Fluororhodamine-based tumor-targeting photothermal reagent, preparation method and application thereof

Abstract

The application belongs to the field of fluorescent probes, and particularly relates to a tumor-targeting photothermal reagent based on fluoro-rhodamine, and a preparation method and application thereof. To solve the problems that most of existing photothermal reagents have difficulty in post-modification due to complex synthesis, poor biological compatibility due to large molecular weight, and lack of tumor targeting of these molecules, and generally need to be made into nanoparticles to enrich to the tumor site by EPR effect, the application develops a tumor-targeting photothermal reagent Tag-880-NHS based on fluoro-rhodamine, which has the characteristics of simple synthesis, good water solubility, good light stability, high photothermal conversion efficiency, and an absorption wavelength located in the NIR region, and can be used for labeling a specific targeting epidermal growth factor monoclonal antibody-panitumumab, and can be used in the fields of tumor imaging and treatment, and has important application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of fluorescent probes, and particularly relates to a tumor-targeting photothermal reagent based on fluoro-rhodamine, and a preparation method and application thereof. BACKGROUND

[0002] Photothermal therapy (PTT) is a non-invasive tumor treatment method that uses photothermal agents (PTAs) to cause local heating of tumor tissue sites under external light irradiation, thereby killing tumor cells. During the treatment process, fluorescence or photoacoustic signals generated during light irradiation can be monitored to guide and assist the treatment in a multi-modal 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, the performance of which can be evaluated by parameters such as absorption / emission wavelength and photothermal conversion efficiency (PCE). Near-infrared light (NIR 700-1700 nm) has strong tissue penetration, low tissue damage, and high spatiotemporal resolution, and 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 evaluating the performance of photothermal agents. Photothermal agents with high PCE can maximize the transfer of absorbed light energy in the form of heat, thereby enhancing the effect of photothermal therapy. However, most of the photothermal agents reported at present have the problems of difficulty in post-modification due to complex synthesis and poor biocompatibility due to large molecular weight. In addition, these molecules lack tumor targeting, and usually need to be made into nanoparticles to enrich in tumor sites by 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

[0003] In view of the above problems, the present application provides a tumor-targeting photothermal reagent based on fluoro-rhodamine, and a preparation method and application thereof. The photothermal reagent has the characteristics of simple synthesis, good water solubility, good light stability, high photothermal conversion efficiency, and absorption wavelength in the NIR region. To improve the tumor targeting of the reagent, it is further functionalized with NHS ester (Tag-880-NHS), so that it can label the specific targeting epidermal growth factor monoclonal antibody—Panitumumab (Pan), and be used for tumor imaging and treatment.

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

[0005] The first aspect of the present application is a tumor-targeting photothermal reagent based on fluorene rhodamine, and its structural formula is:

[0006]

[0007] The second aspect of the present application is a preparation method of the tumor-targeting photothermal reagent based on fluorene rhodamine in the first aspect, comprising the following steps:

[0008] (1) 3,6-dibromofluorene ketone, 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene, and cesium carbonate are sequentially dissolved in 1,4-dioxane, methyl-tert-butyl carbamate and palladium acetate are added to the above solution under a nitrogen environment, and the mixture is refluxed at 95 DEG C for 4 hours; after the reaction is completed, the reaction solution is cooled to room temperature, residual solid is removed by suction filtration, the solvent is spin-dried, and the crude product is purified by column chromatography to obtain yellow solid compound 3;

[0009] (2) Under the protection of nitrogen, 2-bromo-1,3-dimethoxybenzene is dissolved in anhydrous tetrahydrofuran, and n-butyllithium is added dropwise after cooling to-78 DEG C; after reaction for 30 minutes, compound 3 is added dropwise to the above mixture, and the system is continuously stirred for 4 hours after being restored to room temperature; after the reaction is completed, water is added to the reaction solution to quench the reaction, the mixture is reacted for 15 minutes, and then extracted with dichloromethane; the organic phases are combined and the solvent is spin-dried; a dichloromethane / trifluoroacetic acid mixed solution is added under ice bath, and stirred for 1.5 hours; the solvent is spin-dried, and the crude product is purified by column chromatography to obtain brown-yellow solid compound 4;

[0010] (3) Under the protection of nitrogen, 4-bromo-3,5-dimethoxy-tert-butyl benzoate is dissolved in anhydrous tetrahydrofuran, and n-butyllithium is added dropwise after cooling to-78 DEG C; after reaction for 30 minutes at this temperature, compound 3 is added dropwise to the above mixture, and the system is continuously stirred for 4 hours after being restored to room temperature; after the reaction is completed, water is added to quench the reaction, the mixture is reacted for 15 minutes, and then extracted with dichloromethane; the organic phases are combined and the solvent is spin-dried; a dichloromethane / trifluoroacetic acid mixed solution is added under ice bath, and stirred for 1.5 hours; the solvent is spin-dried, and the crude product is purified by column chromatography to obtain brown-yellow solid compound 5;

[0011] (4) Compound 5, N-hydroxysuccinimide, and N-(3-dimethylaminopropyl)-N'-ethyl carbodiimide hydrochloride are dissolved in dichloromethane, and reacted at room temperature for 3 hours; after the reaction is completed, the solvent is spin-dried, and the crude product is purified by column chromatography to obtain yellow solid compound Tag-880-NHS.

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

[0013] Furthermore, the developing solvent used in step (1) for column chromatography separation and purification is a mixture of petroleum ether and ethyl acetate with a volume ratio of 5:1.

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

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

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

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

[0018] Furthermore, in step (4), the developing solvent for column chromatography separation and purification is a mixture of dichloromethane and methanol with a volume ratio of 20:1.

[0019] The third aspect of the present invention is the application of the fluorescein-based tumor-targeting 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] Traditional photothermal materials often suffer from problems such as large molecular weight, complex synthesis, poor biocompatibility, poor tumor targeting, and low photothermal conversion efficiency, which limits their further structural modification and biological applications. The tumor-targeting photothermal reagent based on fluorenazine developed in this invention possesses excellent properties such as small molecular weight, simple synthesis, absorption wavelength in the near-infrared region, and high photothermal conversion efficiency. Further functionalization with NHS esters can enable tumor imaging and treatment. Attached Figure Description

[0022] Figure 1 For compound 3 (A) 1 H-NMR spectrum, (B) 13 C1-NMR and (C)HRMS plots;

[0023] Figure 2 (A) is the HRMS graph of compound 4; (B) is the H-NMR graph, (C) is the C-NMR graph and (D) is the HRMS graph of compound 5; 1 H-NMR graph, (B) 13 C-NMR graph and (C) HRMS graph;

[0024] Figure 3 (A) is the HRMS graph of compound 4; (B) is the H-NMR graph, (C) is the C-NMR graph and (D) is the HRMS graph of compound 5; 1 H-NMR graph, (B) 13 C-NMR graph and (C) HRMS graph;

[0025] Figure 4 (A) is the HRMS graph of compound 4; (B) is the H-NMR graph, (C) is the C-NMR graph and (D) is the HRMS graph of compound 5;

[0026] Figure 5 (A) is the UV-Vis absorption spectrum of compound 4 in PBS (10 mM, pH = 7.4) as a function of concentration; (B) is the linear fitting graph of absorbance value at 880 nm of compound 4 versus concentration;

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

[0028] Figure 7 (A) is the photothermal imaging graph and (B) is the photothermal heating curve of compound 4 at different concentrations under continuous irradiation of 915 nm laser lamp (1.0 W / cm 2 ) in PBS (10 mM, pH = 7.4);

[0029] Figure 8 (A) is the photothermal imaging graph and (B) is the photothermal heating curve of compound 4 at different concentrations under continuous irradiation of 915 nm laser lamp (1.0 W / cm 2 ) in PBS (10 mM, pH = 7.4);

[0030] Figure 9 (A) is the photothermal imaging graph and (B) is the photothermal heating curve of compound 4 at different concentrations under continuous irradiation of 915 nm laser lamp (1.0 W / cm 2 ) in PBS (10 mM, pH = 7.4);

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

[0032] Figure 11Figure 1 shows the therapeutic effect of Pan-Tag-880 on A549 tumor-bearing mice. DETAILED DESCRIPTION

[0033] For the purposes of promoting an understanding of the principles of the application, reference will now be made to the embodiments illustrated in the drawings. It is expressly understood that the application can be practiced otherwise than as set forth in the accompanying figures. In other words, the figures are provided for the purpose of illustration and description only and should not be viewed as limiting the scope of the application.

[0034] Example 1

[0035] A fluorene rhodamine-based tumor-targeting photothermal reagent, the structural formula of which is:

[0036]

[0037] A preparation method of a fluorene rhodamine-based tumor-targeting photothermal reagent, comprising the following steps:

[0038]

[0039] (1) Synthesis of compound 3

[0040] Dissolve 3,6-dibromofluorene (compound 1, 2 g, 5.95 mmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (687 mg, 1.19 mmol), and cesium carbonate (390 mg, 11.91 mmol) in 1,4-dioxane, respectively, and add methyl-t-butyl carbamate (compound 2, 3.32 mL, 23.82 mmol) and palladium acetate (134 mg, 0.60 mmol) to the above solution under a nitrogen environment. Reflux the mixture at 95°C for 4 hours. After the reaction is completed, cool the reaction solution to room temperature, remove the residue by suction filtration, spin dry the solvent, and purify the crude product by column chromatography (petroleum ether / ethyl acetate, 5:1 by volume) to obtain yellow solid compound 3 (2.29 g, yield 88%). 1 H NMR (600 MHz, Chloroform-d) δ 7.60 (d, J = 8.0 Hz, 2H), 7.47 (d, J = 1.9 Hz, 2H), 7.15 (dd, J = 8.0, 1.9 Hz, 2H), 3.33 (s, 6H), 1.50 (s, 18H); 13 CNMR (150 MHz, Chloroform-d) δ 191.97, 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] Compound 4 was prepared according to the procedure described for compound 3, using compound 2 as starting material. Compound 4 was obtained as a yellow solid (183 mg, 75% yield). 1 H NMR (600 MHz, DMSO-d6) δ 9.04 (s, 2H), 7.47 (t, J = 8.5 Hz, 1H), 7.05 (s, 2H), 6.83 (d, J = 8.6 Hz, 2H), 6.77 (d, J = 7.8 Hz, 2H), 6.19 (d, J = 8.8 Hz, 2H), 3.73 (s, 6H), 3.04 (s, 6H); 13 C NMR (150 MHz, DMSO-d6) δ 165.49, 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] Compound 5 was prepared according to the procedure described for compound 3, using tert-butyl 4-bromo-3,5-dimethoxybenzoate as starting material. Compound 5 was obtained as a yellow solid (44 mg, 16% yield).1 H NMR (600 MHz, Methanol-d4) δ 7.40 (s, 2H), 6.88 (s, 2H), 6.75 (d, J = 8.7 Hz, 2H), 6.14 (d, J = 8.7 Hz, 2H), 3.82 (s, 6H), 3.07 (s, 6H); 13 C NMR (150 MHz, Methanol-d4) δ 167.47, 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 evaporated and the crude product was purified by column chromatography (dichloromethane / methanol 20:1 by volume) to obtain compound Tag-880-NHS (12 mg, yield 48%) as a yellow solid. ESI-MS [M] + : calcd for 500.1817, Found 500.1819.

[0047] Example 2

[0048] 1. Test solution preparation

[0049] Compound 4 and Tag-880-NHS were prepared as 2 mM stock solutions in DMSO and then diluted with the solvent to be tested to the test concentration.

[0050] 2. UV absorption spectrum measurement

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

[0052] 3. Photothermal performance test

[0053] Next, the photothermal performance of compound 4 was tested. First, the temperature rising ability of compound 4 at different concentrations was tested. The stock solution of compound 4 was diluted with PBS buffer to concentrations of 5, 10, 20 μmol / L, and was continuously irradiated with a 915 nm laser lamp (1.0 W / cm 2 ) for 15 min, while the temperature of the solution was recorded every 1 min with a thermal imager, and the relationship between temperature change and irradiation time was plotted. As shown in Figure 6 , the temperature of the solution at different concentrations gradually increased with the increase of irradiation time, and reached equilibrium at 10 min; the temperature of the solution gradually increased with the increase of the concentration of the photothermal reagent, and the temperature at 20 μmol / L could be as high as 61.1°C.

[0054] Subsequently, the influence of different intensities of laser on the temperature rise of compound 4 was tested. The stock solution of compound 4 was diluted with PBS buffer to a concentration of 20 μmol / L, and was continuously irradiated with a 915 nm laser lamp at intensities of 0.6, 0.8, 1.0 W / cm 2 for 15 min, while the temperature of the solution was recorded every 1 min with a thermal imager, and the relationship between temperature change and irradiation time was plotted. As shown in Figure 7 , when the solution was irradiated with different intensities of laser, the temperature of the solution gradually increased with the increase of laser power at the same irradiation time, and the temperature of the solution irradiated with a 0.6 W / cm 2 laser could still be raised by more than 50°C.

[0055] The photothermal conversion efficiency is a parameter that can intuitively measure the performance of the photothermal material. Further, the photothermal conversion efficiency of compound 4 was calculated. Compound 4 (20 μmol / L) or PBS solution was continuously irradiated for 15 min with a 915 nm laser lamp (1.0 W / cm 2 The light source was turned off and the solution was cooled at room temperature for 10 min. The temperature of the solution was recorded every 1 min with a thermal imager during the whole process, and a temperature-time curve was drawn. Figure 8 The temperature-rising and cooling curve of compound 4 and the linear fitting curve of -ln(θ) and time are shown in FIG. 6. It was calculated that the PCE of compound 4 in PBS was 89.3%.

[0056] 4. Light / heat stability test

[0057] In order to test the thermal stability of the photothermal reagent, compound 4 (20 μmol / L) was continuously irradiated for 15 min with a 915 nm laser lamp (1.0 W / cm 2 The light source was turned off and the solution was cooled at room temperature for 10 min. The temperature of the solution was recorded every 1 min with a thermal imager during the whole process, and a temperature-time curve was drawn. As shown in FIG. 7, the maximum temperature of the photothermal reagent during the four temperature-rising and cooling cycles was above 57℃, indicating that compound 4 has excellent light / heat stability and can be reused. Figure 9

[0058] 5. Image tumor test

[0059] The epidermal growth factor receptor (EGFR) is a huge transmembrane glycoprotein that is overexpressed on the membrane surface of various cancer cells. Panitumumab is a monoclonal antibody of epidermal growth factor receptor, so compound 4 can be covalently linked to panitumumab to enable it to target tumor cells. First, Tag-880-NHS was reacted with panitumumab in PBS (pH = 8.0) by using a standard protein labeling method, and was purified by a PD-10 column and a size exclusion column in sequence to obtain a "dye-antibody" tumor targeting reagent Pan-Tag-880. It was calculated that when the molar ratio of Tag-880-NHS to panitumumab was 30:1, the degree of labeling (DOL) was 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 shown in FIG. 8, the fluorescence signal at the tumor site of the A549 tumor-bearing mice was significantly enhanced 2 h after injection, reached a maximum at 6 h, and almost no fluorescence was observed at the tumor site 20 h after injection. Therefore, 6 h after injection of Pan-Tag-880 was selected for photothermal therapy. Figure 10 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 shown in FIG. 8, the fluorescence signal at the tumor site of the A549 tumor-bearing mice was significantly enhanced 2 h after injection, reached a maximum at 6 h, and almost no fluorescence was observed at the tumor site 20 h after injection. Therefore, 6 h after injection of Pan-Tag-880 was selected for photothermal therapy.​

[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 PBS / illumination group and Pan-Tag-880 / illumination group, and the tumor sites of tumor-bearing nude mice were injected with PBS and Pan-Tag-880 in situ, respectively, and then irradiated with 915nm(1W / cm 2 ) laser for 15 minutes. As shown in Figure 11 , the tumors of tumor-bearing mice in the PBS / illumination group increased significantly over time, indicating that PBS plus illumination had no inhibitory effect on tumor growth, on the contrary, the tumors of tumor-bearing mice in the Pan-Tag-880 / illumination group gradually ablated over time and basically disappeared after 12 days of illumination; in addition, the body weight of all tumor-bearing nude mice did not decrease significantly during photodynamic therapy. The above results show 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 application develops a photothermal reagent based on fluorene rhodamine dye, which has a very high photothermal conversion rate(89.3%); in addition, the photothermal reagent also has the advantages of small molecular weight, absorption wavelength in the near-infrared region(880nm), good biocompatibility and easy modification. In order to improve its tumor targeting, the photothermal reagent is NHS ester functionalized to obtain Tag-880-NHS, which is further covalently linked with panitumumab to generate Pan-Tag-880, and Pan-Tag-880 can specifically target the tumor site of A549 tumor-bearing mice after tail vein injection, and efficiently ablate the tumor under 915nm laser irradiation.

[0064] The above only serves to better explain the embodiments of the present application, and is not a limitation thereof, any modification or equivalent replacement without departing from the spirit and scope of the present application shall fall within the scope covered by the present application.

Claims

1. A tumor-targeting photothermal agent based on fluoresamine, characterized in that, Its structural formula is: 。 2. The preparation method of the tumor-targeting photothermal reagent based on fluoresamine according to claim 1, characterized in that, Includes the following steps: (1) 3,6-dibromofluorenone, 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene and cesium carbonate were dissolved in 1,4-dioxane in sequence. Tert-butyl methylcarbamate and palladium acetate were added to the above solution under nitrogen atmosphere. The mixture was refluxed at 95°C for 4 hours. After the reaction was completed, the reaction solution was cooled to room temperature, the residue was removed by suction filtration, the solvent was evaporated, and the crude product was purified by column chromatography to obtain yellow solid compound 3. (2) Under nitrogen protection, tert-butyl 4-bromo-3,5-dimethoxybenzoate was dissolved in anhydrous tetrahydrofuran. After the temperature was controlled to -78°C by liquid nitrogen, n-butyllithium was added dropwise. After reacting at this temperature for 30 min, compound 3 was added dropwise to the mixture. After the system was restored to room temperature, the reaction was stirred for 4 hours. After the reaction was completed, 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 evaporated. A dichloromethane / trifluoroacetic acid mixed solution was added under ice bath and stirred for 1.5 hours. The solvent was evaporated. The crude product was purified by column chromatography to obtain brownish-yellow solid compound 5. (3) Compound 5, N -hydroxysuccinimide and N -(3-Dimethylaminopropyl)- N' 1-Ethylcarbodiimide hydrochloride was dissolved in dichloromethane and reacted at room temperature for 3 hours. After the reaction was completed, the solvent was evaporated and the crude product was purified by column chromatography to obtain the yellow solid compound Tag-880-NHS.

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

1.

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

1.

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

1.

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

1.

7. The method for preparing the tumor-targeting photothermal reagent based on fluoresamine according to claim 2, characterized in that, In step (3), compound 5, N -Hydroxysuccinimide, N -(3-Dimethylaminopropyl)- N' The molar ratio of ethylcarbodiimide hydrochloride is 1:3:

3.

8. The method for preparing the tumor-targeting photothermal reagent based on fluoresamine according to claim 2, characterized in that, In step (3), the developing solvent for column chromatography separation and purification is a mixture of dichloromethane and methanol with a volume ratio of 20:

1.

9. The use of the fluorescein-based tumor-targeting photothermal reagent according to claim 1 in the preparation of A549 tumor imaging and therapeutic drugs.

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