Preparation method and application of AIE type photosensitizer mainly used for type I photodynamic therapy

Abstract

This invention belongs to the field of antitumor drug technology, specifically disclosing a method for preparing and applying an AIE-type photosensitizer primarily for type I photodynamic therapy. The chemical structure of the AIE-type photosensitizer of this invention is shown in formula (Ⅰ). Due to the heavy atom effect of the sulfur atom, the photosensitizer of this invention can increase the coupling constant, which is beneficial for intersystem crossing of excitons, increasing the yield of reactive oxygen species generated by the photosensitizer™, resulting in good photodynamic therapy effects. The photosensitizer™ skeleton contains an R1-substituted tetraphenylethylene unit, exhibiting significant aggregation-induced emission (AIE) properties. The AIE-type photosensitizer can rapidly generate cytotoxic superoxide anion radicals (·O2) through electron transfer. ‑ This invention kills tumor cells, achieving the therapeutic goal of inhibiting tumor growth. The photosensitizer prepared using readily available raw materials, with mild synthesis conditions, a simple preparation method, convenient purification, and is easy to implement, showing great application potential.

Description

Technical Field

[0001] This invention belongs to the field of antitumor drug technology, specifically relating to a preparation method and application of an AIE-type photosensitizer mainly used for type I photodynamic therapy. Background Technology

[0002] Current cancer treatment primarily relies on traditional surgical resection, supplemented by chemotherapy and radiotherapy. While traditional methods offer some effectiveness, they also have limitations, such as a high recurrence rate and significant drug side effects. Photodynamic therapy (PDT) complements traditional methods to some extent. For example, PDT offers advantages such as good tissue selectivity, non-invasiveness, rapid onset of action, resistance to drug resistance, fewer systemic side effects, and the ability to be repeated, and it can also synergize with other treatment methods. Thanks to its excellent optical properties and aggregation-induced reactive oxygen species (ROS) generation enhancement characteristics, photosensitizers with AIE properties have shown outstanding performance in fluorescence-visualized PDT of tumors, attracting widespread attention from researchers. PDT involves converting excited-state photosensitizers through singlet-triple-state phototransformation, allowing ground-state oxygen to gain energy or through photo-oxidation to form singlet oxygen (Type II mechanism), or reacting with other biomolecules to form numerous reactive oxygen species (ROS, Type I mechanism), which are then used to kill cancer cells. A schematic diagram of the mechanism is shown below. Figure 1 (Yablonsky diagram)

[0003] Most reported AIE-type photosensitizers are type II photosensitizers that primarily generate singlet oxygen. However, the ROS generation process of type II photosensitizers is highly dependent on oxygen, and their ROS generation efficiency is often limited by the hypoxic conditions of tumor tissue. In contrast, type I photosensitizers have been shown to have better hypoxia tolerance, enabling them to fully utilize the limited oxygen in the tumor microenvironment during photodynamic therapy. Furthermore, based on their crucial role in maintaining cell growth, resisting cell death, and activating cancer cell invasion and metastasis, the cell nucleus is considered the optimal target for photodynamic therapy. Therefore, developing AIE-type photosensitizers with type I photodynamic therapy capabilities can overcome the hypoxia problem faced by traditional oxygen-dependent type II photosensitizers in tumor photodynamic therapy, fully leveraging the photodynamic therapeutic efficacy of type I photosensitizers, and is of great significance for improving the treatment effect of hypoxic solid tumors. Summary of the Invention

[0004] In order to overcome the shortcomings and deficiencies of the prior art, the primary objective of this invention is to provide an AIE-type photosensitizer mainly used for type I photodynamic therapy.

[0005] Another object of the present invention is to provide a method for preparing the above-mentioned AIE-type photosensitizer mainly used for type I photodynamic therapy.

[0006] Another object of the present invention is to provide the application of the above-mentioned AIE type photosensitizer in the preparation of photosensitizers for tumor photodynamic therapy, especially in the preparation of photosensitizers mainly for tumor type I photodynamic therapy.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] An AIE-type photosensitizer, primarily used for type I photodynamic therapy, is named TM, and its chemical structure is shown in formula (I):

[0009]

[0010] In the formula, R is a straight-chain or branched alkyl group with 1-30 carbon atoms, R1 is hydrogen or a straight-chain or branched alkyl group with 1-30 carbon atoms, and R1 is in the para position.

[0011] Preferably, R is a straight-chain or branched alkyl group having 1-12 carbon atoms, R1 is hydrogen or a straight-chain or branched alkyl group having 1-8 carbon atoms, and R1 is in the para position.

[0012] More preferably, R is a straight-chain or branched alkyl group having 6-12 carbon atoms, R1 is hydrogen or a straight-chain or branched alkyl group having 1-4 carbon atoms, and R1 is in the para position.

[0013] More preferably, R is a straight-chain or branched alkyl group having 6, 8, or 12 carbon atoms, and R1 is hydrogen, methyl, or tert-butyl, with R1 in the para position, corresponding to the structural formula of formula (Ⅰ)TM as follows:

[0014]

[0015]

[0016] In the most preferred embodiment, R is n-hexyl, n-octyl, isooctyl or 2-butyloctyl, R1 is hydrogen, methyl or tert-butyl, and R1 is in the para position.

[0017] The preparation method of the above-mentioned AIE-type photosensitizer, mainly used for type I photodynamic therapy, includes the following steps:

[0018] (1) 5,8-Dibromodithiopheno[3',2':3,4;2”,3”:5,6]benzo[1,2-c][1,2,5]thiadiazole was dissolved in toluene and reacted with (4-R-ylthiophen-2-yl)trimethyltinane at 80 °C for 8 hours using tetra(triphenylphosphine)palladium as a catalyst. After purification, compound 5,8-bis(4-R-ylthiophen-2-yl)dithiopheno[3',2':3,4;2”,3”:5,6]benzo[1,2-c][1,2,5]thiadiazole (compound 1) was obtained.

[0019] (2) 5,8-bis(4-R-thiophen-2-yl)dithieno[3',2':3,4;2”,3”:5,6]benzo[1,2-c][1,2,5]thiadiazole (compound 1) underwent bromination under the action of N-bromosuccinimide (NBS), and after purification, compound 5,8-bis(5-bromo-4-R-thiophen-2-yl)dithieno[3',2':3,4;2”,3”:5,6]benzo[1,2-c][1,2,5]thiadiazole (compound 2) was obtained;

[0020] (3) 5,8-bis(5-bromo-4-R-thiophene-2-yl)dithienro[3',2':3,4;2”,3”:5,6]benzo[1,2-c][1,2,5]thiadiazole (compound 2) and 4,4,5,5-tetramethyl-2-(4-(1,2,2-triR1-phenylvinyl)phenyl)-1,3,2-dioxaborane under toluene as solvent and in the presence of tetra(triphenylphosphine)palladium and potassium carbonate catalysts underwent a Suzuki coupling reaction. The reaction was carried out at 80 °C for 12 hours. After purification, compound 5,8-bis(4-R-5-(4-(1,2,2-triR1-phenylvinyl)phenyl)thiophene-2-yl)dithienro[3',2':3,4;2”,3’:5,6]benzo[1,2-c][1,2,5]thiadiazole (TM) was obtained.

[0021] Further, in step (1), the molar ratio of 5,8-dibromodithiopheno[3',2':3,4;2”,3”:5,6]benzo[1,2-c][1,2,5]thiadiazole, (4-R-ylthiophene-2-yl)trimethylstanane, and tetra(triphenylphosphine)palladium is 1:2 to 4:0.03 to 0.07, preferably 1:3:0.05, and the molar amount of 5,8-dibromodithiopheno[3',2':3,4;2”,3”:5,6]benzo[1,2-c][1,2,5]thiadiazole to the volume ratio of toluene is 1 mmol:(5 to 10) mL, preferably 1 mmol:7.5 mL;

[0022] Further, in step (2), the molar ratio of 5,8-bis(4-R-thiophene-2-yl)dithiopheno[3',2':3,4;2”,3”:5,6]benzo[1,2-c][1,2,5]thiadiazole to N-bromosuccinimide is 1:0.3 to 0.7, preferably 1:0.5;

[0023] Further, in step (3), the molar ratio of 5,8-bis(5-bromo-4-R-thiophene-2-yl)dithiopheno[3',2':3,4;2”,3”:5,6]benzo[1,2-c][1,2,5]thiadiazole, 4,4,5,5-tetramethyl-2-(4-(1,2,2-triR1-ylphenylvinyl)phenyl)-1,3,2-dioxaborane, tetra(triphenylphosphine)palladium, and potassium carbonate is... The molar ratio of 5,8-bis(5-bromo-4-R-thiophene-2-yl)dithiopheno[3',2':3,4;2”,3”:5,6]benzo[1,2-c][1,2,5]thiadiazole to toluene is 1 mmol:(15-30) mL, preferably 1 mmol:20 mL.

[0024] The specific preparation route for the AIE-type photosensitizers, primarily used for type I photodynamic therapy, is as follows:

[0025]

[0026] The above-mentioned AIE-type photosensitizers and the AIE-type photosensitizers synthesized by the above methods are used in the preparation of photosensitizers for tumor photodynamic therapy, especially in the preparation of photosensitizers mainly for type I tumor photodynamic therapy.

[0027] In a specific application, the photosensitizer and the amphiphilic polymer are dissolved in an organic solvent, then ultrapure water is added to the system, and the system is sonicated to make it uniformly dispersed. Then the organic solvent is removed to obtain water-soluble photosensitizer nanoparticles, wherein the apparent concentration of the water-soluble photosensitizer nanoparticles is ≥210μg / mL.

[0028] Furthermore, the organic solvent is selected from one of tetrahydrofuran, 1,4-dioxane, N,N-dimethylformamide, and ethyl acetate.

[0029] Furthermore, the amphiphilic polymer is F127 (an amphoteric triblock polymer formed by ethoxy-propoxy), DSPE-mPEG2000 (distearylphosphatidylethanolamine-polyethylene glycol 2000), or DSPE-mPEG5000 (distearylphosphatidylethanolamine-polyethylene glycol 5000).

[0030] Furthermore, the tumor is a subcutaneous tumor.

[0031] This invention relates to an AIE-type photosensitizer primarily used for type I photodynamic therapy. Due to the heavy atom effect of sulfur atoms, it can increase the coupling constant, facilitating intersystem crossing of excitons and improving the yield of reactive oxygen species generated by the photosensitizer™, resulting in better photodynamic therapy efficacy. The photosensitizer™ backbone contains R1-substituted tetraphenylethylene units, exhibiting significant aggregation-induced emission (AIE) properties. Under illumination, the AIE-type photosensitizer rapidly generates cytotoxic superoxide anion radicals (·O2) via a photochemical pathway of electron transfer. - This invention kills tumor cells, thereby inhibiting tumor growth. The photosensitizer prepared using readily available raw materials, with mild synthesis conditions, a simple preparation method, convenient purification, and is easy to implement, showing great application potential.

[0032] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0033] 1. The photosensitizer TM of this invention, due to the heavy atom effect of sulfur atoms, can expand the spin-orbit coupling constant between singlet and triplet states, which is conducive to intersystem crossing of excitons, improves the yield of reactive oxygen species generated by photosensitizer TM, and has a good photodynamic therapy effect.

[0034] 2. The photosensitizer™ contains an R1-substituted tetraphenylethylene unit, exhibiting typical aggregation-induced emission (AIE) properties.

[0035] 3. The photosensitizer™ of this invention is a photosensitizer that can be used for photodynamic therapy of tumors. It is non-toxic to cancer cells under normal culture conditions and has good biocompatibility. Under xenon lamp irradiation (wavelength range of 300-750nm), it has a high damaging effect on cancer cells. The cells are cancer cells, especially HeLa cells.

[0036] 4. Photosensitizers rapidly generate superoxide anion radicals (·O2) through electron transfer. - It kills tumor cells to achieve the therapeutic goal of inhibiting tumor growth, and mainly uses type I photodynamic therapy, which has good hypoxia resistance characteristics.

[0037] 5. The preparation method of the photosensitizer TM of the present invention uses readily available raw materials, has mild synthesis conditions, is simple to prepare, and is convenient to purify. Attached Figure Description

[0038] Figure 1 : A schematic diagram of Yablonsky.

[0039] Figure 2 Fluorescence emission spectra of TM1 nanoparticles in systems with different water contents.

[0040] Figure 3: UV-Vis absorption spectrum of ABDA under TM1 nanoparticle and illumination conditions.

[0041] Figure 4 : Photodynamic therapy results of HeLa cells by photosensitizer™1 nanoparticles.

[0042] Figure 5 Fluorescence emission spectrum of DHE under TM1 nanoparticle and illumination conditions. Detailed Implementation

[0043] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the implementation of the present invention is not limited thereto.

[0044] Example 1: Preparation of Compound TM1

[0045] (1) Synthesis of 5,8-bis(4-hexylthiophene-2-yl)dithieno[3',2':3,4;2”,3”:5,6]benzo[1,2-c][1,2,5]thiadiazole (compound 1)

[0046] 5,8-Dibromodithiopheno[3',2':3,4;2”,3”:5,6]benzo[1,2-c][1,2,5]thiadiazole (8.12 g, 20 mmol), (4-hexylthiophene-2-yl)trimethylstanane (19.87 g, 60 mmol), tetrakis(triphenylphosphine)palladium (1.16 g, 1 mmol) and 150 mL of toluene were added to a 500 mL two-necked flask. The reaction was carried out under nitrogen protection at 80 °C for 8 hours. Stirring was stopped and the mixture was cooled to room temperature. The mixture was extracted with dichloromethane, washed with water, and purified by silica gel column chromatography (eluent: petroleum ether:dichloromethane = 4:1, v / v) to give 9.36 g of the product, yield 81%. MS (APCI) (C 30 H 32 N2S5): 580.90. MS results showed that the obtained compound was the target product, and the chemical reaction equation for the preparation process is as follows:

[0047]

[0048] (2) Synthesis of 5,8-bis(5-bromo-4-hexylthiophene-2-yl)dithieno[3',2':3,4;2”,3”:5,6]benzo[1,2-c][1,2,5]thiadiazole (compound 2)

[0049] 5,8-bis(4-hexylthiophene-2-yl)dithienro[3',2':3,4;2”,3”:5,6]benzo[1,2-c][1,2,5]thiadiazole (9.36 g, 16.11 mmol) and N-bromosuccinimide (1.43 g, 8.06 mmol) were added to a 250 mL single-necked flask. After reacting at room temperature for 5 hours, the catalyst N-bromosuccinimide was quenched with ammonium chloride solution, and then purified by silica gel column chromatography (eluting agent: petroleum ether: dichloromethane = 6:1, v / v); 6.53 g of product was obtained, with a yield of 70%. MS (APCI)(C 30 H 30 Br2N2S5): 738.69. MS results showed that the obtained compound was the target product, and the chemical reaction equation for the preparation process is as follows:

[0050]

[0051] (3) Preparation of photosensitizer (TM1)

[0052] Under an argon atmosphere, 5,8-bis(5-bromo-4-hexylthiophene-2-yl)dithienro[3',2':3,4;2”,3”:5,6]benzo[1,2-c][1,2,5]thiadiazole (6.53 g, 8.84 mmol), 4,4,5,5-tetramethyl-2-(4-(1,2,2-triphenylvinyl)phenyl)-1,3,2-dioxaborane (10.13 g, 22.10 mmol), tetra(triphenylphosphine)palladium (0.51 g, 0.44 mmol), potassium carbonate (9.77 g, 70.72 mmol), and 176.80 mL of toluene were added to a 500 mL two-necked flask. The mixture was heated to 80 °C and reacted for 12 hours. After the reaction was stopped, the reaction was quenched with water, then extracted with dichloromethane and dried with anhydrous magnesium sulfate. The solution was concentrated and then purified by silica gel column chromatography using a mixed solvent of petroleum ether and dichloromethane (volume ratio of petroleum ether to dichloromethane 4:1) as the eluent. After drying, compound TM1 was obtained in 75% yield. MS and elemental analysis results showed that the obtained compound was the target product. The chemical reaction equations for the preparation process are shown below:

[0053]

[0054] TM1 (chemical structural formula is C) 82 H 68 The theoretical molecular weight of N2S5 is 1241.78, and the result measured by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF) is 1240.40 [M+H]. +The contents of C, H, and N elements in TM1 were determined using a PerkinElmer elemental analyzer (model EA-2400II). The theoretical values ​​were: C: 79.24%; H: 5.47%; N: 2.25%; S: 12.88%. The measured values ​​were: C: 79.32%; H: 5.52%; N: 2.26%; S: 12.91%. The mass spectrometry and elemental analysis results were close to the theoretical values, proving that the synthesized product was indeed the target product.

[0055] (4) Preparation of nanoparticles

[0056] To adapt to the complex aquatic environment in organisms, 10 mg of oil-soluble photosensitizer TM1 and 100 mg of amphiphilic polymer F127 (an amphoteric triblock polymer formed by ethoxy-propoxy) were completely dissolved in 1.5 mL of tetrahydrofuran solution. Under sonication, the mixture was quickly added to 20 mL of ultrapure water and sonicated for another 10 min to ensure uniform dispersion. Nitrogen gas was then bubbled into the sample to remove the tetrahydrofuran. Finally, the sample was stored at 4 °C for later use.

[0057] The apparent concentration of the prepared water-soluble TM1 nanoparticles (TM1 NPs) was 500 μg / mL. The size of the TM1 NPs was measured using a Malvern laser particle size analyzer (Mastersizer 3000), and the results showed that the particle size was 86 nm and the polydispersity index (PDI) was 0.17.

[0058] Example 2: Preparation of Compound TM7

[0059] (1) Synthesis of 5,8-bis(4-(2-butyl)octylthiophene-2-yl)dithienro[3',2':3,4;2”,3”:5,6]benzo[1,2-c][1,2,5]thiadiazole (compound 1)

[0060] 5,8-Dibromodithiopheno[3',2':3,4;2”,3”:5,6]benzo[1,2-c][1,2,5]thiadiazole (8.12 g, 20 mmol), (4-(2-butyl)octylthiophene-2-yl)trimethylstanane (24.92 g, 60 mmol), tetrakis(triphenylphosphine)palladium (1.16 g, 1 mmol) and 150 mL of toluene were added to a 500 mL two-necked flask. The reaction was carried out under nitrogen protection at 80 °C for 8 hours. Stirring was stopped and the mixture was cooled to room temperature. The mixture was extracted with dichloromethane, washed with water, and purified by silica gel column chromatography (eluent: petroleum ether:dichloromethane = 4:1, v / v) to give 11.94 g of product, 80% yield. MS (APCI) (C 42 H 56 N2S5): 749.22. MS results showed that the obtained compound was the target product, and the chemical reaction equation for the preparation process is:

[0061]

[0062] (2) Synthesis of 5,8-bis(5-bromo-4-(2-butyl)octylthiophene-2-yl)dithienro[3',2':3,4;2”,3”:5,6]benzo[1,2-c][1,2,5]thiadiazole (compound 2)

[0063] 5,8-bis(4-(2-butyl)octylthiophene-2-yl)dithienro[3',2':3,4;2”,3”:5,6]benzo[1,2-c][1,2,5]thiadiazole (11.94 g, 15.94 mmol) and N-bromosuccinimide (1.42 g, 7.97 mmol) were added to a 250 mL single-necked flask. After reacting at room temperature for 5 hours, the catalyst N-bromosuccinimide was quenched with ammonium chloride solution, and then purified by silica gel column chromatography (eluting agent: petroleum ether: dichloromethane = 6:1, v / v); 10.21 g of product was obtained, yield 71%. MS (APCI)(C 42 H 54 Br2N2S5): 907.02. MS results showed that the obtained compound was the target product, and the chemical reaction equation for the preparation process is as follows:

[0064]

[0065] (3) Preparation of photosensitizer (TM7)

[0066] Under an argon atmosphere, 5,8-bis(5-bromo-4-(2-butyl)octylthiophene-2-yl)dithienro[3',2':3,4;2”,3”:5,6]benzo[1,2-c][1,2,5]thiadiazole (10.21 g, 10.36 mmol), 4,4,5,5-tetramethyl-2-(4-(1,2,2-triphenylvinyl)phenyl)-1,3,2-dioxaborane (11.87 g, 25.90 mmol), tetra(triphenylphosphine)palladium (0.60 g, 0.52 mmol), potassium carbonate (11.45 g, 82.88 mmol), and 207.20 mL of toluene were added to a 500 mL two-necked flask. The mixture was heated to 80 °C and reacted for 12 hours. After the reaction was stopped, the reaction was quenched with water, then extracted with dichloromethane and dried with anhydrous magnesium sulfate. The solution was concentrated and then purified by silica gel column chromatography using a mixed solvent of petroleum ether and dichloromethane (volume ratio of petroleum ether to dichloromethane 4:1) as the eluent. After drying, compound TM7 was obtained in 72% yield. MS and elemental analysis results showed that the obtained compound was the target product. The chemical reaction equations for the preparation process are shown below:

[0067]

[0068] TM7 (chemical structural formula is C) 94 H 92 The theoretical molecular weight of N2S5 is 1410.18, and the result measured by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF) is 1408.59 [M+H]. + The contents of C, H, and N elements in TM1 were determined using a PerkinElmer elemental analyzer (model EA-2400II). The theoretical values ​​were: C: 79.99%; H: 6.52%; N: 1.99%; S: 11.35%. The measured values ​​were: C: 80.07%; H: 6.58%; N: 1.99%; S: 11.37%. The mass spectrometry and elemental analysis results were close to the theoretical values, proving that the synthesized product was indeed the target product.

[0069] (4) Preparation of nanoparticles

[0070] To adapt to the complex aquatic environment in organisms, 10 mg of oil-soluble photosensitizer TM7 and 100 mg of amphiphilic polymer F127 (an amphoteric triblock polymer formed by ethoxy-propoxy) were completely dissolved in 1.0 mL of tetrahydrofuran solution. Under sonication, the mixture was quickly added to 20 mL of ultrapure water and sonicated for another 10 min to ensure uniform dispersion. Nitrogen gas was then bubbled into the sample to remove the tetrahydrofuran. Finally, the sample was stored at 4 °C for later use.

[0071] The apparent concentration of the prepared water-soluble TM7 nanoparticles (TM7 NPs) was 500 μg / mL. The size of the TM7 NPs was measured using a Malvern laser particle size analyzer (Mastersizer 3000), and the results showed that the particle size was 75 nm and the polydispersity index (PDI) was 0.18.

[0072] Comparing photosensitizers TM1 and TM7, they have the same conjugated skeleton and the same photophysical properties. The difference in the side chain alkyl chain leads to the difference in solubility between TM1 and TM7. The processes for preparing nanoparticles are slightly different, specifically reflected in the different volumes of dissolved photosensitizers.

[0073] The photosensitizer TM contains an R1-substituted tetraphenylethylene unit, exhibiting typical aggregation-induced emission (AIE) properties. Experiments show that a formulation of 10... -3 The mother liquor (with TM1 as solute and acetonitrile as solvent) was diluted to a concentration of 10 mol / L. -5 Solutions with a water content of 0%, 20%, 40%, 60%, 80%, and 99% (mol / L) were sonicated in an ultrasonic instrument for 30 seconds. The absorption wavelengths were then measured using fluorescence emission spectroscopy. The test results are as follows: Figure 2 .from Figure 2It can be seen that the fluorescence emission wavelength of TM1 with different water contents is around 560nm. Moreover, as the water content increases, the fluorescence emission wavelength of TM1 first increases, then decreases, and then increases again, indicating that the photosensitizer TM has good AIE performance.

[0074] The photodynamic properties of TM1 nanoparticles are measured by the production of reactive oxygen species (ROS). 9,10-Anthracenediyl-di(methylene)dimalonic acid (ABDA) is a commonly used probe for singlet oxygen (a type of ROS). TM1 nanoparticles generate singlet oxygen under illumination, which can oxidize ABDA. Therefore, the amount of ABDA in the system is consumed, and the absorbance decreases accordingly. If TM1 NPs cannot generate singlet oxygen under illumination, the absorbance of ABDA remains unchanged. A 3 mL test sample was prepared, containing 60 μg / mL of TM1 NPs and 50 μmol / L of ABDA. Figure 3 This is the UV-Vis absorption spectrum of TM1 NPs under illumination (xenon lamp) for ABDA, with an illumination wavelength of 300–450 nm and an illumination power of 80 mW / cm². -2 As shown in the figure, the absorbance of ABDA at its characteristic peak at 399 nm gradually decreases with varying illumination time, exhibiting a time-dependent effect. After 300 seconds of illumination, the absorbance decreased from 0.791 before illumination to 0.507, a reduction of 36%. This indicates that photosensitizer TM1 is a type II photosensitizer, capable of efficiently generating reactive oxygen species upon illumination, and thus suitable for photodynamic therapy.

[0075] The cytotoxicity of TM1 and its photodynamic therapeutic effect on HeLa cells were detected using the CCK-8 assay. The specific experimental steps are as follows:

[0076] 1) Dilute the 500 μg / mL nanoparticle stock solution to concentrations of 10 μg / mL, 30 μg / mL, 50 μg / mL, 70 μg / mL, and 90 μg / mL using complete culture medium (DMEM, containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin).

[0077] 2) HeLa cells (human cervical cancer cells, ATCC) in the logarithmic growth phase were digested with 0.25% trypsin and the cells were uniformly diluted to a concentration of 5 × 10⁻⁶. 4 Cells / mL.

[0078] 3) Add the cell solution to each well of a 96-well plate (100 μL), shake gently to mix, and then incubate at 37°C and 5% CO2 for 24 h.

[0079] 4) Add 100 μL of complete culture medium containing different concentrations of TM1 nanoparticles to each well of a 96-well plate. Each concentration is used to prepare 10 wells, with 5 wells forming two groups: a light-controlled group and a non-light-controlled group. A control group of 0 μg / mL is included. Incubate the 96-well plates in an incubator for 12 hours.

[0080] 5) Remove the 96-well plate from the illumination group and use a xenon lamp (power 80W / cm²). 2 After irradiation for 5.0 min, place in an incubator and continue incubation for 12 h. The 96-well plates in the non-illuminated group do not require light treatment and are directly incubated for 24 h.

[0081] 6) Wash away the waste culture medium in the 96-well plates of the light group and the non-light group, add 100 μL of complete culture medium containing 10% CCK-8 to each well, and then put them back into the incubator for 1 hour.

[0082] 7) Place the 96-well plates of the light-exposed and non-light-exposed groups into a microplate reader and measure the absorbance of each well at the absorption peak at 450 nm. Calculate the average and standard deviation of the absorbance from the five wells in each group, and then calculate the cancer cell survival rate. The CCK-8 assay results are shown below. Figure 4 .

[0083] from Figure 4 It was found that under light-free conditions, the survival rate of HeLa cells remained above 94% at different concentrations of TM1 NPs. This indicates that TM1 NPs exhibit excellent biocompatibility under xenon lamp irradiation. However, under light-induced conditions, cell survival rate was related to the concentration of TM1 NPs; the higher the concentration of TM1 NPs, the lower the cell survival rate, meaning that the cytotoxic effect was stronger under xenon lamp irradiation. At a concentration of 10 μg / mL, TM1 NPs killed 14.4% of HeLa cells; at 30 μg / mL, TM1 NPs killed 28.7% of HeLa cells; at 50 μg / mL, TM1 NPs killed 41.5% of HeLa cells; at 70 μg / mL, TM1 NPs killed 54.4% of HeLa cells; and at 90 μg / mL, TM1 NPs killed 66.8% of HeLa cells. This demonstrates that TM1 NPs have excellent photodynamic therapy effects on HeLa cells.

[0084] The photodynamic therapy effect of TM7 NPs was evaluated using the same method described above, except that the complete culture medium containing different concentrations of TM1 NPs in step 4) was replaced with a complete culture medium containing different concentrations of TM7 NPs. The results showed that HeLa cells maintained a survival rate of over 90% under light-free conditions with different concentrations of TM7 NPs. This indicates that TM7 NPs have no cytotoxicity and good biocompatibility under light-free conditions. However, under light-free conditions, cell survival rate was also related to the concentration of TM7 NPs; the higher the concentration of TM7 NPs, the lower the cell survival rate, meaning that the cytotoxic effect was stronger under xenon lamp irradiation. At a concentration of 10 μg / mL, TM7 NPs killed 13.4% of HeLa cells; at 30 μg / mL, they killed 26.3%; at 50 μg / mL, 34.2%; at 70 μg / mL, 52.9%; and at 90 μg / mL, 67.5%. This indicates that TM7 NPs also exhibit excellent photodynamic therapy efficacy against HeLa cells, comparable to that of TM1 NPs.

[0085] AIE-type photosensitizers can rapidly generate cytotoxic superoxide anion free radicals (·O2-) through electron transfer, killing tumor cells and achieving the therapeutic goal of inhibiting tumor growth.

[0086] DHE (Dihydroethidium) is a cell membrane-permeable blue probe (λEx / λEm: 370 / 420nm) that can be used to detect superoxide anion radicals (·O2-). The decrease in fluorescence indicates the amount and changes in cellular ROS levels. To verify that TM1 is a type I photosensitizer for photodynamic therapy, 100 μL of DHE (10 μol / L) was added to 3.0 mL of TM1 NPs solution at a concentration of 40 μg / mL, and the sample was tested using a fluorescence spectrometer. The specific absorption spectrum is shown in [the image / image / etc.]. Figure 5 Compared to the group without DHE, the sample with DHE showed a rapid response and a decrease in fluorescence in the 380-500 nm range. This indicates that TM1 is a type I photosensitizer suitable for photodynamic therapy. Figure 3 The experiment used ABDA indicator to determine that photosensitizer TM1 was a type II photosensitizer. This indicator has a long test response time of 300 s; while Figure 5 The photosensitizer TM1 was determined to be a type I photosensitizer using DHE indicator, with a test response time of only 5 seconds. This indicates that photosensitizer TM1 is an AIE-type fluorescent molecule primarily used for type I photodynamic therapy.

[0087] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An AIE-type photosensitizer primarily used for type I photodynamic therapy, the chemical structure of which is shown in formula (Ⅰ): In the formula, R is a straight-chain or branched alkyl group with 1-30 carbon atoms, R1 is hydrogen or a straight-chain or branched alkyl group with 1-30 carbon atoms, and R1 is in the para position.

2. The AIE-type photosensitizer according to claim 1, primarily used for type I photodynamic therapy, is characterized in that: In formula (Ⅰ), R is a straight-chain or branched alkyl group with 1-12 carbon atoms, R1 is hydrogen or a straight-chain or branched alkyl group with 1-8 carbon atoms, and R1 is in the para position.

3. The AIE-type photosensitizer according to claim 1, primarily used for type I photodynamic therapy, is characterized in that: In formula (Ⅰ), R is a straight-chain or branched alkyl group with 6-12 carbon atoms, R1 is hydrogen or a straight-chain or branched alkyl group with 1-4 carbon atoms, and R1 is in the para position.

4. The AIE-type photosensitizer according to claim 1, primarily used for type I photodynamic therapy, characterized in that: The structural formula of equation (Ⅰ)TM is:

5. The AIE-type photosensitizer according to claim 1, primarily used for type I photodynamic therapy, characterized in that: In formula (Ⅰ), R is n-hexyl, n-octyl, isooctyl or 2-butyloctyl, R1 is hydrogen, methyl or tert-butyl, and R1 is in the para position.

6. The method for preparing the AIE-type photosensitizer as described in any one of claims 1-5, primarily for type I photodynamic therapy, characterized in that, The preparation route of formula (Ⅰ)TM is as follows: Wherein, the R and R1 groups are defined in the same way as the groups in the corresponding claims 1-5.

7. The method for preparing an AIE-type photosensitizer primarily for type I photodynamic therapy according to claim 6, characterized in that, Includes the following steps: (1) 5,8-Dibromodithiopheno[3',2':3,4;2”,3”:5,6]benzo[1,2-c][1,2,5]thiadiazole was dissolved in toluene and reacted with tetra(triphenylphosphine)palladium as a catalyst at 80 °C for 8 hours. After purification, the compound 5,8-bis(4-R-thiopheno-2-yl)dithiopheno[3',2':3,4;2”,3”:5,6]benzo[1,2-c][1,2,5]thiadiazole was obtained; wherein the R group is the same as the group defined in the corresponding claims 1-5; (2) 5,8-bis(4-R-thiophen-2-yl)dithieno[3',2':3,4;2”,3”:5,6]benzo[1,2-c][1,2,5]thiadiazole under the action of N-bromosuccinimide was brominated to give the compound 5,8-bis(5-bromo-4-R-thiophen-2-yl)dithieno[3',2':3,4;2”,3”:5,6]benzo[1,2-c][1,2,5]thiadiazole; (3) 5,8-bis(5-bromo-4-R-thiophene-2-yl)dithiopheno[3',2':3,4;2”,3”:5,6]benzo[1,2-c][1,2,5]thiadiazole reacts with 4,4,5,5-tetramethyl-2-(4-(1,2,2-triR1-phenylvinyl)phenyl)-1,3,2-dioxaborane in toluene as solvent under the action of tetra(triphenylphosphine)palladium and potassium carbonate catalysts. The Suzuki coupling reaction was carried out at 80°C for 12 hours, and after purification, the compound 5,8-bis(4-R-yl-5-(4-(1,2,2-triR1-ylphenylvinyl)phenyl)thiophene-2-yl)dithiopheno[3',2':3,4;2”,3”:5,6]benzo[1,2-c][1,2,5]thiadiazole was obtained; wherein the R1 group is the same as the group defined in the corresponding claims 1-5.

8. The method for preparing an AIE-type photosensitizer primarily for type I photodynamic therapy according to claim 7, characterized in that: In step (1), the molar ratio of 5,8-dibromodithiopheno[3',2':3,4;2”,3”:5,6]benzo[1,2-c][1,2,5]thiadiazole, (4-R-ylthiophene-2-yl)trimethylstanane, and tetra(triphenylphosphine)palladium is 1:2~4:0.03~0.07, and the molar amount of 5,8-dibromodithiopheno[3',2':3,4;2”,3”:5,6]benzo[1,2-c][1,2,5]thiadiazole to the volume ratio of toluene is 1 mmol:(5~10) mL; In step (2), the molar ratio of 5,8-bis(4-R-thiophene-2-yl)dithiopheno[3',2':3,4;2”,3”:5,6]benzo[1,2-c][1,2,5]thiadiazole to N-bromosuccinimide is 1:0.3~0.7; In step (3), the molar ratio of 5,8-bis(5-bromo-4-R-thiophen-2-yl)dithiopheno[3',2':3,4;2”,3”:5,6]benzo[1,2-c][1,2,5]thiadiazole, 4,4,5,5-tetramethyl-2-(4-(1,2,2-triR1-phenylvinyl)phenyl)-1,3,2-dioxaborane, tetra(triphenylphosphine)palladium, and potassium carbonate is 1:2~3:0.02~0.2:5~10, and the molar amount of 5,8-bis(5-bromo-4-R-thiophen-2-yl)dithiopheno[3',2':3,4;2”,3”:5,6]benzo[1,2-c][1,2,5]thiadiazole to the volume ratio of toluene is 1 mmol:(15~30) mL.

9. The method for preparing an AIE-type photosensitizer primarily for type I photodynamic therapy according to claim 8, characterized in that: In step (1), the molar ratio of 5,8-dibromodithiopheno[3',2':3,4;2”,3”:5,6]benzo[1,2-c][1,2,5]thiadiazole, (4-R-ylthiophene-2-yl)trimethylstanane, and tetra(triphenylphosphine)palladium is 1:3:0.05, and the molar amount of 5,8-dibromodithiopheno[3',2':3,4;2”,3”:5,6]benzo[1,2-c][1,2,5]thiadiazole to the volume ratio of toluene is 1 mmol:7.5 mL; In step (2), the molar ratio of 5,8-bis(4-R-thiophene-2-yl)dithiopheno[3',2':3,4;2”,3”:5,6]benzo[1,2-c][1,2,5]thiadiazole to N-bromosuccinimide is 1:0.5; In step (3), the molar ratio of 5,8-bis(5-bromo-4-R-thiophen-2-yl)dithiopheno[3',2':3,4;2”,3”:5,6]benzo[1,2-c][1,2,5]thiadiazole, 4,4,5,5-tetramethyl-2-(4-(1,2,2-triR1-phenylvinyl)phenyl)-1,3,2-dioxaborane, tetra(triphenylphosphine)palladium, and potassium carbonate is 1:2.5:0.05:8, and the molar amount of 5,8-bis(5-bromo-4-R-thiophen-2-yl)dithiopheno[3',2':3,4;2”,3”:5,6]benzo[1,2-c][1,2,5]thiadiazole to the volume ratio of toluene is 1 mmol:20 mL.

10. The use of the AIE-type photosensitizer according to any one of claims 1-5 or the AIE-type photosensitizer prepared by any one of claims 6-9 in the preparation of photosensitizers for tumor photodynamic therapy.

11. The use of the AIE-type photosensitizer according to any one of claims 1-5 or the AIE-type photosensitizer prepared by any one of claims 6-9 in the preparation of photosensitizers mainly for type I photodynamic therapy of tumors.

Images