Tumor targeting photothermal reagent based on fluorene rhodamine as well as preparation method and application of tumor targeting photothermal reagent

Abstract

The invention belongs to the field of fluorescent probes, and particularly relates to a tumor targeting photo-thermal reagent based on fluorene rhodamine as well as a preparation method and application of the tumor targeting photo-thermal reagent. In order to solve the problems that most of existing photo-thermal reagents are difficult in post-modification due to complex synthesis and poor in biocompatibility due to large molecular weight, and the molecules are generally required to be prepared into nano-particles and then enriched to tumor parts by utilizing an EPR effect due to lack of tumor targeting property, the existing photo-thermal reagents are generally required to be prepared into nano-particles. The tumor targeting photothermal reagent Tag-880-NHS based on fluorene rhodamine is developed, has the characteristics of simple synthesis, good water solubility, good light stability, high photothermal conversion efficiency, absorption wavelength in an NIR region and the like, can be used for marking a monoclonal antibody-panitumab of a specific targeting epidermal growth factor, can be applied to the fields of tumor imaging and treatment, and has broad application prospects. The important application value is realized.

Description

Technical Field

[0001] The present invention belongs to the field of fluorescent probes, and particularly relates to a tumor-targeted photothermal reagent based on fluororhodamine, its preparation method and application. Background Art

[0002] Photothermal therapy (PTT) is a non-invasive tumor treatment method that uses a photothermal agent (PTA) to locally heat the tumor tissue site under the irradiation of an external light source, thereby killing tumor cells. During the treatment process, the fluorescence or photoacoustic signals generated during illumination can be monitored to guide and assist the treatment in a multimodal imaging manner. Compared with other tumor treatment methods, photothermal therapy has the advantages of non-invasiveness, low toxicity, and high spatiotemporal resolution. The key and core of photothermal therapy is the photothermal agent, and its performance can be evaluated by parameters such as absorption / emission wavelength and photothermal conversion efficiency (PCE). Near-infrared light (NIR 700-1700nm) has strong tissue penetration, low tissue damage, and high spatiotemporal resolution. Photothermal agents with absorption / emission wavelengths in the NIR region have more advantages during photothermal therapy; PCE refers to the efficiency of converting light energy into heat energy and is an important indicator for measuring the quality of photothermal agents. Photothermal agents with high PCE can transfer the absorbed light energy in the form of heat to the greatest extent, thereby enhancing the photothermal therapy effect. However, most of the currently reported photothermal agents with the above advantages have problems such as difficult post-modification due to complex synthesis and poor biocompatibility due to large molecular weight; in addition, these molecules themselves lack tumor targeting and usually need to be made into nanoparticles and enriched in tumor sites using the enhanced permeability and retention effect (EPR). Therefore, it is of great significance to develop small molecule photothermal agents with simple synthesis, high photothermal conversion rate, good biocompatibility, and long absorption wavelength. Summary of the Invention

[0003] In view of the above problems, the present invention provides a tumor-targeted photothermal reagent based on fluororhodamine, its preparation method and application. This photothermal reagent has the characteristics of simple synthesis, good water solubility, good photostability, high photothermal conversion efficiency, and absorption wavelength in the NIR region; to improve the tumor targeting of this reagent, it is further NHS ester functionalized (Tag-880-NHS) so that it can label the monoclonal antibody - Panitumumab (Pan) that specifically targets epidermal growth factor and is used for tumor imaging and treatment.

[0004] To achieve the above object, the technical solution of the present invention is as follows:

[0005] The first aspect of the present invention is a tumor-targeted photothermal reagent based on fluororhodamine, and its structural formula is:

[0006]

[0007] The second aspect of the present invention is a preparation method of the tumor-targeted photothermal reagent based on fluororhodamine described in the first aspect, including the following steps:

[0008] (1) Dissolve 3,6-dibromofluorone, 4,5-bis(diphenylphosphino)-9,9-dimethyloxanthrene, and cesium carbonate in 1,4-dioxane in sequence. Under a nitrogen atmosphere, add tert-butyl methylcarbamate and palladium acetate to the above solution. The mixture is refluxed at 95 °C for 4 hours; after the reaction is completed, cool the reaction solution to room temperature, filter off the residue by suction, rotary evaporate the solvent, and purify the crude product by column chromatography to obtain a yellow solid compound 3;

[0009] (2) Under nitrogen protection, dissolve 2-bromo-1,3-dimethoxybenzene in anhydrous tetrahydrofuran. After cooling to -78 °C, dropwise add n-butyllithium. After reacting for 30 minutes, dropwise add compound 3 to the above mixture. After the system returns to room temperature, continue stirring and reacting for 4 hours; after the reaction is completed, add water to quench the reaction. After the mixture reacts for 15 minutes, extract with dichloromethane. Combine the organic phases and rotary evaporate the solvent. Add a dichloromethane / trifluoroacetic acid mixed solution under an ice bath, stir for 1.5 hours, rotary evaporate the solvent, and purify the crude product by column chromatography to obtain a brown-yellow solid compound 4;

[0010] (3) Under nitrogen protection, dissolve tert-butyl 4-bromo-3,5-dimethoxybenzoate in anhydrous tetrahydrofuran. Control the temperature with liquid nitrogen to -78 °C and then dropwise add n-butyllithium. After reacting at this temperature for 30 min, dropwise add compound 3 to the above mixture. After the system returns to room temperature, continue stirring and reacting for 4 hours; after the reaction is completed, add water to quench the reaction. After the mixture reacts for 15 minutes, extract with dichloromethane. Combine the organic phases and rotary evaporate the solvent. Add a dichloromethane / trifluoroacetic acid mixed solution under an ice bath, stir for 1.5 hours, rotary evaporate the solvent, and purify the crude product by column chromatography to obtain a brown-yellow solid compound 5;

[0011] (4) Dissolve compound 5, N-hydroxysuccinimide, and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride in dichloromethane and react at room temperature for 3 hours. After the reaction is completed, rotary evaporate the solvent, and purify the crude product by column chromatography to obtain a yellow solid compound Tag-880-NHS.

[0012] Further, in the step (1), the molar ratio of 3,6-dibromofluorene-9-one, 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene, cesium carbonate, tert-butyl methylcarbamate, and palladium acetate is 1:0.2:2:4:0.1.

[0013] Further, in the step (1), the eluent for column chromatography separation and purification is a mixed solution of petroleum ether / ethyl acetate with a volume ratio of 5:1.

[0014] Further, in the step (2), the molar ratio of 2-bromo-1,3-dimethoxybenzene, n-butyllithium, and compound 3 is 1:1:0.25.

[0015] Further, in the step (3), the molar ratio of tert-butyl 4-bromo-3,5-dimethoxybenzoate, n-butyllithium, and compound 3 is 4:4:1.

[0016] Further, in the steps (2) and (3), the volume ratio of dichloromethane to trifluoroacetic acid in the dichloromethane / trifluoroacetic acid mixed solution is 1:1; the eluent for column chromatography separation and purification is a mixed solution of dichloromethane / methanol with a volume ratio of 15:1.

[0017] Further, in the step (4), the molar ratio of compound 5, N-hydroxysuccinimide, and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride is 1:3:3.

[0018] Further, in the step (4), the eluent for column chromatography separation and purification is a mixed solution of dichloromethane / methanol with a volume ratio of 20:1.

[0019] The third aspect of the present invention is the application of the fluororhodamine-based tumor-targeted photothermal reagent described in the first aspect in the preparation of tumor imaging and therapeutic drugs.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] Most traditional photothermal materials have problems such as large molecular weight, complex synthesis, poor biocompatibility, poor tumor targeting, and low photothermal conversion efficiency, which limit their further structural modification and biological applications to a certain extent. The fluororhodamine-based tumor-targeted photothermal reagent developed by the present invention has excellent characteristics such as small molecular weight, simple synthesis, absorption wavelength in the near-infrared region, and high photothermal conversion rate. After further NHS ester functionalization, it can achieve tumor imaging and treatment. Description of the Drawings

[0022] Figure 1 For compound 3, (A) 1 1H-NMR spectrum, (B) 13 13C-NMR spectrum, and (C) HRMS spectrum;

[0023] Figure 2 For (A) the 1 1H-NMR spectrum, (B) the 13 13C-NMR spectrum and (C) the HRMS spectrum of Compound 4;

[0024] Figure 3 For (A) the 1 1H-NMR spectrum, (B) the 13 13C-NMR spectrum and (C) the HRMS spectrum of Compound 5;

[0025] Figure 4 For the HRMS spectrum of Compound Tag-880-NHS;

[0026] Figure 5 (A) Plot of the UV-Vis absorption spectrum of Compound 4 in PBS (10 mM, pH = 7.4) versus concentration; (B) Linear fitting plot of the absorbance value of Compound 4 at 880 nm versus concentration;

[0027] Figure 6 For the photothermal imaging plot (A) and photothermal heating curve plot (B) of different concentrations of Compound 4 under continuous irradiation of a 915 nm laser lamp (1.0 W / cm 2 ) in PBS (10 mM, pH = 7.4);

[0028] Figure 7 For the photothermal heating curve plot of Compound 4 (20 μmol / L) under continuous irradiation of a 915 nm laser lamp with different optical densities in PBS (10 mM, pH = 7.4);

[0029] Figure 8 For the heating-cooling curve and linear fitting plot of -ln(θ) versus time of Compound 4 (20 μmol / L) under irradiation of a 915 nm laser lamp (1.0 W / cm 2 ) in PBS (10 mM, pH = 7.4);

[0030] Figure 9 For the heating-cooling cycle plot of Compound 4 (20 μmol / L) under irradiation of a 915 nm laser lamp (1.0 W / cm 2 ) in PBS (10 mM, pH = 7.4);

[0031] Figure 10 For the in vivo imaging plot (λex = 808 nm, λem = 1000 nm) of A549 tumor-bearing mice at different time points after tail vein injection of Pan-Tag-880 (100 μg);

[0032] Figure 11Treatment diagram of Pan-Tag-880 on A549 tumor-bearing mice. Detailed implementation mode

[0033] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0034] Example 1

[0035] A tumor-targeted photothermal reagent based on fluororhodamine, and its structural formula is:

[0036]

[0037] A preparation method of a tumor-targeted photothermal reagent based on fluororhodamine, comprising the following steps:

[0038]

[0039] (1) Synthesis of compound 3

[0040] Dissolve 3,6-dibromofluorone (compound 1, 2 g, 5.95 mmol), 4,5-bis(diphenylphosphino)-9,9-dimethyloxanthrene (687 mg, 1.19 mmol), and cesium carbonate (390 mg, 11.91 mmol) in 1,4-dioxane in sequence. Under a nitrogen atmosphere, add tert-butyl methylcarbamate (compound 2, 3.32 mL, 23.82 mmol) and palladium acetate (134 mg, 0.60 mmol) to the above solution. The mixture is refluxed at 95 °C for 4 hours. After the reaction is completed, cool the reaction solution to room temperature, filter off the residue by suction, rotary evaporate the solvent, and purify the crude product by column chromatography (petroleum ether / ethyl acetate volume ratio 5:1) to obtain yellow solid compound 3 (2.29 g, yield 88%). 1 H NMR(600MHz,Chloroform-d)δ7.60(d,J=8.0Hz,2H),7.47(d,J=1.9Hz,2H),7.15(dd,J=8.0,1.9Hz,2H),3.33(s,6H),1.50(s,18H); 13 CNMR(150MHz,Chloroform-d)δ191.97,154.24,149.80,144.67,131.25,124.97,124.64,117.18,81.40,37.20,28.45.

[0041] (2) Synthesis of compound 4

[0042] Under nitrogen protection, 2-bromo-1,3-dimethoxybenzene (742 mg, 3.44 mmol) was dissolved in anhydrous tetrahydrofuran. After cooling to -78 °C, n-butyllithium (2.5 M, 1.41 mL, 3.44 mmol) was added dropwise. After reacting for 30 minutes, compound 3 (300 mg, 0.68 mmol) was added dropwise to the above mixture. After the system was restored to room temperature, the reaction was continued with stirring for 4 hours. After the reaction was completed, 15 mL of water was added to the reaction solution to quench the reaction. After the mixture reacted for 15 minutes, it was extracted with dichloromethane. The organic phases were combined and the solvent was evaporated. Under ice bath, a mixed solution of dichloromethane / trifluoroacetic acid with a volume ratio of 1:1 was added, and the mixture was stirred for 1.5 hours. The solvent was evaporated, and the crude product was purified by column chromatography (dichloromethane / methanol volume ratio 15:1) to obtain a yellowish-brown solid compound 4 (183 mg, yield 75%). 1 H NMR(600MHz,DMSO-d 6 )δ9.04(s,2H),7.47(t,J=8.5Hz,1H),7.05(s,2H),6.83(d,J=8.6Hz,2H),6.77(d,J=7.8Hz,2H),6.19(d,J=8.8Hz,2H),3.73(s,6H),3.04(s,6H); 13 C NMR(150MHz,DMSO-d 6 )δ165.49,158.36,157.80,134.06,132.76,129.23,118.28,116.29,108.41,104.75,55.95,40.06,30.33;ESI-MS[M] + :calcd for 395.1755,Found 359.1754.

[0043] (3) Synthesis of compound 5

[0044] Under nitrogen protection, tert-butyl 4-bromo-3,5-dimethoxybenzoate (867.9 mg, 2.75 mmol) was dissolved in anhydrous tetrahydrofuran. After controlling the temperature with liquid nitrogen to -78 °C, n-butyllithium (1.6 M, 1.71 mL, 2.75 mmol) was added dropwise. After reacting at this temperature for 30 min, compound 3 (300 mg, 0.68 mmol) was added dropwise to the above mixture. After the system was restored to room temperature, the reaction was continued with stirring for 4 hours. After the reaction was completed, 15 mL of water was added to quench the reaction. After the mixture reacted for 15 minutes, it was extracted with dichloromethane. The organic phases were combined and the solvent was rotary evaporated. Under an ice bath, a mixed solution of dichloromethane / trifluoroacetic acid with a volume ratio of 1:1 was added, and the mixture was stirred for 1.5 hours. The solvent was rotary evaporated, and the crude product was purified by column chromatography (dichloromethane / methanol volume ratio 15:1) to obtain the yellowish-brown solid compound 5 (44 mg, yield 16%). 1 H NMR(600MHz,Methanol-d 4 )δ7.40(s,2H),6.88(s,2H),6.75(d,J=8.7Hz,2H),6.14(d,J=8.7Hz,2H),3.82(s,6H),3.07(s,6H); 13 C NMR(150MHz,Methanol-d 4 )δ167.47,166.68,159.15,158.12,147.24,134.49,130.18,112.89,105.08,62.85,55.24,29.36;ESI-MS[M] + :calcd for 403.1653,Found 403.1651.

[0045] (4) Synthesis of compound Tag-880-NHS

[0046] Compound 5 (20 mg, 0.050 mmol), N-hydroxysuccinimide (17 mg, 0.15 mmol) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (28 mg, 0.15 mmol) were dissolved in dichloromethane and reacted at room temperature for 3 hours. After the reaction was completed, the solvent was rotary evaporated, and the crude product was purified by column chromatography (dichloromethane / methanol volume ratio 20:1) to obtain the yellow solid compound Tag-880-NHS (12 mg, yield 48%). ESI-MS[M] + :calcd for 500.1817,Found500.1819.

[0047] Example 2

[0048] 1. Preparation of test solution

[0049] Prepare a 2 mM stock solution of Compound 4 and Tag-880-NHS in DMSO, and then dilute it to the test concentration with the solvent to be tested.

[0050] 2. Ultraviolet absorption spectroscopy

[0051] Since Tag-880-NHS is an active ester and unstable, Compound 4 was selected as a model compound to test its photothermal properties in vitro. First, using a UV-visible spectrophotometer, the absorption wavelength and water solubility of Compound 4 were tested. The stock solution of Compound 4 was diluted with PBS buffer to solutions with concentrations of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 μmol / L, respectively. The absorption spectra in the range of 300 - 1200 nm were scanned, and the linear fitting curve of the absorbance at the maximum absorption wavelength versus the concentration was plotted. As Figure 5 shown, the maximum absorption wavelength of Compound 4 in PBS is 880 nm. As the concentration gradually increases, the absorbance value at 880 nm increases uniformly, and there is a good linear relationship with the concentration (R 2 = 0.9991), indicating that this photothermal reagent exhibits good solubility in water.

[0052] 3. Photothermal property test

[0053] Next, the photothermal properties of Compound 4 were tested. First, its heating ability at different concentrations was tested. The stock solution of Compound 4 was diluted with PBS buffer to solutions with concentrations of 5, 10, 20 μmol / L. It was continuously irradiated with a 915 nm laser lamp (1.0 W / cm 2 ) for 15 min, and at the same time, the temperature of the solution was recorded every 1 min with an infrared thermal imager, and the relationship between the temperature change and the irradiation time was plotted. As Figure 6 shown, the temperatures of solutions with different concentrations all gradually increase with the increase of the irradiation time and reach equilibrium at 10 min; the temperature of the solution gradually increases with the increase of the concentration of the photothermal reagent, and the highest temperature can reach 61.1 °C at 20 μmol / L.

[0054] Subsequently, the heating effect of lasers with different intensities on Compound 4 was tested. The stock solution of Compound 4 was diluted with PBS buffer to a solution with a concentration of 20 μmol / L, and it was continuously irradiated with 915 nm lasers of 0.6, 0.8, 1.0 W / cm 2 for 15 min, and at the same time, the temperature of the solution was recorded every 1 min with an infrared thermal imager, and the relationship between the temperature change and the irradiation time was plotted. As Figure 7As shown, when irradiating the solution with lasers of different intensities, the temperature of the solution gradually increases with the increase of the laser power under the same illumination time. The laser with a power of 0.6 W / cm 2 can still raise the temperature of the photothermal reagent by more than 50 °C.

[0055] The photothermal conversion efficiency is a parameter that can intuitively measure the performance of photothermal materials. Furthermore, the photothermal conversion efficiency of Compound 4 was calculated. A 915 nm laser lamp (1.0 W / cm 2 ) was used to continuously irradiate Compound 4 (20 μmol / L) or PBS solution for 15 min. After turning off the light source, it was cooled at room temperature for 10 min. During the whole process, the temperature of the solution was recorded every 1 min with an infrared thermal imager, and the relationship between temperature and time was plotted. Figure 8 is the heating-cooling curve of Compound 4 and the linear fitting curve of -ln(θ) vs. time. After calculation, the PCE of Compound 4 in PBS is 89.3%.

[0056] 4. Photo / Thermal Stability Test

[0057] To test the thermal stability of the photothermal reagent, a 915 nm laser lamp (1.0 W / cm 2 ) was used to continuously irradiate Compound 4 (20 μmol / L) for 15 min. After turning off the light source, it was cooled at room temperature for 10 min. This process was repeated four times. During the whole process, the temperature of the solution was recorded every 1 min with an infrared thermal imager, and the relationship between temperature and time was plotted. As Figure 9 shown, the maximum temperature of the photothermal reagent can reach above 57 °C during the four heating-cooling cycles, indicating that Compound 4 has excellent photo / thermal stability and can be reused.

[0058] 5. Imaging Tumor Test

[0059] Epidermal growth factor receptor (EGFR) is a large transmembrane glycoprotein that is overexpressed on the membrane surface of various cancer cells. Panitumumab is a monoclonal antibody against epidermal growth factor receptor. Therefore, Compound 4 can be covalently linked to panitumumab to endow it with the ability to target tumor cells. First, using the standard protein labeling method, Tag-880-NHS was reacted with panitumumab in PBS (pH = 8.0) and purified successively through a PD-10 column and a size exclusion column to obtain the "dye-antibody" tumor targeting reagent Pan-Tag-880. After calculation, when the molar ratio of Tag-880-NHS to panitumumab is 30:1, the degree of labeling (DOL) is 2.

[0060] Subsequently, Pan-Tag-880 (100 μg) was injected into A549 tumor-bearing mice via the tail vein, and imaging was performed at 2, 4, 6, 8, 10, 12, 14, and 20 h after injection. As Figure 10 shown, the fluorescence signal at the tumor site of A549 tumor-bearing mice was significantly enhanced 2 h after injection and reached the maximum at 6 h. After 20 h of injection, almost no fluorescence was observed at the tumor site. Therefore, photothermal therapy was performed 6 h after injecting Pan-Tag-880.

[0061] 6. Photothermal therapy test

[0062] To verify the photothermal therapy effect of Pan-Tag-880 in tumor-bearing nude mice, A549 tumor-bearing mice were divided into a PBS / light irradiation group and a Pan-Tag-880 / light irradiation group. After the tumor sites of the tumor-bearing nude mice were injected with PBS and Pan-Tag-880 in situ, respectively, they were irradiated with a 915 nm (1 W / cm 2 ) laser for 15 minutes. As Figure 11 shown, the tumors of the tumor-bearing mice in the PBS / light irradiation group increased significantly over time, indicating that PBS plus light irradiation had no inhibitory effect on tumor growth. On the contrary, the tumors of the tumor-bearing mice in the Pan-Tag-880 / light irradiation group gradually ablated over time and basically disappeared 12 days after light irradiation; in addition, during the photodynamic therapy, the body weights of all tumor-bearing nude mice did not decrease significantly. The above results indicate that Pan-Tag-880 can specifically target the tumor site of A549 tumor-bearing mice, has good biocompatibility and negligible side effects, and can efficiently kill cancer cells under near-infrared light irradiation.

[0063] In summary, the present invention developed a photothermal reagent based on fluororhodamine dye, which has a very high photothermal conversion rate (89.3%); in addition, the photothermal reagent also has excellent properties such as a small molecular weight, an absorption wavelength in the near-infrared region (880 nm), good biocompatibility, and easy modification. To improve its tumor targeting ability, the photothermal reagent was functionalized with NHS ester, and the obtained Tag-880-NHS was further covalently linked with panitumumab to generate Pan-Tag-880. After intravenous injection via the tail vein, Pan-Tag-880 can specifically target the tumor site of A549 tumor-bearing mice and efficiently ablate tumors under 915 nm laser irradiation.

[0064] The above are only examples for better explaining the present invention and are not intended to limit it. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention falls within the scope covered by the present invention.

Claims

1. A tumor-targeting photothermal agent based on fluorene rhodamine, characterized in that: Its structural formula is:

2. The method for preparing the fluorene rhodamine-based tumor-targeted photothermal agent according to claim 1, characterized in that: The following steps are involved: (1) 3,6-dibromofluorenone, 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene, and cesium carbonate were dissolved in 1,4-dioxane in sequence, methyl-tert-butyl carbamate and palladium acetate were added to the above solution under a nitrogen environment, and the mixture was refluxed at 95° C. for 4 hours; after the reaction, the reaction solution was cooled to room temperature, the residue was removed by suction filtration, the solvent was dried by spin drying, and the crude product was purified by column chromatography to obtain a yellow solid compound 3; (2) Under nitrogen protection, 2-bromo-1,3-dimethoxybenzene was dissolved in anhydrous tetrahydrofuran, and the temperature was lowered to -78°C, and n-butyl lithium was added dropwise. After reacting for 30 minutes, compound 3 was added dropwise to the above mixture. After the system returned to room temperature, stirring and reacting for 4 hours was continued. After the reaction was completed, water was added to the reaction solution to quench the reaction. After the mixture reacted for 15 minutes, it was extracted with dichloromethane, the organic phases were combined and the solvent was dried by spin-drying, and a dichloromethane / trifluoroacetic acid mixed solution was added under ice bath, stirred for 1.5 hours, and the solvent was dried by spin-drying. The crude product was purified by column chromatography to obtain brown solid compound 4; (3) Under nitrogen protection, tert-butyl 4-bromo-3,5-dimethoxybenzoate was dissolved in anhydrous tetrahydrofuran, and n-butyl lithium was added dropwise after the temperature was controlled to -78°C with liquid nitrogen. After reacting at this temperature for 30 minutes, compound 3 was added dropwise to the above mixture. After the system returned to room temperature, stirring and reacting for 4 hours; after the reaction was completed, water was added to quench the reaction, and the mixture was extracted with dichloromethane after reacting for 15 minutes. The organic phases were combined and the solvent was dried by spin-drying, and a dichloromethane / trifluoroacetic acid mixed solution was added under ice bath, stirred for 1.5 hours, and the solvent was dried by spin-drying. The crude product was purified by column chromatography to obtain a brown-yellow solid compound 5; (4) Compound 5, N-hydroxysuccinimide and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride were dissolved in dichloromethane and reacted at room temperature for 3 hours. After the reaction was completed, the solvent was dried and the crude product was purified by column chromatography to obtain a yellow solid compound Tag-880-NHS.

3. The method for preparing a tumor-targeting photothermal agent based on fluorene rhodamine according to claim 2, characterized in that: In the step (1), the molar ratio of 3,6-dibromofluorenone, 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene, cesium carbonate, methyl-tert-butyl carbamate and palladium acetate is 1:0.2:2:4:0.

1.

4. The method for preparing a tumor-targeting photothermal agent based on fluorene rhodamine according to claim 2, characterized in that: The developing solvent for column chromatography separation and purification in step (1) is a petroleum ether / ethyl acetate mixture with a volume ratio of 5:

1.

5. The method for preparing a tumor-targeting photothermal agent based on fluorene rhodamine according to claim 2, characterized in that: In the step (2), the molar ratio of 2-bromo-1,3-dimethoxybenzene, n-butyl lithium and compound 3 is 1:1:0.

25.

6. The method for preparing a tumor-targeting photothermal agent based on fluorene rhodamine according to claim 2, characterized in that: In the step (3), the molar ratio of tert-butyl 4-bromo-3,5-dimethoxybenzoate, n-butyl lithium and compound 3 is 4:4:

1.

7. The method for preparing a tumor-targeting photothermal agent based on fluorene rhodamine according to claim 2, characterized in that: In the steps (2) and (3), the volume ratio of dichloromethane to trifluoroacetic acid in the dichloromethane / trifluoroacetic acid mixed solution is 1:1; and the developing solvent for column chromatography separation and purification is a dichloromethane / methanol mixed solution with a volume ratio of 15:

1.

8. The method for preparing a tumor-targeting photothermal agent based on fluorene rhodamine according to claim 2, characterized in that: In the step (4), the molar ratio of compound 5, N-hydroxysuccinimide and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride is 1:3:

3.

9. The method for preparing a tumor-targeting photothermal agent based on fluorene rhodamine according to claim 2, characterized in that: The developing solvent for column chromatography separation and purification in step (4) is a dichloromethane / methanol mixture with a volume ratio of 20:

1.

10. Use of the fluorene rhodamine-based tumor-targeted photothermal agent according to claim 1 in the preparation of tumor imaging and therapeutic drugs.

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