, tetra(triphenylphosphine)palladium, and triphenylamine 4-borate to 4,7-dibromo-2,1,3-benzothiadiazole is 0.004:0.016:0.44:1–0.033:0.053:1.133:

1.

3. The tumor diagnostic and therapeutic agent with targeting function and its preparation method as described in claim 1, characterized in that: In step (2), the volume ratio of toluene to methanol is 1:1 to 1:5, and the concentration of intermediate I is 11.76 to 36.36 g / mL.

4. The tumor diagnostic and therapeutic agent with targeting function and its preparation method as described in claim 1, characterized in that: In step (2), the mass ratio of pyridine 4-borate, potassium carbonate, and palladium dichlorobis(1,1'-bis(diphenylphosphino)ferrocene) to intermediate I is 0.21: 0.71: 0.005:1 to 0.69: 1.64: 0.15:

1.

5. The tumor diagnostic and therapeutic agent with targeting function and its preparation method as described in claim 1, characterized in that: In step (3), the concentration of TBP is 16.67 to 50.0 g / mL, and the mass ratio of TBP to 1,2-bis(2-iodoethoxy)ethane is 1.3:1 to 3.5:

1.

6. The tumor diagnostic and therapeutic agent with targeted function and its preparation method as described in claim 1, characterized in that: In step (4), the concentration of TBP-1 is 4.17 to 10.67 g / mL, and the mass ratio of TBP-1 to triethylamine is 1.5:1 to 4.4:

1.

7. The tumor diagnostic and therapeutic agent with targeting function and its preparation method as described in claim 1, characterized in that: In step (5), the volume ratio of acetone to methanol is 1:1 to 1:5, the concentration of TBP-2 is 8.4 to 33.4 g / mL, and the mass ratio of potassium hexafluorophosphate to TBP-2 is 69.6:1 to 278.5:

1.

8. The tumor diagnostic and therapeutic agent with targeting function prepared by the preparation method according to claim 1, characterized in that: The fluorescent targeted therapeutic agent TBPPM not only has good precision in targeting tumor cell plasma membranes, but also has superior aggregation-induced emission performance that is significantly higher than that of traditional commercial photosensitizers, thereby achieving precise targeting of tumor cells.

9. The tumor diagnostic and therapeutic agent with targeting function prepared by the preparation method according to claim 1, characterized in that: The fluorescent targeted therapeutic agent TBPPM has excellent singlet oxygen generation capacity, thus exhibiting strong tumor cell killing ability.

10. The tumor diagnostic and therapeutic agent with targeting function prepared by the preparation method according to claim 1, characterized in that: The fluorescent targeted therapeutic agent provides new materials for the precise localization and treatment of tumor cells under fluorescence imaging guidance, and also provides new ideas for the development of a targeted diagnosis and treatment platform for breast cancer.

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