Nanometer material for treating bladder cancer through near-infrared two-region AIE probe and light activation

Abstract

The invention provides a near-infrared second-region AIE probe and a nanometer material for treating bladder cancer through light activation, belongs to the technical field of material chemistry, and designs an AIE probe capable of performing efficient photo-thermal conversion in a near-infrared second region aiming at the problem that the treatment effect of a photo-thermal therapy on the bladder cancer is poor. On the basis, an AIE probe and a thermosensitive NO donor are blended, and photo-thermal conversion is induced by near-infrared light to release NO; the cell membrane surface PMCA calcium channel blocking peptide and the tumor surface FGFR1 receptor targeting peptide are modified after being packaged by an amphiphilic polymer, the cell membrane surface PMCA calcium channel blocking peptide and the tumor surface FGFR1 receptor targeting peptide jointly act with NO released by a thermosensitive NO donor, a calcium ion pathway is adjusted through endogenous and exogenous combination, tumor calcium overload is caused, and tumor cell apoptosis is induced; the surface-modified tumor surface FGFR1 receptor targeting peptide can realize efficient enrichment of the nano material at a tumor site, and the treatment effect is improved. The material provided by the invention provides a new thought for treatment of bladder cancer.

Description

Technical Field

[0001] The invention belongs to the technical field of material chemistry. Background Art

[0002] Bladder cancer (BCa) is a global health problem. BCa is the second most common urinary tract malignancy, accounting for 6% of all cancer cases in men and 3% in women. According to the depth of invasion, BCa can be further divided into non-muscle invasive bladder cancer (NMIBC) and muscle invasive bladder cancer (MIBC). MIBC has always been a difficult problem to treat due to its high metastasis rate, high recurrence rate and high mortality rate. In the past decade, the treatment of bladder cancer has shifted from radical surgery to organ preservation, but the efficacy is often unsatisfactory. Therefore, there is an urgent need to develop new diagnostic and treatment methods to improve the survival rate of BCa patients.

[0003] Photothermal therapy (PTT) is a safe cancer treatment method that can convert light energy into heat energy to ablate tumor cells. A large number of reports have confirmed that the advantages of PTT are non-invasiveness, local treatment and good therapeutic effects. However, simple PTT is difficult to completely eradicate tumors in solid tumors, which is limited by the limited light irradiation area and insufficient local accumulation of photothermal agents, which often leads to tumor recurrence at the edge of the radiation range. In recent years, photosensitizers with aggregation-induced emission (AIE) characteristics have been widely explored in the field of tumor treatment because of their outstanding advantages such as large Stokes shift, good photostability and strong biocompatibility.

[0004] BCa is a typical deep tissue tumor. Common near-infrared light (NIR) has limited penetration depth and is difficult to reach tumors deep in the bladder wall. In addition, the targeting ability of photosensitizers alone to bladder tumor tissue is often unsatisfactory, which will not only reduce the efficacy of PTT but also increase the risk of normal tissue damage. Summary of the invention

[0006] In order to solve the above problems, the present invention designs a near-infrared second-zone AIE probe and a light-activated nanomaterial for treating bladder cancer. While having the ability to penetrate deep tumor tissue, it can also achieve good targeting of tumor tissue, thereby greatly enhancing the efficacy of PTT and reducing the occurrence of toxic side effects.

[0007] The present invention provides a near-infrared second-zone AIE probe, wherein the probe is one of SF, FK and BD, and the molecular structures of SF, FK and BD are as follows: The synthesis method of the second near-infrared region AIE probes SF, FK and BD of the present invention comprises the following steps: (1) 9-hexyl-7-(4-hexyl-5-(tributyltin)thiophene-2-yl)-N,N-diphenyl-9H-carbazole-2-amine, 4,7-dibromo-5,6-dinitrobenzo[c][1,2,5]thiadiazole and Pd(PPh3)4 were mixed in an argon atmosphere, and then anhydrous tetrahydrofuran (THF) was added. The mixture was heated and stirred. The resulting mixture was extracted three times with CH2Cl2, dried with MgSO4 and concentrated. After purification, 7,7'-((5,6-dinitrobenzo[c][1,2,5]thiadiazole-4,7-diyl)bis(4-hexylthiophene-5,2-diyl))bis(9-hexyl-N,N-diphenyl-9H-carbazole-2-amine) was obtained; 9-hexyl-7-(4-hexyl-5-(tributyltin)thiophen-2-yl)-N,N-diphenyl-9H-carbazole-2-amine, 4,7-dibromo-5,6-dinitrobenzo[c][1,2,5]thiadiazole and Pd(PPh3)4 in a molar ratio of 2:1:0.04; (2) 7,7'-((5,6-dinitrobenzo[c][1,2,5]thiadiazole-4,7-diyl)bis(4-hexylthiophene-5,2-diyl))bis(9-hexyl-N,N-diphenyl-9H-carbazole-2-amine) and glacial acetic acid are mixed, iron powder is added to the mixture, heated and stirred. The resulting mixture is extracted three times with CH2Cl2. The organic phase is dried over MgSO4 and concentrated. The resulting crude product of 4,7-bis(5-(7-(diphenylamine)-9-hexyl-9H-carbazole-2-yl)-3-hexylthiophene-2-yl)benzo[c][1,2,5]thiadiazole-5,6-diamine can be used without further purification; The molar ratio of 7,7'-((5,6-dinitrobenzo[c][1,2,5]thiadiazol-4,7-yl)bis(4-hexylthiophene-5,2-diyl))bis(9-hexyl-N,N-diphenyl-9H-carbazole-2-amine) to iron powder is 1:30; (3) 4,7-bis(5-(7-(diphenylamine)-9-hexyl-9H-carbazole-2-yl)-3-hexylthiophene-2-yl)benzo[c][1,2,5]thiadiazole-5,6-diamine, chloroform and acetic acid are mixed in an argon atmosphere, and then benzo[1,2-b:6,5-b']dithiophene-4,5-dione, phenanthroquinone or 1,10-phenanthroline-5,6-dione is added. The mixture is heated to reflux and stirred continuously. The resulting mixture is extracted three times with CH2Cl2. The collected organic phase is dried over MgSO4 and concentrated. After purification, the final product AIE probe SF, FK or BD is obtained.

[0008] The molar ratio of 4,7-bis(5-(7-(diphenylamine)-9-hexyl-9H-carbazole-2-yl)-3-hexylthiophene-2-yl)benzo[c][1,2,5]thiadiazole-5,6-diamine to benzo[1,2-b:6,5-b']dithiophene-4,5-dione, phenanthronequinone or 1,10-phenanthroline-5,6-dione is 1:1.5.

[0009] Preferably, in step (1), 9-hexyl-7-(4-hexyl-5-(tributyltin)thiophene-2-yl)-N,N-diphenyl-9H-carbazole-2-amine is prepared by the following method: In an argon environment, 9-hexyl-7-(4-hexylthiophene-2-yl)-N,N-diphenyl-9H-carbazole-2-amine was dissolved in anhydrous tetrahydrofuran, the mixture was cooled to -78°C and maintained for 30 minutes, then a hexane solution of n-butyl lithium was added, and the mixture was stirred at -78°C for 2 hours, then tri-n-butyltin chloride was added, and then the temperature was slowly raised to room temperature and stirred for 8-12 hours. Subsequently, water was added to quench the reaction, and the resulting mixture was extracted three times with CH2Cl2, and the obtained organic phase was dried with MgSO4 and concentrated to obtain 9-hexyl-7-(4-hexyl-5-(tributyltin)thiophene-2-yl)-N,N-diphenyl-9H-carbazole-2-amine; Among them, the molar ratio of 9-hexyl-7-(4-hexylthiophene-2-yl)-N,N-diphenyl-9H-carbazole-2-amine, n-butyl lithium, and tri-n-butyltin chloride is 1:(1~1.2):(1~1.2).

[0010] Further preferably, the preparation method of 9-hexyl-7-(4-hexylthiophene-2-yl)-N,N-diphenyl-9H-carbazole-2-amine is: In an argon atmosphere, 7-bromo-9-hexyl-N,N-diphenyl-9H-carbazole-2-amine and Pd(pph3)4 are added to tri-n-butyl(4-hexylthiophene-2-yl)stannane and mixed, and then anhydrous tetrahydrofuran is added; the mixture is heated to reflux and stirred continuously for 24 hours, and after cooling to room temperature, water is added, and the resulting mixture is extracted three times with CH2Cl2, and the collected organic phase is dried with MgSO4 and concentrated to obtain 9-hexyl-7-(4-hexylthiophene-2-yl)-N,N-diphenyl-9H-carbazole-2-amine after purification; Among them, the molar ratio of tri-n-butyl (4-hexylthiophene-2-yl) stannane to 7-bromo-9-hexyl-N,N-diphenyl-9H-carbazole-2-amine is (1.2~1):1.

[0011] Further preferably, the preparation method of tri-n-butyl (4-hexylthiophen-2-yl) stannane is: In an argon environment, 3-hexylthiophene was dissolved in 30 mL of anhydrous tetrahydrofuran, the mixture was cooled to -78 ° C and maintained for 30 minutes, then a hexane solution of n-butyl lithium was added, and stirred at -78 ° C for 2 hours, then tri-n-butyltin chloride was added, and then the temperature was slowly raised to room temperature and stirred for 8-12 hours. Subsequently, water was added to quench the reaction, and the resulting mixture was extracted three times with CH2Cl2, and the obtained organic phase was dried with MgSO4 and concentrated to obtain tri-n-butyl (4-hexylthiophene-2-yl) tin ane; the molar ratio of 3-hexylthiophene, n-butyl lithium, and tri-n-butyltin chloride was 1: (1-1.2): (1-1.2); Further preferably, the preparation method of 7-bromo-9-hexyl-N,N-diphenyl-9H-carbazole-2-amine is: In an argon environment, 2,7-dibromo-9-hexyl-9H-carbazole and diphenylamine were mixed in toluene, and then cesium carbonate, palladium acetate, and tributylphosphine were added, and the mixture was heated to reflux and stirred continuously for 24 hours. After cooling to room temperature, water was added, and the resulting mixture was extracted three times with CH2Cl2. The collected organic phase was dried with MgSO4 and concentrated, and purified to obtain 7-bromo-9-hexyl-N,N-diphenyl-9H-carbazole-2-amine (Compound 2); wherein the molar ratio of 2,7-dibromo-9-hexyl-9H-carbazole, diphenylamine, cesium carbonate, and palladium acetate was 2:2:3:0.1; Further preferably, the preparation method of 2,7-dibromo-9-hexyl-9H-carbazole is: 2,7-Dibromocarbazole and sodium hydride were mixed in anhydrous N,N-dimethylformamide (DMF). Under argon protection, hexyl bromide was added after 30 minutes and stirred for 20 hours. Subsequently, water was added to quench the reaction. The resulting mixture was extracted three times with CH2Cl2. The obtained organic phase was dried with MgSO4 and concentrated. After purification, 2,7-dibromo-9-hexyl-9H-carbazole was obtained. The molar ratio of 2,7-dibromocarbazole, sodium hydride and hexyl bromide was 1:1.5:1.3.

[0012] The present invention also provides a nanomaterial for light-activated treatment of bladder cancer. The material is prepared by blending the AIE probe with a thermosensitive nitric oxide donor, encapsulating it with an amphiphilic polymer, and modifying its surface with a tumor surface FGFR1 receptor targeting peptide and a cell membrane surface PMCA calcium ion channel blocking peptide, thereby forming a nanomaterial for light-activated treatment of bladder cancer.

[0013] Preferably, the thermosensitive nitric oxide donor is S-nitroso-N-acetylpenicillamine, the tumor surface FGFR1 receptor targeting peptide is FGL peptide, the cell membrane surface PMCA calcium ion channel blocking peptide is Caloxin 2A1, and the amphiphilic polymer is DSPE-PEG 2000 .

[0014] The preparation method of the light-activated nanomaterial for treating bladder cancer in the present invention is as follows: 1) The tumor FGFR1 receptor targeting peptide FGL and the cell membrane surface PMCA calcium channel blocking peptide Caloxin 2A1 were subjected to thiol addition reaction with DSPE-PEG-MAL in a water / methanol solution with a volume ratio of 90:10 at room temperature, and then the residual peptides were removed by centrifugal filter, and the products obtained by freeze-drying were recorded as FR and PM; the mass ratio of tumor FGFR1 receptor targeting peptide FGL to DSPE-PEG-MAL was (0.9~1):3, and the mass ratio of cell membrane surface PMCA calcium channel blocking peptide Caloxin 2A1 to DSPE-PEG-MAL was (1~1.1):3; 2) The FR and PM obtained in step 1) were mixed with AIE probe, S-nitroso-N-acetylpenicillamine, DSPE-PEG 2000 Dispersed in THF, after complete dissolution, the above stock solution was dropped into 10 mL of purified water and stirred vigorously. The organic solvent was stirred and evaporated in a fume hood and then concentrated through an ultrafiltration filter. The obtained light-activated nanomaterials for the treatment of bladder cancer; among them, AIE probe, S-nitroso-N-acetylpenicillamine, DSPE-PEG 2000 The mass ratio of FR and PM is 1:2:9:2.4:2.6.

[0015] The beneficial effects of the present invention are: 1. The AIE provided by the present invention is an aggregation-induced emission molecule containing thiophene substitution, which can achieve a strong near-infrared light-induced photothermal conversion effect in the near-infrared II region (NIR-II, 1000 ~ 1700 nm) with a longer wavelength and lower energy. NIRII has been proven to have smaller scattering and greater tissue penetration ability at the in vivo level, and can reach deeper into the bladder to achieve better therapeutic effects.

[0016] 2. The photoactivated nanomaterial SF / S@NPs-FR / PM for treating bladder cancer provided by the present invention is a nanomaterial. AIE is mixed with a thermosensitive nitric oxide (NO) donor SNAP to release NO by photothermal conversion induced by near-infrared light. The PMCA calcium channel blocking peptide Caloxin 2A1 (VSNSNWPSFPSSGGG-NH2) on the cell membrane surface acts together with the released NO to regulate calcium ions (Ca) by combining endogenous and exogenous 2+ ) pathway, causing tumor calcium overload and inducing tumor cell apoptosis; the surface-modified tumor surface FGFR1 receptor targeting peptide FGL (EVYVVAENQQGKSKA) can achieve efficient enrichment of the nanomaterial at the tumor site, providing a new idea for BCa treatment.

[0017] 3. The present invention utilizes the release of photosensitizer and NO gas in nanomaterials to activate intracellular calcium ion pathways, thereby affecting biological processes such as tumor cell proliferation and apoptosis. Therefore, the combination of near-infrared probe technology and NO gas treatment strategy is expected to achieve the goal of precision medicine in tumor treatment and improve patient prognosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Density functional theory (DFT) calculation diagram of the three AIE molecules of the present invention.

[0019] Figure 2 (a) Absorption spectra, (b) fluorescence emission spectra, and (c) photoluminescence PL intensities of the three AIE nanomaterials in different solvents.

[0020] Figure 3 The photothermal properties of the three nanomedicines (50 µM) of the present invention under 730 nm laser irradiation at different powers (a, c, e); the photothermal properties of the three nanomedicines at different concentrations under 730 nm laser irradiation (0.8 W / cm 2 )’s photothermal properties (b, d, f).

[0021] Figure 4 The water and SF / S@NPs-FR / PM solution of the present invention were respectively illuminated by 730 nm laser (0.8 W / cm 2) (a) Temperature curve and (b) photothermal conversion efficiency fitting curve after irradiation for different time periods.

[0022] Figure 5 The SF / S@NPs-FR / PM and SNAP of the present invention were illuminated by 730 nm laser (0.8 W / cm 2 ) NO release after irradiation for different time periods.

[0023] Figure 6 These are photos of the enrichment of the non-tumor targeting group (a) SF / S@NPs and the tumor targeting group (b) SF / S@NPs-FR / PM in MB49 tumor cells.

[0024] Figure 7 The killing ability of the tumor targeting group SF / S@NPs-FR / PM of the present invention against MB49 tumor cells at different concentrations.

[0025] Figure 8 The results of the Calcein-AM / PI live-dead staining experiments on MB49 tumor cells by various drug treatment groups of the present invention are shown.

[0026] Fig. 9 The results of the present invention are the results of the calcium ion concentration test in MB49 tumor cells of each drug treatment group by CLSM.

[0027] Fig.10 In the present invention, CLSM was used to detect the release amount of cytochrome C in MB49 tumor cells in each drug treatment group.

[0028] Fig.11 The present invention provides feedback through hemolysis experiments on (a) the hemolysis rate of each drug treatment group and (b) the hemolysis rate after treatment with nanodrug SF / S@NPs-FR / PM at different concentrations. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is fully described below in conjunction with the accompanying drawings of the present invention.

[0030] Example 1 Synthesis of near-infrared region II AIE probes SF, FK and BD (1) Synthesis of compound 1: 10 g (30.77 mmol) of 2,7-dibromocarbazole and 1.85 g (46.16 mmol) of sodium hydride were mixed in 120 mL of anhydrous N,N-dimethylformamide (DMF). Under argon protection, 6.6 g (39.98 mmol) of bromohexane were added after 30 minutes. The mixture was stirred for 20 hours. Subsequently, water was added to quench the reaction. The resulting mixture was extracted three times with CH2Cl2, and the resulting organic phase was dried with MgSO4 and concentrated. The crude product was purified by silica gel column chromatography using hexane as the elution solvent to obtain 2,7-dibromo-9-hexyl-9H-carbazole (Compound 1).

[0031] 1 H NMR (400 MHz, Chloroform- d ) δ 7.83 (d, J = 8.2 Hz, 2H), 7.49 (d, J =1.6 Hz, 2H), 7.30 (dd, J = 8.2, 1.6 Hz, 2H), 4.12 (t, J = 7.4 Hz, 2H), 1.83 –1.75 (m, 2H), 1.34 – 1.25 (m, 6H), 0.90 – 0.84 (m, 3H). 13 C NMR (101 MHz, Chloroform- d ) δ 141.38, 122.55, 121.49, 121.31, 119.73,112.02, 43.37, 31.55,28.81, 26.90, 22.60, 14.05. (2) Synthesis of compound 2 In an argon atmosphere, 10 g (24.4 mmol) of compound 1 (2,7-dibromo-9-hexyl-9H-carbazole) and 4.2 g (24.4 mmol) of diphenylamine were mixed in 100 mL of toluene, followed by the addition of 11.92 g (36.6 mmol) of cesium carbonate, 273.8 mg (1.22 mmol) of palladium acetate, and 2.68 mL (0.22 mmol) of tributylphosphine. The mixture was heated to reflux and stirred continuously for 24 hours. After cooling to room temperature, water was added and the resulting mixture was extracted three times with CH2Cl2. The collected organic phases were dried over MgSO4 and concentrated. The crude product was purified by silica gel column chromatography using CH2Cl2 / hexane (v / v 1:8) as the elution solvent to obtain 7-bromo-9-hexyl-N,N-diphenyl-9H-carbazole-2-amine (compound 2).

[0032] 1 H NMR (400 MHz, Chloroform- d ) δ 7.94 (d, J = 8.4 Hz, 1H), 7.86 (d, J =8.3 Hz, 1H), 7.54 (d, J = 1.7 Hz, 1H), 7.36 – 7.29 (m, 5H), 7.23 (dt, J = 8.4,1.6 Hz, 4H), 7.18 (d, J = 1.9 Hz, 1H), 7.12 – 7.02 (m, 3H), 4.11 (t, J = 7.1 Hz,2H), 1.82 – 1.75 (m, 2H), 1.37 – 1.26 (m, 6H), 0.96 – 0.87 (m, 3H). 13 C NMR(101 MHz, Chloroform-d) δ 148.31, 146.80,141.88, 141.77, 129.34, 124.15,122.74, 122.18, 122.04, 121.05, 120.96, 118.51, 118.19, 117.53, 111.70,105.03, 43.11, 31.61, 28.85, 26.90, 22.61, 14.16. (3) Synthesis of compound 3 In an argon atmosphere, 605.9 mg (3.6 mmol) of 3-hexylthiophene was dissolved in 30 mL of anhydrous tetrahydrofuran (THF), and the mixture was cooled to -78 °C and kept for 30 minutes, then 2.7 mL of 1.6 M (4.32 mmol) n-butyllithium (n-BuLi) hexane solution was added, stirred at -78 °C for 2 hours, and then 1.2 mL (4.32 mmol) of tri-n-butyltin chloride was added, and then slowly warmed to room temperature and stirred overnight. Subsequently, water was added to quench the reaction, and the resulting mixture was extracted three times with CH2Cl2, and the obtained organic phase was dried with MgSO4 and concentrated to obtain tri-n-butyl (4-hexylthiophene-2-yl) tin ane (compound 3). The obtained crude product can be used without any purification.

[0033] (4) Synthesis of compound 4: In an argon atmosphere, 1.5 g (3 mmol) of compound 2 (7-bromo-9-hexyl-N,N-diphenyl-9H-carbazole-2-amine) and 138.7 mg (0.12 mmol) of tetrakis(triphenylphosphine)palladium (Pd(pph3)4) were added to compound 3 (tri-n-butyl(4-hexylthiophen-2-yl)stannane) obtained in step (3) and mixed, followed by the addition of anhydrous THF. The mixture was heated to reflux and stirred for 24 hours. After cooling to room temperature, water was added and the resulting mixture was extracted three times with CH2Cl2. The collected organic phases were dried over MgSO4 and concentrated. The crude product was purified by silica gel column chromatography using CH2Cl2 / hexane (v / v 1:7) as the elution solvent to obtain 9-hexyl-7-(4-hexylthiophen-2-yl)-N,N-diphenyl-9H-carbazole-2-amine (compound 4).

[0034] 1 H NMR (400 MHz, Chloroform- d ) δ 8.00 (dd, J = 14.4, 8.2 Hz, 2H), 7.61(d, J = 1.4 Hz, 1H), 7.55 (dd, J = 8.1, 1.5 Hz, 1H), 7.34 (dd, J = 9.1, 6.5 Hz,5H), 7.26 (d, J = 7.8 Hz, 4H), 7.21 (d, J = 1.8 Hz, 1H), 7.14 – 7.04 (m, 3H), 6.95 (s, 1H), 4.22 (t, J= 7.1 Hz, 2H), 2.73 (t, J = 7.7 Hz, 2H), 1.86 (t, J = 7.2Hz, 2H), 1.78 (t, J = 7.8 Hz, 2H), 1.51 – 1.30 (m, 12H), 1.05 – 0.98 (m, 3H), 0.98 – 0.90 (m, 3H). 13 C NMR (101 MHz, Chloroform- d ) δ 148.33, 146.31, 145.39,144.39, 142.21, 141.31, 131.68, 129.24, 124.35, 123.96, 122.51, 122.41,120.92, 120.10, 119.13, 118.65, 117.91,117.61, 117.35, 105.63, 105.11, 42.90,31.83, 31.61, 30.83, 30.57, 29.17, 28.90, 26.91, 22.75, 22.59, 14.25, 14.14. (5) Synthesis of compound 5: In an argon atmosphere, 1.2 g (2.1 mmol) of compound 4 (9-hexyl-7-(4-hexylthiophen-2-yl)-N,N-diphenyl-9H-carbazole-2-amine) was dissolved in anhydrous THF, the mixture was cooled to -78°C and kept for 30 minutes, then 1.6 mL (2.5 mmol) of 1.6 M n-butyl lithium (n-BuLi) hexane solution was added, stirred at -78°C for 2 hours, then 0.68 mL (2.5 mmol) of tri-n-butyltin chloride was added, and then the temperature was slowly raised to room temperature and stirred overnight. Subsequently, water was added to quench the reaction, the resulting mixture was extracted three times with CH2Cl2, the resulting organic phase was dried with MgSO4, and concentrated to obtain 9-hexyl-7-(4-hexyl-5-(tributyltin)thiophen-2-yl)-N,N-diphenyl-9H-carbazole-2-amine (compound 5). The crude product was used without any purification.

[0035] (6) Synthesis of compound 6: In an argon atmosphere, 403 mg (1.05 mmol) of 4,7-dibromo-5,6-dinitrobenzo[c][1,2,5]thiadiazole and 48.5 mg (0.042 mmol) of Pd(PPh3)4 were added to the compound 5 obtained in step (5) (9-hexyl-7-(4-hexyl-5-(tributyltin)thiophen-2-yl)-N,N-diphenyl-9H-carbazole-2-amine) and mixed, followed by the addition of anhydrous THF. The mixture was heated to reflux and stirred for 24 hours. After cooling to room temperature, water was added and the resulting mixture was extracted three times with CH2Cl2. The collected organic phases were dried over MgSO4 and concentrated. The crude product was purified by silica gel column chromatography using CH2Cl2 / hexane (v / v 1:2) as the eluting solvent to give 7,7'-((5,6-dinitrobenzo[c][1,2,5]thiadiazole-4,7-diyl)bis(4-hexylthiophene-5,2-diyl))bis(9-hexyl-N,N-diphenyl-9H-carbazole-2-amine) (Compound 6) as a dark purple solid.

[0036] 1 H NMR (400 MHz, Chloroform- d ) δ 7.91 (d, J = 8.1 Hz, 2H), 7.84 (d, J =8.4 Hz, 2H), 7.47 (s, 2H), 7.42 (d, J = 8.2 Hz, 2H), 7.33 (s, 2H), 7.18 (t, J =7.9 Hz, 8H), 7.08 (d, J = 8.0 Hz, 8H), 7.03 (d, J = 1.8 Hz, 2H), 6.98 – 6.87 (m,6H), 4.09 (t, J = 7.0 Hz, 4H), 2.40 (h, J = 7.3, 6.1 Hz, 4H), 1.70 (p, J = 7.1 Hz,4H), 1.57 (p, J = 7.5 Hz, 4H), 1.22 – 1.13 (m, 24H), 0.76 (t, J = 6.7 Hz, 12H). 13 CNMR (101 MHz, Chloroform- d) δ 153.42, 148.21, 146.67, 146.61, 142.34, 141.14,130.11, 129.23, 124.70, 124.03, 123.24, 122.60, 121.05, 120.19, 118.33,117.70, 117.36,105.94, 104.91, 42.96, 31.63, 31.56, 28.87, 26.87, 22.56,22.53, 14.12, 14.09. (7) Synthesis of the final product: (7.1) Synthesis of compound 7 (AIE molecule SF): 50 mg (0.036 mmol) of compound 6 [7,7'-((5,6-dinitrobenzo[c][1,2,5]thiadiazole-4,7-diyl)bis(4-hexylthiophene-5,2-diyl))bis(9-hexyl-N,N-diphenyl-9H-carbazole-2-amine)] and 30 mL of glacial acetic acid were mixed, and then 60.5 mg (1.08 mmol) of iron powder was added to the mixture, which was then heated to 80°C and stirred for 0.5 hours. After cooling to room temperature, water was added, and the resulting mixture was extracted three times with CH2Cl2. The resulting organic phase was dried over MgSO4 and concentrated. The resulting crude 4,7-bis(5-(7-(diphenylamino)-9-hexyl-9H-carbazol-2-yl)-3-hexylthiophen-2-yl)benzo[c][1,2,5]thiadiazole-5,6-diamine product was used without further purification.

[0037] 4,7-Bis(5-(7-(diphenylamine)-9-hexyl-9H-carbazole-2-yl)-3-hexylthiophene-2-yl)benzo[c][1,2,5]thiadiazole-5,6-diamine, 15 mL of chloroform and 15 mL of acetic acid were mixed under an argon atmosphere, followed by the addition of 11.89 mg (0.054 mmol) of benzo[1,2-b:6,5-b']dithiophene-4,5-dione. The mixture was heated to reflux and stirred continuously for 12 hours. After cooling to room temperature, water was added and the resulting mixture was extracted three times with CH2Cl2. The collected organic phases were dried over MgSO4 and concentrated. The crude product was purified by silica gel column chromatography using CH2Cl2 / hexane (v / v 4:7) as the eluting solvent to obtain the final product, compound 7, as a dark green solid.

[0038] 1 H NMR (400 MHz, Chloroform- d ) δ 8.28 (d,J = 5.3 Hz, 2H), 8.03 (d, J =8.1 Hz, 2H), 7.94 (d, J = 8.4 Hz, 2H), 7.75 – 7.62 (m, 6H), 7.45 (d, J = 5.2 Hz,2H), 7.29 – 7.23 (m, 8H), 7.21 – 7.14 (m, 8H), 7.13 (d, J = 1.9 Hz, 2H), 7.04 –6.97 (m, 6H), 4.19 (t, J = 7.1 Hz, 4H), 2.59 (t, J = 7.8 Hz, 4H), 1.80 (q, J = 7.2Hz, 4H), 1.66 – 1.61 (m, 4H), 1.31 – 1.27 (m, 6H), 1.26 – 1.21 (m, 8H), 1.09(t, J = 7.4 Hz, 4H), 1.01 – 0.98 (m 6H), 0.85 – 0.78 (m, 6H), 0.63 (t, J = 6.8Hz, 6H). 13 C NMR (101 MHz, Chloroform- d ) δ 152.96, 148.28, 147.61, 146.37,145.49, 142.27, 141.60, 141.34, 138.34, 137.57,135.02, 131.56, 129.32,129.22, 126.21, 124.90, 124.80, 124.09, 123.95, 122.62, 122.50, 120.93,120.14, 118.66, 117.59, 117.36, 105.72, 105.07, 42.98, 31.61,31.52, 30.60,30.31, 29.06, 28.93, 26.92, 22.55, 22.44, 14.10, 13.96. (7.2) Synthesis of compound 8 (AIE molecule FK): 50 mg (0.036 mmol) of compound 6 [7,7'-((5,6-dinitrobenzo[c][1,2,5]thiadiazole-4,7-diyl)bis(4-hexylthiophene-5,2-diyl))bis(9-hexyl-N,N-diphenyl-9H-carbazole-2-amine)] and 30 mL of glacial acetic acid were mixed, and then 60.5 mg (1.08 mmol) of iron powder was added to the mixture, which was then heated to 80°C and stirred for 0.5 hours. After cooling to room temperature, water was added, and the resulting mixture was extracted three times with CH2Cl2. The resulting organic phase was dried over MgSO4 and concentrated. The resulting crude 4,7-bis(5-(7-(diphenylamino)-9-hexyl-9H-carbazol-2-yl)-3-hexylthiophen-2-yl)benzo[c][1,2,5]thiadiazole-5,6-diamine product was used without further purification.

[0039] The crude product of 4,7-bis(5-(7-(diphenylamine)-9-hexyl-9H-carbazole-2-yl)-3-hexylthiophene-2-yl)benzo[c][1,2,5]thiadiazole-5,6-diamine, 15 mL of chloroform and 15 mL of acetic acid were combined under an argon atmosphere, followed by the addition of 11.2 mg (0.054 mmol) of phenanthrenequinone. The mixture was heated to reflux and stirred continuously for 12 hours. After cooling to room temperature, water was added and the resulting mixture was extracted three times with CH2Cl2. The collected organic phases were dried over MgSO4 and concentrated. The crude product was purified by silica gel column chromatography using CH2Cl2 / hexane (v / v 1:2.5) as the eluting solvent to give the final product, compound 8, as a dark green solid.

[0040] 1 H NMR (400 MHz, Chloroform- d ) δ 9.11 (d, J = 6.6 Hz, 2H), 8.37 (d, J =8.1 Hz, 2H), 7.97 (d, J = 8.0 Hz, 2H), 7.87 (d, J = 8.4 Hz, 2H), 7.72 – 7.66 (m,2H), 7.64 – 7.55 (m, 6H), 7.22 – 7.17 (m, 8H), 7.10 (dd, J = 7.6, 1.2 Hz, 8H),7.05 (d, J = 1.9 Hz, 2H), 6.99 – 6.89 (m, 6H), 4.12 (t, J = 7.1 Hz, 4H), 2.53 (t,J = 7.7 Hz, 4H), 1.73 (q, J = 7.1 Hz, 4H), 1.58 – 1.53 (m, 4H), 1.22 – 1.19 (m,6H), 1.18 – 1.16 (m, 8H), 1.01 (t, J = 7.1 Hz, 4H), 0.93 – 0.88 (m, 6H), 0.76 –0.73 (m, 6H), 0.53 (t, J = 6.8 Hz, 6H). 13 C NMR (101 MHz, Chloroform- d ) δ 152.17,147.21, 146.46, 145.30, 144.33, 143.35, 141.21, 140.28, 137.72, 131.99,130.54, 130.50,129.27, 128.14, 127.47, 126.67, 123.89, 123.22, 122.88,122.05, 121.55, 121.42, 119.85, 119.08, 117.58, 116.50, 116.29, 104.66,104.00, 41.91, 30.52, 30.42,29.38, 29.27, 28.67, 27.94, 27.85, 25.83, 21.47,21.35, 13.10, 13.01, 12.86. (7.3) Synthesis of compound 9 (AIE molecule BD): 50 mg (0.036 mmol) of compound 6 [7,7'-((5,6-dinitrobenzo[c][1,2,5]thiadiazole-4,7-diyl)bis(4-hexylthiophene-5,2-diyl))bis(9-hexyl-N,N-diphenyl-9H-carbazole-2-amine)] and 30 mL of glacial acetic acid were mixed, and then 60.5 mg (1.08 mmol) of iron powder was added to the mixture, which was then heated to 80°C and stirred for 0.5 hours. After cooling to room temperature, water was added, and the resulting mixture was extracted three times with CH2Cl2. The resulting organic phase was dried over MgSO4 and concentrated. The resulting crude 4,7-bis(5-(7-(diphenylamino)-9-hexyl-9H-carbazol-2-yl)-3-hexylthiophen-2-yl)benzo[c][1,2,5]thiadiazole-5,6-diamine product was used without further purification.

[0041] The crude product of 4,7-bis(5-(7-(diphenylamine)-9-hexyl-9H-carbazole-2-yl)-3-hexylthiophene-2-yl)benzo[c][1,2,5]thiadiazole-5,6-diamine, 15 mL of chloroform and 15 mL of acetic acid were combined under an argon atmosphere, followed by the addition of 11.4 mg (0.054 mmol) of 1,10-phenanthroline-5,6-dione. The mixture was heated to reflux and stirred continuously for 12 hours. After cooling to room temperature, water was added and the resulting mixture was extracted three times with CH2Cl2. The collected organic phases were dried over MgSO4 and concentrated. The crude product was purified by silica gel column chromatography using ethyl acetate / hexane (v / v 1:3) as the eluting solvent to obtain the final product, compound 9, as a dark green solid.

[0042] 1 H NMR (400 MHz, Chloroform- d ) δ 8.03 (s, 2H), 7.98 (d, J = 8.0 Hz,2H), 7.64 (s, 2H), 7.60 (d, J = 3.5 Hz, 2H), 7.47 – 7.45 (m, 2H), 7.19 (d, J =5.4 Hz, 8H), 7.10 (d, J = 7.9 Hz, 8H), 7.05 (s, 2H), 6.99 – 6.91 (m, 6H), 4.15(t, J = 7.1 Hz, 4H), 2.53 (t, J = 7.7 Hz, 4H), 1.74 (q, J = 7.3 Hz, 4H), 1.61 –1.58 (m, 4H), 1.37 – 1.34 (m, 6H), 1.25 – 1.23 (m, 8H), 1.01 (t, J = 7.2 Hz,4H), 0.93 – 0.90 (m, 6H), 0.77 – 0.75 (m, 6H), 0.54 (t, J = 6.6 Hz, 6H). Figure 1The molecular structures and electron distributions of SF, FK, and BD were calculated by density functional theory (DFT). The HOMO is distributed along the entire molecule, and the LUMO is mainly located in the electron-deficient part, showing an effective ICT effect. The energy band gap of SF is 1.46 eV, the energy band gap of BD is 1.43 eV, and the energy band gap of FK is 1.53 eV.

[0043] Weigh 1 mg of AIE molecules (SF, FK, BD) and 4 mg of DSPE-PEG 2000 (i.e. AIE: DSPE-PEG 2000 The mass ratio is 1:4), placed in a centrifuge tube, and 1 mL of THF was added. 9 mL of ultrapure water was added to a 50 mL centrifuge tube and placed in an ultrasonic cell disruptor. The pre-prepared THF solution was added dropwise to 9 mL of ultrapure water under probe ultrasonic conditions for self-assembly. THF was removed in a fume hood, concentrated to 1 mg / mL by ultrafiltration centrifugation at 6000 rpm / min, and stored in a 4°C refrigerator for later use. The absorption spectrum of the AIE nanomaterial was then measured using a UV spectrophotometer, and its fluorescence spectrum was measured using a fluorescence spectrophotometer.

[0044] The photoluminescence (PL) properties of the molecules in different aggregation states were measured using a fluorescence spectrophotometer. The three AIE molecules were placed in THF / water mixtures with different water fractions (fw), with THF as a good solvent for the molecules and water as a poor solvent. The PL intensity of the AIE molecules in THF / water mixtures with different water fractions (fw) was measured.

[0045] like Figure 2 As shown in Figure 2, the maximum absorption wavelengths of SF NPs, BD NPs, and FK NPs are 728 nm, 740 nm, and 707 nm, respectively, and the maximum fluorescence wavelengths are 950 nm, 1029 nm, and 938 nm, respectively. The PL intensities of SF, BD, and FK molecules in THF / water mixtures with different water fractions (%) ( I / I 0 I0 and I represent the maximum PL intensity in THF and THF / water mixture, respectively. Both show typical AIE characteristics.

[0046] A thermal imager was used to monitor the temperature changes of the three nanomaterials over time. First, a 50 µM AIE nanoparticle solution was prepared and a 730 nm laser (0.2 W / cm 2 , 0.4 W / cm 2 , 0.6 W / cm 2 , 0.8 W / cm 2 , 1.0 W / cm 2) laser irradiation for 5 min, recording every 30 s. 0 µM, 12.5 µM, 25 µM, 50 µM, and 75 µM AIE nanoparticle solutions were prepared and laser irradiated at 730 nm (0.8 W / cm 2 ) were used to irradiate solutions of different concentrations for 5 min and record every 30 s.

[0047] Figure 3 Figures a, c, and e show the photothermal performance of three AIE NPs at the same concentration (50 µM) under 730 nm laser irradiation with different powers. Compared with the other two NPs, SF NPs showed the best photothermal effect. Figures b, d, and f show the photothermal performance of three AIE NPs at different concentrations under 730 nm laser irradiation with the same power (0.8 W / cm 2 ) Under laser irradiation, SF NPs exhibited the best photothermal performance compared with the other two NPs.

[0048] Example 2 Preparation of photoactivated nanomaterials (SF / S@NPs-FR / PM) for treating bladder cancer 0.9 mg of tumor surface FGFR1 receptor targeting peptide FGL (EVYVVAENQQGKSKA) and 1.1 mg of cell membrane PMCA calcium channel blocking peptide Caloxin 2A1 (GPLGLPG-VSNSNWPSFPSSGGG-NH2) were added to DSPE-PEG 2000 -MAL was subjected to thiol addition reaction in water / methanol solution (90: 10, v / v) at room temperature for 8 hours. The residual peptide was then removed by centrifugal filter and the final products were obtained by lyophilization and were recorded as FR and PM, respectively.

[0049] 1 mg SF (i.e., compound 7), 2 mg SNAP, 9 mg DSPE-PEG 2000 2.4 mg FR and 2.6 mg PM were dispersed in 1 mL THF and stirred. After complete dissolution, the above stock solution was dropped into 10 mL purified water and stirred vigorously. The organic solvent was evaporated in a fume hood and then concentrated through an ultrafiltration filter. The obtained SF / S@NPs-FR / PM was stored in a refrigerator at 4 °C for subsequent experiments.

[0050] Using 730 nm (0.8 W / cm 2 ) laser irradiation, evaluate the photothermal performance of water and SF / S@NPs-FR / PM nanoparticles, then remove the laser and let the sample cool naturally. The slope of the irradiation time of SF / S@NPs-FR / PM versus -ln(θ) represents its system time constant (τs), and the photothermal conversion efficiency can be calculated. Figure 4As shown, the photothermal conversion efficiency of SF / S@NPs-FR / PM is 31.57%.

[0051] The concentration of NO released under 730 nm laser irradiation was quantitatively determined using the Griess detection kit. SNAP and SF / S@NPs-FR / PM solutions were irradiated with 730 nm laser (0.8 W / cm 2 ) for different time periods. After irradiation, the NPs were precipitated and separated by centrifugation. The supernatant was incubated with Griess reagent at 37 °C for 20 min. The absorbance at 540 nm (OD 540 nm) was measured using a microplate reader to analyze the nitrite concentration in the solution. The NO concentration was calculated based on the calibration curve of nitrite ion solutions with different concentrations. Figure 5 As shown in the figure, with the increase of illumination time, the NO release effect of SF / S@NPs-FR / PM continued to increase.

[0052] Figure 6 The cellular uptake of nanomedicines was observed by confocal laser scanning microscope (CLSM) to explore whether the use of FGFR1 receptor targeting peptide FGL can promote the intracellular enrichment of nanomedicines. Before the experiment, due to the NIRII characteristics of the photosensitizer SF (λ em = 950 nm, which is beyond the detection range of conventional fluorescence microscopes (400-730 nm). Therefore, we encapsulated the commercial photosensitizer dihydrochlorin (Chlorin e6, Ce6; λ em = 669 nm‌) (the final concentration of Ce6 in the nanomedicine was 1 mg / mL) for intracellular imaging observation. At the same dosing concentration (20 μg / mL), observations were performed at the 1st hour, 4th hour, and 8th hour, respectively. The results showed that the tumor targeting group SF / S@NPs-FR / PM ( Figure 6 b) Compared with the non-tumor targeting group SF / S@NPs ( Figure 6 a) The red fluorescence intensity in MB49 tumor cells was higher at each time period, indicating more significant intracellular enrichment.

[0053] Figure 7 For cytotoxicity study: MB49 tumor cells were inoculated in 96-well plates. After the cells adhered to the wall, the culture medium was replaced with serum-free 1640 medium, and different concentrations of SF / S@NPs-FR / PM were prepared and incubated in the dark at 37 °C for 4 hours. After 4 hours, the serum-free 1640 medium was removed and replaced with 1640 complete medium (containing 10% fetal bovine serum). The illumination group (withlight) used a 730 nm laser (0.33 W / cm 2) irradiated cells for 3 min, and the non-light group (without light) was kept in a dark environment without light treatment. After 12 hours, the absorbance at 450 nm was detected by CCK8 reagent to evaluate cell viability. The results showed that with the continuous increase in the concentration of nanodrugs, the killing ability of SF / S@NPs-FR / PM against MB49 tumor cells increased. When the drug concentration was 32 μg / mL, the growth of more than 80% of tumor cells was inhibited, confirming the good efficacy of nanodrugs.

[0054] Figure 8 This is a Calcein-AM / PI live and dead cell staining experiment. The Calcein-AM / PI live and dead staining assay kit was used to evaluate the proportion of live and dead cells. MB49 tumor cells were seeded in a 6-well plate. After the cells adhered to the wall, the culture medium was replaced with serum-free 1640 medium. 20 μg / mL of SF NPs, SNAP NPs or SF / S@ NPs-FR / PM was added to each treatment group and incubated in the dark at 37°C for 4 hours. After 4 hours, the serum-free 1640 medium was removed and replaced with 1640 complete medium. The illumination groups (G5, G6, G7) were irradiated with 730 nm laser (0.33 W / cm 2 ) irradiated cells for 3 min, and the non-illuminated groups (G1, G2, G3, G4) were kept in a dark environment without laser irradiation. After the treatment, the cells were washed with PBS, trypsinized, centrifuged, and the cell pellets were collected. Subsequently, the cell pellets were resuspended in 1 mL PBS, and 1 μL of Calcein-AM and 1 μL of PI dye were added and incubated in the incubator for 30 minutes. Live cells were imaged by CLSM using an excitation wavelength of 488 nm, showing green fluorescence; dead cells were imaged by CLSM using an excitation wavelength of 633 nm, showing red fluorescence. The experimental results showed that at the same dosing concentration (20 μg / mL), compared with the non-targeted groups SF NPs + L and SF / SNAP NPs + L, the targeted treatment group SF / S@NPs-FR / PM+ L treated group showed the strongest tumor cell killing effect under light; (G1: Con, G2: Con + L, G3: SF NPs, G4: SNAPNPs, G5: SF NPs + L, G6:SF / SNAP NPs + L, G7: SF / S@ NPs-FR / PM + L; L: Laser).

[0055] Fig. 9 Through Fluo-4AM (λ ex =494 nm, λ em=516 nm) calcium ion concentration probe was used to evaluate the intracellular calcium ion level. MB49 tumor cells were inoculated in a confocal dish. After the cells adhered to the wall, the culture medium was replaced with serum-free 1640 culture medium. 20 μg / mL of SF NPs, SNAP NPs or SF / S@ NPs-FR / PM was added to each treatment group and incubated in the dark at 37 °C for 4 hours. After 4 hours, the serum-free 1640 culture medium was removed and replaced with 1640 complete culture medium. The illumination groups (G5, G6, G7) were irradiated with a 730 nm laser (0.33 W / cm 2 ) irradiated cells for 3 min, and the non-illuminated groups (G1, G2, G3, G4) were kept in the dark without laser irradiation. After 2 hours, the original culture medium was removed, the confocal dish was washed with PBS, and Fluo-4AM probe (2 μM) and Mito-tracker (50 nM) diluted in serum-free culture medium were added and incubated in the incubator for 30 minutes. The intracellular calcium ions were imaged by CLSM using an excitation wavelength of 488 nm, showing green fluorescence; the mitochondria of the cells were imaged by CLSM using an excitation wavelength of 633 nm, showing red fluorescence. The intracellular calcium ion concentration detection experiment showed that at the same dosage concentration (20 μg / mL), CLSM observations showed that SF NPs + L, SF / SNAP NPs + L and SF / S@ NPs-FR / PM + L could effectively increase the intracellular calcium ion concentration. Among them, compared with other treatment groups, the fluorescence intensity of the SF / S@NPs-FR / PM + L treatment group increased most significantly, indicating that the PMCA calcium channel blocking peptide Caloxin2A1 can further promote the increase in intracellular calcium ion concentration, leading to calcium overload; (G1: Con, G2: Con + L, G3: SF NPs, G4: SNAPNPs, G5: SF NPs + L, G6: SF / SNAP NPs + L, G7: SF / S@ NPs-FR / PM + L; L: Laser).

[0056] Fig.10Cytochrome C immunofluorescence detection reflects the occurrence of cell apoptosis caused by calcium overload. Cytochrome C is an important marker for cell apoptosis and is often used to reflect the level of cell apoptosis. MB49 tumor cells were inoculated in a confocal dish. After the cells adhered to the wall, the culture medium was replaced with serum-free 1640 medium. 20 μg / mL of SF NPs, SNAP NPs or SF / S@ NPs-FR / PM were added to each treatment group and incubated in the dark at 37 °C for 4 hours. After 4 hours, the serum-free 1640 medium was removed and replaced with 1640 complete medium. The illumination groups (G5, G6, G7) were irradiated with 730 nm laser (0.33 W / cm 2 ) irradiated cells for 3 min, and the non-illuminated groups (G1, G2, G3, G4) were kept in the dark without laser irradiation. After 2 hours, the original culture medium was removed, the confocal dish was washed with PBS, and the cells were fixed with 4% paraformaldehyde on ice for 20 min. The cells were then incubated with anti-cytochrome C antibody at 4 °C overnight. Thereafter, the cells were further incubated with donkey anti-rabbit IgG H&L (AlexaFluor® 647) at room temperature for 90 min, and then the nuclei were stained with DAPI for 5 min. The intracellular cytochrome C was imaged by CLSM using an excitation wavelength of 633 nm, showing red fluorescence. The release of intracellular cytochrome C detected by CLSM confirmed that at the same dosage concentration (20 μg / mL), SF NPs + L, SF / SNAPNPs + L and SF / S@NPs-FR / PM + L could effectively promote the release of cytochrome C, confirming the occurrence of cell apoptosis. Among them, compared with other treatment groups, the SF / S@NPs-FR / PM + L treatment group had the highest red fluorescence signal intensity, indicating the most significant increase in cytochrome C levels; (G1: Con, G2: Con + L, G3: SF NPs, G4: SNAP NPs, G5: SF NPs + L, G6: SF / SNAP NPs + L, G7: SF / S@ NPs-FR / PM + L; L: Laser).

[0057] Fig.11The hemolysis experiment reflects the biosafety of nanomedicines by rupturing and dissolving red blood cells. Before the experiment, whole blood of mice was collected, placed in EDTA anticoagulant tubes, and mixed thoroughly. After centrifugation at 12000 rpm for 15 minutes in a centrifuge, the supernatant was discarded and resuspended with 1.5 mL PBS; centrifugation continued at 8000 rpm for 5 minutes, and the process was repeated twice; finally, centrifugation was performed at 8000 rpm for 10 minutes, at which time the supernatant of red blood cells was almost colorless. Take 100 μl of red blood cells and add 5 mL PBS to mix and dilute to make a red blood cell suspension. A negative control group (500 μl PBS + 500 ul red blood cell suspension), a positive control group (500 μl 1% Triton + 500 μl red blood cell suspension) and a treatment group (500 μl nanomedicine + 500 ul red blood cell suspension) were set up respectively. After standing at room temperature for 4 hours, centrifuge at 10,000 rpm for 15 minutes; take photos, take the supernatant, and measure the absorbance at 540 nm. Fig.11 a shows that after treatment with nanomedicines in different treatment groups, the red blood cell hemolysis rate was less than 2%, which was within the safe range. Fig.11 b shows that as the concentration of SF / S@NPs-FR / PM increases, even when the drug concentration is as high as 100 μg / mL, the red blood cell hemolysis rate is still less than 2%, which is within the safe range, confirming that the nanomedicine has good safety; The above embodiments are only specific implementations of the present invention, but the protection scope of the present invention is not limited thereto. For technical workers in this technical field, changes or replacements that can be easily thought of are also considered as the protection scope of the present invention.

Claims

1. A near-infrared second region AIE probe, characterized in that: The AIE probe molecular structure is one of SF, FK and BD, and the molecular structures of SF, FK and BD are as follows: 。 2. The method for synthesizing the near-infrared second region AIE probe according to claim 1, characterized in that: The method comprises the following steps: (1) 9-hexyl-7-(4-hexyl-5-(tributyltin)thiophene-2-yl)-N,N-diphenyl-9H-carbazole-2-amine, 4,7-dibromo-5,6-dinitrobenzo[c][1,2,5]thiadiazole and Pd(PPh3)4 were mixed in an argon atmosphere, and then anhydrous tetrahydrofuran was added; the mixture was heated and stirred, and the resulting mixture was extracted three times with CH2Cl2, dried with MgSO4 and concentrated, and purified to obtain 7,7'-((5,6-dinitrobenzo[c][1,2,5]thiadiazole-4,7-diyl)bis(4-hexylthiophene-5,2-diyl))bis(9-hexyl-N,N-diphenyl-9H-carbazole-2-amine); Among them, the molar ratio of 9-hexyl-7-(4-hexyl-5-(tributyltin)thiophen-2-yl)-N,N-diphenyl-9H-carbazole-2-amine, 4,7-dibromo-5,6-dinitrobenzo[c][1,2,5]thiadiazole and Pd(PPh3)4 is (1.8~2.2):(0.9~1.1):0.04; (2) 7,7'-((5,6-dinitrobenzo[c][1,2,5]thiadiazole-4,7-diyl)bis(4-hexylthiophene-5,2-diyl))bis(9-hexyl-N,N-diphenyl-9H-carbazole-2-amine) and glacial acetic acid are mixed, iron powder is added to the mixture, heated and stirred; the resulting mixture is extracted three times with CH2Cl2, the organic phase is dried with MgSO4, and concentrated, and the resulting crude product of 4,7-bis(5-(7-(diphenylamine)-9-hexyl-9H-carbazole-2-yl)-3-hexylthiophene-2-yl)benzo[c][1,2,5]thiadiazole-5,6-diamine can be used without further purification; Wherein, the molar ratio of 7,7'-((5,6-dinitrobenzo[c][1,2,5]thiadiazol-4,7-yl)bis(4-hexylthiophene-5,2-diyl))bis(9-hexyl-N,N-diphenyl-9H-carbazole-2-amine) to iron powder is (0.9~1.1):30; (3) 4,7-bis(5-(7-(diphenylamine)-9-hexyl-9H-carbazole-2-yl)-3-hexylthiophene-2-yl)benzo[c][1,2,5]thiadiazole-5,6-diamine, chloroform and acetic acid are mixed in an argon atmosphere, and then benzo[1,2-b:6,5-b']dithiophene-4,5-dione, phenanthrenequinone or 1,10-phenanthroline-5,6-dione is added; the mixture is heated to reflux and stirred continuously; the obtained mixture is extracted three times with CH2Cl2, and the collected organic phase is dried with MgSO4 and concentrated, and the final product AIE probe SF, FK or BD is obtained after purification; Among them, the molar ratio of 4,7-bis(5-(7-(diphenylamine)-9-hexyl-9H-carbazole-2-yl)-3-hexylthiophene-2-yl)benzo[c][1,2,5]thiadiazole-5,6-diamine to benzo[1,2-b:6,5-b']dithiophene-4,5-dione, phenanthronequinone or 1,10-phenanthroline-5,6-dione is 1:(1.4~1.6).

3. The method for synthesizing the near-infrared second region AIE probe according to claim 2, characterized in that: In step (1), 9-hexyl-7-(4-hexyl-5-(tributyltin)thiophene-2-yl)-N,N-diphenyl-9H-carbazole-2-amine is prepared by the following method: In an argon environment, 9-hexyl-7-(4-hexylthiophene-2-yl)-N,N-diphenyl-9H-carbazole-2-amine was dissolved in anhydrous tetrahydrofuran, the mixture was cooled to -78°C and maintained for 30 minutes, then a hexane solution of n-butyl lithium was added, and the mixture was stirred at -78°C for 2 hours, then tri-n-butyltin chloride was added, and then the temperature was slowly raised to room temperature and stirred for 8-12 hours. Subsequently, water was added to quench the reaction, and the resulting mixture was extracted three times with CH2Cl2, and the obtained organic phase was dried with MgSO4 and concentrated to obtain 9-hexyl-7-(4-hexyl-5-(tributyltin)thiophene-2-yl)-N,N-diphenyl-9H-carbazole-2-amine; Among them, the molar ratio of 9-hexyl-7-(4-hexylthiophene-2-yl)-N,N-diphenyl-9H-carbazole-2-amine, n-butyl lithium, and tri-n-butyltin chloride is 1:(1~1.2):(1~1.2).

4. The method for synthesizing the near-infrared second region AIE probe according to claim 3, characterized in that: The preparation method of 9-hexyl-7-(4-hexylthiophene-2-yl)-N,N-diphenyl-9H-carbazole-2-amine is as follows: In an argon atmosphere, 7-bromo-9-hexyl-N,N-diphenyl-9H-carbazole-2-amine and Pd(pph3)4 are added to tri-n-butyl(4-hexylthiophene-2-yl)stannane and mixed, and then anhydrous tetrahydrofuran is added; the mixture is heated to reflux and stirred continuously for 24 hours, and after cooling to room temperature, water is added, and the resulting mixture is extracted three times with CH2Cl2, and the collected organic phase is dried with MgSO4 and concentrated to obtain 9-hexyl-7-(4-hexylthiophene-2-yl)-N,N-diphenyl-9H-carbazole-2-amine after purification; Among them, the molar ratio of tri-n-butyl (4-hexylthiophene-2-yl) stannane to 7-bromo-9-hexyl-N,N-diphenyl-9H-carbazole-2-amine is (1.2~1):

1.

5. The method for synthesizing the near-infrared second region AIE probe according to claim 4, characterized in that: The preparation method of the tri-n-butyl (4-hexylthiophene-2-yl) stannane is: In an argon environment, 3-hexylthiophene was dissolved in 30 mL of anhydrous tetrahydrofuran, the mixture was cooled to -78 ° C and maintained for 30 minutes, then a hexane solution of n-butyl lithium was added, and stirred at -78 ° C for 2 hours, then tri-n-butyltin chloride was added, and then the temperature was slowly raised to room temperature and stirred for 8-12 hours. Subsequently, water was added to quench the reaction, and the resulting mixture was extracted three times with CH2Cl2, and the obtained organic phase was dried with MgSO4 and concentrated to obtain tri-n-butyl (4-hexylthiophene-2-yl) tin ane; the molar ratio of 3-hexylthiophene, n-butyl lithium, and tri-n-butyltin chloride was 1: (1-1.2): (1-1.2); The preparation method of 7-bromo-9-hexyl-N,N-diphenyl-9H-carbazole-2-amine is as follows: In an argon atmosphere, 2,7-dibromo-9-hexyl-9H-carbazole and diphenylamine were mixed in toluene, and then cesium carbonate, palladium acetate, and tributylphosphine were added. The mixture was heated to reflux and stirred continuously for 24 hours. After cooling to room temperature, water was added and the resulting mixture was extracted three times with CH2Cl2; The collected organic phase was dried over MgSO4 and concentrated, and purified to obtain 7-bromo-9-hexyl-N,N-diphenyl-9H-carbazole-2-amine (Compound 2); wherein the molar ratio of 2,7-dibromo-9-hexyl-9H-carbazole, diphenylamine, cesium carbonate, and palladium acetate was 2:2:3:0.

1.

6. The method for synthesizing the near-infrared second region AIE probe according to claim 5, characterized in that: The preparation method of 2,7-dibromo-9-hexyl-9H-carbazole is: 2,7-Dibromocarbazole and sodium hydride were mixed in anhydrous N,N-dimethylformamide (DMF). Under argon protection, hexyl bromide was added after 30 minutes and stirred for 20 hours. Subsequently, water was added to quench the reaction. The resulting mixture was extracted three times with CH2Cl2. The obtained organic phase was dried with MgSO4 and concentrated. After purification, 2,7-dibromo-9-hexyl-9H-carbazole was obtained. The molar ratio of 2,7-dibromocarbazole, sodium hydride and hexyl bromide was 1:1.5:1.

3.

7. A light-activated nanomaterial for treating bladder cancer comprising the AIE probe according to claim 1, characterized in that: The material is blended with the AIE probe and a thermosensitive nitric oxide donor, encapsulated with an amphiphilic polymer, and the surface is modified with a tumor surface FGFR1 receptor targeting peptide and a cell membrane surface PMCA calcium ion channel blocking peptide, thereby forming a nanomaterial for light-activated treatment of bladder cancer.

8. The AIE probe light-activated nanomaterial for treating bladder cancer according to claim 7, characterized in that: The thermosensitive nitric oxide donor is S-nitroso-N-acetylpenicillamine, the tumor surface FGFR1 receptor targeting peptide is FGL, and the cell membrane surface PMCA calcium ion channel blocking peptide is Caloxin 2A1.

9. The AIE probe light-activated nanomaterial for treating bladder cancer according to claim 8, characterized in that: The amphiphilic polymer is DSPE-PEG 2000 .

10. The method for preparing a light-activated nanomaterial for treating bladder cancer according to claim 9, characterized in that: The specific steps of this method are as follows: 1) The tumor FGFR1 receptor targeting peptide FGL and the cell membrane surface PMCA calcium channel blocking peptide Caloxin 2A1 were subjected to thiol addition reaction with DSPE-PEG-MAL in a water / methanol solution with a volume ratio of 90:10 at room temperature, and then the residual peptides were removed by centrifugal filter, and the products obtained by freeze-drying were recorded as FR and PM; the mass ratio of tumor FGFR1 receptor targeting peptide FGL to DSPE-PEG-MAL was (0.9~1):3, and the mass ratio of cell membrane surface PMCA calcium channel blocking peptide Caloxin 2A1 to DSPE-PEG-MAL was (1~1.1):3; 2) The FR and PM obtained in step 1) were mixed with AIE probe, S-nitroso-N-acetylpenicillamine, DSPE-PEG 2000 Dispersed in tetrahydrofuran, after complete dissolution, the above stock solution was dropped into 10 mL of purified water and stirred vigorously. The organic solvent was stirred and evaporated in a fume hood and then concentrated through an ultrafiltration filter. The obtained light-activated nanomaterials for the treatment of bladder cancer were: AIE probe, S-nitroso-N-acetylpenicillamine, DSPE-PEG 2000 The mass ratio of FR and PM is 1:2:9:2.4:2.6.

Images