An aggregation-induced luminescence mitochondrial-targeted near-infrared absorption photosensitizer, its preparation method and application

Abstract

This invention discloses an aggregation-induced emission mitochondrial-targeting near-infrared absorption photosensitizer, its preparation method, and its applications. The photosensitizer of this invention has a D-π-A structure, using triphenylamine as a donor group, 3,4-ethylenedioxythiophene as a π-bridge, and a quinoline cation salt as an acceptor. The photosensitizer of this invention can effectively target mitochondria and, under 660 nm excitation light, efficiently generate type I and type II reactive oxygen species, thereby effectively killing tumor cells, demonstrating its potential for developing highly efficient photodynamic therapy technology.

Description

Technical Field

[0001] This invention belongs to the field of optical diagnostic and therapeutic technology, specifically relating to an aggregation-induced emission mitochondrial-targeted near-infrared absorption photosensitizer, its preparation method, and its application. Background Technology

[0002] Traditional cancer treatments include chemotherapy, surgery, immunotherapy, and radiotherapy. These methods each have their limitations and cannot solve some of the current challenges in cancer treatment. Photodynamic therapy (PDT), with its advantages of being non-invasive, low-invasiveness, and highly spatiotemporally selective, has attracted widespread attention from researchers in recent years. PDT requires three key elements: oxygen, excitation light, and a photosensitizer. The treatment method involves irradiating a photosensitizer with excitation light. The photosensitizer absorbs the energy of the excitation light, generating cytotoxic reactive oxygen species (ROS) that kill cancer cells. By delivering the photosensitizer to the tumor site and activating the photodynamic therapy through light irradiation, PDT significantly reduces toxicity to normal tissues, resulting in high biocompatibility.

[0003] Reactive oxygen species (ROS), the primary weapon used by photosensitizers to kill tumors, typically have short lifespans and short propagation distances, making them unable to be released at critical sites within cancer cells, thus significantly reducing the effectiveness of photodynamic therapy (PDT). Therefore, photosensitizers targeting subcellular organelles tend to have better PDT efficacy. Among subcellular organelles, mitochondria possess a highly dynamic structure, constantly undergoing fusion and fission, providing a site for cellular life activities. Designing mitochondrial-targeting photosensitizers can enhance the PDT performance of existing photosensitizers. Mitochondria in cancer cells are often swollen and fragmented; mitochondria in the S phase (DNA synthesis phase) appear tubular, while those in the M phase (mitosis phase) exhibit a uniformly fragmented state. Furthermore, compared to normal cells (-140mV), cancer cells have a higher degree of mitochondrial membrane potential polarization (MMP, -220mV). Therefore, the uptake of lipophilic cationic compounds can increase 100-1000 times compared to extracellular compounds. Consequently, more mitochondrial-targeting drugs can be taken up by the mitochondria of cancer cells.

[0004] Traditional photosensitizers typically possess large, rigid planar structures and hydrophobic structures, often forming aggregates in biological environments. This leads to strong π-π interactions, causing the excited-state energy to dissipate rapidly through nonradiative transitions, resulting in decreased fluorescence and ROS generation efficiency. In contrast, aggregation-induced emission (AIE) materials possess molecular rotor structures. In their single-molecule state, molecular rotation facilitates the dissipation of excited-state energy through nonradiative transitions. However, when AIE materials are aggregated, the rotation of these molecular rotors is suppressed, preventing the dissipation of excited-state energy through nonradiative transitions. This allows the excited state of the photosensitizer to cross over to the triplet state, generating ROS via electron or energy transfer. Therefore, AIE photosensitizers exhibit higher photodynamic therapy capabilities. Furthermore, designing photosensitizers with near-infrared absorption can increase skin penetration depth, thereby achieving the goal of treating deep tumors.

[0005] In summary, designing aggregation-induced emission mitochondrial-targeted near-infrared absorption photosensitizers is a promising photosensitizer design approach. Summary of the Invention

[0006] To address the problems existing in current photosensitizers, the present invention aims to provide an aggregation-induced emission mitochondrial-targeted near-infrared absorption photosensitizer. The photosensitizer material of the present invention has a DA structure, can be excited by 660nm excitation light, and efficiently generates ROS. Under 660nm excitation light, this photosensitizer can generate large quantities and efficiently of Type-I (hydroxyl radicals, superoxide anions) and Type-II (singlet oxygen) ROS, and can effectively target cancer cell mitochondria, maintaining targeting on the mitochondria for up to 12 hours, thereby efficiently killing tumor cells.

[0007] Another objective of this invention is to provide a method for preparing the above-mentioned aggregation-induced luminescence mitochondrial-targeted near-infrared absorption photosensitizer.

[0008] Another object of the present invention is to provide the application of the above-mentioned aggregation-induced luminescence mitochondrial-targeted near-infrared absorption photosensitizer.

[0009] The objective of this invention is achieved through the following technical solution:

[0010] An aggregation-induced luminescent mitochondrial-targeted near-infrared absorption photosensitizer has the following general structural formula:

[0011]

[0012] Where n is an independent integer from 0 to 16 (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16); X is an anion.

[0013] Preferably, X is independently selected from at least one of fluoride ions, chloride ions, bromide ions, and iodide ions, and the quantity is determined according to the positive charge.

[0014] Preferably, the structural formula of the aggregation-induced emission mitochondrial-targeted near-infrared absorption photosensitizer is as follows:

[0015]

[0016] The aggregation-induced emission mitochondrial-targeted near-infrared absorption photosensitizer of the present invention has a D-π-A structure, with triphenylamine as the donor group, 3,4-ethylenedioxythiophene as the π-bridge, quinoline cationic salt as the acceptor, and is constructed with a propyl bond structure.

[0017] The above-mentioned method for preparing the aggregation-induced emission mitochondrial-targeted near-infrared absorption photosensitizer includes the following steps:

[0018] The reaction of 4-boronic acid triphenylamine and 5-bromo-2-(3,4-vinyldioxythiophene)formaldehyde yields intermediate 1. The reaction of 4-methylquinoline and its halogenated derivative yields N-substituted 4-methylquinoline. The reaction of intermediate 1 and N-substituted 4-methylquinoline yields an aggregation-induced luminescence mitochondrial-targeted near-infrared absorption photosensitizer.

[0019] Preferably, the preparation method of the aggregation-induced emission mitochondrial-targeted near-infrared absorption photosensitizer includes the following steps:

[0020] (1) Triphenylamine borate, 5-bromo-2-(3,4-vinyldioxythiophene)carboxaldehyde, tetra(triphenylphosphine)palladium and cesium carbonate were added to a mixed solvent of THF and water under a protective atmosphere, and the mixture was refluxed and purified to obtain intermediate product 1.

[0021] (2) Preparation of DTEVQ

[0022] (2.1) 4-Methylquinoline and 1,3-diiodopropane were added to ethanol, refluxed, and purified to obtain intermediate product 2;

[0023] (2.2) Intermediate product 1 and intermediate product 2 were added to ethanol and piperidine, refluxed, and purified to obtain DTEVQ;

[0024] (3) Preparation of TEVQ-1

[0025] (3.1) 4-Methylquinoline and iodomethane were added to ethanol, refluxed, and purified to obtain intermediate product 3;

[0026] (3.2) Intermediate product 1 and intermediate product 3 were added to ethanol and piperidine, refluxed, and purified to obtain TEVQ-1;

[0027] (4) Preparation of TEVQ-2

[0028] (4.1) 4-Methylquinoline and 1-iodopropane were added to ethanol, refluxed, and purified to give intermediate product 4.

[0029] (4.2) Intermediate product 1 and intermediate product 4 were added to ethanol and piperidine, refluxed, and purified to obtain TEVQ-2.

[0030] (5) Preparation of TEVQ-3

[0031] (5.1) 4-Methylquinoline and (3-bromopropyl)trimethylammonium bromide were added to ethanol, refluxed, and purified to give intermediate product 5;

[0032] (5.2) Intermediate product 1 and intermediate product 5 were added to ethanol and piperidine, refluxed, and purified to obtain TEVQ-3.

[0033] More preferably, in step (1), the molar ratio of 4-boronic acid triphenylamine, 5-bromo-2-(3,4-vinyldioxythiophene)carboxaldehyde, tetra(triphenylphosphine)palladium and cesium carbonate is 11-15:10:0.02-0.05:20-30;

[0034] More preferably, in step (1), the volume ratio of THF to water is 20-27:3-10;

[0035] More preferably, in step (1), the molar volume ratio of 5-bromo-2-(3,4-vinyldioxythiophene)formaldehyde to the mixed solvent is 10 mmol: 30-50 mL;

[0036] More preferably, in step (1), the reflux reaction temperature is 70-80°C and the time is 24-36 h;

[0037] More preferably, in step (1), the protective atmosphere is nitrogen.

[0038] More preferably, in step (2.1), the molar ratio of 4-methylquinoline to 1,3-diiodopropane is 10 to 15:4;

[0039] More preferably, in step (2.1), the molar volume ratio of 1,3-diiodopropane to ethanol is 4 mmol: 5-20 mL;

[0040] More preferably, in step (2.1), the reflux reaction temperature is 80–90°C and the time is 12–36 h;

[0041] More preferably, in step (2.2), the molar ratio of intermediate product 1 to intermediate product 2 is 1 to 1.5: 0.4;

[0042] More preferably, in step (2.2), the ratio of intermediate product 2, ethanol and piperidine is 0.4 mmol: 15-30 mL: 15-30 μL;

[0043] More preferably, in step (2.2), the reflux reaction temperature is 80–90°C and the time is 12–36 h.

[0044] More preferably, in step (3.1), the molar ratio of 4-methylquinoline to iodomethane is 5 to 10:4;

[0045] More preferably, in step (3.1), the molar volume ratio of iodomethane to ethanol is 4 mmol: 5-20 mL;

[0046] More preferably, in step (3.1), the reflux reaction temperature is 80–90°C and the time is 12–36 h;

[0047] More preferably, in step (3.2), the molar ratio of intermediate product 1 to intermediate product 3 is 1 to 1.5:1;

[0048] More preferably, in step (3.2), the ratio of intermediate product 3, ethanol and piperidine is 1 mmol: 15-30 mL: 15-30 μL;

[0049] More preferably, in step (3.2), the reflux reaction temperature is 80–90°C and the time is 12–36 h.

[0050] More preferably, in step (4.1), the molar ratio of 4-methylquinoline to 1-iodopropane is 5 to 10:4;

[0051] More preferably, in step (4.1), the molar volume ratio of 1-iodopropane to ethanol is 4 mmol: 5-20 mL;

[0052] More preferably, in step (4.1), the reflux reaction temperature is 80–90°C and the time is 12–36 h.

[0053] More preferably, in step (4.2), the molar ratio of intermediate product 1 to intermediate product 4 is 1 to 1.5:1;

[0054] More preferably, in step (4.2), the ratio of intermediate product 4, ethanol and piperidine is 1 mmol: 15-30 mL: 15-30 μL;

[0055] More preferably, in step (4.2), the reflux reaction temperature is 80–90°C and the time is 12–36 h.

[0056] More preferably, in step (5.1), the molar ratio of 4-methylquinoline and (3-bromopropyl)trimethylammonium bromide is 5 to 10:4;

[0057] More preferably, in step (5.1), the molar volume ratio of (3-bromopropyl)trimethylammonium bromide to ethanol is 4 mmol: 5-20 mL;

[0058] More preferably, in step (5.1), the reflux reaction temperature is 80–90°C and the time is 12–36 h.

[0059] More preferably, in step (5.2), the molar ratio of intermediate product 1 to intermediate product 5 is 1 to 1.5:1;

[0060] More preferably, in step (5.2), the ratio of intermediate product 5, ethanol and piperidine is 1 mmol: 15-30 mL: 15-30 μL;

[0061] More preferably, in step (5.2), the reflux reaction temperature is 80–90°C and the time is 12–36 h.

[0062] The above-mentioned aggregation-induced emission mitochondrial-targeting near-infrared absorption photosensitizer is used in the preparation of photodynamic therapy reagents for cancer cells. The photosensitizer of this invention possesses near-infrared absorption capability, efficient reactive oxygen species (ROS) generation capability, and mitochondrial targeting capability. It effectively generates type I and type II ROS under 660nm laser irradiation and can be applied in photodynamic therapy of cancer cells.

[0063] Compared with the prior art, the present invention has the following beneficial effects:

[0064] (1) The photosensitizer of the present invention has near-infrared absorption capability and can be excited by 660nm excitation light; and has the ability to efficiently generate type I ROS and type II ROS.

[0065] (2) The photosensitizer of the present invention can efficiently target the mitochondria of cancer cells and efficiently kill cancer cells under 660nm excitation light, and has low toxicity to cells (negligible toxicity) under non-light conditions.

[0066] (3) The photosensitizer of the present invention has a simple structure and is easy to synthesize. Attached Figure Description

[0067] Figure 1 This is the molecular structural formula of the photosensitizer DTEVQ.

[0068] Figure 2 The synthesis equation for the photosensitizer DTEVQ is shown below.

[0069] Figure 3 The molecular structure of the photosensitizer TEVQ-1 is shown.

[0070] Figure 4 The synthesis equation for the photosensitizer TEVQ-1 is shown below.

[0071] Figure 5 The molecular structure of the photosensitizer TEVQ-2 is shown below.

[0072] Figure 6 The synthesis equation for the photosensitizer TEVQ-2 is shown below.

[0073] Figure 7 The molecular structure of the photosensitizer TEVQ-3 is shown below.

[0074] Figure 8 The synthesis equation for the photosensitizer TEVQ-3 is shown below.

[0075] Figure 9 The molecular structure and synthesis equation of the photosensitizer TTVP are shown.

[0076] Figure 10 The molecular structure and synthesis equation of the photosensitizer TEVP are shown.

[0077] Figure 11 The molecular structure and synthesis equation of the photosensitizer TTVQ are shown.

[0078] Figure 12 The UV-Vis absorption spectra of photosensitizers DTEVQ, TEVQ-3, TTVQ, TEVP, and TTVP are shown.

[0079] Figure 13 The UV-Vis absorption and fluorescence emission spectra of photosensitizers DTEVQ, TEVQ-1, TEVQ-2, and TEVQ-3 are shown.

[0080] Figure 14 Fluorescence emission spectra of photosensitizers DTEVQ, TEVQ-2, and TEVQ-3 in DMSO / toluene mixtures with different toluene fractions.

[0081] Figure 15 The graph shows the fluorescence response of photosensitizers DTEVQ, TEVQ-1, TEVQ-2, TEVQ-3 and Ce6 to DCFH after 660nm laser irradiation at different times and the comparison of fluorescence intensity at 60s.

[0082] Figure 16 The graph shows the fluorescence response of photosensitizers DTEVQ, TEVQ-1, TEVQ-2, TEVQ-3 and Ce6 to DHR123 after 660nm laser irradiation at different times and the comparison of fluorescence intensity at 60s.

[0083] Figure 17The graph shows the fluorescence response of photosensitizers DTEVQ, TEVQ-1, TEVQ-2, and TEVQ-3 to HPF after 660nm laser irradiation at different times and the comparison of fluorescence intensity at 300s.

[0084] Figure 18 The graph shows the absorption response of photosensitizers DTEVQ, TEVQ-1, TEVQ-2, and TEVQ-3 to ABDA after 660nm laser irradiation at different times and the comparison of fluorescence intensity at 40s.

[0085] Figure 19 Images showing the colocalization of mitochondria in 4TI cells by photosensitizers DTEVQ, TEVQ-2, and TEVQ-3.

[0086] Figure 20 The image shows the cell viability results of 4T1 cells after treatment with 660nm laser irradiation under photosensitizers DTEVQ, TEVQ-2, and TEVQ-3 with and without 660nm laser irradiation.

[0087] Figure 21 The graph shows the fluorescence response of photosensitizers DTEVQ, TEVQ-1, TEVQ-2, TEVQ-3, and the commercial photosensitizer Ce6 to DCFH after white light irradiation at different times and the comparison of fluorescence intensity at 110s.

[0088] Figure 22 The graph shows the fluorescence response of photosensitizers DTEVQ, TEVQ-1, TEVQ-2, and TEVQ-3 to DHR123 after white light irradiation at different times and the comparison of fluorescence intensity at 90s.

[0089] Figure 23 The graph shows the fluorescence response of photosensitizers DTEVQ, TEVQ-1, TEVQ-2, and TEVQ-3 to HPF after white light irradiation at different times and the comparison of fluorescence intensity at 360s.

[0090] Figure 24 The graph shows the degradation rate of ABDA by photosensitizers DTEVQ, TEVQ-1, TEVQ-2, and TEVQ-3 after white light irradiation at different times, and the fluorescence intensity comparison at 180s.

[0091] Figure 25 The graph shows the fluorescence response of photosensitizers DTEVQ, TEVQ-3, TEVP, and TTVP to DCFH after white light irradiation at different times and the comparison of fluorescence intensity at 60s.

[0092] Figure 26 This is a schematic diagram of the structure of the photosensitizer DTEVQ and a schematic diagram of the process by which it generates ROS after being irradiated by a 660nm laser, thereby affecting the mitochondria of cancer cells and effectively killing them. Detailed Implementation

[0093] To better understand the technical solution of the present invention, the embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples.

[0094] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments can be obtained through conventional commercial channels unless otherwise specified.

[0095] Example 1

[0096] Synthesis of DTEVQ, a bridging intramolecular dimer aggregation-induced luminescence mitochondrial-targeted near-infrared photosensitizer.

[0097] (1) Triphenylamine 4-borate (11 mmol, 3.18 g), 5-bromo-2-(3,4-vinyldioxythiophene)carbaldehyde (10 mmol, 2.49 g), 2% equivalent tetra(triphenylphosphine)palladium (0.2 mmol, 231.1 mg), and cesium carbonate (20 mmol, 6.52 g) were added to a 250 mL double-necked reaction flask, purged with nitrogen three times, and 27 mL of THF and 3 mL of ultrapure water were added under nitrogen protection. The mixture was heated to reflux at 70 °C for 24 h, and the reaction progress was detected by thin-layer chromatography. After the reaction was completed, the reaction solution was cooled to room temperature, THF was removed by rotary evaporation, and the organic phase was extracted with dichloromethane and saturated brine. The organic phase was collected and purified by silica gel chromatography using dichloromethane / petroleum ether as eluent (1:1). The mixture was dried under vacuum to obtain a yellow solid powder, which was intermediate 1.

[0098] (2) Add 4-methylquinoline (10 mmol, 1.43 g) and 1,3-diiodopropane (4 mmol, 1.18 g) to a 100 mL double-necked reaction flask, add 5 mL of ethanol, heat to reflux at 80 °C, and react overnight. After the reaction is complete, cool the reaction solution to room temperature, add diethyl ether dropwise to the reaction solution, precipitate a solid, filter quickly, and dry under vacuum to obtain a pale yellow solid powder, which is intermediate product 2;

[0099] (3) Intermediate product 1 (1 mmol, 413.5 mg) and intermediate product 2 (0.4 mmol, 233 mg) were added to a 50 mL double-necked reaction flask, along with 15 mL of ethanol and 1-2 drops of piperidine. The mixture was heated to reflux at 80 °C overnight. After the reaction was complete, the reaction solution was cooled to room temperature, the solvent was removed using a rotary evaporator, and the solution was purified by silica gel chromatography using dichloromethane / methanol as the eluent (20:1). The solution was then dried under vacuum to obtain a blue-purple solid powder, DTEVQ. Its molecular structure is as follows: Figure 1 As shown, the synthesis equation is as follows: Figure 2 As shown.

[0100] Example 2

[0101] Synthesis of aggregation-induced luminescence mitochondrial-targeted near-infrared absorption photosensitizer TEVQ-1.

[0102] (1) Prepare intermediate product 1 by referring to step (1) of Example 1;

[0103] (2) Add 4-methylquinoline (5 mmol, 716 mg) and iodomethane (4 mmol, 568 mg) to a 100 mL double-necked reaction flask, add 5 mL of ethanol, heat to reflux at 80 °C, and react overnight. After the reaction is complete, cool the reaction solution to room temperature, add diethyl ether dropwise to the reaction solution, precipitate a solid, filter quickly, and dry under vacuum to obtain a pale yellow solid powder, which is intermediate product 3;

[0104] (3) Intermediate product 1 (1.1 mmol, 454.9 mg) and intermediate product 3 (1 mmol, 285 mg) were added to a 50 mL double-necked reaction flask, along with 15 mL of ethanol and 1-2 drops of piperidine. The mixture was heated to reflux at 80 °C and reacted overnight. After the reaction was complete, the reaction solution was cooled to room temperature, the solvent was removed using a rotary evaporator, and purification was performed using silica gel chromatography with dichloromethane / methanol as the eluent (20:1). The solution was dried under vacuum to obtain a blue-purple solid powder, TEVQ-1. Its molecular structure is as follows: Figure 3 As shown, the synthesis equation is as follows: Figure 4 As shown.

[0105] Example 3

[0106] Synthesis of aggregation-induced luminescence mitochondrial-targeted near-infrared absorption photosensitizer TEVQ-2.

[0107] (1) Prepare intermediate product 1 by referring to step (1) of Example 1;

[0108] (2) Add 4-methylquinoline (5 mmol, 716 mg) and 1-iodopropane (4 mmol, 680 mg) to a 100 mL double-necked reaction flask, add 5 mL of ethanol, heat to reflux at 80 °C, and react overnight. After the reaction is complete, cool the reaction solution to room temperature, add diethyl ether dropwise to the reaction solution, precipitate a solid, filter quickly, and dry under vacuum to obtain a pale yellow solid powder, which is intermediate product 4;

[0109] (3) Intermediate product 1 (1.1 mmol, 454.9 mg) and intermediate product 4 (1 mmol, 313.2 mg) were added to a 50 mL double-necked reaction flask, along with 15 mL of ethanol and 1-2 drops of piperidine. The mixture was heated to reflux at 80 °C and reacted overnight. After the reaction was complete, the reaction solution was cooled to room temperature, the solvent was removed using a rotary evaporator, and the solution was purified by silica gel chromatography using dichloromethane / methanol as the eluent (20:1). The solution was then dried under vacuum to obtain a blue-purple solid powder, TEVQ-2. Its molecular structure is as follows: Figure 5 As shown, the synthesis equation is as follows: Figure 6 As shown.

[0110] Example 4

[0111] Synthesis of aggregation-induced luminescence mitochondrial-targeted near-infrared absorption photosensitizer TEVQ-3.

[0112] (1) Prepare intermediate product 1 by referring to step (1) of Example 1;

[0113] (2) Add 4-methylquinoline (5 mmol, 716 mg) and (3-bromopropyl)trimethylammonium bromide (4 mmol, 1044 mg) to a 100 mL double-necked reaction flask, add 5 mL of ethanol, heat to reflux at 80 °C, and react overnight. After the reaction is complete, cool the reaction solution to room temperature, add diethyl ether dropwise to the reaction solution, precipitate a solid, filter quickly, and dry under vacuum to obtain a pale yellow solid powder, which is intermediate product 5;

[0114] (3) Intermediate product 1 (1.1 mmol, 454.9 mg) and intermediate product 5 (1 mmol, 404.2 mg) were added to a 50 mL double-necked reaction flask, along with 15 mL of ethanol and 1-2 drops of piperidine. The mixture was heated to reflux at 80 °C overnight. After the reaction was complete, the reaction solution was cooled to room temperature, the solvent was removed using a rotary evaporator, and the solution was purified by silica gel chromatography using dichloromethane / methanol as the eluent (20:1). The solution was then dried under vacuum to obtain a blue-purple solid powder, TEVQ-3. Its molecular structure is as follows: Figure 7 As shown, the synthesis equation is as follows: Figure 8 As shown.

[0115] Comparative Example 1

[0116] Synthesis of aggregation-induced luminescence mitochondrial-targeted near-infrared photosensitizer TTVP.

[0117] (1) Referring to step (1) of Example 1, intermediate product 6 was prepared using 5-bromothiophene-2-carboxaldehyde;

[0118] (2) Referring to step (2) of Example 4, intermediate product 7 was prepared using 4-methylpyridine;

[0119] (3) Referring to step (3) of Example 4, intermediates 6 and 7 were used to obtain a red solid powder, TTVP. Its molecular structure and synthesis equation are as follows: Figure 9 As shown.

[0120] Comparative Example 2

[0121] Synthesis of aggregation-induced luminescence mitochondrial-targeted near-infrared photosensitizer TEVP.

[0122] (1) Prepare intermediate product 1 by referring to step (1) of Example 1;

[0123] (2) Referring to step (2) of Example 4, intermediate product 7 was prepared using 4-methylpyridine;

[0124] (3) Referring to step (3) of Example 4, intermediate product 1 and intermediate product 7 were used to obtain a red solid powder, TEVP. Its molecular structure and synthetic equation are as follows: Figure 10 As shown.

[0125] Comparative Example 3

[0126] Synthesis of aggregation-induced luminescence mitochondrial-targeted near-infrared photosensitizer TTVQ.

[0127] (1) Referring to step (1) of Comparative Example 1, intermediate product 6 was prepared using 5-bromothiophene-2-carboxaldehyde;

[0128] (2) Following step (2) of Example 4, intermediate product 5 was prepared;

[0129] (3) Referring to step (3) of Example 4, intermediate product 6 and intermediate product 5 were used to obtain a red solid powder, TTVQ. Its molecular structure and synthesis equation are as follows: Figure 11 As shown.

[0130] Example 5

[0131] The UV-Vis absorption spectra of aggregation-induced luminescence mitochondrial-targeted near-infrared absorption photosensitizers DTEVQ, TEVQ-3, TTVQ, TEVP, and TTVP.

[0132] The absorbance of solutions of DTEVQ, TEVQ-3, TTVQ, TEVP, and TTVP was measured at room temperature using a Shimadzu UV-2600 UV spectrophotometer (Japan). The solvent was dimethyl sulfoxide (DMSO), with molecular concentrations of 5 μM for DTEVQ and 10 μM for TEVQ, TTVQ, TEVP, and TTVP. Data were collected from 300 to 800 nm.

[0133] Figure 12 The UV-Vis absorption spectra of photosensitizers DTEVQ, TEVQ-3, TTVQ, TEVP, and TTVP are shown. TTVP, TEVP, TTVQ, and DTEVQ exhibit a gradual redshift compared to TEVQ-3, confirming that improvements in the π-bridge and acceptor components lead to a redshift trend in the photosensitizers.

[0134] Example 6

[0135] Photophysical properties of bridging intramolecular dimer aggregation-induced emission mitochondrial-targeted near-infrared absorption photosensitizer DTEVQ and aggregation-induced emission mitochondrial-targeted near-infrared absorption photosensitizers TEVQ-1, TEVQ-2, and TEVQ-3 were tested.

[0136] The absorbance of solutions of DTEVQ, TEVQ-1, TEVQ-2, and TEVQ-3 was measured at room temperature using a Shimadzu UV-2600 UV spectrophotometer (Japan). The solvent was dimethyl sulfoxide (DMSO), with molecular concentrations of 5 μM for DTEVQ and 10 μM for TEVQ-1, TEVQ-2, and TEVQ-3. Data were collected from 300 to 800 nm.

[0137] The fluorescence emission of DTEVQ, TEVQ-1, TEVQ-2, and TEVQ-3 in their aggregated state was measured at room temperature using a Horiba FluoroMax-4 high-sensitivity integrated fluorescence spectrometer manufactured by HORIBA Scientific, USA. The solvent was a dimethyl sulfoxide / toluene mixture (99:1), with molecular concentrations of 5 μM for DTEVQ and 10 μM for TEVQ-1, TEVQ-2, and TEVQ-3. The excitation slit width was 4 nm, the emission slit width was 4 nm, the excitation wavelength was 560 nm, and the data collection range was 600–900 nm. Fluorescence emission values ​​were normalized.

[0138] The fluorescence emission of DTEVQ, TEVQ-2, and TEVQ-3 in a dimethyl sulfoxide / toluene mixed solvent was measured at room temperature using a Horiba FluoroMax-4 high-sensitivity integrated fluorescence spectrometer manufactured by HORIBA Scientific, USA. The toluene content in the solvent mixture ranged from 0% to 99%. The molecular concentrations were 5 μM for DTEVQ and 10 μM for TEVQ-2 and TEVQ-3. The excitation slit width was 4 nm, the emission slit width was 4 nm, the excitation wavelength was 560 nm, and the data collection range was 600 to 900 nm.

[0139] Figure 13 The images show the UV-Vis absorption and fluorescence emission spectra of the photosensitizers DTEVQ, TEVQ-1, TEVQ-2, and TEVQ-3. All materials exhibit strong absorption in the near-infrared region.

[0140] Figure 14The fluorescence emission spectra of photosensitizers DTEVQ, TEVQ-2, and TEVQ-3 in dimethyl sulfoxide / toluene mixtures with different toluene fractions are shown. It can be observed that as the toluene content increases, the photosensitizers gradually aggregate in the solvent, and the fluorescence emission gradually increases, demonstrating that the material exhibits a significant aggregation-induced emission effect.

[0141] Example 7

[0142] ROS generation under 660nm laser irradiation of the bridging intramolecular dimer aggregation-induced emission mitochondrial-targeted near-infrared absorption photosensitizer DTEVQ and aggregation-induced emission mitochondrial-targeted near-infrared absorption photosensitizers TEVQ-1, TEVQ-2, and TEVQ-3.

[0143] (1) Total ROS generation test:

[0144] First, the total ROS generation capacity was detected using 2′,7′-dichlorodihydrofluorescein (DCFH) as an indicator. A 1 mM DCFH-DA stock solution was prepared with anhydrous ethanol. 0.1 mL of this stock solution was added to 0.9 mL of anhydrous ethanol and 4 mL of 0.01 M NaOH solution. After standing at room temperature in the dark for 30 min, 15 mL of PBS (pH = 7.4) was added to obtain a final DCFH solution with a concentration of 50 μM. A 1 mM photosensitizer stock solution was added to the DCFH solution to prepare a final photosensitizer solution with DTEVQ concentration of 0.25 μM and TEVQ-1, TEVQ-2, and TEVQ-3 concentrations of 0.5 μM. Irradiation was performed using a 660 nm laser at a power of 0.5 W / cm². 2 The total illumination time was 60 seconds, with fluorescence intensity measured every 5 seconds. Changes in fluorescence intensity were monitored using a Horiba Fluoromax-4 fluorescence spectrometer. Excitation was performed at 480 nm, and the emission intensity at 525 nm was recorded. The same testing procedure was also applied to the control photosensitizer Ce6.

[0145] (2) Superoxide cation generation test

[0146] Detection of superoxide anion (O2) using dihydrorhodamine 123 (DHR123) as an indicator ·- Generation capacity. A 10 mM DHR123 stock solution was prepared using DMF, and a final DHR123 solution with a concentration of 20 μM was obtained using ultrapure water. A 1 mM photosensitizer stock solution was added to the DHR123 solution to prepare a final photosensitizer solution with a DEVQ concentration of 2.5 μM and TEVQ-1, TEVQ-2, and TEVQ-3 concentrations of 5 μM. Irradiation was performed using a 660 nm laser at a power of 0.5 W / cm². 2The total illumination time was 60 s, with intervals of 5 s, and fluorescence intensity was measured at each interval. Changes in fluorescence intensity were monitored using a Horiba Fluoromax-4 fluorescence spectrometer. Excitation was performed at 480 nm, and the emission intensity at 525 nm was recorded.

[0147] (3) Hydroxyl radical generation test

[0148] The ability to generate hydroxyl radicals (·OH) was detected using hydroxyphenyl fluorescein (HPF) as an indicator. A 5 mM HPF stock solution was prepared using DMF, and then ultrapure water was used to prepare a final HPF solution with a concentration of 10 μM. A 1 mM photosensitizer stock solution was added to the HPF solution to prepare a final photosensitizer solution with concentrations of 2.5 μM for DTEVQ and 5 μM for TEVQ-1, TEVQ-2, and TEVQ-3. Irradiation was performed using a 660 nm laser at a power of 0.5 W / cm². 2 The total illumination time was 300 s, with intervals of 5 s, 10 s, 30 s, and 60 s, and fluorescence intensity was measured at each interval. Changes in fluorescence intensity were monitored using a Horiba Fluoromax-4 fluorescence spectrometer. Excitation was performed at 480 nm, and the emission intensity at 514 nm was recorded.

[0149] (4) Singlet oxygen generation test:

[0150] Using 9,10-anthracene dimethylbis(methylene)-dimalonic acid (ABDA) as an indicator for the detection of singlet oxygen (… 1 O2 generation capacity. A 10 mM ABDA stock solution was prepared using DMF, and an ABDA solution with a final concentration of 50 μM was obtained using ultrapure water. A 1 mM photosensitizer stock solution was added to the ABDA solution to prepare a final photosensitizer solution with DTEVQ concentration of 2.5 μM and TEVQ-1, TEVQ-2, and TEVQ-3 concentrations of 5 μM. Irradiation was performed using a 660 nm laser at a power of 0.5 W / cm². 2 The total illumination time was 40 seconds, with intervals of 5 seconds. Absorption intensity was measured at each interval. A Shimadzu UV-2600 ultraviolet spectrophotometer from Japan was used to monitor the absorbance change of ABDA at 400 nm. Background absorption by the photosensitizer itself should be subtracted before testing.

[0151] Figure 15 This image shows the fluorescence response of photosensitizers DTEVQ, TEVQ-1, TEVQ-2, TEVQ-3, and the commercial photosensitizer Ce6 to DCFH after illumination at different times, and a comparison of fluorescence intensity at 60 seconds. Figure 15 As shown, the total ROS generation capacity of the four photosensitizers is greater than that of the commercial photosensitizer Ce6.

[0152] Figure 16This image shows the fluorescence response of photosensitizers DTEVQ, TEVQ-1, TEVQ-2, and TEVQ-3 to DHR123 after illumination at different times, and a comparison of fluorescence intensity at 60 seconds. Figure 16 As shown, DTEVQ, TEVQ-1, and TEVQ-2 have good superoxide anion generation capabilities.

[0153] Figure 17 This image shows the fluorescence response of photosensitizers DTEVQ, TEVQ-1, TEVQ-2, and TEVQ-3 to HPF after illumination at different times, and a comparison of fluorescence intensity at 300 s. Figure 17 As shown, all four photosensitizers have good hydroxyl radical generation capabilities.

[0154] Figure 18 This image shows the degradation rate of ABDA by photosensitizers DTEVQ, TEVQ-1, TEVQ-2, and TEVQ-3 after illumination at different times, and a comparison of fluorescence intensity at 40 seconds. Figure 18 As shown, the order of singlet oxygen generation capacity of the four photosensitizers is: DTEVQ > TEVQ-1 > TEVQ-2 > TEVQ-3.

[0155] Example 8

[0156] DTEVQ, TEVQ-2, and TEVQ-3 were selected for subsequent biological experiments.

[0157] (1) Mitochondrial colocalization

[0158] In mitochondrial colocalization imaging, 4T1 cells were used as a cancer cell model. 4T1 cells were co-incubated with DTEVQ, TEVQ-2, and TEVQ-3 for 12 h, with DTEVQ at a concentration of 3 μM and TEVQ-2 and TEVQ-3 at concentrations of 6 μM. Then, they were co-incubated with Mito-Tracker Green for 30 min at a concentration of 1 μM. The intracellular fluorescence of DTEVQ, TEVQ-2, and TEVQ-3, and their overlap with Mito-Tracker Green fluorescence, were observed using a Zeiss LSM710 confocal laser scanning biomicroscope (Germany).

[0159] (2) Effects of photodynamic therapy

[0160] In the cytotoxicity assay, 4T1 cells were used as a cancer cell model. 4T1 cells were co-incubated with DTEVQ, TEVQ-2, and TEVQ-3. Medium containing 0, 0.5, 1, 1.5, 2, 2.5, and 3 μM DTEVQ and 0, 1, 2, 3, 4, 5, and 6 μM TEVQ-2 and TEVQ-3 were added to 96-well plates containing 4T1 cells, respectively. After 12 h of culture, the cells were irradiated with a 660 nm laser (0.1 W cm⁻²) for 10 min, and then cultured for another 12 h. The old medium was then discarded, the 4T1 cells were washed with PBS, and the medium was replaced with medium containing 0.5 mg / mL MTT. The cells were cultured in the dark for another 3 h, and then the medium was replaced with DMSO. Finally, the 96-well plates were shaken for 5 min using a multi-mode microplate reader, and the absorbance at 570 nm was measured in each well.

[0161] Figure 19 Laser confocal cell imaging of 4T1 cells after incubation with photosensitizers DTEVQ, TEVQ-2, and TEVQ-3 and the commercial mitochondrial dye Mito-Tracker Green. It can be seen that all three photosensitizers, DTEVQ, TEVQ-2, and TEVQ-3, exhibit good mitochondrial targeting ability.

[0162] Figure 20 This chart shows the dark toxicity of different concentrations of photosensitizers DTEVQ, TEVQ-2, and TEVQ-3, as well as statistical data on in vitro photodynamic therapy. DTEVQ, TEVQ-2, and TEVQ-3 can effectively kill 4T1 cells under light irradiation, with DTEVQ showing the best therapeutic effect. Furthermore, without laser irradiation, DTEVQ, TEVQ-2, and TEVQ-3 exhibit negligible toxicity to 4T1 cells.

[0163] Example 9

[0164] ROS generation under white light by bridging intramolecular dimer aggregation-induced emission mitochondrial targeted near-infrared absorption photosensitizer DTEVQ and aggregation-induced emission mitochondrial targeted near-infrared absorption photosensitizers TEVQ-1, TEVQ-2, and TEVQ-3.

[0165] (1) Total ROS generation test:

[0166] First, the total ROS generation capacity was detected using 2′,7′-dichlorodihydrofluorescein (DCFH) as an indicator. A 1 mM DCFH-DA stock solution was prepared with anhydrous ethanol. 0.1 mL of this stock solution was added to 0.9 mL of anhydrous ethanol and 4 mL of 0.01 M NaOH solution. After standing at room temperature in the dark for 30 min, 15 mL of PBS (pH = 7.4) was added to obtain a final DCFH solution with a concentration of 50 μM. A 1 mM photosensitizer stock solution was added to the DCFH solution to prepare a final photosensitizer solution with a DTEVQ concentration of 0.5 μM and TEVQ-1, TEVQ-2, and TEVQ-3 concentrations of 1 μM. White light illumination at a power of 20 mW / cm² was used. 2 The total illumination time was 110 s, with fluorescence intensity measured every 10 s. Changes in fluorescence intensity were monitored using a Horiba Fluoromax-4 fluorescence spectrometer. Excitation was performed at 480 nm, and the emission intensity at 525 nm was recorded. The same testing procedure was also applied to the control photosensitizer Ce6.

[0167] (2) Superoxide cation generation test

[0168] Detection of superoxide anion (O2) using dihydrorhodamine 123 (DHR123) as an indicator ·- Generation capacity. A 10 mM DHR123 stock solution was prepared using DMF, and a final DHR123 solution with a concentration of 20 μM was obtained using ultrapure water. A 1 mM photosensitizer stock solution was added to the DHR123 solution to prepare a final photosensitizer solution with concentrations of 5 μM for DTEVQ and 10 μM for TEVQ-1, TEVQ-2, and TEVQ-3. White light irradiation was used at a power of 20 mW / cm². 2 The total illumination time was 90 s, with intervals of 10 s and 30 s, and fluorescence intensity was measured at each interval. Changes in fluorescence intensity were monitored using a Horiba Fluoromax-4 fluorescence spectrometer. Excitation was performed at 480 nm, and the emission intensity at 525 nm was recorded.

[0169] (3) Hydroxyl radical generation test

[0170] The ability to generate hydroxyl radicals (·OH) was detected using hydroxyphenyl fluorescein (HPF) as an indicator. A 5 mM HPF stock solution was prepared using DMF, and ultrapure water was used to prepare a final HPF solution with a concentration of 10 μM. A 1 mM photosensitizer stock solution was added to the HPF solution to prepare a final photosensitizer solution with concentrations of 2.5 μM for DTEVQ and 5 μM for TEVQ-1, TEVQ-2, and TEVQ-3. White light illumination at a power of 20 mW / cm² was used. 2The total illumination time was 360 s, with intervals of 5 s, 10 s, 30 s, and 60 s, and fluorescence intensity was measured at each interval. Changes in fluorescence intensity were monitored using a Horiba Fluoromax-4 fluorescence spectrometer. Excitation was performed at 480 nm, and the emission intensity at 514 nm was recorded.

[0171] (4) Singlet oxygen generation test:

[0172] Using 9,10-anthracene dimethylbis(methylene)-dimalonic acid (ABDA) as an indicator for the detection of singlet oxygen (… 1 O2 generation capacity. A 10 mM ABDA stock solution was prepared using DMF, and an ABDA solution with a final concentration of 50 μM was obtained using ultrapure water. A 1 mM photosensitizer stock solution was added to the ABDA solution to prepare a final photosensitizer solution with DTEVQ concentration of 2.5 μM and TEVQ-1, TEVQ-2, and TEVQ-3 concentrations of 5 μM. White light irradiation was used at a power of 20 mW / cm². 2 The total illumination time was 180 seconds, with intervals of 10 seconds and 30 seconds. Absorption intensity was measured at each interval. A Shimadzu UV-2600 ultraviolet spectrophotometer from Japan was used to monitor the absorbance change of ABDA at 400 nm. Background absorption from the photosensitizer itself was subtracted before testing.

[0173] Figure 21 The graph shows the fluorescence response of photosensitizers DTEVQ, TEVQ-1, TEVQ-2, TEVQ-3, and the commercial photosensitizer Ce6 to DCFH after white light irradiation at different times, and the fluorescence intensity at 110 s. The total ROS generation capacity of the four photosensitizers is greater than that of the commercial photosensitizer Ce6.

[0174] Figure 22 The graph shows the fluorescence response of photosensitizers DTEVQ, TEVQ-1, TEVQ-2, and TEVQ-3 to DHR123 after white light irradiation at different times, and a comparison of fluorescence intensity at 90 s. DTEVQ, TEVQ-1, and TEVQ-2 exhibit good superoxide anion generation capabilities.

[0175] Figure 23 The graph shows the fluorescence response of photosensitizers DTEVQ, TEVQ-1, TEVQ-2, and TEVQ-3 to HPF after white light exposure at different times and a comparison of fluorescence intensity at 360 s. All four photosensitizers exhibit good hydroxyl radical generation capabilities.

[0176] Figure 24 The graphs show the degradation rate of ABDA by photosensitizers DTEVQ, TEVQ-1, TEVQ-2, and TEVQ-3 after white light irradiation at different times, and the fluorescence intensity comparison at 180 s. All four photosensitizers exhibit good singlet oxygen generation capabilities.

[0177] Example 10

[0178] The total ROS production of bridging intramolecular dimer aggregation-induced luminescence mitochondrial targeted near-infrared absorption photosensitizers DTEVQ, TEVQ-3, TEVP, and TTVP under white light.

[0179] Total ROS generation capacity was detected using 2′,7′-dichlorodihydrofluorescein (DCFH) as an indicator. A 1 mM DCFH-DA stock solution was prepared with anhydrous ethanol. 0.1 mL of this stock solution was added to 0.9 mL of anhydrous ethanol and 4 mL of 0.01 M NaOH solution. After standing at room temperature in the dark for 30 min, 15 mL of PBS (pH = 7.4) was added to obtain a final DCFH solution with a concentration of 50 μM. A 1 mM photosensitizer stock solution was added to the DCFH solution to prepare a final photosensitizer solution with DTEVQ concentration of 1.25 μM and TEVQ-3, TEVP, and TTVP concentrations of 2.5 μM. White light illumination at a power of 20 mW / cm² was used. 2 The total illumination time was 60 s, with fluorescence intensity measured every 10 s. Changes in fluorescence intensity were monitored using a Horiba Fluoromax-4 fluorescence spectrometer. Excitation was performed at 480 nm, and the emission intensity at 525 nm was recorded.

[0180] Figure 25 The graph shows the fluorescence response of photosensitizers DTEVQ, TEVQ-3, TEVP, and TTVP to DCFH after white light irradiation at different times and a comparison of fluorescence intensity at 60 s. The order of total ROS generation capacity of the four photosensitizers is: DTEVQ > TEVQ-3 > TEVP > TTVP, confirming that the total ROS generation capacity of the photosensitizers is enhanced with improvements in the π-bridge and acceptor components.

[0181] Figure 26 This is a schematic diagram of the structure of the photosensitizer DTEVQ and a schematic diagram of the process by which it generates ROS after being irradiated by a 660nm laser, thereby affecting the mitochondria of cancer cells and effectively killing them.

[0182] The embodiments described above are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. An aggregation-induced luminescence mitochondrial-targeted near-infrared absorption photosensitizer, characterized in that, The chemical structural formula is: The preparation method of the DTEVQ includes the following steps: (1) Triphenylamine 4-borate, 5-bromo-2-(3,4-vinyldioxythiophene)carboxaldehyde, tetrakis(triphenylphosphine)palladium and cesium carbonate were added to a mixed solvent of THF and water under a protective atmosphere, and the mixture was refluxed. After purification, intermediate product 1 was obtained. The structural formula of intermediate product 1 is as follows: ; (2) Add 4-methylquinoline and 1,3-diiodopropane to ethanol, reflux the reaction, and purify to obtain intermediate 2. The structural formula of intermediate 2 is: ; (3) Add intermediate product 1 and intermediate product 2 to ethanol and piperidine, reflux and purify to obtain DTEVQ.

2. The aggregation-induced emission mitochondrial-targeted near-infrared absorption photosensitizer according to claim 1, characterized in that, In step (1), the molar ratio of 4-boronic acid triphenylamine, 5-bromo-2-(3,4-vinylhydrothiophene)carboxaldehyde, tetra(triphenylphosphine)palladium and cesium carbonate is 11~15:10:0.02~0.05:20~30; the volume ratio of THF and water is 20~27:3~10.

3. The application of the aggregation-induced emission mitochondrial-targeted near-infrared absorption photosensitizer according to claim 1 in the preparation of photodynamic therapy reagents for cancer cells.

Images