Radical photoinitiators with fluorescent "light-on" properties, methods of making and use thereof

Abstract

The application discloses a free radical photosensitizer with a fluorescent 'lighting' property, and a preparation method and application thereof. 4,7-dibromo-2,1,3-benzothiazole and triphenylamine derivatives are used as raw materials, and an intermediate product 1 is obtained through a Suzuki coupling reaction; the obtained intermediate product 1 and 4-aminobenzoic acid pinacol ester are subjected to a Suzuki coupling reaction to obtain an intermediate product 2; and the obtained intermediate product 2 and maleic anhydride are subjected to a reaction to obtain a final product. Advantages of the application include: maleimide with specific recognition to Cys and strong electron acceptor is introduced into a photosensitizer molecular skeleton, specific recognition of the photosensitizer to Cys is realized, the photosensitizer is 'lighted' by fluorescence, and the free radical ROS generation efficiency is improved, and the cancer cells have a good photodynamic therapy effect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of photosensitizer preparation, and particularly relates to application of a photosensitizer in preparation of an antitumor drug. BACKGROUND

[0002] Cysteine is an important biological thiol, which widely exists in cells and has multiple functions. Cysteine level is related to various diseases, and abnormal concentration can cause edema, weakness, lethargy, hair loss, slow growth, liver damage, skin damage, etc. Cancer is a disease with high mortality, and it has been reported that cysteine is essential for promoting cancer cell proliferation and survival, and is highly expressed in tumors.

[0003] Photodynamic therapy (PDT) has unique advantages such as non-invasive / minimally invasive, strong controllability, good curative effect and difficulty in developing drug resistance, and has important application value in clinical aspects such as cancer treatment, wound healing promotion and skin infection resistance. The active oxygen (ROS) generated by the photosensitizer under the irradiation of the excitation light can effectively kill cancer cells, thereby achieving the effect of treating diseases. ROS can be divided into two categories, one is singlet oxygen which is mainly energy transfer, and has the problem of high oxygen dependence, and the photosensitizer, oxygen and excitation light source are the three important components of PDT for generating this type of ROS; and the other is free radical type ROS which is mainly electron transfer, and can still efficiently generate free radical type ROS even under low oxygen concentration.

[0004] Therefore, developing a photosensitizer which can specifically recognize intracellular Cys and generate free radical type ROS is expected to realize precise diagnosis and efficient treatment of tumors. SUMMARY

[0005] The application provides a free radical type photosensitizer with a fluorescent "lighting" property and a preparation method and application thereof, so as to solve the problem of lack of antitumor drugs with non-invasive / minimally invasive, strong controllability, good curative effect and difficulty in developing drug resistance in the prior art.

[0006] To achieve the above object, the application provides the following technical solutions.

[0007] The free radical type photosensitizer with the fluorescent "lighting" property has the following structural formula:

[0008] .

[0009] The application further provides a preparation method of the photosensitizer, comprising the following steps:

[0010] (1) taking 4,7-dibromo-2,1,3-benzothiadiazole and triphenylamine derivative as raw materials, and obtaining an intermediate product 1 through Suzuki coupling reaction;

[0011] (2) the intermediate product 1 obtained and 4-aminophenylboronic acid pinacol ester are subjected to Suzuki coupling reaction to obtain an intermediate product 2;

[0012] (3) the intermediate product 2 obtained and maleic anhydride are subjected to reaction to obtain the final product TBZMA.

[0013] Further, in the step (1), the reaction of 4,7-dibromo-2,1,3-benzothiadiazole and triphenylamine derivative requires the addition of a catalyst and a salt, and the molar ratio of 4,7-dibromo-2,1,3-benzothiadiazole, triphenylamine derivative, catalyst and salt is 1: (1-1.2): (0.05-0.1): 12;

[0014] In the step (2), the reaction of the intermediate product 1 and 4-aminophenylboronic acid pinacol ester requires the addition of a catalyst and a salt, and the molar ratio of the intermediate product 1, 4-aminophenylboronic acid pinacol ester, catalyst and salt is 1: (1-1.2): (0.05-0.1): 18;

[0015] In the step (3), the reaction of the intermediate product 2 and maleic anhydride requires the addition of anhydrous sodium acetate and acetic anhydride, and the molar ratio of the intermediate product 2, maleic anhydride, anhydrous sodium acetate and acetic anhydride is 1: (1.8-2.2): (7.6-7.8): (330-335).

[0016] Further, the detailed process of the step (1) comprises: 4,7-dibromo-2,1,3-benzothiadiazole 1.5 mmol, 4-boronic acid triphenylamine 1.8 mmol, Pd(PPh3)4 0.06 mmol and anhydrous potassium carbonate 18 mmol are added into a mixed solvent of tetrahydrofuran and water, the volume ratio of tetrahydrofuran and water is 20 mL: 8 mL, and TBZ-Br is obtained by reaction;

[0017] The detailed process of the step (2) comprises: TBZ-Br 1 mmol, 4-aminophenylboronic acid pinacol ester 1.2 mmol, Pd(PPh3)4 0.06 mmol and anhydrous potassium carbonate 18 mmol are added into a mixed solvent of tetrahydrofuran and water, the volume ratio of tetrahydrofuran and water is 20 mL: 8 mL, and TBZ-NH2 is obtained by reaction;

[0018] The detailed process of the step (3) comprises: TBZ-NH2 0.64 mmol and maleic anhydride 1.28 mmol are added into tetrahydrofuran and stirred at room temperature overnight, after evaporation of the solvent, anhydrous sodium acetate 0.4 g and acetic anhydride 20 ml are added and stirred at 75 ℃ for 6 h, and after the mixture is cooled, purification is performed to obtain orange yellow powder TBZMA.

[0019] Further, in the step (1) and step (2), the Suzuki coupling reaction condition is 100 ℃, under the condition of nitrogen, refluxing for 12 h; in the step (3), the reaction condition of the intermediate product 2 and maleic anhydride is 75 ℃, and the reaction time is 6 h.

[0020] Further, the triphenylamine derivative is 4-boronic acid triphenylamine or a derivative of 4-boronic acid triphenylamine; the catalyst is tetrakis triphenylphosphine palladium; and the salt is one of K2CO3 and Na2CO3.

[0021] The application further provides an application of the photosensitizer in preparation of a tumor treatment drug.

[0022] The application further provides a preparation method of the nanopolymer containing the photosensitizer, comprising the following steps:

[0023] The photosensitizer and the amphiphilic macromolecular liposome are dissolved in an organic solvent to obtain a mixed solution; the obtained mixed solution is mixed with water and then ultrasonically treated until uniformly dispersed, and the organic solvent is removed, so that the amphiphilic macromolecular liposome coats the photosensitizer to obtain the nanopolymer containing the photosensitizer.

[0024] Further, the amphiphilic macromolecular liposome is DSPE-PEG 2000 , and the mass ratio of the photosensitizer and DSPE-PEG 2000 is 1:2.

[0025] The application further provides an application of the nanopolymer containing the photosensitizer obtained by the method in preparation of a tumor treatment drug.

[0026] Compared with the prior art, the application has the following advantages and technical effects:

[0027] Maleimide with specific recognition to Cys and strong electron acceptor is introduced into the molecular skeleton of the photosensitizer, the fluorescence signal is enhanced after the photosensitizer reacts with the thiol group of Cys, the photosensitizer is lighted up, the electron donor and the electron acceptor are optimized, the generation of singlet oxygen is inhibited, and the generation of free radical type ROS is promoted, so that the photosensitizer is specifically recognized to Cys and lighted up by fluorescence, and the efficiency of free radical type ROS generation is improved, and the cancer cells have good photodynamic treatment effect.

[0028] The AIE photosensitizer nanopolymer (TBZMA NPs) prepared by coating the above free radical type photosensitizer with the amphiphilic macromolecular liposome has good water dispersibility, higher solubility and faster dissolution speed, and has longer circulation time in blood flow, and the permeability and retention effect in tumor tissues are enhanced. The TBZMA NPs can be quickly phagocytosed by cells and show bright red fluorescence, and can also effectively generate 1O2 and O2 •- , OH and other radical type ROS. The results of cytotoxicity evaluation show that TBZMA NPs have good biocompatibility to cells without white light irradiation, but most of the cancer cells are killed after white light irradiation with appropriate power. The results of in vivo tumor experiments show that TBZMA NPs can effectively inhibit the growth of mouse tumors. BRIEF DESCRIPTION OF DRAWINGS

[0029] The accompanying drawings, which form a part of the present application, are intended to provide further understanding of the present application and are incorporated herein in conjunction with the description. The detailed description of the exemplary embodiments of the present application and its illustrations serve to explain the present application and do not constitute undue limitations on the present application. In the drawings:

[0030] Figure 1 A schematic diagram of the synthesis process of the AIE photosensitizer-containing nanopolymer;

[0031] Figure 2 A graph of the results of UV absorption spectrum and fluorescence spectrum of the photosensitizer prepared in Examples 1-4 in tetrahydrofuran solvent;

[0032] Figure 3 A graph of the fluorescence intensity of the photosensitizer prepared in Examples 1-4 in dimethyl sulfoxide / water mixed solvents with different water contents;

[0033] Figure 4 A graph of the fluorescence intensity change amplitude of the photosensitizer prepared in Examples 1-4 after light irradiation in DMSO / water solution containing H2DCF-DA probe, DHR123 probe, HPF probe, and ABDA probe, respectively;

[0034] Figure 5 A graph of the fluorescence change results of the AIE photosensitizer prepared in Examples 1-4 after light irradiation with Cys;

[0035] Figure 6 A graph of the fluorescence intensity change amplitude of the photosensitizer after light irradiation with or without Cys in DMSO / water solution containing H2DCF-DA probe, DHR123 probe, HPF probe, and ABDA probe;

[0036] Figure 7 A graph of the size distribution test results of the nanopolymer TBZMA NPs;

[0037] Figure 8 A graph of the fluorescence staining effect of TBZMA NPs (10 µM) incubated 4T1 cells;

[0038] Figure 9 A graph of the fluorescence staining effect of TBZMA NPs (10 µM) incubated 4T1 cells containing 10 µM H2DCF-DA;

[0039] Figure 10 Figure of MTT detection result of photodynamic therapy of 4T1 cells incubated with TBZMA NPs of different culture concentrations;

[0040] Figure 11 Figure of calcein acetoxymethyl ester (AM) / propidium iodide (PI) live and dead staining effect of 4T1 cells incubated with 10 μM TBZMA NPs and subjected to photodynamic therapy;

[0041] Figure 12 Figure of fluorescence imaging of mice after TBZMA NPs injection into tumors and statistical diagram of tumor growth and body weight change, wherein A is a fluorescence imaging diagram of TBZMA NPs injection into tumors at different time lengths, B is a statistical diagram of tumor volume change of different groups, C is a tumor diagram of different groups, and D is a statistical diagram of body weight change of mice in different groups. DETAILED DESCRIPTION

[0042] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0043] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the present application will be further described in detail below with reference to the drawings and specific embodiments.

[0044] In the embodiments of the present application, room temperature refers to “25 ± 2 ℃”.

[0045] Unless otherwise specified, the raw materials in the embodiments of the present application are obtained by commercial purchase.

[0046] Embodiment 1

[0047] The present embodiment provides a free radical type photosensitizer with a fluorescent “lighting” property, which is an aggregation-induced emission type photosensitizer (AIE photosensitizer) that lights up fluorescence of Cys and improves the efficiency of generation of free radical type reactive oxygen species, and the structural formula is as follows:

[0048]

[0049] The synthesis route is as follows:

[0050]

[0051] The specific preparation process is as follows:

[0052] (1) Synthesis of compound TBZ-Br

[0053] To a mixture of 4,7-dibromo-2,1,3-benzothiadiazole (440 mg, 1.5 mmol), 4-aminobenzeneboronic acid pinacol ester (260 mg, 1.2 mmol), Pd(PPh3)4(68 mg, 0.06 mmol) and anhydrous potassium carbonate (2.4 g, 18 mmol) in a mixture of tetrahydrofuran and water (volume ratio of tetrahydrofuran and water is 20 mL : 8 mL), reflux at 100 °C under nitrogen for 12 h. After the mixture was cooled, it was extracted with dichloromethane (DCM) three times, then washed with brine, and the organic layer was dried over anhydrous sodium sulfate, and column chromatography was performed using DCM and petroleum ether (volume ratio of DCM and petroleum ether is 1:30) as eluent to obtain TBZ-Br (754 mg) as a bright yellow powder.

[0054] The characterization data of the obtained TBZ-Br are as follows: 1 H NMR (500 MHz, CDCl3) δ 7.90 (d, 1H), 7.80(d, 2H), 7.54 (d, 1H), 7.32-7.27 (m, 4H), 7.21-7.14 (m, 6H), 7.10-7.05 (m,2H).

[0055] (2) Synthesis of compound TBZ-NH2

[0056] To a mixture of TBZ-Br (460 mg, 1 mmol), 4-aminobenzeneboronic acid pinacol ester (260 mg, 1.2 mmol), Pd(PPh3)4(68 mg, 0.06 mmol) and anhydrous potassium carbonate (2.4 g, 18 mmol) in a mixture of tetrahydrofuran and water (volume ratio of tetrahydrofuran and water is 20 mL : 8 mL), reflux at 100 °C under nitrogen for 12 h. After the mixture was cooled, it was extracted with dichloromethane (DCM) three times, then washed with brine, and the organic layer was dried over anhydrous sodium sulfate, and column chromatography was performed using DCM and petroleum ether (volume ratio of DCM and petroleum ether is 1:30) as eluent to obtain TBZ-NH2 (438 mg) as a yellow powder.

[0057] The characterization data of the obtained TBZ-NH2 are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 8.08 (d, 2H), 7.92(d, 4H), 7.49 (d, 2H), 7.32 (t, 4H), 7.20 (s, 2H), 7.08 (d, 8H).

[0058] (3) Synthesis of compound TBZMA

[0059] TBZ-NH2 (300 mg, 0.64 mmol) and maleic anhydride (125 mg, 1.28 mmol) were added to tetrahydrofuran and stirred overnight at room temperature. After the solvent evaporated, anhydrous sodium acetate (0.4 g) and acetic anhydride (20 ml) were added and stirred at 75 °C for 6 h. After the mixture was cooled, it was extracted three times with dichloromethane (DCM), washed with brine, and then the organic layer was dried with anhydrous sodium sulfate. Column chromatography was then performed using DCM and petroleum ether (DCM to petroleum ether volume ratio of 1:20) as eluent to obtain orange-yellow powder TBZMA (308 mg).

[0060] The characterization data of the obtained TBZMA are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 8.08 (d, 2H), 7.92(d, 4H), 7.49 (d, 2H), 7.32 (t, 4H), 7.20 (s, 2H), 7.08 (d, 8H).

[0061] (4) Synthesis of TBZMA NPs

[0062] like Figure 1 As shown, TBZMA (1.0 mg) and DSPE-PEG were used. 2000 (2.0 mg) was fully dissolved in 1 mL of tetrahydrofuran, then added to 10 mL of distilled water and sonicated until uniformly dispersed. The resulting solution was stirred for 24 h until tetrahydrofuran was removed, and a clear aqueous solution was obtained, which is the nanopolymer TBZMA NPs.

[0063] Example 2

[0064] This embodiment provides a free radical photosensitizer with fluorescent "lighting" properties. It is an aggregation-induced emission photosensitizer (AIE photosensitizer) that uses Cys to light up fluorescence and improves the efficiency of free radical reactive oxygen species generation. Its structural formula is as follows:

[0065]

[0066] The synthetic route is as follows:

[0067]

[0068] (1) Synthesis of compound MTBZ-Br

[0069] To a mixture of 4,7-dibromo-2,1,3-benzothiadiazole (440 mg, 1.5 mmol), 4-boronic acid-4',4'-dimethoxytriphenylamine (630 mg, 1.8 mmol), Pd(PPh3)4(68 mg, 0.06 mmol) and anhydrous potassium carbonate (2.4 g, 18 mmol) in a mixture of tetrahydrofuran and water (volume ratio of tetrahydrofuran and water is 20 mL:8 mL), reflux at 100 °C under nitrogen for 12 h. After the mixture was cooled, extracted with dichloromethane (DCM) three times, washed with brine, the organic layer was dried over anhydrous sodium sulfate, and then column chromatography was performed using DCM and petroleum ether (volume ratio of DCM and petroleum ether is 1:30) as eluent to obtain orange powder MTBZ-Br (558 mg).

[0070] The characterization data of the obtained MTBZ-Br are as follows: 1 H NMR (400 MHz, CD2Cl2) δ 7.88 (d, 1H), 7.75(s, 2H), 7.52 (d, 1H), 7.31-6.70 (m, 10H), 3.80 (s, 6H).

[0071] (2) Synthesis of compound MTBZ-NH2

[0072] To a mixture of MTBZ-Br (500 mg, 0.96 mmol), 4-aminobenzoic acid pinacol ester (260 mg, 1.2 mmol), Pd(PPh3)4(68 mg, 0.06 mmol) and anhydrous potassium carbonate (2.4 g, 18 mmol) in a mixture of tetrahydrofuran and water (volume ratio of tetrahydrofuran and water is 20 mL:4 mL), reflux at 100 °C under nitrogen for 12 h. After the mixture was cooled, extracted with dichloromethane (DCM) three times, washed with brine, the organic layer was dried over anhydrous sodium sulfate, and then column chromatography was performed using DCM and petroleum ether (volume ratio of DCM and petroleum ether is 1:25) as eluent to obtain red powder MTBZ-NH2 (362 mg).

[0073] The characterization data of the obtained MTBZ-NH2 are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 7.82 (d, 2H),7.74 (dd, 4H), 7.07 (d, 4H), 6.92 (d, 4H), 6.86 (d, 2H), 6.68 (d, 2H), 5.42(s, 2H), 3.73 (s, 6H).

[0074] (3) Synthesis of compound MTBZMA

[0075] MTBZ-NH2(300 mg, 0.56 mmol) and maleic anhydride (120 mg, 1.2 mmol) were added to tetrahydrofuran and stirred at room temperature overnight. After evaporation of the solvent, anhydrous sodium acetate (0.4 g) and acetic anhydride (20 ml) were added and stirred at 75 °C for 6 h. After cooling, the mixture was extracted with dichloromethane (DCM) three times, then washed with brine, and then the organic layer was dried over anhydrous sodium sulfate, and column chromatography was performed using DCM and petroleum ether (volume ratio of DCM to petroleum ether was 1:20) as eluent to obtain red powder MTBZMA (281 mg).

[0076] The characterization data of the obtained MTBZMA are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 8.13-8.09 (m, 1H), 7.99-7.87 (m, 2H), 7.56-7.50 (m, 1H), 7.24 (d, 1H), 7.16-7.09 (m, 2H), 7.00-6.94 (m, 2H), 6.90 (d, 1H), 3.77 (s, 3H).

[0077] (4) Synthesis of MTBZMA NPs

[0078] As shown in Figure 1 , MTBZMA (1.0 mg) and DSPE-PEG 2000 (2.0 mg) were fully dissolved in 1 mL of tetrahydrofuran, then put into 10 mL of distilled water, and ultrasonically treated until uniformly dispersed. The obtained solution was stirred for 24 h until the tetrahydrofuran was removed, to obtain a clear aqueous solution, which was the nano-polymer MTBZMA NPs.

[0079] Example 3

[0080] The present embodiment provides a free radical photosensitizer with a fluorescent "lighting" property, which is an aggregation-induced emission photosensitizer (AIE photosensitizer) that lights up the fluorescence of Cys and improves the efficiency of free radical active oxygen generation, and has the following structural formula:

[0081]

[0082] The synthesis route is as follows:

[0083]

[0084] (1) Synthesis of compound TNZ-Br

[0085] To a mixture of 4,9-dibromonaphtho[2,3-c][l,2,5]thiadiazole (500 mg, 1.45 mmol), 4-aminobenzeneboronic acid pinacol ester (260 mg, 1.2 mmol), Pd(PPh3)4(68 mg, 0.06 mmol) and anhydrous potassium carbonate (2.4 g, 18 mmol) in a mixture of tetrahydrofuran and water (volume ratio of tetrahydrofuran and water is 20 mL : 8 mL), reflux at 100 °C under nitrogen for 12 h. After the mixture was cooled, extracted with dichloromethane (DCM) three times, washed with brine, the organic layer was dried over anhydrous sodium sulfate, and column chromatography was performed using DCM and petroleum ether (volume ratio of DCM and petroleum ether is 1 : 30) as eluent to obtain red powder TNZ-Br (520 mg).

[0086] The characterization data of the obtained TNZ-Br are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 8.01 (d, 1H), 7.94(d, 1H), 7.47 (d, 2H), 7.40-7.23 (m, 8H), 7.18-7.01 (m, 8H), 6.75 (d, 2H),5.38 (s, 2H).

[0087] (2) Synthesis of compound TNZ-NH2

[0088] To a mixture of 4,9-dibromonaphtho[2,3-c][l,2,5]thiadiazole (500 mg, 1.45 mmol), 4-aminobenzeneboronic acid pinacol ester (260 mg, 1.2 mmol), Pd(PPh3)4(68 mg, 0.06 mmol) and anhydrous potassium carbonate (2.4 g, 18 mmol) in a mixture of tetrahydrofuran and water (volume ratio of tetrahydrofuran and water is 20 mL : 8 mL), reflux at 100 °C under nitrogen for 12 h. After the mixture was cooled, extracted with dichloromethane (DCM) three times, washed with brine, the organic layer was dried over anhydrous sodium sulfate, and column chromatography was performed using DCM and petroleum ether (volume ratio of DCM and petroleum ether is 1 : 25) as eluent to obtain purple red powder TNZ-NH2 (186 mg).

[0089] The characterization data of the obtained TNZ-NH2 are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 8.01 (d, 1H), 7.94(d, 1H), 7.47 (d, 2H), 7.40-7.23 (m, 8H), 7.18-7.01 (m, 8H), 6.75 (d, 2H),5.38 (s, 2H).

[0090] (3) Synthesis of compound TNZMA

[0091] TNZ-NH2(300 mg, 0.58 mmol) and maleic anhydride (120 mg, 1.2 mmol) were added to tetrahydrofuran and stirred at room temperature overnight. After evaporation of the solvent, anhydrous sodium acetate (0.4 g) and acetic anhydride (20 ml) were added and stirred at 75 °C for 6 h. After cooling, the mixture was extracted with dichloromethane (DCM) three times, then washed with brine, and then the organic layer was dried over anhydrous sodium sulfate, and column chromatography was performed using DCM and petroleum ether (volume ratio of DCM to petroleum ether was 1:20) as eluent to obtain red-black powder TNZMA (176 mg).

[0092] The characterization data of the obtained TNZMA are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 8.09-8.05 (m, 1H), 7.94-7.90 (m, 1H), 7.79-7.74 (m, 2H), 7.62-7.56 (m, 4H), 7.53-7.49 (m, 2H), 7.40 (dd, 4H), 7.28 (s, 2H), 7.23-7.17 (m, 6H), 7.14 (t, 2H).

[0093] (4) Synthesis of TNZMA NPs

[0094] As shown in Figure 1 , TNZMA (1.0 mg) and DSPE-PEG 2000 (2.0 mg) were fully dissolved in 1 mL of tetrahydrofuran, then placed in 10 mL of distilled water, and ultrasonically treated until uniformly dispersed. The obtained solution was stirred for 24 h until the tetrahydrofuran was removed, to obtain a clear aqueous solution, which was the nano-polymer TNZMA NPs.

[0095] Example 4

[0096] This example provides a free radical photosensitizer with a fluorescent “lighting” property, which is an aggregation-induced emission photosensitizer (AIE photosensitizer) that lights up the fluorescence of Cys and improves the efficiency of free radical active oxygen generation, and its structural formula is:

[0097]

[0098] The synthesis route is as follows:

[0099]

[0100] (1) Synthesis of compound MTNZ-Br

[0101] To a mixture of 4,9-dibromonaphtho[2,3-c][l,2,5]thiadiazole (500 mg, 1.45 mmol), 4-boronic acid-4',4'-dimethoxytriphenylamine (630 mg, 1.8 mmol), Pd(PPh3)4(68 mg, 0.06 mmol) and anhydrous potassium carbonate (2.4 g, 18 mmol) in a mixture of tetrahydrofuran and water (volume ratio of tetrahydrofuran and water is 20 mL : 8 mL) was added, then refluxed at 100 °C under nitrogen for 12 h, the mixture was cooled, extracted with dichloromethane (DCM) three times, then washed with brine, the organic layer was dried over anhydrous sodium sulfate, then column chromatography was performed using DCM and petroleum ether (volume ratio of DCM and petroleum ether is 1 : 30) as eluent to obtain red powder MTNZ-Br (518 mg).

[0102] The characterization data of the obtained MTNZ-Br are as follows: 1 H NMR (500 MHz, CD2Cl2) δ 8.47-8.24 (m, 1H), 8.14-8.00 (m, 1H), 7.61-7.49 (m, 1H), 7.43-7.35 (m, 3H), 7.21 (t, 4H), 7.04 (dd, 2H), 6.90 (d, 4H), 3.81 (d, 6H).

[0103] (2) Synthesis of compound MTNZ-NH2

[0104] To a mixture of MTNZ-Br (500 mg, 0.88 mmol), 4-aminophenylboronic acid pinacol ester (260 mg, 1.2 mmol), Pd(PPh3)4(68 mg, 0.06 mmol) and anhydrous potassium carbonate (2.4 g, 18 mmol) in a mixture of tetrahydrofuran and water (volume ratio of tetrahydrofuran and water is 20 mL : 8 mL) was added, then refluxed at 100 °C under nitrogen for 12 h, the mixture was cooled, extracted with dichloromethane (DCM) three times, then washed with brine, the organic layer was dried over anhydrous sodium sulfate, then column chromatography was performed using DCM and petroleum ether (volume ratio of DCM and petroleum ether is 1 : 25) as eluent to obtain purple red powder MTNZ-NH2 (168 mg).

[0105] The characterization data of the obtained MTNZ-NH2 are as follows: 1H NMR (400 MHz, DMSO-d6) δ 8.00 (dd, 2H), 7.61 (dd, 2H), 7.45-7.36 (m, 4H), 7.30 (dd, 3H), 7.17 (d, 3H), 7.03-6.92 (m, 5H), 6.76 (d, 2H), 6.54 (d, 1H), 3.74 (s, 6H).

[0106] (3) Synthesis of compound MTNZMA

[0107] MTNZ-NH2(300 mg, 0.52 mmol) and maleic anhydride (120 mg, 1.2 mmol) were added to tetrahydrofuran and stirred at room temperature overnight. After evaporation of the solvent, anhydrous sodium acetate (0.4 g) and acetic anhydride (20 ml) were added and stirred at 75 °C for 6 h. After the mixture was cooled, it was extracted with dichloromethane (DCM) three times, then washed with brine, and then the organic layer was dried over anhydrous sodium sulfate and column chromatography was performed using DCM and petroleum ether (volume ratio of DCM to petroleum ether was 1:20) as eluent to obtain red-black powder MTNZMA (172 mg).

[0108] The characterization data of the obtained MTNZMA are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 8.11-8.04 (m, 1H), 7.94-7.87 (m, 1H), 7.84-7.72 (m, 2H), 7.69-7.36 (m, 7H), 7.29-7.16 (m, 5H), 7.05-6.90 (m, 6H), 3.84-3.72 (m, 6H).

[0109] (4) Synthesis of MTNZMA NPs

[0110] As shown in Figure 1 , MTNZMA (1.0 mg) and DSPE-PEG 2000 (2.0 mg) were fully dissolved in 1 mL of tetrahydrofuran, and then put into 10 mL of distilled water, and ultrasonic treatment was performed until uniform dispersion. The obtained solution was stirred for 24 h until the tetrahydrofuran was removed, to obtain a clear aqueous solution, which was the nanopolymer MTNZMA NPs.

[0111] Experiment 1

[0112] The ultraviolet absorption spectrum and fluorescence spectrum of the photosensitizer prepared in Examples 1-4 in tetrahydrofuran solvent were tested. From Figure 2As can be seen, the maximum absorption peaks of TBZMA, MTBZMA, TNZMA, and MTNZMA are at 438 nm, 458 nm, 500 nm, and 520 nm, respectively, while the maximum fluorescence peaks are at 605 nm, 663 nm, 658 nm, and 578 nm, respectively. The Stokes shifts of TBZMA, MTBZMA, TNZMA, and MTNZMA are 167 nm, 205 nm, 158 nm, and 58 nm, respectively, preliminarily indicating that TBZMA, MTBZMA, and TNZMA molecules all possess large Stokes shifts.

[0113] Experiment 2

[0114] The photosensitizers prepared in Examples 1-4 were tested in dimethyl sulfoxide / water mixed solvents with water contents (v / v) of 0%, 20%, 40%, 60%, and 80%, respectively, to determine their fluorescence intensity. The fluorescence intensity amplification and fluorescence effect are as follows: Figure 3 As shown.

[0115] Experiment 3

[0116] Test in H2DCF-DA ( Figure 4 The fluorescence intensity changes after irradiation of DMSO / water solutions containing the abbreviations DCFH, DHR123, HPF, and ABDA probes (each with the photosensitizers prepared in Examples 1-4) were measured. The results are as follows: Figure 4 As shown, where, Figure 4 A shows the fluorescence intensity changes after illumination of DMSO / water containing H2DCF-DA probe with the addition of TBZMA, MTBZMA, TNZMA, and MTNZMA, respectively. λ ex =488 nm; Figure 4 B shows the fluorescence intensity changes after illumination of DMSO / water containing the DHR123 probe with the addition of TBZMA, MTBZMA, TNZMA, and MTNZMA, respectively. ex =490 nm; Figure 4 C represents the fluorescence intensity changes after illumination of DMSO / water containing HPF probe with the addition of TBZMA, MTBZMA, TNZMA, and MTNZMA, respectively. λ ex =490 nm; Figure 4 D represents the fluorescence intensity variation after illumination of DMSO / water containing ABDA probe with the addition of TBZMA, MTBZMA, TNZMA, and MTNZMA, respectively. λ abs =400 nm. From Figure 4As can be seen from the figure, the four AIE photosensitizers TBZMA, MTBZMA, TNZMA and MTNZMA all produce reactive oxygen species, and all produce free radical type reactive oxygen species. Among them, MTBZMA, TNZMA and MTNZMA only produce free radical type reactive oxygen species, while TBZMA produces both free radical type reactive oxygen species and singlet oxygen, and TBZMA has the highest efficiency of producing reactive oxygen species.

[0117] Experiment 4

[0118] The AIE photosensitizers prepared in Examples 1-4 were tested for fluorescence changes after adding Cys and light irradiation, and the results are shown in Figure 5 It was found that only photosensitizer TBZMA had obvious fluorescence enhancement after adding Cys and light irradiation. Subsequent experiments were carried out using photosensitizer TBZMA.

[0119] Experiment 5

[0120] The fluorescence intensity changes of photosensitizer TBZMA after adding Cys and light irradiation in DMSO / water solution containing H2DCF-DA probe, DHR123 probe, HPF probe or ABDA probe were tested. The results are shown in Figure 6 As can be seen from the figure, photosensitizer TBZAM quenches ·OH and 1 O2 after adding Cys, only produces O2 ·- , realizing the conversion of ROS.

[0121] Experiment 6

[0122] The size distribution of nanopolymer TBZMA NPs was tested using a laser particle size analyzer, and the results are shown in Figure 7 The size of nanopolymer TBZMA NPs is mainly concentrated at about 118 nm, indicating that it has a high surface area to volume ratio, thereby ensuring higher solubility and faster dissolution rate. Such small particles have a longer circulation time in the blood stream, enhanced permeability and retention in tumor tissues.

[0123] Experiment 7

[0124] 10 µM TBZMA NPs were incubated with 4T1 cells, and the fluorescence staining effect was observed at incubation times of 0.5 h, 3 h, 4 h and 6 h, respectively. The results are shown in Figure 8 It can be seen that the photosensitizer TBZMA NPs can quickly enter the 4T1 cells.

[0125] Experiment 8

[0126] 10 µM TBZMA NPs incubated 4T1 cells containing 10 µM H2DCF-DA, respectively, in the incubation length of 0 s, 10 s, 20 s, 40 s to observe the fluorescence staining effect. The results are shown in Figure 9 , TBZMA NPs can produce effective reactive oxygen species in 4T1 cells.

[0127] Experiment 9

[0128] The photodynamic therapy effect of TBZMA NPs incubated 4T1 cells with culture concentration of 0 µM, 0.25 µM, 0.5 µM, 1 µM, 2 µM, 4 µM, 8 µM, 16 µM, 32 µM respectively. The results are shown in Figure 10 , when the culture concentration of TBZMA NPs is 1 μM, after white light irradiation, the cell survival rate is only 20%, which shows that TBZMA NPs has good photodynamic therapy effect on cancer cells.

[0129] Experiment 10

[0130] 10 µM TBZMA NPs incubated 4T1 cells and carried out photodynamic therapy, then calcein AM / PI live and dead staining, the results are shown in Figure 11 , TBZMA NPs incubated 4T1 cells, which has no biological dark toxicity to cells, and after white light irradiation, only PI staining, which shows that photosensitizer has good cell phototoxicity, and TBZMA NPs photosensitizer has good photodynamic therapy effect on cancer cells.

[0131] Experiment 11

[0132] TBZMA NPs were injected into the tumor of tumor-bearing mice, and the tumor growth was observed by in vivo fluorescence imaging, the results are shown in Figure 12 , from Figure 12 A can be seen that after 2 h of TBZMA NPs injection into the tumor, intratumoral fluorescence imaging is obvious, and the signal-to-noise ratio is low, which shows that TBZMA NPs has good in vivo fluorescence imaging effect. From Figure 12 B, Figure 12 C can be seen that TBZMA NPs without photodynamic therapy do not light group of tumor proliferation rapidly, while after photodynamic therapy, it can effectively inhibit the growth of tumor, which shows that TBZMA NPs still has good effect in in vivo tumor treatment. In addition, Figure 12 D shows that the body weight growth trend of mice in all experimental groups is the same, which shows that TBZMA NPs does not cause adverse effects on mice, and has good biological safety.

[0133] The above merely provides the preferred embodiments of the present application, and the protection scope of the present application is not limited thereto, and any changes or substitutions within the technical scope disclosed by the present application should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A free radical photosensitizer with fluorescent "lighting" properties, characterized in that, The structural formula is: 。 2. A method for preparing the photosensitizer as described in claim 1, characterized in that, Includes the following steps: (1) TBZ-Br was obtained by Suzuki coupling reaction using 4,7-dibromo-2,1,3-benzothiadiazole and triphenylamine 4-boronic acid as raw materials. The structural formula is: ； (2) TBZ-Br and pinacol 4-aminophenylboronic acid were subjected to a Suzuki coupling reaction to obtain TBZ-NH2, with the following structural formula: ； (3) TBZ-NH2 and maleic anhydride are reacted to obtain the final product TBZMA.

3. The preparation method according to claim 2, characterized in that, In step (1), a catalyst and salt are added to react 4,7-dibromo-2,1,3-benzothiadiazole with triphenylamine 4-boronic acid. The molar ratio of 4,7-dibromo-2,1,3-benzothiadiazole, triphenylamine 4-boronic acid, catalyst, and salt is 1: (1-1.2): (0.05-0.1):

12. In step (2), TBZ-Br reacts with 4-aminophenylboronic acid pinacol ester with the addition of a catalyst and a salt. The molar ratio of TBZ-Br, 4-aminophenylboronic acid pinacol ester, catalyst and salt is 1: (1-1.2): (0.05-0.1):

18. In step (3), anhydrous sodium acetate and acetic anhydride are added to react TBZ-NH2 with maleic anhydride. The molar ratio of TBZ-NH2, maleic anhydride, anhydrous sodium acetate and acetic anhydride is 1: (1.8-2.2): (7.6-7.8): (330-335).

4. The preparation method according to claim 3, characterized in that, The detailed process of step (1) includes: adding 1.5 mmol of 4,7-dibromo-2,1,3-benzothiadiazole, 1.8 mmol of triphenylamine 4-boronic acid, 0.06 mmol of Pd(PPh3)4 and 18 mmol of anhydrous potassium carbonate to a mixed solvent of tetrahydrofuran and water, with a volume ratio of 20 mL : 8 mL, to obtain TBZ-Br; The detailed process of step (2) includes: adding 1 mmol of TBZ-Br, 1.2 mmol of 4-aminophenylboronic acid pinacol ester, 40.06 mmol of Pd(PPh3)4 and 18 mmol of anhydrous potassium carbonate to a mixed solvent of tetrahydrofuran and water, with a volume ratio of 20 mL : 8 mL, and reacting to obtain TBZ-NH2; The detailed process of step (3) includes: adding 0.64 mmol of TBZ-NH2 and 1.28 mmol of maleic anhydride to tetrahydrofuran and stirring overnight at room temperature. After the solvent evaporates, 0.4 g of anhydrous sodium acetate and 20 ml of acetic anhydride are added and stirred at 75 °C for 6 h. After cooling the mixture, it is purified to obtain orange-yellow powder TBZMA.

5. The preparation method according to claim 2, characterized in that, In steps (1) and (2), the Suzuki coupling reaction conditions are 100 °C and reflux under nitrogen for 12 h; in step (3), the reaction conditions of TBZ-NH2 and maleic anhydride are 75 °C and 6 h.

6. The preparation method according to claim 3, characterized in that, The catalyst is tetrakis(triphenylphosphine)palladium; the salt is one of K₂CO₃ and Na₂CO₃.

7. The use of the photosensitizer as described in claim 1 in the preparation of a tumor-treating drug, wherein the tumor-treating drug specifically recognizes intracellular cysteine ​​and generates free radical ROS.

8. A method for preparing a nanopolymer containing the photosensitizer of claim 1, characterized in that, Includes the following steps: Photosensitizer and amphiphilic polymer DSPE-PEG 2000 Dissolve in an organic solvent to obtain a mixed solution; mix the obtained mixed solution with water and sonicate until uniformly dispersed, remove the organic solvent, and encapsulate the photosensitizer with amphiphilic polymer liposomes to obtain a photosensitizer-containing nanopolymer.

9. The method for preparing the photosensitizer-containing nanopolymer according to claim 8, characterized in that, The photosensitizer and DSPE-PEG 2000 The mass ratio is 1:

2.

10. The use of a photosensitizer-containing nanopolymer obtained by the method of any one of claims 8-9 in the preparation of a tumor-treating drug, wherein the tumor-treating drug specifically recognizes intracellular cysteine ​​and generates free radical ROS.

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