An aggregation-induced emission active photosensitizer, and a preparation method and application thereof

Abstract

The application provides an aggregation-induced emission active photosensitizer, a preparation method and application thereof, and belongs to the technical field of biomedical materials and photodynamic therapy.The photosensitizer is TPA-Py, TPA-S-Py and TPA-D-Py, wherein TPA-D-Py is dominated by a PICT effect in an excited state, can realize molecular conformation planarization, and can significantly improve a molar absorption coefficient, a fluorescence quantum yield and a type I / type II active oxygen generation efficiency.Cell experiments show that TPA-D-Py has mitochondrion targeting, low dark toxicity, and can efficiently kill GL261 glioma cells and has a high anti-drug resistant bacteria effect under white light irradiation.The application overcomes the defects of low light capture efficiency and dependence on oxygen to generate ROS of a traditional AIE photosensitizer, and has important application prospects in the field of fluorescence imaging guided photodynamic therapy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and photodynamic therapy technology, and particularly relates to an aggregation-induced emission active photosensitizer, its preparation method and application. Background Technology

[0002] As one of the major challenges facing global public health, the innovation and optimization of cancer treatment strategies have always been at the forefront of scientific research. Among numerous treatment methods, photodynamic therapy (PDT) has become a promising paradigm for cancer treatment due to its non-invasiveness, high spatiotemporal selectivity, and low systemic toxicity. The core mechanism of PDT relies on the synergistic effect of three key components: photosensitizer (PS), light source, and molecular oxygen. When irradiated with light of a specific wavelength, the photosensitizer in its ground state absorbs photon energy and is excited to a singlet excited state, followed by intersystem crossing (ISC) to enter a longer-lived triplet state. The excited photosensitizer then transfers energy or electrons to the surrounding triplet oxygen, producing highly cytotoxic reactive oxygen species (ROS). These substances can irreversibly damage tumor cells and induce apoptosis or necrosis. Therefore, developing ideal photosensitizers is crucial for promoting the clinical translation of PDT. However, traditional PS, such as porphyrins and their derivatives, often aggregate in the physiological environment through intermolecular π-π stacking interactions, leading to severe fluorescence quenching and a significant decrease in ROS generation efficiency. This phenomenon is known as aggregation-induced quenching (ACQ). The concept of aggregation-induced emission (AIE) has revolutionized the design of photosensitizers. AIE-active photosensitizers exhibit weak emission intensity and produce almost no reactive oxygen species (ROS) in their dispersed state; however, when aggregated, their internal molecular motion is restricted, blocking non-radiative decay pathways, resulting in significantly enhanced fluorescence and highly efficient ROS generation. Therefore, AIE-active photosensitizers have successfully solved the problem of activity loss in the aggregated state, achieving highly efficient fluorescence imaging-guided phototherapy (PDT). Despite significant progress in AIE-active photosensitizers, their performance, especially in terms of photophysical parameters, still has room for further improvement. For example, the molecular molar absorptivity determines the photon capture efficiency, while the conversion pathway and efficiency of the lowest singlet (S1) energy determine fluorescence brightness and ROS efficiency; these factors directly affect imaging quality and therapeutic efficacy. Typically, AIE-active PSs have a low molar absorptivity due to their torsional molecular conformation. Furthermore, traditional AIE-active PSs usually produce singlet oxygen (S1)... 1 O2), these types of ROS require large amounts of oxygen to be continuously generated. However, the microenvironment of tumors and bacterial biofilms is hypoxic, which inevitably leads to the presence of oxygen in tumors or bacterial biofilms. 1 A decrease in O2 affects treatment efficacy. In contrast, type I photosensitizers are less dependent on oxygen and can efficiently generate superoxide anions (O2). •-Phosphorus compounds, such as hydroxyl radicals (•OH), have shown great potential for use in practical phototherapy (PDT) for tumor treatment and bacterial eradication. Therefore, improving the molar absorptivity of active PS for type I AIE is particularly urgent.

[0003] To improve the molecular molar absorptivity of AIE-active PS, introducing planar rigid conjugated ring units has proven to be an effective method. However, this approach also easily induces strong intermolecular π-π stacking interactions, leading to fluorescence quenching and a decrease in ROS efficiency. Although combining the AIE mechanism and enhancing intramolecular charge transfer (ICT) or heavy atom effects can effectively suppress nonradiative thermal motion at the S1 level, the distortion of the molecular structure inevitably reduces the light absorption efficiency. Therefore, achieving simultaneous improvement in light absorption efficiency, radiative transition efficiency, and ISC efficiency in type I AIE-active PS remains a challenge, which is crucial for preparing AIE-active PS with high fluorescence quantum efficiency. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes an aggregation-induced emission (AIE) active photosensitizer, its preparation method, and its application. Based on the excited-state planar intramolecular charge transfer (PICT) strategy, the AIE active photosensitizer prepared by this invention can simultaneously improve light capture efficiency (molar absorptivity), fluorescence quantum yield, and type I / II reactive oxygen species generation efficiency, and achieve highly efficient photodynamic therapy in mitochondrial targeting and hypoxic environments.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides an aggregation-induced emission active photosensitizer with the following general chemical formula: ; In the formula, R is selected from any one of carbon-carbon single bonds, styryl, and diphenylbutadienyl.

[0006] Specifically, when R is a carbon-carbon single bond, the chemical structural formula of the aggregation-induced emission photosensitizer is: denoted as TPA-Py; when R is styrene, the chemical structural formula of the aggregation-induced emission photosensitizer is... It is denoted as TPA-S-Py; when R is diphenylbutadiene, the chemical structural formula of the aggregation-induced emission photosensitizer is... It is denoted as TPA-D-Py.

[0007] Furthermore, the aggregation-induced emission photosensitizer is preferably TPA-D-Py, which exhibits a dominant PICT effect in its excited state, a distorted conformation in its ground state, and a planarized molecular conformation in its excited state. It possesses aggregation-induced emission properties, with significantly enhanced fluorescence in the aggregated state, and the ability to generate type I and type II reactive oxygen species. It can target mitochondria and generate reactive oxygen species under white light irradiation to kill tumor cells. It can achieve specific fluorescence imaging of Gram-positive bacteria and generate reactive oxygen species under white light irradiation to kill drug-resistant bacteria.

[0008] The present invention also provides a method for preparing the above-mentioned aggregation-induced emission active photosensitizer, comprising the following steps: 4-Bromotriphenylamine, compound A, potassium carbonate, and a catalyst are added to a solvent, and an intermediate is obtained by coupling reaction, wherein compound A is selected from 4-pyridineboronic acid, 4-formylphenylboronic acid, and 4-bromocinnamaldehyde; more specifically, when the starting material is 4-bromotriphenylamine, compound A is 4-pyridineboronic acid or 4-formylphenylboronic acid; when the starting material is 4-(diphenylamino)phenylboronic acid, compound A is 4-bromocinnamaldehyde; The intermediate is mixed with an iodide and reacted under alkaline conditions to obtain the aggregation-induced emission active photosensitizer, wherein the iodide is selected from iodomethane or 1,4-dimethylpyridine-1-iodide.

[0009] More specifically, when the aggregation-induced emission photosensitizer is TPA-Py, the compound A used is 4-pyridineboronic acid, and the chemical structural formula of the intermediate prepared is as follows: (denoted as TPA-P), when the aggregation-induced emission photosensitizer is TPA-S-Py, the compound A used is 4-formylphenylboronic acid, and the chemical structure of the intermediate prepared is as follows: (denoted as TPA-Ph-CHO), when the aggregation-induced emission photosensitizer is TPA-D-Py, compound A used is 4-bromocinnamaldehyde, and the chemical structure of the intermediate prepared is as follows: (Written as TPA-Ph-dCHO).

[0010] Furthermore, the molar ratio of 4-bromotriphenylamine or 4-(diphenylamino)phenylboronic acid, compound A, and catalyst is 20:20:1.

[0011] Furthermore, the catalyst is tetrakis(triphenylphosphine)palladium (Pd(PPh3)4).

[0012] Furthermore, the solvent is a mixture of tetrahydrofuran and water.

[0013] Furthermore, the coupling reaction is carried out at a temperature of 90°C for 12 hours.

[0014] Furthermore, the molar ratio of the intermediate to the iodide is 1:(1~2.5).

[0015] Furthermore, the reaction temperature under alkaline conditions was 80℃, and the reaction time was 12h.

[0016] The present invention also provides the application of the above-mentioned aggregation-induced emission active photosensitizer in the preparation of fluorescent imaging agents or photodynamic therapy drugs.

[0017] Furthermore, the photodynamic therapy drug is used for photodynamic therapy of cancer cells or photodynamic clearance of Gram-positive bacterial infections.

[0018] Compared with the prior art, the present invention has the following advantages and technical effects: 1. This invention is the first to propose the design of AIE active photosensitizers using the excited-state planar intramolecular charge transfer (PICT) strategy, overcoming the low light capture efficiency of the traditional TICT strategy.

[0019] 2. The AIE-active photosensitizer compounds of the present invention, especially TPA-D-Py, achieve molecular conformation planarization in the excited state, significantly improving the molar absorptivity and effectively suppressing the nonradiative internal conversion of S1 energy, thereby simultaneously improving the fluorescence quantum yield and ROS yield.

[0020] 3. The AIE-active photosensitizer of the present invention also possesses type I (O2) properties. •- and •OH) and type II ( 1 Its ability to generate O2 and ROS is particularly suitable for hypoxic tumor microenvironments.

[0021] 4. Cell experiments show that the AIE-active photosensitizer of the present invention, especially TPA-D-Py, has good mitochondrial targeting ability and low dark toxicity. Under white light irradiation, it exhibits highly efficient photodynamic killing effect on GL261 glioma cells and Gram-positive bacteria, and has broad application prospects in fluorescence imaging-guided photodynamic therapy. Attached Figure Description

[0022] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 The 1H NMR spectrum of the intermediate TPA-P prepared in Example 1; Figure 2 The 1H NMR spectrum of the intermediate TPA-Ph-CHO prepared in Example 2; Figure 3 The 1H NMR spectrum of the intermediate TPA-Ph-dCHO prepared in Example 3; Figure 4 The 1H NMR spectrum of TPA-Py prepared in Example 1; Figure 5 The 1H NMR spectrum of TPA-S-Py prepared in Example 2; Figure 6 The 1H NMR spectrum of TPA-D-Py prepared in Example 3; Figure 7 The high-resolution mass spectrum of TPA-Py prepared in Example 1; Figure 8 The high-resolution mass spectrum of TPA-S-Py prepared in Example 2; Figure 9 The high-resolution mass spectrum of TPA-D-Py prepared in Example 3; Figure 10 The theoretical calculation results of the electronic transition from HOMO to LUMO of the active photosensitizer molecules prepared in Examples 1-3 in THF solvent (A) TPA-Py, (B) TPA-S-Py, (C) TPA-D-Py, and the absorption and fluorescence spectra in THF solvent ((D) TPA-Py, (E) TPA-S-Py, (F) TPA-D-Py). Figure 11 The theoretical calculation results of the excited-state molecular conformations of the photosensitizers TPA-Py, TPA-S-Py, and TPA-D-Py prepared in Examples 1-3 in different solvents such as toluene, tetrahydrofuran (THF), and dimethylformamide (DMF) are shown in the inset. The inset shows the fluorescence spectra and images of the emitters of the photosensitizers in toluene, THF, and DMF. Figure 12 (A) shows the absorption spectrum of the photosensitizers prepared in Examples 1-3 in THF solvent; (B) shows the fluorescence intensity of TPA-D-Py in a toluene / DMSO mixed solution as a function of the toluene ratio; (C) shows the fluorescence intensity of the three emitters in toluene / DMSO mixed solutions with different toluene ratios; (D) shows the fluorescence spectrum of TPA-Py; (E) shows the fluorescence spectrum of TPA-S-Py; and (F) shows the fluorescence spectrum of TPA-D-Py in different organic solvents. Figure 13(A) shows the absolute fluorescence quantum efficiency of the photosensitizers prepared in Examples 1-3 in DMSO and powder; (B) shows the theoretical calculation of the excited state energy levels; (C) shows the change in fluorescence intensity of the DCFH probe with or without photosensitizer added and exposed to white light at different time points (DCFH = 4 μM, photosensitizer = 2 μM); (D) shows the change in absorption intensity of the ABDA probe with or without photosensitizer added and exposed to white light at different time points (ABDA = 20 μM, photosensitizer = 2 μM); (E) shows the change in fluorescence intensity of the DHR123 probe with or without photosensitizer added and exposed to white light at different time points (DHR123 = 4 μM, photosensitizer = 10 μM); (F) shows the test results of •OH formation. Figure 14 The image shows a CLSM image of GL261 cells after being treated with the photosensitizer TPA-D-Py prepared in Example 3 for 24 hours and then co-stained with Mitotracker green for 30 minutes. The scale bar is 100 µm, and the right side is a fluorescence intensity distribution curve (the horizontal axis represents the spatial distance (μm) along the white straight line, and the vertical axis represents the fluorescence intensity). Figure 15 (A) shows the survival rate of GL261 cells in the dark; (B) shows the survival rate of GL261 cells under light conditions; (C) shows the CLSM image of live / dead cells (Calcein-AM / PI double staining), where green fluorescence represents live cells and red fluorescence represents dead cells. Figure 16 The fluorescence staining effect of the photosensitizer TPA-D-Py prepared in Example 3 on Gram-positive bacteria S. Aureus and MRSA is shown in the figure. Figure 17 Visual representation of the photodynamic effects of the photosensitizer TPA-D-Py prepared in Example 3 on the survival of Gram-positive bacteria S. Aureus and MRSA before and after photodynamic treatment at different concentrations and under light conditions: (A) Photodynamic killing effect of TPA-D-Py on Gram-positive bacteria S. Aureus, (B) Photodynamic killing effect of TPA-D-Py on Gram-positive bacteria MRSA. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0026] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0027] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0028] This invention provides an aggregation-induced emission photosensitizer with the following general chemical formula: ; In the formula, R is selected from any one of carbon-carbon single bonds, styryl, and diphenylbutadienyl.

[0029] Specifically, when R is a carbon-carbon single bond, the chemical structural formula of the aggregation-induced emission photosensitizer is: denoted as TPA-Py; when R is styrene, the chemical structural formula of the aggregation-induced emission photosensitizer is... It is denoted as TPA-S-Py; when R is diphenylbutadiene, the chemical structural formula of the aggregation-induced emission photosensitizer is... It is denoted as TPA-D-Py.

[0030] This invention also provides a method for preparing the above-mentioned aggregation-induced emission photosensitizer, comprising the following steps: 4-Bromotriphenylamine or 4-(diphenylamino)phenylboronic acid is added to a solvent along with compound A, potassium carbonate and a catalyst, and an intermediate is obtained by coupling reaction, wherein compound A is selected from 4-pyridineboronic acid, 4-formylphenylboronic acid and 4-bromocinnamaldehyde. The intermediate is mixed with an iodide and reacted under alkaline conditions to obtain an aggregation-induced emission active photosensitizer. The iodide is selected from iodomethane or 1,4-dimethylpyridine-1-iodide.

[0031] More specifically, when the aggregation-induced emission photosensitizer is TPA-Py, the preparation method includes the following steps: 4-bromotriphenylamine (chemical structural formula: ) and 4-pyridineboronic acid (chemical structural formula is Potassium carbonate and catalyst were added to a solvent, and the intermediate TPA-P was obtained by coupling reaction at 90°C for 12 h. The intermediate was mixed with iodomethane and reacted at 80°C for 12 h under alkaline conditions to obtain the aggregation-induced emission active photosensitizer TPA-Py.

[0032] When the aggregation-induced emission photosensitizer is TPA-S-Py, the preparation method includes the following steps: 4-Bromotriphenylamine was reacted with 4-formylphenylboronic acid (chemical structural formula: Potassium carbonate and catalyst were added to a solvent, and the intermediate TPA-Ph-CHO was obtained by coupling reaction at 90°C for 12 h. The intermediate was mixed with 1,4-dimethylpyridine-1-iodide and reacted at 80°C for 12 h under alkaline conditions to obtain the aggregation-induced emission active photosensitizer TPA-S-Py.

[0033] When the aggregation-induced emission photosensitizer is TPA-D-Py, the preparation method includes the following steps: 4-(diphenylamino)phenylboronic acid was reacted with 4-bromocinnamaldehyde (chemical structural formula: Potassium carbonate and catalyst were added to a solvent, and the intermediate TPA-Ph-dCHO was obtained by coupling reaction at 90°C for 12 h. The intermediate was reacted with 1,4-dimethylpyridine-1-iodide (chemical structural formula: The mixture was reacted at 80°C for 12 hours under alkaline conditions to obtain the aggregation-induced emission active photosensitizer TPA-D-Py.

[0034] In a preferred embodiment of the present invention, the molar ratio of 4-bromotriphenylamine or 4-(diphenylamino)phenylboronic acid, compound A and catalyst is 20:20:1.

[0035] In a preferred embodiment of the present invention, the catalyst is tetrakis(triphenylphosphine)palladium (Pd(PPh3)4).

[0036] In a preferred embodiment of the present invention, the solvent is a mixture of tetrahydrofuran and water.

[0037] In a preferred embodiment of the present invention, the molar ratio of the intermediate to the iodide is 1:(1~2.5).

[0038] This invention also provides an application of the above-mentioned aggregation-induced emission active photosensitizer in the preparation of fluorescent imaging agents or photodynamic therapy drugs.

[0039] In a preferred embodiment of the present invention, the photodynamic therapy drug is used for photodynamic therapy of tumors or photodynamic clearance of Gram-positive bacterial infections.

[0040] All raw materials used in the embodiments of this invention were purchased through commercial channels.

[0041] The column chromatography purification method used in this embodiment of the invention is a conventional technique in the field, and therefore will not be described in detail here.

[0042] The technical solution of the present invention will be further illustrated by the following embodiments.

[0043] Example 1 The chemical structural formula of the aggregation-induced emission photosensitizer (TPA-Py) is as follows: ; The synthetic route for TPA-Py is as follows: ; The specific preparation process is as follows: S1. Synthesis of intermediate TPA-P: In a two-necked reaction flask, 4-bromotriphenylamine (5.4 mmol, 1.74 g), 4-pyridineboronic acid (5.4 mmol, 0.66 g), potassium carbonate (34 mmol, 4.8 g), and Pd(PPh3)4 (0.27 mmol, 312 mg) were added. The reaction flask was evacuated and rinsed three times with nitrogen to ensure a nitrogen atmosphere. Tetrahydrofuran (40 mL) and water (16 mL) were added to the flask, and the mixture was refluxed and stirred at 90 °C for 12 hours. After cooling to room temperature, the reaction mixture was extracted with dichloromethane and water. The organic solvent was removed by rotary evaporation, and the product was purified by column chromatography to give a yellow to green powder (1.35 g), which was intermediate TPA-P, in 77.6% yield.

[0044] Figure 1 The image shows the 1H NMR spectrum of the intermediate TPA-P prepared in Example 1. Figure 1The 1H NMR spectrum showed that TPA-P has a theoretical number of 19 hydrogen atoms, and the actual 1H NMR spectrum results showed that the compound has 19 hydrogen atoms, indicating the successful synthesis of TPA-P.

[0045] The characterization data of the obtained TPA-P are as follows: 1 H NMR (400 MHz, DMSO- d6 ) δ 8.63 (d, J = 4 Hz,2H), 7.78 – 7.70 (m, 4H), 7.41 – 7.39 (t, J = 12 Hz, 2H), 7.18 – 7.07 (m, 8H). S2. Synthesis of product TPA-Py: TPA-P (0.862 mmol, 277 mg) and iodomethane (0.862 mmol, 122 mg) were added to a two-necked reaction flask. Then, 4-methylpiperidine (100 μL) and ethanol (60 mL) were added to the flask, and the mixture was refluxed at 80 °C for 12 hours with stirring. After cooling to room temperature, the mixture was directly filtered and washed with ethyl acetate to obtain 193 mg of an orange-red powder, which was the product TPA-Py, with a yield of 48.4%.

[0046] Figure 4 and Figure 7 The 1H NMR spectrum and high-resolution mass spectrum of TPA-Py prepared in Example 1 are shown below. Figure 4 The 1H NMR spectrum shows that TPA-Py theoretically has 21 hydrogen atoms, and the actual 1H NMR spectrum results confirm that the compound has 21 hydrogen atoms. Furthermore, Figure 7 High-resolution mass spectra show that the theoretical molecular weight is 465.0822 and the actual molecular weight is 465.2221, indicating the successful synthesis of TPA-Py.

[0047] The characterization data of TPA-Py obtained are as follows: 1 H NMR (400 MHz, DMSO- d6 ) δ 8.77-8.75 (d, J =8.0 Hz, 2H), 8.13-8.12 (d, J = 4.0 Hz, 2H), 7.95-7.90 (d, J = 20.0 Hz, 1H), 7.62-7.60 (d, J =8.0 Hz, 2H), 7.38-7.27 (m, 6H), 7.12 (m, 6H), 6.94-6.91 (d, J= 12.0Hz, 2H). HRMS (ESI) m / z: [M + H + calcd for C 30 H 21 N2I, 465.0822; found, 465.2221. Example 2 The chemical structural formula of the aggregation-induced emission photosensitizer (TPA-S-Py) is as follows: ; The synthetic route of TPA-S-Py is as follows: ; The specific preparation process is as follows: S1. Synthesis of intermediate TPA-Ph-CHO: 4-Bromotriphenylamine (5.4 mmol, 1.74 g), 4-formylphenylboronic acid (5.4 mmol, 0.81 g), potassium carbonate (34 mmol, 4.8 g), and Pd(PPh3)4 (0.27 mmol, 312 mg) were placed in a two-necked reaction flask. The reaction flask was evacuated three times under nitrogen to ensure a nitrogen atmosphere. Tetrahydrofuran (40 mL) and water (16 mL) were added to the reaction flask, and the mixture was stirred and refluxed at 90 °C for 12 hours. After cooling to room temperature, the reaction mixture was extracted with dichloromethane and water. The organic solvent was removed by rotary evaporation, and the product was purified by column chromatography to give 1.35 g of yellow to green powder, which was intermediate TPA-Ph-CHO, in 71.8% yield.

[0048] Figure 2 The above is the 1H NMR spectrum of the intermediate TPA-Ph-CHO prepared in Example 2. Figure 2 The 1H NMR spectrum shows that the theoretical number of hydrogen atoms in TPA-Ph-CHO is 19, and the actual 1H NMR spectrum results show that the compound has 19 hydrogen atoms, indicating the successful synthesis of TPA-Ph-CHO.

[0049] The characterization data of TPA-Ph-CHO obtained are as follows: 1 H NMR (400 MHz, DMSO- d6 ) δ 10.02 (s, 1H),7.97-7.95 (d, J = 8.0 Hz, 2H), 7.88-7.86 (d, J = 8.0 Hz, 2H), 7.71-7.69 (d, J = 8.0Hz, 2H), 7.37-7.33 (t, J= 16.0 Hz, 4H), 7.11-7.08 (m, 6H), 7.05-7.03 (d, J = 8.0Hz, 2H). S2. Synthesis of product TPA-S-Py: In a two-necked reaction flask, TPA-Ph-CHO (1.145 mmol, 400 mg) and 1,4-dimethylpyridine-1-iodide (2.29 mmol, 538 mg) were added. Then, 4-methylpiperidine (100 μL) and ethanol (60 mL) were added, and the mixture was refluxed and stirred at 80 °C for 12 hours. After cooling to room temperature, the mixture was directly filtered and washed with ethyl acetate to give an orange-red powder (370 mg), which was the product TPA-S-Py, with a yield of 57.1%.

[0050] Figure 5 and Figure 8 The 1H NMR spectrum and high-resolution mass spectrum of TPA-S-Py prepared in Example 2 are shown below. Figure 5 The 1H NMR spectrum shows that the theoretical number of hydrogen atoms in TPA-S-Py is 27, and the actual 1H NMR spectrum results confirm that the compound has 27 hydrogen atoms. Furthermore, Figure 8 High-resolution mass spectra show that the theoretical molecular weight is 567.1292 and the actual molecular weight is 567.1306, indicating the successful synthesis of TPA-S-Py.

[0051] The characterization data of TPA-S-Py obtained are as follows: 1 H NMR (400 MHz, DMSO- d6 ) δ 8.88-8.86 (d, J =8.0 Hz, 2H), 8.24-8.23 (d, J = 4.0 Hz, 2H), 8.08-8.04 (d, J = 16.0 Hz, 1H), 7.84-7.78 (m, 4H), 7.71-7.69 (d, J = 8.0 Hz, 2H), 7.58-7.54 (d, J = 16.0 Hz, 1H), 7.37-7.33 (t, J = 16.0 Hz, 4H), 7.12 – 7.04 (m, 8H), 4.26 (s, 3H). HRMS (ESI)m / z: [M + H + calcd for C 32 H 27 N2I, 567.1292; found, 567.1306. Example 3 The chemical structural formula of the aggregation-induced emission photosensitizer (TPA-D-Py) is as follows: ; The synthetic route of TPA-D-Py is as follows: ; The specific preparation process is as follows: S1. Synthesis of intermediate TPA-Ph-dCHO: In a two-necked reaction flask, 4-(diphenylamino)phenylboronic acid (5.4 mmol, 1.56 g), 4-bromocinnamaldehyde (5.4 mmol, 1.13 g), potassium carbonate (34 mmol, 4.8 g), and Pd(PPh3)4 (0.27 mmol, 312 mg) were added. The reaction flask was evacuated three times under nitrogen atmosphere to ensure the formation of a nitrogen atmosphere. Tetrahydrofuran (40 mL) and water (16 mL) were added to the flask, and the mixture was refluxed and stirred at 90 °C for 12 hours. After cooling to room temperature, the reaction mixture was extracted with dichloromethane and water. The organic solvent was removed by rotary evaporation, and the product was purified by column chromatography to give 1.2 g of orange-yellow powder, which was intermediate TPA-Ph-dCHO, with a yield of 59.3%.

[0052] Figure 3 The image shows the 1H NMR spectrum of the intermediate TPA-Ph-dCHO prepared in Example 3. Figure 3 The 1H NMR spectrum shows that the theoretical number of hydrogen atoms in TPA-Ph-dCHO is 21, and the actual 1H NMR spectrum results show that the compound has 21 hydrogen atoms, indicating the successful synthesis of TPA-Ph-dCHO.

[0053] The characterization data of the obtained TPA-Ph-dCHO are as follows: 1 H NMR (400 MHz, DMSO- d6 ) δ 9.70 (s, 1H),7.82–7.74 (m, 5H), 7.69–7.67 (d, J = 8.0 Hz, 2H), 7.36–7.32 (t, J = 16.0 Hz, 4H), 7.11–7.02 (m, 8H), and 6.92–6.88 (m, 1H). S2. Synthesis of product TPA-D-Py: TPA-Ph-dCHO (1.067 mmol, 400 mg) and 1,4-dimethylpyridine-1-iodide (2.133 mmol, 501 mg) were added to a two-necked reaction flask. 4-methylpiperidine (100 μL) and ethanol (60 mL) were added to the flask, and the mixture was stirred and refluxed at 80 °C for 12 hours. After cooling to room temperature, the mixture was directly filtered and washed with ethyl acetate to obtain 193 mg of an orange-red powder, which was the product TPA-D-Py, in a yield of 30.5%.

[0054] Figure 6 and Figure 9 The images show the 1H NMR spectrum and high-resolution mass spectrum of TPA-D-Py prepared in Example 3. Figure 6 The 1H NMR spectrum shows that the theoretical number of hydrogen atoms in TPA-D-Py is 29, and the actual 1H NMR spectrum results confirm that the compound has 29 hydrogen atoms. Furthermore, Figure 9 High-resolution mass spectra show that the theoretical molecular weight is 593.1454 and the actual molecular weight is 593.1448, indicating the successful synthesis of TPA-D-Py.

[0055] The characterization data of TPA-D-Py obtained are as follows: 1 H NMR (400 MHz, DMSO- d6 ) δ 8.78-8.76 (d, J =8.0 Hz, 2H), 8.12-8.10 (d, J = 8.0 Hz, 2H), 7.85-7.79 (m,1H), 7.67 (s, 4H),7.64-7.62 (d, J = 8.0 Hz, 2H), 7.33-7.28 (m, 5H), 7.08 – 7.00 (m, 9H), 6.91-6.87 (d, J = 16.0 Hz, 1H), 4.20 (s, 3H). HRMS (ESI) m / z: [M + H + ] calcd forC 34 H 29 N2I, 593.1454; found, 593.1448. Performance testing (1) Theoretical calculations and basic photophysical properties To gain a deeper and more intuitive understanding of the electronic transition modes of these three active photosensitizer molecules, theoretical calculations were performed on the electron distributions of the highest occupied molecular orbitals (HOMO) and lowest unoccupied molecular orbitals (LUMO) of the photosensitizers TPA-Py, TPA-S-Py, and TPA-D-Py prepared in Examples 1-3. Density functional theory (DFT) and time-dependent density functional theory (TD-DFT) simulations were conducted using Gaussian 09 software at the MO-2X / 6-31G(d,p) level. Figure 10 Theoretical calculation results of the electronic transition from HOMO to LUMO of the active photosensitizer molecules prepared in Examples 1-3 in THF solvent: (A) TPA-Py, (B) TPA-S-Py, (C) TPA-D-Py; absorption and fluorescence spectra in THF solvent: (D) TPA-Py, (E) TPA-S-Py, (F) TPA-D-Py.

[0056] like Figure 10 As shown in (A)-(C), the HOMO electrons of TPA-S-Py and TPA-D-Py are mainly distributed on the TPA unit, with only a small contribution from the central benzene ring; while their LUMO electrons are mainly concentrated on the charged pyridine heterocycle, with a smaller contribution from the central benzene ring. This phenomenon indicates that TPA-S-Py and TPA-D-Py have significant intramolecular charge transfer (ICT) effects. In contrast, the TPA-Py model exhibits significant local excitation (LE) transitions due to the large electronic overlap between HOMO and LUMO. Furthermore, the UV-Vis absorption and fluorescence spectra of the three active photosensitizer molecules in THF were measured. Figure 10 (D-F) Absorption spectra show that the maximum absorption wavelengths of TPA-Py, TPA-S-Py, and TPA-D-Py are 457 nm, 465 nm, and 367 nm, respectively, while their maximum emission wavelengths are 635 nm, 643 nm, and 497 nm, respectively. Therefore, the Stokes shifts of TPA-Py, TPA-S-Py, and TPA-D-Py are calculated to be 178 nm, 178 nm, and 130 nm, respectively. The smaller Stokes shift of TPA-D-Py may reflect a smaller conformational change from the ground state to the excited state, which can be explained by the two "locks" of the C=C bond severely restricting intramolecular motion. In addition, TPA-D-Py exhibits more severe fluorescence self-absorption in the 400 nm to 550 nm region, while TPA-Py and TPA-S-Py only have weak fluorescence self-absorption. This result also shows that TPA-D-Py has a more rigid structure than TPA-Py and TPA-S-Py.

[0057] (2) Characteristic analysis of the PICT effect To confirm the excited-state characteristics of torsional intramolecular charge transfer (TICT) and planar intramolecular charge transfer (PICT) in the photosensitizers TPA-Py, TPA-S-Py and TPA-D-Py prepared in Examples 1-3, the molecular DA dihedral angles of these photosensitizers in different polar organic solvents (low polarity toluene, medium polarity tetrahydrofuran THF and high polarity dimethylformamide DMF) were calculated. Figure 11 The theoretical calculation results of the excited-state molecular conformations of the photosensitizers TPA-Py, TPA-S-Py, and TPA-D-Py prepared in Examples 1-3 in different solvents such as toluene, tetrahydrofuran (THF), and dimethylformamide (DMF) are shown in the illustrations. The illustrations show the fluorescence spectra and images of the emitters of the photosensitizers in toluene, THF, and DMF.

[0058] like Figure 11 As shown, the critical dihedral angle of the TPA-Py model molecule gradually increases with increasing solvent polarity (from 22.0° in toluene to 25.0° in THF, and then to 28.0° in DMF). Furthermore, the fluorescence spectra of TPA-Py in the three solvents show a significant redshift of the maximum emission wavelength (586 nm in toluene, 632 nm in THF, and 658 nm in DMF), indicating that TPA-Py possesses a distinct TICT characteristic. When a C=C structure is introduced into TPA-Py, the maximum fluorescence wavelength of TPA-S-Py also shows a slight redshift from toluene towards DMF, but its DA dihedral angle gradually decreases from toluene to DMF. For example, in toluene, the DA dihedral angles of α2 and α3 are 1.1° and 2.2°, respectively, while in DMF they decrease to 0.6° and 0.9°, respectively. This result suggests that TPA-S-Py may simultaneously possess both TICT and PICT effects. However, when two C=C groups are introduced into TPA-Py, the maximum fluorescence wavelength of TPA-D-Py exhibits a blue shift from toluene to DMF, and the DA dihedral angle decreases from toluene to DMF. For example, in toluene, the DA dihedral angles for α2, α3, and α4 are 1.3º, 0.8º, and 1.1º, respectively, while in DMF they decrease to 0.3º, 0.1º, and 0.9º, respectively. Based on these results, we can infer that the PICT effect dominates in the excited state of TPA-D-Py, while the TICT effect dominates in the excited state of TPA-Py. For TPA-S-Py, the TICT and PICT effects compete with each other. Therefore, introducing rigid C=C groups helps to modulate the TICT and PICT effects in the excited state.

[0059] (3) Characterization of molar absorption coefficient and AIE properties Figure 12(A) shows the absorption spectrum of the photosensitizers prepared in Examples 1-3 in THF solvent; (B) shows the fluorescence intensity of TPA-D-Py in a toluene / DMSO mixed solution as a function of the toluene ratio; (C) shows the fluorescence intensity of the three emitters in toluene / DMSO mixed solutions with different toluene ratios; (D) shows the fluorescence spectrum of TPA-Py; (E) shows the fluorescence spectrum of TPA-S-Py; and (F) shows the fluorescence spectrum of TPA-D-Py in different organic solvents.

[0060] Compared to the TICT effect, molecules exhibiting the PICT effect present a planar conformation in the excited state, which helps to increase the absorption cross-section of the molecule, thereby enhancing the molar absorption effect. For example... Figure 12 As shown in Figure (A), at the same detection concentration, the absorption efficiency of TPA-D-Py is higher than that of TPA-S-Py and TPA-Py, indicating that the PICT effect can indeed improve the absorption efficiency and promote the generation of S1 energy. For further characterization of the AIE properties of the luminescent material, dimethyl sulfoxide (DMSO) was chosen as an excellent solvent, while toluene was chosen as a poor solvent. Figure 12 As shown in (B) and (C), the fluorescence intensity of TPA-D-Py and TPA-S-Py gradually increased with increasing toluene content, indicating a significant AIE phenomenon. However, TPA-Py did not exhibit obvious AIE characteristics. This difference suggests that the PICT effect contributes to the realization of AIE properties: due to the relatively rigid molecular conformation in the excited state, molecular motion is reduced, suppressing the nonradiative internal conversion of S1 energy. Ultimately, the ICT effect was confirmed. Figure 12 As shown in (D)-(F), TPA-Py and TPA-S-Py exhibit a significant red shift in emission wavelength during the transition from low-polarity toluene to high-polarity DMF, indicating that both photosensitizers possess the ICT effect. However, TPA-D-Py shows a blue shift in maximum emission wavelength, attributed to the introduction of more C=C groups, which weakens the ICT effect. In summary, TPA-D-Py has the largest molar absorptivity and the highest efficiency in suppressing nonradiative internal conversion of the S1 level, theoretically favoring the generation of reactive oxygen species (ROS) and the emission of strong fluorescence.

[0061] (4) Characterization of fluorescence and reactive oxygen species (ROS) efficiency To demonstrate the positive role of the PICT effect in suppressing the nonradiative internal conversion of S1 energy, the photoluminescence quantum yields of the photosensitizers TPA-Py, TPA-S-Py, and TPA-D-Py prepared in Examples 1-3 were measured. Figure 13(A) shows the absolute fluorescence quantum efficiency of the photosensitizers prepared in Examples 1-3 in DMSO and powder; (B) shows the theoretical calculation of the excited state energy levels; (C) shows the change in fluorescence intensity of the DCFH probe (DCFH = 4 μM, photosensitizer = 2 μM) with or without photosensitizer added at different time points and subjected to white light irradiation; (D) shows the change in absorption intensity of the ABDA probe (ABDA = 20 μM, photosensitizer = 2 μM) with or without photosensitizer added at different time points and subjected to white light irradiation; (E) shows the change in fluorescence intensity of the DHR123 probe (DHR123 = 4 μM, photosensitizer = 10 μM) with or without photosensitizer added at different time points and subjected to white light irradiation. The white light power is 30 mW / cm². -2 (F) represents the test results for the formation of •OH.

[0062] like Figure 13 As shown in (A), when the photosensitizer is fully dissolved in DMSO solvent, it emits almost no fluorescence; however, it emits significant fluorescence in the aggregated state. The PLQY of TPA-Py is lower than that of TPA-S-Py and TPA-D-Py, which provides strong evidence that the PICT effect helps suppress the nonradiative internal conversion of S1 energy. Next, the excited state energy levels of the photosensitizer were calculated, as shown in Figure (A). Figure 13 As shown in (B), the energy difference (ΔE) between S1 and T2 of TPA-D-Py and TPA-Py is... S1T2 The values ​​are 0.13 eV and 0.16 eV, respectively, while the ΔE of TPA-S-Py is... S1T2 The value was 0.39 eV. This result suggests that TPA-D-Py and TPA-Py may have higher triplet energy generation efficiency, which is conducive to the generation of reactive oxygen species (ROS). Subsequently, the ROS efficiency and types of these photosensitizers were detected using ROS-specific probes such as 2′,7′-dichlorodihydrofluorescein diacetate (DCFH), 9,10-anthraquinone diimine bis(methylene)isophthalic acid (ABDA), hydroxyphenylfluorescein (HPF), and rhodamine 123 dihydrate (DHR123). Figure 13As shown in Figure (C), the total ROS generation of PSs was detected using a DCFH probe. After the addition of TPA-D-Py and illumination, the fluorescence intensity of DCFH significantly increased, while that of TPA-S-Py and TPA-Py only increased slightly, indicating that TPA-D-Py has a strong ROS generation capacity. This result can be explained by the fact that TPA-D-Py possesses an efficient internal conversion (ISC) process, and due to its PICT effect, it inhibits the nonradiative internal conversion of S1 energy, thereby promoting the generation of triplet energy and thus enhancing ROS generation. However, TPA-Py and TPA-S-Py exhibit a significant TICT effect, leading to strong nonradiative energy dissipation of S1 energy. The singlet oxygen (S1) level was measured using an ABDA probe. 1 O2), such as Figure 13 As shown in (D), the characteristic absorption intensity of the pure ABDA probe did not change, indicating that no formation occurred. 1 O2. After the addition of PSs and exposure to light, the absorption intensity of ABDA decreased significantly, indicating that all three photosensitizers could be effectively generated. 1 O2. In addition, TPA-D-Py's 1 The O2 generation efficiency was higher than that of TPA-S-Py and TPA-Py. Subsequently, under white light irradiation, the superoxide anion radical (O2•) was detected using a DHR123 probe. - The generation status of ) such as Figure 13 As shown in Figure (E), the fluorescence intensity of the pure DHR123 probe increased only slightly. After adding a photosensitizer, the fluorescence intensity increased rapidly upon the addition of TPA-D-Py, while the other two photosensitizers did not increase the fluorescence intensity of the DHR123 probe, indicating that only TPA-D-Py can generate O2• - Finally, the formation of •OH was detected using an HPF probe. Figure 13 (F)). The results showed that all three photosensitizers could generate •OH under light irradiation. Therefore, it can be confirmed that TPA-D-Py has the best efficiency in generating type I and type II reactive oxygen species (ROS) among the three photosensitizers. This indicates that introducing two carbon-carbon double bonds into the DA structure can effectively improve the ROS generation efficiency, which is due to the PICT effect.

[0063] (5) Evaluation of in vitro cell imaging and photodynamic therapy (PDT) Figure 14The image shows a CLSM image of GL261 cells treated with the photosensitizer TPA-D-Py prepared in Example 3 for 24 hours, followed by co-staining with Mitotracker green for 30 minutes. The concentration of TPA-D-Py was 10 µM, with an excitation wavelength Ex = 532 nm, an emission wavelength Em = 600-660 nm, and a laser intensity of 5%. The concentration of Mitotracker green FM was 200 nM, with an excitation wavelength Ex = 488 nm and an emission wavelength Em = 500-530 nm. The scale bar is 100 µm. The right side shows the fluorescence intensity distribution curves (the horizontal axis represents the spatial distance (μm) along the white line, and the vertical axis represents the fluorescence intensity). The green curve represents the green fluorescence signal intensity distribution of the commercial mitochondrial probe (MitoTracker Green); the red curve represents the red fluorescence signal intensity distribution of TPA-D-Py.

[0064] Figure 15 (A) represents the survival rate of GL261 cells in the dark; (B) represents the survival rate of GL261 cells under light conditions. The live / dead cell co-staining assay used fluorescent probes Calcein-AM and PI to detect TPA-D-Py (10 µM); [PI] = 1 mg·mL⁻¹ -1 The excitation wavelength is 488 nm and the emission wavelength is 600-650 nm; [Calcein-AM] = 1 µM, the excitation wavelength is 488 nm and the emission wavelength is 500-560 nm; (C) is a CLSM image of live / dead cells co-stained (Calcein-AM / PI double staining), where green fluorescence represents live cells and red fluorescence represents dead cells.

[0065] Due to its high efficiency in generating reactive oxygen species (ROS), TPA-D-Py was selected for subsequent cellular fluorescence imaging and photodynamic therapy (PDT) evaluation. First, this invention investigated the intracellular targeting ability of TPA-D-Py to mitochondria. For example... Figure 14 As shown, subcellular co-localization analysis was performed using confocal laser scanning microscopy (CLSM). TPA-D-Py (red fluorescence) and the mitochondrial-specific probe MitoTracker Green (green fluorescence) were co-incubated in cells. In the merged channel, the red fluorescence of TPA-D-Py and the green fluorescence of MitoTracker Green showed high overlap, forming a distinct yellow signal. Further analysis of the linear distribution of fluorescence intensity showed that the peak positions of the red and green fluorescence almost completely overlapped. This result strongly demonstrates that TPA-D-Py can specifically accumulate in the mitochondria of GL261 cells.

[0066] The photodynamic effects of GL261 cells were evaluated using the CCK-8 assay. Figure 15 As shown in (A), under dark conditions, even with a TPA-D-Py concentration as high as 10 µM, the survival rate of GL261 cells remained significantly unchanged, indicating that TPA-D-Py itself has good biocompatibility and low dark toxicity. However, after white light irradiation, the cell survival rate of the treated group decreased sharply ( Figure 15 (B) This result clearly demonstrates that the anticancer activity of TPA-D-Py is entirely dependent on light exposure, thus exhibiting a significant photodynamic therapy effect. To visually verify the above cell viability and observe cell death status, this invention further employs Calcein-AM and propidium iodide (PI) for double staining of live and dead cells. Figure 15 As shown in (C), cells exposed to light only exhibited green fluorescence, indicating that they were in a healthy state. After the addition of TPA-D-Py, a broad red fluorescence signal appeared, accompanied by a significant decrease in green fluorescence. This directly indicates that TPA-D-Py can effectively induce GL261 cell death under light conditions, and the results are highly consistent with those obtained by the CCK-8 assay.

[0067] The staining effect of the photosensitizer TPA-D-Py prepared in Example 3 on Gram-positive bacteria was characterized using fluorescence confocal microscopy. Figure 16 As shown, S. aureus and MRSA were selected as research subjects. When the photosensitizer was excited at a wavelength of 488 nm, both bacteria showed bright red light, indicating that the photosensitizer TPA-D-Py could be well phagocytosed by the two Gram-positive bacteria.

[0068] The photodynamic antibacterial effect of the photosensitizer TPA-D-Py prepared in Example 3 was further evaluated. Figure 17 Visual representations of the photodynamic effects of photosensitizer TPA-D-Py on Gram-positive bacteria *S. aureus* and *MRSA* before and after photodynamic therapy at different concentrations and under light conditions ((A) photodynamic killing effect of TPA-D-Py on *S. aureus*, (B) photodynamic killing effect of TPA-D-Py on *MRSA*). With increasing photosensitizer concentration, under lightless conditions, the photosensitizer showed almost no toxicity to bacteria. However, under white light conditions (power 30 mW cm⁻¹), the toxicity decreased significantly. -2 As the concentration of the photosensitizer increased, the bacterial survival rate decreased significantly. Furthermore, the photosensitizer TPA-D-Py showed a more significant photodynamic killing effect on *S. aureus* bacteria. Therefore, TPA-D-Py in Example 3 also exhibited good photodynamic efficacy against Gram-positive bacteria.

[0069] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. An aggregation-induced emission photosensitizer, characterized in that, The general chemical structural formula is: ； In the formula, R is selected from any one of carbon-carbon single bonds, styryl, and diphenylbutadienyl.

2. The aggregation-induced emission photosensitizer according to claim 1, characterized in that, The chemical structural formula is: 。 3. A method for preparing the aggregation-induced emission active photosensitizer according to any one of claims 1 to 2, characterized in that, Includes the following steps: 4-Bromotriphenylamine or 4-(diphenylamino)phenylboronic acid is added to a solvent along with compound A, potassium carbonate and a catalyst, and an intermediate is obtained by coupling reaction, wherein compound A is selected from 4-pyridineboronic acid, 4-formylphenylboronic acid and 4-bromocinnamaldehyde; The intermediate is mixed with an iodide and reacted under alkaline conditions to obtain the aggregation-induced emission active photosensitizer, wherein the iodide is selected from iodomethane or 1,4-dimethylpyridine-1-iodide.

4. The method for preparing the aggregation-induced emission photosensitizer according to claim 3, characterized in that, The molar ratio of 4-bromotriphenylamine or 4-(diphenylamino)phenylboronic acid, compound A, and catalyst is 20:20:

1.

5. The method for preparing the aggregation-induced emission photosensitizer according to claim 4, characterized in that, The catalyst is tetra(triphenylphosphine)palladium.

6. The method for preparing the aggregation-induced emission photosensitizer according to claim 3, characterized in that, The coupling reaction was carried out at a temperature of 90°C for 12 hours.

7. The method for preparing the aggregation-induced emission photosensitizer according to claim 3, characterized in that, The molar ratio of the intermediate to the iodide is 1:(1~2.5).

8. The method for preparing the aggregation-induced emission photosensitizer according to claim 3, characterized in that, The reaction temperature under alkaline conditions is 80℃, and the reaction time is 12h.

9. The use of the aggregation-induced emission active photosensitizer according to any one of claims 1 to 2 in the preparation of a fluorescent imaging agent or a photodynamic therapy drug.

10. The application according to claim 9, characterized in that, The photodynamic therapy drug is used for photodynamic therapy of tumors or photodynamic clearance of Gram-positive bacterial infections.

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