A near-infrared two-zone AIE probe and a nano material for photoactivated treatment of bladder cancer

Abstract

The present application provides a near-infrared two-region AIE probe and a nano material for light-activated treatment of bladder cancer, and belongs to the technical field of material chemistry. In view of the poor treatment effect of photothermal therapy on bladder cancer, the present application designs an AIE probe capable of efficient photo-thermal conversion in the near-infrared two-region, and on the basis of the AIE probe, the AIE probe is blended with a heat-sensitive NO donor to release NO through near-infrared light-induced photo-thermal conversion. After being encapsulated by an amphiphilic polymer, the surface of the cell membrane is modified with PMCA calcium ion channel blocking peptides and tumor surface FGFR1 receptor targeting peptides. The PMCA calcium ion channel blocking peptides on the surface of the cell membrane and the NO released by the heat-sensitive NO donor jointly act on the calcium ion channel through endogenous and exogenous combined regulation, causing calcium overload in the tumor and inducing tumor cell apoptosis. The tumor surface FGFR1 receptor targeting peptides on the surface of the cell membrane can realize efficient enrichment of the nano material at the tumor site, thereby improving the treatment effect. The material provided by the present application provides a new idea for the treatment of bladder cancer.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of material chemistry. BACKGROUND

[0002] Bladder cancer (BCa) is a global health problem, BCa is the second most common urological 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 in treatment due to its high metastasis rate, high recurrence rate and high mortality. In the past decade, the treatment for bladder cancer has shifted from radical surgery to organ preservation, but the efficacy is often unsatisfactory. Therefore, it is urgent to develop new diagnostic and therapeutic 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-invasive, local treatment and good treatment effect. However, pure PTT is difficult to completely eradicate tumors in solid tumors, which is limited by limited light irradiation area and insufficient accumulation of local photothermal agents, which often leads to tumor recurrence at the edge of the irradiation range. In recent years, photosensitizers with aggregation-induced emission (AIE) characteristics have been widely explored in the field of tumor treatment due to their outstanding advantages such as large Stokes shift, good light stability, and strong biocompatibility.

[0004] BCa is a typical deep tissue tumor, and the common near-infrared light (NIR) has limited penetration depth, making it difficult to reach tumors located deep in the bladder wall. In addition, the targeting ability of pure photosensitizers to bladder tumor tissue is often unsatisfactory, which not only leads to a decline in the efficacy of PTT, but also increases the risk of damage to normal tissues. SUMMARY

[0005] In order to solve the above problems, the present application designs a near-infrared two-zone AIE probe and a nano material for light-activated treatment of bladder cancer, which has the ability to penetrate deep tumor tissue and also can achieve good targeting of tumor tissue, thereby greatly enhancing the efficacy of PTT and reducing the occurrence of toxic side effects.

[0006] The application provides a near-infrared two-region AIE probe, the probe is one of SF, FK and BD, and molecular structural formulas of SF, FK and BD are as follows:

[0007]

[0008]

[0009]

[0010] The synthesis method of the near-infrared two-region AIE probe SF, FK and BD in the application comprises the following steps:

[0011] (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 are mixed in an argon atmosphere, and then anhydrous tetrahydrofuran (THF) is added. The mixture is heated and stirred. The obtained mixture is extracted with CH2Cl2 three times, 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) is obtained;

[0012] 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 are mixed in an argon atmosphere, and then anhydrous tetrahydrofuran (THF) is added. The mixture is heated and stirred. The obtained mixture is extracted with CH2Cl2 three times, 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) is obtained;

[0013] (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, and heating and stirring are performed. The obtained mixture is extracted with CH2Cl2 three times. The organic phase is dried with MgSO4, and concentrated. The obtained 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 crude product can be used without further purification;

[0014] 7,7'-((5,6-dinitrobenzo[c][l,2,5]thiadiazol-4,7-yl)bis(4-hexylthiophene-5,2-diyl))bis(9-hexyl-N,N-diphenyl-9H-carbazol-2-amine) and iron powder in a molar ratio of 1:30;

[0015] (3) 4,7-bis(5-(7-(diphenylamine)-9-hexyl-9H-carbazol-2-yl)-3-hexylthiophene-2-yl)benzo[c][l,2,5]thiadiazole-5,6-diamine, chloroform and acetic acid are mixed in an argon atmosphere, then benzo[l,2-b:6,5-b']dithiophene-4,5-dione or phenanthrenequinone or 1,10-phenanthroline-5,6-dione is added. The mixture is heated to reflux with continuous stirring. The resulting mixture is extracted with CH2Cl2three times. The collected organic phase is dried with MgSO4and concentrated. The end product AIE probe SF, FK or BD is obtained after purification.

[0016] 4,7-bis(5-(7-(diphenylamine)-9-hexyl-9H-carbazol-2-yl)-3-hexylthiophene-2-yl)benzo[c][l,2,5]thiadiazole-5,6-diamine, benzo[l,2-b:6,5-b']dithiophene-4,5-dione, phenanthrenequinone or 1,10-phenanthroline-5,6-dione in a molar ratio of 1:1.5.

[0017] Preferably, 9-hexyl-7-(4-hexyl-5-(tributyltin)thiophene-2-yl)-N,N-diphenyl-9H-carbazol-2-amine in step (1) is prepared by the following method:

[0018] 9-hexyl-7-(4-hexylthiophene-2-yl)-N,N-diphenyl-9H-carbazol-2-amine is dissolved in anhydrous tetrahydrofuran in an argon environment, the mixture is cooled to -78°C and kept for 30 minutes, then n-butyllithium hexane solution is added, stirred at -78°C for 2 hours, then tri-n-butyltin chloride is added, then slowly warmed to room temperature, stirred for 8~12h, then the reaction is quenched with water, the resulting mixture is extracted with CH2Cl2three times, the obtained organic phase is dried with MgSO4and concentrated to obtain 9-hexyl-7-(4-hexyl-5-(tributyltin)thiophene-2-yl)-N,N-diphenyl-9H-carbazol-2-amine;

[0019] wherein the molar ratio of 9-hexyl-7-(4-hexylthiophene-2-yl)-N,N-diphenyl-9H-carbazol-2-amine, n-butyllithium, tri-n-butyltin chloride is 1:(1~1.2):(1~1.2).

[0020] Further preferably, the preparation method of 9-hexyl-7-(4-hexylthiophene-2-yl)-N,N-diphenyl-9H-carbazol-2-amine is:

[0021] In an argon atmosphere, tri-n-butyl(4-hexylthiophen-2-yl)stannane is added with 7-bromo-9-hexyl-N,N-diphenyl-9H-carbazol-2-amine and Pd(pph3)4 and mixed, then anhydrous tetrahydrofuran is added; the mixture is heated to reflux, continuously stirred for 24 hours, after cooling to room temperature, water is added, the obtained mixture is extracted with CH2Cl2 three times, the collected organic phase is dried with MgSO4, and concentrated, after purification, 9-hexyl-7-(4-hexylthiophen-2-yl)-N,N-diphenyl-9H-carbazol-2-amine is obtained;

[0022] The molar ratio of tri-n-butyl(4-hexylthiophen-2-yl)stannane to 7-bromo-9-hexyl-N,N-diphenyl-9H-carbazol-2-amine is (1.2~1):1.

[0023] Further preferably, the preparation method of the tri-n-butyl(4-hexylthiophen-2-yl)stannane is:

[0024] In an argon environment, 3-hexylthiophene is dissolved in 30 mL of anhydrous tetrahydrofuran, the mixture is cooled to -78℃ and kept for 30 minutes, then n-butyllithium hexane solution is added, stirred at -78℃ for 2 hours, then tri-n-butyltin chloride is added, then slowly warmed to room temperature, stirred for 8~12 hours, then the reaction is quenched by adding water, the obtained mixture is extracted with CH2Cl2 three times, the obtained organic phase is dried with MgSO4, and concentrated to obtain tri-n-butyl(4-hexylthiophen-2-yl)stannane; the molar ratio of 3-hexylthiophene, n-butyllithium, tri-n-butyltin chloride is 1:(1~1.2):(1~1.2);

[0025] Further preferably, the preparation method of 7-bromo-9-hexyl-N,N-diphenyl-9H-carbazol-2-amine is:

[0026] In an argon environment, 2,7-dibromo-9-hexyl-9H-carbazole, diphenylamine are mixed in toluene, then cesium carbonate, palladium acetate, tributylphosphine are added, the mixture is heated to reflux, continuously stirred for 24 hours. After cooling to room temperature, water is added, the obtained mixture is extracted with CH2Cl2 three times. The collected organic phase is dried with MgSO4, and concentrated, after purification, 7-bromo-9-hexyl-N,N-diphenyl-9H-carbazol-2-amine is obtained; wherein the molar ratio of 2,7-dibromo-9-hexyl-9H-carbazole, diphenylamine, cesium carbonate, palladium acetate is 2:2:3:0.1;

[0027] Further preferably, the preparation method of 2,7-dibromo-9-hexyl-9H-carbazole is:

[0028] 2,7-dibromo-9-hexyl-9H-carbazole was obtained after purification by mixing 2,7-dibromo-carbazole, sodium hydride and bromohexane in anhydrous N,N-dimethylformamide (DMF) under argon protection, adding bromohexane after 30 minutes, stirring for 20 hours, then quenching the reaction by adding water, extracting the obtained mixture with CH2Cl2 three times, drying the obtained organic phase with MgSO4 and concentrating, and purifying; the molar ratio of 2,7-dibromo-carbazole, sodium hydride and bromohexane was 1:1.5:1.3.

[0029] The application also provides a nano material for photo-activated treatment of bladder cancer, which is obtained by blending the AIE probe and a heat-sensitive nitric oxide donor, encapsulating with an amphiphilic polymer, and modifying the surface with a tumor surface FGFR1 receptor targeting peptide and a cell membrane surface PMCA calcium ion channel blocking peptide.

[0030] Preferably, the heat-sensitive 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 .

[0031] The preparation method of the nano material for photo-activated treatment of bladder cancer in the application is as follows:

[0032] 1) The tumor FGFR1 receptor targeting peptide FGL and the cell membrane surface PMCA calcium ion channel blocking peptide Caloxin 2A1 are subjected to thiol addition reaction with DSPE-PEG-MAL in a water / methanol solution with a volume ratio of 90:10 at room temperature, then a centrifugal filter is used to remove residual peptides, and the product is obtained by freeze-drying and is recorded as FR and PM; the mass ratio of the tumor FGFR1 receptor targeting peptide FGL to DSPE-PEG-MAL is (0.9-1):3, and the mass ratio of the cell membrane surface PMCA calcium ion channel blocking peptide Caloxin 2A1 to DSPE-PEG-MAL is (1-1.1):3;

[0033] 2) The FR and PM obtained in step 1) are dispersed in THF, and after complete dissolution, the above stock solution is dropped into 10 mL of purified water, and stirred vigorously, and the organic solvent is evaporated in a fume hood, and then concentrated by ultrafiltration filter; the obtained nano material for photo-activated treatment of bladder cancer; wherein the mass ratio of the AIE probe, S-nitroso-N-acetylpenicillamine, DSPE-PEG 2000 , FR and PM is 1:2:9:2.4:2.6. 2000

[0034] The application has the following beneficial effects:​

[0035] 1. The AIE provided by the application is a thienyl-substituted aggregation-induced emission molecule, which can achieve strong near-infrared light-induced photothermal conversion effect in the long-wavelength and low-energy near-infrared two region (NIR-II, 1000 ~ 1700 nm). NIR-II has been proved to have small scattering and large tissue penetration ability at the level of living body, which can reach the deep part of bladder and obtain better treatment effect.

[0036] 2. The nano material SF / S@NPs-FR / PM for photoactivated treatment of bladder cancer provided by the application releases NO by blending AIE with a heat-sensitive nitric oxide (NO) donor SNAP and near-infrared light-induced photothermal conversion; the cell membrane surface PMCA calcium ion channel blocking peptide Caloxin 2A1 (VSNSNWPSFPSSGGG-NH2) and the released NO jointly act on the calcium ion (Ca 2+ ) channel, causing tumor calcium overload and inducing tumor cell apoptosis; the tumor surface FGFR1 receptor targeting peptide FGL (EVYVVAENQQGKSKA) on the surface of the nano material can realize efficient enrichment of the nano material at the tumor site, providing a new idea for BCa treatment.

[0037] 3. The application utilizes the release of photosensitizer and NO gas in the nano material to activate the intracellular calcium ion channel, thereby affecting the biological processes such as proliferation and apoptosis of tumor cells. 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 the prognosis of patients. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 The figure is the density functional theory (DFT) calculation diagram of the three AIE molecules of the application.

[0039] Figure 2 The figure is the (a) absorption spectrum, (b) fluorescence emission spectrum and (c) photoluminescence PL intensity of the three AIE nano materials.

[0040] Figure 3 The figure is the photothermal performance of the three nano drugs (50 µM) of the application under the irradiation of 730 nm laser of different powers (a, c, e); the photothermal performance of the three nano drugs under the irradiation of 730 nm laser (0.8 W / cm 2 ) of different concentrations (b, d, f).

[0041] Figure 4 The figure is the photothermal performance of the SF / S@NPs-FR / PM solution of the application under the irradiation of 730 nm laser (0.8 W / cm2 Temperature curves and (b) photo-thermal conversion efficiency fitting curves after irradiation for different times.

[0042] Figure 5 The SF / S@NPs-FR / PM and the SNAP of the present application were irradiated by a 730 nm laser (0.8 W / cm 2 NO release amount after irradiation for different times.

[0043] Figure 6 Photos of 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.

[0044] Figure 7 Killing ability of the tumor targeting group SF / S@NPs-FR / PM of the present application against MB49 tumor cells at different concentrations.

[0045] Figure 8 Calcein-AM / PI live and dead cell staining experiment results of MB49 tumor cells in each drug treatment group of the present application.

[0046] Figure 9 CLSM detection experiment results of calcium ion concentration in MB49 tumor cells in each drug treatment group of the present application.

[0047] Figure 10 Cytochrome C release amount in MB49 tumor cells in each drug treatment group detected by CLSM of the present application.

[0048] Figure 11 Hemolysis rate of (a) each drug treatment group and (b) SF / S@NPs-FR / PM after treatment at different concentrations by hemolysis experiment of the present application. DETAILED DESCRIPTION

[0049] The technical solutions of the present application will be described in detail below in combination with the accompanying drawings of the present application.

[0050] Example 1 Synthesis of near-infrared two-region AIE probes SF, FK and BD

[0051] (1) Synthesis of compound 1:

[0052]

[0053] 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 was added after 30 minutes, and the mixture was stirred for 20 hours. Subsequently, the reaction was quenched with water. The resulting mixture was extracted three times with CH2Cl2, and the organic phase was dried over MgSO4 and concentrated. The crude product was purified by silica gel column chromatography using hexane as the eluent to give 2,7-dibromo-9-hexyl-9H-carbazole (compound 1).

[0054] 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.

[0055] (2) Synthesis of compound 2

[0056]

[0057] 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 eluent to give 7-bromo-9-hexyl-N,N-diphenyl-9H-carbazole-2-amine (compound 2).

[0058] 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.

[0059] (3) Synthesis of compound 3

[0060]

[0061] In an argon atmosphere, 605.9 mg (3.6 mmol) of 3-hexylthiophene was dissolved in 30 mL of anhydrous tetrahydrofuran (THF). The mixture was cooled to -78 °C and maintained for 30 min. Then, 2.7 mL of a 1.6 M (4.32 mmol) solution of n-butyllithium (n-BuLi)hexane was added, and the mixture was stirred at -78 °C for 2 h. Subsequently, 1.2 mL (4.32 mmol) of tri-n-butyltin chloride was added, and the mixture was slowly heated to room temperature and stirred overnight. The reaction was then quenched with water. The resulting mixture was extracted three times with CH₂Cl₂. The organic phase was dried over MgSO₄ and concentrated to give tri-n-butyl(4-hexylthiophene-2-yl)stanane (compound 3). The crude product was ready for use without any purification.

[0062] (4) Synthesis of compound 4:

[0063]

[0064] 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)stanane) obtained in step (3) and mixed, followed by the addition of anhydrous THF. 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. The crude product was purified by silica gel column chromatography using CH2Cl2 / hexane (v / v 1:7) as the elution solvent to give 9-hexyl-7-(4-hexylthiophen-2-yl)-N,N-diphenyl-9H-carbazole-2-amine (compound 4).

[0065] 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.

[0066] (5) Synthesis of compound 5:

[0067]

[0068] 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 maintained for 30 minutes. Then, 1.6 mL (2.5 mmol) of a 1.6 M n-butyllithium (n-BuLi)hexane solution was added, and the mixture was stirred at -78°C for 2 hours. Subsequently, 0.68 mL (2.5 mmol) of tri-n-butyltin chloride was added, and the mixture was slowly heated to room temperature and stirred overnight. The reaction was then quenched with water. The resulting mixture was extracted three times with CH₂Cl₂. The resulting organic phase was dried over MgSO₄ and concentrated to give 9-hexyl-7-(4-hexyl-5-(tributyltin)thiophen-2-yl)-N,N-diphenyl-9H-carbazole-2-amine (compound 5). The crude product was ready for use without any purification.

[0069] (6) Synthesis of compound 6:

[0070]

[0071] Under 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 compound 5 (9-hexyl-7-(4-hexyl-5-(tributyltin)thiophene-2-yl)-N,N-diphenyl-9H-carbazole-2-amine) obtained in step (5) and mixed, followed by the addition of anhydrous THF. 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. The crude product was purified by silica gel column chromatography using CH2Cl2 / hexane (v / v 1:2) as the elution 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), a dark purple solid.

[0072] 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). 13CNMR (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.

[0073] (7) Synthesis of the final product:

[0074] (7.1) Synthesis of compound 7 (AIE molecule SF):

[0075]

[0076] 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)] was mixed with 30 mL of glacial acetic acid. Then, 60.5 mg (1.08 mmol) of iron powder was added to the mixture. The mixture was then heated to 80 °C and stirred for 0.5 h. 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 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.

[0077] 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 phase was dried over MgSO4 and concentrated. The crude product was purified by silica gel column chromatography using CH2Cl2 / hexane (v / v 4:7) as the elution solvent to give the final product compound 7 as a dark green solid.

[0078] 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.

[0079] (7.2) Synthesis of compound 8 (AIE molecule FK):

[0080]

[0081] 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)] was mixed with 30 mL of glacial acetic acid. Then, 60.5 mg (1.08 mmol) of iron powder was added to the mixture. The mixture was then heated to 80 °C and stirred for 0.5 h. 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 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.

[0082] 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 elution solvent to give the final product compound 8 as a dark green solid.

[0083] 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.

[0084] (7.3) Synthesis of compound 9 (AIE molecule BD):

[0085]

[0086] 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)] was mixed with 30 mL of glacial acetic acid. Then, 60.5 mg (1.08 mmol) of iron powder was added to the mixture. The mixture was then heated to 80 °C and stirred for 0.5 h. 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 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.

[0087] Crude 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 elution solvent to give the final product, compound 9, as a dark green solid.

[0088] 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).

[0089] Figure 1 The molecular structures and electron distributions of SF, FK, and BD were calculated using density functional theory (DFT). The HOMO is distributed throughout the entire molecule, while the LUMO is mainly located in the electron-deficient region, exhibiting an effective ICT effect. The band gap of SF is 1.46 eV, that of BD is 1.43 eV, and that of FK is 1.53 eV.

[0090] Weigh out 1 mg of AIE molecules (SF, FK, BD) and 4 mg of DSPE-PEG respectively. 2000 (i.e., AIE:DSPE-PEG) 2000 The THF solution (at a mass ratio of 1:4) was placed in a centrifuge tube, and 1 mL of THF was added. In a 50 mL centrifuge tube, 9 mL of ultrapure water was added, and the tube was placed in an ultrasonic cell disruptor. Under ultrasonic conditions, the pre-prepared THF solution was added dropwise to the 9 mL of ultrapure water to initiate self-assembly. The THF was removed in a fume hood, and the solution was concentrated to 1 mg / mL by ultrafiltration centrifugation at 6000 rpm / min and stored at 4°C. The absorption spectrum of the AIE nanomaterials was then measured using a UV spectrophotometer, and its fluorescence spectrum was measured using a fluorescence spectrophotometer.

[0091] The photoluminescence (PL) properties of molecules in different aggregation states were measured using a fluorescence spectrophotometer. Three types of AIE molecules were placed in THF / water mixtures with different water fractions (fw), with THF acting as a good solvent and water as a poor solvent. The PL intensity of AIE molecules in the THF / water mixtures with different water fractions (fw) was measured.

[0092] like Figure 2 As shown, 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 photoluminescence (PL) intensities of SF, BD, and FK molecules in mixtures of THF / water with different water fractions (%) are also shown. I / I 0(Ratio). I0 and I represent the maximum PL intensity in THF and THF / water mixtures, respectively. Both exhibit typical AIE characteristics.

[0093] The temperature changes of three nanomaterials over time were monitored using a thermal imager. First, a 50 µM AIE nanosolution was prepared, and then a 730 nm laser (0.2 W / cm²) was used. 2 0.4 W / cm 2 0.6 W / cm 2 0.8 W / cm 2 1.0 W / cm 2 Laser irradiation was performed for 5 minutes, with data recorded every 30 seconds. AIE nanosolutions of 0 µM, 12.5 µM, 25 µM, 50 µM, and 75 µM were prepared using a 730 nm laser (0.8 W / cm²). 2 Different concentrations of solutions were irradiated with light for 5 minutes, and the results were recorded every 30 seconds.

[0094] Figure 3 In Figures a, c, and e, the photothermal performance of three AIE NPs at the same concentration (50 µM) under different laser powers at a 730 nm laser is shown. Compared to the other two NPs, SF NPs exhibit the best photothermal effect. Figures b, d, and f show the photothermal performance of the three AIE NPs at different concentrations under the same power (0.8 W / cm²) at a 730 nm laser. 2 Under laser irradiation, SF NPs exhibit the best photothermal performance compared to the other two types of NPs.

[0095] Example 2: Preparation of nanomaterials (SF / S@NPs-FR / PM) for photoactivated therapy of bladder cancer

[0096] 0.9 mg of tumor surface FGFR1 receptor-targeting peptide FGL (EVYVVAENQQGKSKA), 1.1 mg of cell membrane surface PMCA calcium ion channel blocking peptide Caloxin 2A1 (GPLGLPG-VSNSNWPSFPSSGGG-NH2), and DSPE-PEG were respectively added. 2000 -MAL was subjected to a thiol addition reaction in an aqueous / methanol solution (90:10, v / v) at room temperature for 8 hours. Residual peptides were then removed by centrifugation, and the final products were obtained by lyophilization, denoted as FR and PM, respectively.

[0097] 1 mg SF (i.e., compound 7), 2 mg SNAP, and 9 mg DSPE-PEG were administered. 20002.4 mg FR and 2.6 mg PM were dispersed in 1 mL THF and stirred until completely dissolved. The stock solution was then added dropwise to 10 mL of purified water and stirred vigorously. The organic solvent was evaporated in a fume hood and then concentrated through an ultrafiltration filter. The resulting SF / S@NPs-FR / PM was stored at 4°C for subsequent experiments.

[0098] Using 730 nm (0.8 W / cm) 2 Laser irradiation was used to evaluate the photothermal properties of water and SF / S@NPs-FR / PM nanoparticles. The laser was then removed, and the samples were allowed to cool naturally. A curve showing the irradiation time versus -ln(θ) for SF / S@NPs-FR / PM is presented, with the slope representing the system time constant (τs), from which the photothermal conversion efficiency can be calculated. Figure 4 As shown, the photothermal conversion efficiency of SF / S@NPs-FR / PM is 31.57%.

[0099] The release concentration of NO under 730 nm laser irradiation was quantitatively determined using a Griess assay kit. SNAP and SF / S@NPs-FR / PM solutions were irradiated with a 730 nm laser (0.8 W / cm²). 2 Irradiation was performed for different durations. After irradiation, NPs were precipitated and centrifuged. 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 curves of nitrite ion solutions with different concentrations. Figure 5 As shown, the NO release effect of SF / S@NPs-FR / PM continuously increases with the increase of illumination time.

[0100] Figure 6 This study investigated how the use of the FGFR1 receptor-targeting peptide FGL can promote the intracellular accumulation of nanomedicines by observing cellular uptake of nanomedicines using confocal laser scanning microscopy (CLSM). Prior to the experiment, due to the NIRII properties (λ) of the photosensitizer SF... em = 950 nm, exceeding the detection range of conventional fluorescence microscopy (400-730 nm). Therefore, we encapsulated the commercially available photosensitizer dihydroporphyrin (Chlorin e6, Ce6; λ) in the nanomedicine. em = 669 nm (final Ce6 concentration in the nanomedicine was 1 mg / mL) for intracellular imaging observation. At the same administered concentration (20 μg / mL), observations were performed at 1 hour, 4 hours, and 8 hours, respectively. The results showed that the tumor-targeting group SF / S@NPs-FR / PM (Figure 6 b) Compared to the non-tumor-targeted group SF / S@NPs ( Figure 6 a) The red fluorescence intensity was higher in MB49 tumor cells at all time points, indicating more significant intracellular enrichment.

[0101] Figure 7 For cytotoxicity studies: MB49 tumor cells were seeded in 96-well plates. After cell attachment, the medium was replaced with serum-free 1640 medium, and different concentrations of SF / S@NPs-FR / PM were prepared. The cells were incubated at 37 °C in the dark for 4 hours. After 4 hours, the serum-free 1640 medium was removed and replaced with complete 1640 medium (containing 10% fetal bovine serum). The light-treated group used a 730 nm laser (0.33 W / cm²). 2 Cells were irradiated for 3 min, while the non-light group was kept in darkness without any light treatment. Cell viability was assessed 12 hours later by measuring absorbance at 450 nm using the CCK8 reagent. The results showed that the killing ability of SF / S@NPs-FR / PM against MB49 tumor cells increased with increasing nanodrug concentration. At a drug concentration of 32 μg / mL, over 80% of tumor cell growth was inhibited, confirming the good efficacy of the nanodrug.

[0102] Figure 8 For the Calcein-AM / PI live / dead cell staining assay, the percentage of live and dead cells was evaluated using the Calcein-AM / PI live / dead cell staining assay kit. MB49 tumor cells were seeded in 6-well plates. After cell attachment, the medium was replaced with serum-free 1640 medium. Each treatment group was incubated with 20 μg / mL of SF NPs, SNAP NPs, or SF / S@NPs-FR / PM, respectively, and incubated at 37°C in the dark for 4 hours. After 4 hours, the serum-free 1640 medium was removed and replaced with complete 1640 medium. The light-treated groups (G5, G6, G7) were treated with a 730 nm laser (0.33 W / cm²). 2Cells were irradiated for 3 min. The non-illuminated groups (G1, G2, G3, G4) were kept in darkness without laser irradiation. After treatment, cells were washed with PBS, trypsinized, centrifuged, and cell pellets were collected. The cell pellets were then resuspended in 1 mL PBS, and incubated for 30 min with 1 μL Calcein-AM and 1 μL PI dye. Live cells were imaged using CLSM at an excitation wavelength of 488 nm, showing green fluorescence; dead cells were imaged using CLSM at an excitation wavelength of 633 nm, showing red fluorescence. Experimental results showed that, at the same drug concentration (20 μg / mL), compared with the non-targeted groups SF NPs + L and SF / SNAP NPs + L, the targeted therapy group SF / S@NPs-FR / PM+ L showed the strongest tumor cell killing effect under light irradiation; (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).

[0103] Figure 9 via Fluo-4AM (λ) ex =494 nm, λ em =516 nm) Calcium ion concentration probes were used to evaluate intracellular calcium ion levels. MB49 tumor cells were seeded in confocal microplates. After cell adhesion, the medium was replaced with serum-free 1640 medium. Each treatment group was incubated with 20 μg / mL of SF NPs, SNAP NPs, or SF / S@NPs-FR / PM, respectively, in the dark at 37 ℃ for 4 hours. After 4 hours, the serum-free 1640 medium was removed and replaced with complete 1640 medium. The light-treated groups (G5, G6, G7) were treated with a 730 nm laser (0.33 W / cm²). 2Cells were irradiated for 3 min, while the non-illuminated groups (G1, G2, G3, G4) remained in darkness without laser irradiation. Two hours later, the original culture medium was removed, the confocal dishes were washed with PBS, and serum-free culture medium diluted with Fluo-4AM probe (2 μM) and Mito-tracker (50 nM) was added. The cells were incubated for 30 min. Intracellular calcium ions were imaged using CLSM at an excitation wavelength of 488 nm, showing green fluorescence; mitochondria were imaged using CLSM at an excitation wavelength of 633 nm, showing red fluorescence. The intracellular calcium ion concentration detection experiments showed that, at the same drug concentration (20 μg / mL), CLSM observations showed that SF NPs + L, SF / SNAP NPs + L, and SF / S@NPs-FR / PM + L all effectively increased intracellular calcium ion concentration. Among these, the fluorescence intensity of the SF / S@NPs-FR / PM + L treatment group was the most significantly increased compared to other treatment groups. This indicates that the PMCA calcium channel blocking peptide Caloxin2A1 can further promote the increase of intracellular calcium ion concentration, leading to calcium overload; (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).

[0104] Figure 10 Cytochrome C immunofluorescence assay was used to detect apoptosis induced by calcium overload. Cytochrome C is an important marker of apoptosis and is often used to reflect the level of apoptosis in cells. MB49 tumor cells were seeded in confocal microplates. After cell adhesion, the medium was replaced with serum-free 1640 medium. Each treatment group was incubated with 20 μg / mL of SF NPs, SNAP NPs, or SF / S@NPs-FR / PM, respectively, and incubated in the dark at 37 ℃ for 4 hours. After 4 hours, the serum-free 1640 medium was removed and replaced with complete 1640 medium. The light-treated groups (G5, G6, G7) were treated with a 730 nm laser (0.33 W / cm²). 2Cells were irradiated for 3 min, while the non-illuminated groups (G1, G2, G3, G4) were kept in darkness without laser irradiation. Two hours later, the original culture medium was removed, the confocal dishes were washed with PBS, and the cells were fixed on ice with 4% paraformaldehyde for 20 min. The cells were then incubated overnight at 4 °C with anti-cytochrome C antibody. Subsequently, the cells were further incubated with donkey anti-rabbit IgG H&L (Alexa Fluor® 647) at room temperature for 90 min, followed by nuclear staining with DAPI for 5 min. Intracellular cytochrome C was imaged using CLSM at an excitation wavelength of 633 nm, showing red fluorescence. CLSM detection of intracellular cytochrome C release confirmed that, at the same drug concentration (20 μg / mL), SF NPs + L, SF / SNAP NPs + L, and SF / S@NPs-FR / PM + L all effectively promoted cytochrome C release, confirming the occurrence of apoptosis. Among these, the SF / S@NPs-FR / PM + L treatment group showed the highest red fluorescence signal intensity compared to other treatment groups, indicating the most significant increase in cytochrome C level; (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).

[0105] Figure 11 The hemolysis experiment reflects the biosafety of nanomedicines by the lysis and dissolution of red blood cells. Before the experiment, whole blood was collected from mice, placed in EDTA anticoagulant tubes, and thoroughly mixed. After centrifugation at 12,000 rpm for 15 minutes, the supernatant was discarded, and the cells were resuspended in 1.5 mL of PBS. Centrifugation was then repeated twice at 8,000 rpm for 5 minutes. Finally, centrifugation was performed at 8,000 rpm for 10 minutes, at which point the red blood cell supernatant was almost colorless. 100 μl of red blood cells were diluted with 5 mL of PBS to prepare a red blood cell suspension. A negative control group (500 μl PBS + 500 μl 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 μl red blood cell suspension) were established. After standing at room temperature for 4 hours, the cells were centrifuged at 10,000 rpm for 15 minutes; photographs were taken, the supernatant was collected, and the absorbance at 540 nm was measured. Figure 11 The result of a indicates that the hemolysis rate of red blood cells after treatment with nanomedicine in different treatment groups was less than 2%, which is within the safe range. Figure 11b indicates that as the concentration of SF / S@NPs-FR / PM continues to increase, even when the drug concentration is as high as 100 μg / mL, the hemolysis rate of red blood cells is still less than 2%, which is within the safe range, confirming that the nanomedicine has good safety.

[0106] The above embodiments are merely specific implementations of the present invention, but the scope of protection of the present invention is not limited thereto. For those skilled in the art, any changes or substitutions that can be easily conceived, such improvements and modifications, are also considered to be within the scope of protection of the present invention.

Claims

1. A near-infrared II region AIE probe, characterized in that, The AIE probe molecular structure is one of SF, FK, and BD, and the molecular structural formulas of SF, FK, and BD are as follows: 。 2. The method for synthesizing the near-infrared II region AIE probe as described in claim 1, characterized in that, The method includes the following steps: (1) 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 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-hexylthiophen-5,2-diyl))bis(9-hexyl-N,N-diphenyl-9H-carbazole-2-amine); The molar ratio of 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 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 were mixed, iron powder was added to the mixture, and the mixture was heated and stirred. The resulting mixture was extracted three times with CH2Cl2, the organic phase was dried with MgSO4 and concentrated. 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 could be used without further purification. The molar ratio of 7,7'-((5,6-dinitrobenzo[c][1,2,5]thiadiazole-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) Mix 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 in an argon atmosphere, then add benzo[1,2-b:6,5-b']dithiophene-4,5-dione or phenanthrenequinone or 1,10-phenanthroline-5,6-dione; heat the mixture to reflux and stir continuously; extract the resulting mixture three times with CH2Cl2, dry the collected organic phase with MgSO4, concentrate and purify to obtain the final product AIE probe SF, FK or BD; 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, phenanthrenequinone, or 1,10-phenanthroline-5,6-dione is 1:(1.4~1.6).

3. The method for synthesizing a near-infrared II AIE probe according to claim 2, characterized in that, In step (1), 9-hexyl-7-(4-hexyl-5-(tributyltin)thiophen-2-yl)-N,N-diphenyl-9H-carbazole-2-amine is prepared by the following method: In an argon atmosphere, 9-hexyl-7-(4-hexylthiophen-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-butyllithium was added, and the mixture was stirred at -78°C for 2 hours. Subsequently, tri-n-butyltin chloride was added, and the mixture was slowly heated to room temperature and stirred for 8-12 hours. The reaction was then quenched with water. The resulting mixture was extracted three times with CH2Cl2. The 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. The molar ratio of 9-hexyl-7-(4-hexylthiophen-2-yl)-N,N-diphenyl-9H-carbazole-2-amine, n-butyllithium, and tri-n-butyltin chloride is 1:(1~1.2):(1~1.2).

4. The method for synthesizing a near-infrared II AIE probe according to claim 3, characterized in that, The preparation method of 9-hexyl-7-(4-hexylthiophen-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 were added to tri-n-butyl(4-hexylthiophen-2-yl)stanane and mixed. Anhydrous tetrahydrofuran was then added. The mixture was heated to reflux and stirred continuously for 24 hours. After cooling to room temperature, water was added. The resulting mixture was extracted three times with CH2Cl2. The collected organic phase was dried with MgSO4 and concentrated. After purification, 9-hexyl-7-(4-hexylthiophen-2-yl)-N,N-diphenyl-9H-carbazole-2-amine was obtained. The molar ratio of tri-n-butyl(4-hexylthiophen-2-yl)stanane to 7-bromo-9-hexyl-N,N-diphenyl-9H-carbazole-2-amine is (1.2~1):

1.

5. The method for synthesizing a near-infrared II AIE probe according to claim 4, characterized in that, The preparation method of the tri-n-butyl(4-hexylthiophen-2-yl)tinane is as follows: In an argon atmosphere, 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-butyllithium was added, and the mixture was stirred at -78°C for 2 hours. Subsequently, tri-n-butyltin chloride was added, and the mixture was slowly heated to room temperature and stirred for 8–12 hours. The reaction was then quenched with water. The resulting mixture was extracted three times with CH2Cl2. The organic phase was dried over MgSO4 and concentrated to obtain tri-n-butyl(4-hexylthiophene-2-yl)tinane. The molar ratio of 3-hexylthiophene, n-butyllithium, 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, followed by the addition of cesium carbonate, palladium acetate, and 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 phase was dried with MgSO4, concentrated, and purified to obtain 7-bromo-9-hexyl-N,N-diphenyl-9H-carbazole-2-amine. 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 a near-infrared II AIE probe according to claim 5, characterized in that, The preparation method of 2,7-dibromo-9-hexyl-9H-carbazole is as follows: 2,7-Dibromocarbazole and sodium hydride were mixed in anhydrous N,N-dimethylformamide (DMF). Under argon protection, bromohexane was added after 30 minutes, and the mixture was stirred for 20 hours. Subsequently, the reaction was quenched with water. The resulting mixture was extracted three times with CH2Cl2. The 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 bromohexane was 1:1.5:1.

3.

7. A photoactivated nanomaterial for treating bladder cancer comprising the AIE probe of claim 1, characterized in that, This material is formed 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 photoactivated therapy of bladder cancer. 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.

8. The photoactivated nanomaterial for bladder cancer therapy using the AIE probe according to claim 7, characterized in that, The amphiphilic polymer is DSPE-PEG 2000 .

9. The preparation method of the photoactivated nanomaterial for bladder cancer therapy as described in claim 8, 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 ion channel blocking peptide Caloxin 2A1 were subjected to thiol addition reactions with DSPE-PEG-MAL in a water / methanol solution at a volume ratio of 90:10 at room temperature. The residual peptides were then removed by centrifugation and lyophilization to obtain the products, denoted 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 ion channel blocking peptide Caloxin 2A1 to DSPE-PEG-MAL was (1~1.1):

3. 2) Combine the FR and PM obtained in step 1) with the AIE probe, S-nitroso-N-acetylpenicillamine, and DSPE-PEG. 2000 The organic solvent was dispersed in tetrahydrofuran and completely dissolved. The stock solution was then added dropwise to 10 mL of purified water and stirred vigorously. The organic solvent was evaporated by stirring in a fume hood, and then concentrated through an ultrafiltration filter to obtain a photoactivated nanomaterial for bladder cancer therapy. This nanomaterial contains an AIE probe, S-nitroso-N-acetylpenicillamine, and DSPE-PEG. 2000 The mass ratio of FR and PM is 1:2:9:2.4:2.6.

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