An aggregation-induced emission self-reporting photosensitizer probe, its preparation method and application

Abstract

This invention discloses an aggregation-induced emission (AIE) self-reported photosensitizer probe, its preparation method, and its applications. The structural formula of the AIE self-reported photosensitizer probe is shown in Formula (I), representing a novel type of AIE self-reported photosensitizer probe. The probe is characterized by ease of preparation, high fluorescence intensity, good water solubility, and low fluorescence background; it is a photosensitizer with dual targeting of the cell membrane and nucleus; it can be used to distinguish between live and dead cells, for rapid sensitivity analysis of cells to clinical drugs, and for analyzing drug resistance levels in tumor cells; it can be used for highly efficient photodynamic therapy of tumors, achieving efficient tumor cell killing by targeting and destroying the cell membrane and nucleus, generating ROS at the membrane and nucleus; it can also perform self-reported analysis of its own photodynamic therapy process and possesses two-photon properties, enabling two-photon imaging of cells and tissues.

Description

Technical Field

[0001] This invention relates to the fields of cell detection and photodynamic therapy, and more specifically, to an aggregation-induced emission self-reporting photosensitizer probe, its preparation method, and its application. Background Technology

[0002] Chemotherapy is one of the classic and primary treatments for cancer. However, existing chemotherapy methods often fail to produce a complete or sustained anti-cancer effect, ultimately leading to drug resistance and tumor recurrence. Drug resistance further impacts treatment efficacy and reduces survival rates. Currently, there is a lack of methods in clinical practice for real-time monitoring of treatment efficacy, hindering the timely acquisition of information on tumor cell sensitivity, responsiveness, and the development of drug resistance, thus impeding the diagnosis and treatment of drug-resistant tumors. Better treatment and efficacy monitoring protocols for drug-resistant tumors are urgently needed in clinical practice.

[0003] Photodynamic therapy (PDT) is a clinically approved treatment. It can target and destroy tumor tissue with minimal toxicity to normal tissues and negligible systemic effects. In particular, PDT lacks intrinsic or acquired resistance mechanisms, giving it a significant advantage in treating drug-resistant tumors.

[0004] PDT (Photosensitive Therapy) primarily relies on the interaction of a specific photosensitizer (PS) with oxygen to generate reactive oxygen species (ROS). Currently, most traditional PS are limited by their aggregation-caused quenching (ACQ) properties. Upon activation, they aggregate in aqueous media due to π-π stacking, leading to fluorescence quenching (ACQ effect), which reduces ROS generation and decreases PDT efficacy. Furthermore, in the treatment of drug-resistant tumors, existing photosensitizers may be affected by drug efflux pumps (drμg efflux pμMps) on the cell membrane, resulting in reduced photosensitizer uptake by cells and impacting the final PDT effect. Moreover, because the effective action area of ​​ROS is limited to the subcellular nanoscale, it is necessary to deliver the photosensitizer directly to specific cells or precisely targeted subcellular structures to achieve effective PDT. However, most existing PS do not have specific organelle targeting capabilities, nor do they possess dual-targeting capabilities targeting both the cell membrane and the nucleus.

[0005] The degree or proportion of cancer cell death is one of the most reliable indicators for measuring drug efficacy and is often used in screening anticancer therapies and in mechanistic studies using preclinical models. Existing probes for measuring cell death ratios mostly rely on the detection of cell death biomarkers, such as: 1. MTT: 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoli μM romide, chemical name: 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide, trade name: thiazolium blue; 2. CCK-8: based on WST–8, chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazolium monosodium salt; 3. PI (Propidi μM Iodide): Chinese name: propidium iodide. Since PIs cannot pass through normal, intact cell membranes, cells in necrosis or late-stage apoptosis are labeled with PIs and exhibit red fluorescence, which can be used to distinguish between necrotic and normal cells. However, current probes for measuring cell death require complex washing procedures, suffer from poor photostability and biocompatibility, and are time-consuming, thus limiting their application. Summary of the Invention

[0006] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide a fluorescent compound with aggregation-induced emission properties.

[0007] A second objective of this invention is to provide a method for preparing the fluorescent compound having aggregation-induced emission properties.

[0008] A third object of the present invention is to provide applications of the fluorescent compounds having aggregation-induced emission properties.

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

[0010] A fluorescent compound exhibiting aggregation-induced emission properties has the chemical structural formula shown in formula (I):

[0011]

[0012] Wherein, Ar1 is an aromatic ring; Ar2 is the same or different hydrogen or nitrogen-containing aromatic rings; X1 is an anion; R1 is a substituted or unsubstituted straight-chain, branched, cyclic alkyl chain or straight-chain trimethylammonium having 1 to 20 carbon atoms, wherein the substitution is one or more carbon atoms optionally substituted by an oxygen atom, alkenyl, alkynyl, aryl, carbonyl, hydroxyl, amino, carboxyl, cyano, nitro or ester group.

[0013] This invention constructs a typical donor-acceptor (DA) structure to achieve long-wavelength emission and efficient reactive oxygen species (ROS) generation. The triarylamine group possesses strong electron-donating ability, while the aryl diketone and two positively charged azaaromatic units possess electron-withdrawing ability. The entire structural framework contains multiple flexible rotors or vibrators connected by single or double bonds, thereby ensuring ideal aggregation-induced emission (AIE) performance. The positively charged azaaromatic moiety at the end of the compound tends to interact with the negative transmembrane potential of the cell membrane through electrostatic interactions, anchoring itself to the intact cell membrane of living cells and further restricting intramolecular rotation to enhance fluorescence emission. When the cell membrane integrity is disrupted, the compound molecule enters the cell through the ruptured membrane gap and anchors to the negatively charged nucleus, thus achieving different targeting imaging effects for living and dead cells.

[0014] Preferably, Ar1 is selected from any one of the following chemical structural formulas (1) to (20); Ar2 is selected from any one of the following chemical structural formulas (21) to (27); and X1 is selected from any one of the following chemical structural formulas (28) to (34):

[0015]

[0016] Wherein, R' on Ar1 is hydrogen, a straight-chain, branched, or cyclic alkyl chain having 1 to 20 carbon atoms, substituted or unsubstituted, wherein the substitution is one or more carbon atoms optionally substituted by an oxygen atom, alkenyl, alkynyl, aryl, carbonyl, hydroxyl, amino, carboxyl, cyano, nitro, or ester group; R” on Ar2 is hydrogen, a straight-chain, branched, or cyclic alkyl chain having 1 to 20 carbon atoms, substituted or unsubstituted, wherein the substitution is one or more carbon atoms optionally substituted by an oxygen atom, alkenyl, alkynyl, aryl, carbonyl, hydroxyl, amino, carboxyl, cyano, nitro, or ester group; * indicates the substitution position.

[0017] Preferably, its chemical structural formula is shown in formula (II), formula (III), or formula (IV):

[0018]

[0019] The present invention also provides a method for preparing the above-mentioned fluorescent compound with aggregation-induced emission properties.

[0020] Will and The reaction was prepared to obtain Again with R1X - The reaction yielded the compound shown in formula (I);

[0021] Wherein, Ar1 is an aromatic ring; Ar2 is the same or different hydrogen or nitrogen-containing aromatic rings; X1 is an anion; R1 is a substituted or unsubstituted straight-chain, branched, cyclic alkyl chain or straight-chain trimethylammonium chain having 1 to 20 carbon atoms, wherein the substitution is one or more carbon atoms optionally substituted by an oxygen atom, alkenyl, alkynyl, aryl, carbonyl, hydroxyl, amino, carboxyl, cyano, nitro or ester group.

[0022] Preferably, Ar1 is selected from any one of the following chemical structural formulas (1) to (20); Ar2 is selected from any one of the following chemical structural formulas (21) to (27); and X1 is selected from any one of the following chemical structural formulas (28) to (34):

[0023]

[0024] Wherein, R' on Ar1 is hydrogen, a straight-chain, branched, or cyclic alkyl chain having 1 to 20 carbon atoms, substituted or unsubstituted, wherein the substitution is one or more carbon atoms optionally substituted by an oxygen atom, alkenyl, alkynyl, aryl, carbonyl, hydroxyl, amino, carboxyl, cyano, nitro, or ester group; R” on Ar2 is hydrogen, a straight-chain, branched, or cyclic alkyl chain having 1 to 20 carbon atoms, substituted or unsubstituted, wherein the substitution is one or more carbon atoms optionally substituted by an oxygen atom, alkenyl, alkynyl, aryl, carbonyl, hydroxyl, amino, carboxyl, cyano, nitro, or ester group; * indicates the substitution position.

[0025] The fluorescent compounds of this invention possess a typical donor-acceptor (DA) structure, enabling long-wavelength emission and efficient generation of reactive oxygen species (ROS). The entire structural framework comprises multiple flexible rotors or vibrators connected by single or double bonds, thereby ensuring ideal aggregation-induced emission performance. The positively charged aza-aromatic moiety at the end of the compound tends to interact with the negative transmembrane potential of the cell membrane through electrostatic interactions, anchoring itself to the intact cell membrane of living cells and further restricting intramolecular rotation to enhance fluorescence emission. When the cell membrane integrity is disrupted, the compound molecules enter the cell through the ruptured membrane gaps and anchor to the negatively charged nucleus, thereby achieving different targeting imaging effects for living and dead cells.

[0026] The compounds described above in this invention can generate a large amount of ROS under white light irradiation, and their generation capacity is superior to commercial photosensitizers under the same conditions. They can not only generate ROS rapidly and effectively, but also produce a large amount of singlet oxygen, making them a novel photosensitizer. Simultaneously, these chemicals exhibit dual-targeting imaging effects on cells, specifically imaging the cell membranes of living cells and the nuclei of dead cells, demonstrating a dual-targeting effect. Compared to commercial dyes, they exhibit better photostability and cell membrane imaging performance. Based on their specific dual-targeting nature, visual differentiation between living and dead cells can be achieved, and their performance is far superior to commercial biological probes in all aspects. Based on their excellent visual (fluorescence) recognition capabilities for living and dead cells, these compounds can also be developed into a highly efficient drug sensitivity screening platform, enabling precise analysis of tumor cell viability after drug treatment to determine the efficacy of chemotherapy drugs. They can accurately quantify the ratio of living to dead cells corresponding to each drug treatment, thereby rapidly and accurately reporting the drug efficacy of different chemotherapy drugs on tumor cells, and simultaneously quickly determining the drug resistance status of cancer cells against anti-tumor drugs. Furthermore, any of the compounds described above in this invention also possess two-photon properties, enabling tissue two-photon imaging. They not only exhibit excellent fluorescence imaging capabilities at the cellular level but also demonstrate superior imaging capabilities at the tissue level. The reconstructed 3D images display ultra-clear two-photon tissue structures and local details of different tissues such as the liver, kidneys, heart, and spleen. Due to the unique AIE luminescence advantages of the compounds, they can also achieve long-term in vivo imaging in living animals.

[0027] Furthermore, since the compound can effectively generate a large amount of ROS after light irradiation, and due to its dual targeting ability, it can not only generate ROS at the cell membrane to damage the cell membrane, but also severely damage the cell nucleus structure through photoinduced reactive oxygen species (ROS), and directly destroy the cell membrane integrity and cause cell death through membrane-targeting PS properties, and directly destroy the DNA structure and cause cell death through cell nucleus-targeting PS properties. Therefore, it can achieve highly efficient photodynamic therapy for cancer cells, especially drug-resistant tumors. At the same time, based on its induced luminescence properties, the entire photodynamic process can be self-reported in situ through spatiotemporally resolved fluorescence migration from the cell membrane to the cell nucleus, which can be used to prepare highly efficient tumor therapeutic drugs.

[0028] Therefore, the present invention also provides the following applications of fluorescent compounds having aggregation-induced emission properties as described above:

[0029] The use of any of the fluorescent compounds with aggregation-induced emission properties described above in cell staining or in the preparation of cell staining agents.

[0030] Optionally, the cell staining agent is used to distinguish between live and dead cells.

[0031] Optionally, the cell staining agent is used to evaluate the drug's efficacy on cells.

[0032] Optionally, the cells are tumor cells or drug-resistant tumor cells.

[0033] The application of any of the fluorescent compounds with aggregation-induced emission properties described above in the preparation of two-photon tissue imaging agents.

[0034] The application of any of the fluorescent compounds with aggregation-induced emission properties described above in the preparation of photosensitizers with dual targeting functions of cell membrane and cell nucleus.

[0035] The application of any of the fluorescent compounds with aggregation-induced emission properties described above in the preparation of antitumor drugs.

[0036] The present invention also provides a cell staining agent, tissue imaging agent, photosensitizer or antitumor drug, which contains any of the fluorescent compounds with aggregation-induced emission properties described above.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] This invention provides a novel fluorescent compound with aggregation-induced emission properties, as shown in formula (I). The compound is easy to prepare, exhibits high fluorescence intensity, good water solubility, and low fluorescence background; it is a fluorescent dye and photosensitizer with dual targeting of the cell membrane and cell nucleus; it can be used to distinguish between live and dead cells, for rapid sensitivity analysis of cells to clinical drugs, and for analyzing tumor cell drug resistance levels. It has advantages such as good photostability, excellent biocompatibility, short processing time, simple operation, and wash-free imaging; it can be used for highly efficient photodynamic therapy of tumors, achieving efficient tumor cell killing by targeting and destroying the cell membrane and nucleus, generating ROS at the cell membrane and nucleus. Because it achieves photodynamic therapy on the membrane, it is not affected by membrane-based drug resistance mechanisms, making it particularly suitable for the treatment of drug-resistant tumors; it also allows for self-reporting analysis of its own photodynamic therapy process and possesses two-photon properties, enabling two-photon imaging of cells and tissues. Attached Figure Description

[0039] Figure 1-1 Compound 1 in Example 1 1 H NMR spectrum; Figure 1-2 Compound 1 in Example 1 13 C NMR spectrum; Figure 1-3 The HRMS spectrum of the compound in Example 1; Figure 1-4 The compound ITPM ​​in Example 1 1 H NMR spectrum; Figure 1-5 The compound ITPM ​​in Example 1 13 C NMR spectrum; Figure 1-6 The image shows the HRMS spectrum of compound ITPM ​​from Example 1.

[0040] Figure 2 (A) is the UV-Vis absorption spectrum of compound ITPM ​​in dimethyl sulfoxide; Figure 2 (B) is the fluorescence emission spectrum of compound ITPM ​​in a mixed solution of DMSO and tetrahydrofuran with increasing tetrahydrofuran content; Figure 2 (C) represents the ratio (I / I0) of the fluorescence intensity of compound ITPM ​​in different DMSO / tetrahydrofuran mixed solvents (I) to that in DMSO (I0).

[0041] Figure 3 (A) represents the ROS generation capacity of the compound ITPM ​​aggregates; Figure 3 (B) represents the ability of the compound ITPM ​​to produce singlet oxygen.

[0042] Figure 4 Imaging effect of compound IPTM in dead / live A549 tumor cells.

[0043] Figure 5 Colocalization diagram of compound IPTM with commercial dyes (Dil, Cell mask, PI, and DAPI).

[0044] Figure 6 The photostability of compound IPTM is compared with that of commercial dyes (Dil and Cell mask) and nuclear dyes (PI and DAPI).

[0045] Figure 7 This study compares the staining effects of the compound ITPM ​​with those of commercial dyes (Dil and Cell mask).

[0046] Figure 8 The staining effect of commercial dyes (Dil and Cell mask) on dead cells.

[0047] Figure 9-1 Imaging of the compound ITPM ​​in different tumor cell lines; Figure 9-2 This shows the imaging of the compound ITPM ​​in different normal cell lines.

[0048] Figure 10-1 This enables ultra-rapid and precise evaluation of the anticancer activity of the compound ITPM ​​against chemotherapy drugs. Figure 10-2 This is a flow cytometry quantitative diagram.

[0049] Figure 11 The compound ITPM ​​can be used to determine the degree of resistance of tumor cells to antitumor drugs such as cisplatin or paclitaxel.

[0050] Figure 12Two-photon imaging of the compound ITPM ​​in tumor and normal tissues.

[0051] Figure 13 This is an in vivo imaging image of the compound ITPM ​​at the tumor site in tumor-bearing mice.

[0052] Figure 14-1 The compound ITPM-labeled cells produced a large amount of reactive oxygen species (ROS) after light exposure. Figure 14-2 The cell viability of tumor cells treated with the compound ITPM ​​after exposure to darkness or white light.

[0053] Figure 15-1 A self-reporting PDT process for labeling tumor cells with the compound IPTM; Figure 15-2 To demonstrate the self-reported PDT process of ITPM-labeled tumor cells using PI co-staining fluorescence imaging and flow cytometry.

[0054] Figure 16 This describes the tumor treatment efficacy of the compound IPTM. Detailed Implementation

[0055] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0056] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0057] Example 1: Preparation of compound ITPM

[0058] The preparation of compound ITPM ​​is carried out according to the following reaction:

[0059]

[0060] Regarding the synthesis of the intermediate and target compound ITPM:

[0061] Unless otherwise specified, all operations are performed using standard Schlenk techniques under a dry argon atmosphere. Solvents are pre-dried and distilled under argon before use. Solvents used directly for spectroscopic measurements are spectral grade. 1 H and 13C10 NMR spectra were measured on a Bruker AVIII 400MHz NMR spectrometer using deuterated chloroform or acetonitrile as solvents and tetramethylsilane (TMS; δ = 0) as an internal reference. High-resolution mass spectra (HRMS) were recorded on a GCT Premier CAB048 mass spectrometer system running in MALDI-TOF mode. UV spectra were measured on a Varian CARY 50 UV-Vis spectrophotometer. PL spectra were performed using a Horiba Fluoromax 4 fluorescence spectrophotometer. The specific steps included are as follows:

[0062] (1) Synthesis of compound 1:

[0063] Compound 1 was prepared according to the procedure described in the literature [K. Acharyya, S. Bhattacharyya, H. Sepehrpour, S. Chakraborty, S. Lu, B. Shi, X. Li, P. S. Mukherjee, P. J. S. Tang, J. Am. Chem. Soc. 2019, 141, 14565.]: Target compound 1 (4-[bis[4-(4-pyridyl)phenyl]amino]benzaldehyde) Specific synthetic steps: Under nitrogen protection, 1 mmol of 4-[bis(4-bromophenyl)amino]benzaldehyde, 2.2 mmol of pyridine-4-boronic acid, 0.1 mmol of Pd(PPh3)4 and 3 mmol of K2CO3 were placed in a 60 mL mixed solvent of THF / water (volume ratio: 5 / 1), and the mixture was further reacted at 70 °C for 24 h. After the reaction was complete, the system was injected into 150 mL of water, extracted with dichloromethane, dried over anhydrous sodium sulfate, and the organic phase was concentrated. Further purification was achieved by column chromatography (eluent: dichloromethane / n-hexane) to give product 1 (4-[bis[4-(4-pyridyl)phenyl]amino]benzaldehyde). Yield: 65%.

[0064] (2) Synthesis of compound 2:

[0065] As shown in the reaction equation above, 219 mg of 1,3-indanedione (1.5 mmol) and 409 mg of 4-[bis[4-(4-pyridyl)phenyl]amino]benzaldehyde (1.5 mmol) were dissolved in 30 mL of n-butanol and refluxed for 2 h, with the reaction progress monitored by TLC. During this period, the color of the mixture changed from pale yellow to deep red. After cooling to room temperature, the resulting red precipitate was collected, washed with ethanol, and dried under vacuum to give compound 2 as a red powder. Yield: 766 mg (92%).

[0066] The structural characterization data of compound 2 are as follows: 1H NMR (400MHz, CDCl3), δ (ppm): 8.65 (d, J = 4Hz, 4H), 8.46 (d, J = 8Hz, 2H), 7.97-7.94 (m, 2H), 7.80 (s, 1H) ,7.77-7.75(m,2H),7.64(d,J=8Hz,4H),7.51(d,J=4Hz,4H),7.32(d,J=8Hz,4H),7.16(d,J=8Hz,2H)( Figure 1-1 ); 13 C NMR (100MHz, CDCl3), δ (ppm): 190.9 (C=O), 189.5 (C=O), 151.5, 150.2, 150.0, 147.2, 146.7, 146.0, 142.4,140.0,136.6,135.0,134.8,134.6,128.3,127.2,126.5,126.3,123.0,121.2,121.0,120.9( Figure 1-2 ); HRMS(MALDI-TOF),m / z:calcd.for C 38 H 25 N3O2:555.1947; found:555.1956( Figure 1-3 ).

[0067] (3) Synthesis of compound ITPM:

[0068] A mixture of compound 2 (555 mg, 1.00 mmol) and CH3I (0.3 mL, 5.00 mmol) was stirred in CH3CN (30 mL) for 24 hours (85 °C under argon). After the reaction was complete, the solvent was filtered and the residue was collected. The collected residue was dispersed in 3 mL of DMSO, and then 50 mL of an acetone / CH2Cl2 (4 / 1, v / v) mixture was added to produce a red precipitate. The resulting red precipitate was filtered, washed with acetone, and dried under vacuum to give a red solid ITPM ​​in 89% (747 mg) yield.

[0069] The structural characterization data of compound ITPM ​​are as follows: 1 H NMR (400MHz, DMSO-d6), δ (ppm): 8.99 (d, J = 4Hz, 4H), 8.61 (d, J = 16Hz, 2H), 8.50 (d, J = 8Hz, 4H), 8.17 (d, J =8Hz,4H),7.99-7.94(m,4H),7.82(s,1H),7.42(d,J=8Hz,4H),7.25(d,J=12Hz,2H),4.33(s,6H,–CH3)( Figure 1-4 );13 CNMR(100MHz,DMSO-d6),δ(ppm):189.6(C=O).188.8(C=O),153.0,150.0,148.6,145.4,141.7,1 39.3,136.3,135.8,135.7,129.9,129.3,128.2,127.3,125.6,123.4,122.9,122.6,47.0(–CH3)( Figure 1-5 ); HRMS(MALDI-TOF),m / z:[M-2I] 2+ calcd.for[C 40 H 31 N3O2] 2+ :585.2405; found:585.2385( Figure 1-6 ).

[0070] Example 2 Preparation of compound ITSPM

[0071] The preparation of compound ITPM ​​is carried out according to the following reaction:

[0072]

[0073] Regarding the synthesis of the intermediate and target compound ITSPM:

[0074] (1) Synthesis of compound 3 (4–[phenyl(4-(4-pyridyl)phenyl)amino]benzaldehyde):

[0075] The precursor of compound 3, 4-[4-bromophenyl)(phenyl)amino]benzaldehyde, was obtained by reflux of 4-(N,N-diphenylamino)-benzaldehyde and N-bromosuccinimide (NBS) in CHCl3 overnight. The crude product was filtered, and the solvent was evaporated under vacuum. The purified solid was obtained by recrystallization from benzene / methanol. Yield: 76%. Further, under nitrogen protection, 1 mmol of 4-[4-bromophenyl)(phenyl)amino]benzaldehyde, 12 mmol of pyridine-4-boronic acid, 0.05 mmol of Pd(PPh3)4 and 3 mmol of K2CO3 were placed in a 60 mL mixed solvent of THF / water (v / v ratio: 5 / 1), and the mixture was further reacted at 70 °C for 24 h. After the reaction was complete, the system was injected into 150 mL of water, extracted with dichloromethane, dried over anhydrous sodium sulfate, and the organic phase was concentrated. Further purification was achieved by column chromatography (eluent: dichloromethane / n-hexane) to obtain product 3. Yield: 68%.

[0076] (2) Synthesis of compound 4:

[0077] As shown in the reaction equation above, 219 mg of 1,3-indanedione (1.5 mmol) and 525 mg of 4-[phenyl(4-(4-pyridyl)phenyl)amino]benzaldehyde (1.5 mmol) were dissolved in 30 mL of n-butanol and refluxed for 2 h, with the reaction progress monitored by TLC. During this period, the color of the mixture changed from pale yellow to deep red. After cooling to room temperature, the resulting red precipitate was collected, washed with ethanol, and dried under vacuum to give compound 4 as a red powder. Yield: 700 mg (97%).

[0078] (3) Synthesis of compound ITSPM:

[0079] A mixture of compound 4 (478 mg, 1.00 mmol) and CH3I (0.2 mL, 2.50 mmol) was stirred in CH3CN (30 mL) for 24 hours (85 °C under argon). After the reaction was complete, the solvent was filtered and the residue was collected. The collected residue was dispersed in 3 mL of DMSO, and then 50 mL of an acetone / CH2Cl2 (4 / 1, v / v) mixture was added to produce a red precipitate. The resulting red precipitate was filtered, washed with acetone, and dried under vacuum to give a red solid ITSPM in 92% (570 mg) yield.

[0080] Example 3: Preparation of compound ITPA

[0081] The preparation of compound ITPA is carried out according to the following reaction formula:

[0082]

[0083] As shown in the reaction equation above, a mixture of compound 2 (555 mg, 1.00 mmol) and (3-bromopropyl)trimethylammonium bromide (516 mg, 2.00 mmol) was stirred in CH3CN (30 mL) for 24 hours (85 °C under argon). After the reaction was complete, the solvent was filtered and the residue was collected. The collected residue was dispersed in 3 mL of DMSO, and then 50 mL of an acetone / CH2Cl2 (4 / 1, v / v) mixture was added to produce a red precipitate. The resulting red precipitate was filtered, washed with acetone, and dried under vacuum to give a purple solid ITPA in a yield of 86% (922 mg).

[0084] Example 4: Characterization of the photophysical properties of compound ITPM

[0085] (1) Using dimethyl sulfoxide (DMSO) as solvent, prepare a solution with a final concentration of 2×10⁻⁶. -5 mol·L -1 The compound ITPM ​​solution was placed in a cuvette, and its ultraviolet-absorption spectrum was detected using a Varian CARY 50 ultraviolet spectrometer. Figure 2(A) is the UV-Vis absorption spectrum of compound ITPM ​​in dimethyl sulfoxide.

[0086] (2) Because the compound is soluble in water, THF was used as a poorly soluble solvent to study the aggregation-induced emission effect. Tetrahydrofuran (THF) and dimethyl sulfoxide (DMSO) were mixed in different proportions (THF to DMSO ratio: 0, 20, 40, 60, 80, 90, 95, 99%), and the mixtures were used as solvents to prepare solutions with a final concentration of 2 × 10⁻⁶. -5 mol·L -1 The fluorescence emission spectrum of the compound ITPM ​​solution was detected using a fluorescence spectrometer. Figure 2 (B) is the fluorescence emission spectrum of compound ITPM ​​in a mixed solution of DMSO and tetrahydrofuran with increasing tetrahydrofuran content; Figure 2 (C) represents the ratio (I / I0) of the fluorescence intensity of compound ITPM ​​in different DMSO / tetrahydrofuran mixed solvents (I) to that in DMSO (I0).

[0087] Figure 2 AC analysis revealed that, based on its absorption spectrum, a diluted DMSO solution of ITPM ​​exhibited weak orange emission when excited with 405 nm light. With the addition of tetrahydrofuran as a poor solvent, the luminescence of the resulting system gradually increased, reaching a maximum at a tetrahydrofuran content of 70 vol%. During this process, a slight blue shift in the emission wavelength was observed, which should be attributed to the combined effect of AIE and the distorted intramolecular charge transfer (TICT) effect.

[0088] ITPM has a typical donor-acceptor (DA) structure, which enables long-wavelength emission and efficient generation of reactive oxygen species (ROS). The entire structural framework contains multiple flexible rotors or vibrators connected by single or double bonds, thus ensuring the ideal AIE performance of ITPM.

[0089] (3) Singlet oxygen detection (anthracene dimethylmalonic acid ABDA method): ABDA (9,10-anthracene-diyl-bis(methylene)dimalonic acid) was used as the singlet oxygen detection probe. In the experiment, 10 μL of ABDA stock solution (7.5 mM) was added to 2 mL of ITPM ​​sample suspension (10 μM), and white light (4.2 mW / cm²) was used. -2 ABDA was used as the light source. The absorbance of ABDA at 378 nm was recorded for different irradiation periods to obtain the decay rate of the photosensitization process. The results are as follows: Figure 3As shown in (A), under white light irradiation, ITPM ​​can rapidly and effectively generate a large amount of ROS within 10 seconds, and its generation capacity is significantly better than that of the commercial photosensitizer Rose Bengal (RB) under the same conditions.

[0090] (4) DCFH-DA (2',7'-Dichlorodihydrofluorescein diacetate) was used as a ROS reactive oxygen species detection probe to detect the ROS generation of the synthesized compound ITPM ​​in solution (aggregated state). In the experiment, 10 μL of DCFH-DA activation solution (1.0 mM, activated by adding NaOH to DCF-DA ethanol solution) was added to 2 mL of ITPM ​​(10 μM) solution, and white light (4.2 mW / cm²) was used. -2 As the light source, the absorbance of DCFH-DA at 525 nm was recorded for different irradiation periods. The results are as follows: Figure 3 As shown in (B), when exposed to white light, the absorbance of ABDA rapidly decreases to 0 within 40 seconds. Its production... 1 ITPM's ability to generate O2 (singlet oxygen) is also stronger than that of RB. That is, ITPM ​​can quickly and efficiently generate ROS, especially large amounts of singlet oxygen.

[0091] Example 5: Compound ITPM ​​exhibits dual-targeting imaging effects on cells.

[0092] (1) Cell Culture

[0093] Tumor cell lines: HeLa cells (cervical cancer cells), MCF-7 and T47D cells (breast cancer cells), A549 cells (lung cancer cells), Caco2 and HT29 cells (colorectal cancer cells), MG63 cells (osteosarcoma cells), and B16 mouse melanoma cells; and normal cell lines: 16HBE cells (human bronchial epithelial cells), NCM460 cells (colonial epithelial cells), 293T cells (human kidney epithelial cells), and HVSMC cells (human vascular smooth muscle cells) were purchased from the Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All cells were cultured in DMEM (Gibco) containing 10% fetal bovine serum (Invitrogen) and streptomycin in a 37°C, 21% O2, and 5% CO2 cell culture incubator. Strict aseptic techniques and laboratory protocols were followed during cell handling.

[0094] (2) Cell imaging:

[0095] A549 cells were cultured overnight in 35 mm culture dishes (or confocal dishes) with coverslips. Cells were stained for 2 minutes with cell culture medium containing 10 μM ITPM ​​or PBS (Gibco). The cell coverslips (or the confocal dish) were removed and imaged under a confocal microscope (no washing required). For imaging of dead cells: tumor cells were fixed with pre-chilled methanol for 10 minutes, then washed three times with PBS. Cells were then cultured for 2 minutes in cell culture medium containing 10 μM ITPM ​​or PBS (no washing required). Imaging was performed using a laser scanning confocal microscope (FV3000, Olympus) at 405 nm and 0.1% laser power. Emission filter: 550-650 nm. Results are as follows. Figure 4 As shown, bright red fluorescence on the cell membrane can be clearly seen after 2 minutes of incubation without washing. When cell imaging experiments are performed using dead cells, the nuclei of dead A549 cells show bright red fluorescence. ITPM ​​can specifically image the cell membrane of living cells and the nucleus of dead cells, respectively, providing a dual-targeting effect.

[0096] (3) Co-location assay of ITPM ​​with existing commercial reagents (all purchased from Thermo Fisher Scientific):

[0097] Cell membrane dyes: 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanineperchlorate (Dil) and Cell mask deep red;

[0098] Nuclear dyes: propidi μM iodide (PI) and 4',6-diamidino-2-phenylindole (DAPI);

[0099] A549 cells were incubated for 2 minutes in cell culture medium or PBS containing 10 μM ITPM. Then, different commercial dyes were added for co-staining imaging. For imaging of dead cells: tumor cells were fixed with pre-chilled methanol for 10 minutes, washed three times with PBS, and then stained with ITPM ​​for 2 minutes. Dil (10 μM, 30 min), Cell mask deep red (5 μg / mL, 30 min), PI (10 μM, 10 min), and DAPI (1 μM, 5 min) were added. After washing twice with PBS, imaging was performed under a confocal microscope. Excitation wavelengths: ITPM ​​and DAPI 405 nm, Dil and PI 561 nm, Cell mask deep red 633 nm. Emission wavelengths: ITPM ​​550-650 nm, DAPI 410-450 nm, Cell mask deep red 640-700 nm, Dil 570-630 nm. Results are as follows: Figure 5 As shown, the results indicate that ITPM ​​has a unique dual-targeting capability, specifically targeting cell membrane imaging in living cells while simultaneously targeting cell nucleus imaging in dead cells.

[0100] (4) Light stability test:

[0101] A549 tumor cells were used as an example. A549 cells were incubated with ITPM ​​and commercial dyes (in cell culture medium or PBS containing the corresponding dye concentrations). The incubation conditions were as follows: ITPM: 10 μM, 2 min; Dil: 10 μM, 30 min; Cell mask deep red: 5 μg / mLm, 30 min; PI: 10 μM, 10 min; DAPI: 1 μM, 5 min. After staining, A549 cells with the commercial dyes were washed three times with PBS, while those with ITPM ​​did not require a washing step. A549 cells labeled with each dye were imaged using a confocal microscope (Zeiss LSM710 laser scanning confocal microscope). Conditions: Excitation wavelengths: ITPM ​​and DAPI: 405 nm (2% laser power); Dil: 561 nm (2% laser power); Cell mask deep red: 633 nm (2% laser power). Results are as follows: Figure 6 As shown, the photostability of compound IPTM is compared with that of commercially available membrane dyes (Dil and Cell mask) and nuclear dyes (PI and DAPI). A549 cells labeled with compound ITPM ​​maintained significantly high fluorescence intensity after 60 consecutive laser scans, with the fluorescence signal gradually increasing. However, A549 cells labeled with commercial dyes showed varying degrees of fluorescence signal attenuation under the same laser scanning conditions, indicating that compound ITPM ​​has superior photostability.

[0102] (5) Comparison of imaging performance between ITPM ​​and existing commercial membrane dyes (Dil and Cell mask):

[0103] A549 cells were incubated with ITPM ​​and commercial membrane dyes for different times (30 s, 5 min, 15 min, 0.5 h, 1 h, 3 h, and 5 h). The incubation concentrations were as follows (cell culture medium or PBS containing the corresponding dye concentrations): ITPM: 10 μM; Dil: 10 μM; Cell mask deep red: 5 μg / mL; PI: 10 μM; DAPI: 1 μM. After staining, A549 cells in the commercial dye group were washed three times with PBS, while those in the ITPM ​​group required no washing. Fluorescence imaging was performed using a confocal microscope (Zeiss LSM710 laser scanning confocal microscope). Results are shown below. Figure 7 As shown, compared with the staining effect of commercial membrane dyes, ITPM ​​staining can achieve clear fluorescence imaging of the cell membrane in just 30 seconds. Furthermore, as the incubation time increases, most of the dye remains on the cell membrane, enabling stable imaging. In contrast, existing commercial dyes require longer staining times, and as the incubation time increases, more dye molecules enter the cell, resulting in poorer cell membrane imaging.

[0104] (6) The staining effect of existing commercial membrane dyes (Dil and Cell mask) on dead cells:

[0105] For imaging of dead cells: A549 tumor cells were fixed with pre-chilled methanol for 10 minutes and washed three times with PBS to obtain dead cells. Dead / live A549 cells were incubated with commercial dyes (cell culture medium or PBS containing the corresponding dye concentration). The incubation conditions were as follows: Dil: 10 μM, 30 minutes; Cell mask deep red: 5 μg / mL, 30 minutes. After staining, dead / live A549 cells were washed three times with PBS. Fluorescence imaging was performed using a confocal microscope (Zeiss LSM710 laser scanning confocal microscope). The results are as follows. Figure 8 As shown, the staining effect of commercial membrane dyes on dead cells is such that neither Cell Mask nor Dil can specifically image the cell nucleus of dead cells, and they do not have dual targeting of the cell membrane and cell nucleus.

[0106] Example 6: Compound ITPM ​​enables specific dual-target imaging of different cells.

[0107] (1) The compound ITPM ​​can perform specific dual-target fluorescence imaging on different tumor cells:

[0108] Tumor cells of different types and origins (HeLa cells, MCF-7 cells, T47D cells, A549 cells, Caco2 cells, HT29 cells, MG63 cells, and B16 cells, etc.) were incubated for 2 minutes in cell culture medium containing 10 μM ITPM ​​or PBS. For imaging of dead cells: tumor cells were fixed with pre-chilled methanol for 10 minutes, washed three times with PBS, and then stained with ITPM ​​for 2 minutes. Imaging was performed using a laser scanning confocal microscope (FV3000, Olympus) at 405 nm and 0.1% laser power. Emission filter: 550-650 nm. Results are as follows. Figure 9-1 As shown, the results indicate that ITPM ​​possesses a unique dual-targeting capability, specifically targeting cell membrane imaging in living cells while simultaneously targeting cell nucleus imaging in dead cells. This result has broad applicability to tumor cells.

[0109] (2) The compound ITPM ​​can perform specific dual-target fluorescence imaging on different normal cells:

[0110] Normal cells of different types and origins (16HBE cells, NCM460 cells, 293T cells, and HVSMC cells, etc.) were incubated for 2 minutes in cell culture medium or PBS containing 10 μM ITPM. For imaging of dead cells: tumor cells were fixed with pre-chilled methanol for 10 minutes, washed three times with PBS, and then stained with ITPM ​​for 2 minutes. Imaging was performed using a laser scanning confocal microscope (FV3000, Olympus) at 405 nm and 0.1% laser power. Emission filter: 550-650 nm. Results are as follows. Figure 9-2 As shown, this demonstrates that ITPM ​​possesses a unique dual-targeting capability, specifically targeting cell membrane imaging in living cells while simultaneously targeting cell nucleus imaging in dead cells. The results are universally applicable to normal cells.

[0111] The positively charged pyridinium terminal portion of ITPM ​​tends to interact with the negative transmembrane potential of the cell membrane through electrostatic interactions and anchors itself to the intact cell membrane of a living cell, further restricting the intramolecular rotation of the molecule and thus enhancing fluorescence emission. When the integrity of the cell membrane is disrupted, the ITPM ​​molecule enters the cell through the ruptured cell membrane gap and anchors itself to the negatively charged cell nucleus, thereby achieving different targeting imaging effects for living and dead cells.

[0112] In summary, ITPM ​​is an excellent AIE fluorescent probe capable of specifically imaging the cell membrane of living cells and the nucleus of dead cells, exhibiting a dual-targeting effect. Based on this dual-targeting characteristic, it can achieve visual differentiation between living and dead cells, and its performance far surpasses that of commercial biological probes in all aspects.

[0113] Example 7: Compound ITPM ​​can be used to quantitatively analyze the efficacy of different clinical chemotherapy drugs on tumor cells.

[0114] Given the superior visual (fluorescence) recognition capabilities of the novel AIT probe ITPM ​​for both live and dead cells, ITPM ​​is the perfect choice for developing into a highly efficient drug sensitivity screening platform. Based on ITPM's rapid, wash-free fluorescence screening and high-throughput, accurate flow cytometry analysis capabilities, precise analysis of tumor cell viability after drug treatment can be achieved, thereby determining the efficacy of chemotherapy drugs. The entire test takes only 5 minutes, including 2 minutes of TIPM staining and 3 minutes of flow cytometry.

[0115] The specific steps are as follows:

[0116] (1) Tumor cells (taking A549 cells as an example) were seeded on a sterile 6-well plate and incubated overnight (37°C, 21% O2 and 5% CO2);

[0117] (2) Commonly used clinical chemotherapy drugs were pre-diluted to the corresponding working concentration (diluted in cell culture medium) and incubated with tumor cells in a cell culture incubator at 37°C for 24 hours; after incubation, the cells were washed 3 times with PBS; the cells were digested with trypsin (trypsin-EDTA (0.05%), containing phenol red; Gibco) to obtain cell suspension.

[0118] The drug information is as follows (the drug is dissolved in DMSO):

[0119] Lonidamine: 200 μM;

[0120] Cisplatin: 200μM;

[0121] Rapamycin: 20 μM;

[0122] Paclitaxel: 20μM;

[0123] Erlotinib: 20 μM;

[0124] Verapamil: 200μM;

[0125] Pemetrexed: 100μM;

[0126] Vincristine: 100μM;

[0127] Gemcitabine: 20 μM;

[0128] (3) Treat the cells in (2) with the compound ITPM ​​for 2 minutes;

[0129] (4) Rapid and wash-free fluorescent screening of cell viability was performed by flow cytometry (BD FACS AriaⅢ).

[0130] Drug sensitivity tests were performed on a variety of commonly used clinical chemotherapy drugs using this method. The results are as follows: Figure 10-1 and Figure 10-2 As shown, ITPM ​​can precisely quantify the ratio of live to dead cells corresponding to each drug treatment, thereby quickly and accurately reporting the efficacy of different chemotherapy drugs on A549 tumor cells. Figure 10-2 The corresponding statistical histogram for flow cytometry provides a more intuitive view of the drug sensitivity test results for different drugs: high red bars indicate high mortality rates, which correspond to high drug sensitivity and effective anticancer effects, and vice versa.

[0131] It is evident that ITPM ​​can be used to quantitatively analyze the efficacy of different clinical chemotherapy drugs on tumor cells, providing a new fluorescence detection method to further guide clinicians in selecting the best treatment drugs for cancer.

[0132] Example 8: ITPM ​​can rapidly determine the drug resistance status of cancer cells against anti-tumor drugs.

[0133] Accurate and real-time assessment of cancer cell resistance to drugs can guide clinical treatment plans and dosage regimens. Taking non-small cell lung cancer (NSCLC) as an example, resistance to cisplatin and paclitaxel is common in clinical lung cancer cases, making it crucial to rapidly determine a patient's resistance / sensitivity to the chemotherapy drugs cisplatin or paclitaxel. Using the aforementioned developed ITPM, sensitive NSCLC cell lines A549 and resistant NSCLC cell lines A549 / DDP (cisplatin-resistant) and A549 / TAX (paclitaxel-resistant) can be used to simulate the drug sensitivity of different tumor cells. Cisplatin and paclitaxel were used to treat the corresponding normal and resistant A549 cells, respectively, and their respective efficacy was compared to assess cellular drug sensitivity.

[0134] A549 / DDP (cisplatin-resistant) and A549 / TAX (paclitaxel-resistant) cells were cultured in DMEM (Gibco) complete medium containing 10% fetal bovine serum (Invitrogen) supplemented with 1 μg / mL cisplatin or paclitaxel.

[0135] (1) A549 cells, A549 / DDP cells and A549 / TAX cells were seeded on sterile 6-well plates and incubated overnight (37°C, 21% O2 and 5% CO2);

[0136] (2) Cisplatin and paclitaxel were pre-diluted to their respective working concentrations (diluted in cell culture medium: Cisplatin: 200 μM; Paclitaxel: 20 μM) and incubated with A549 cells, A549 / DDP cells and A549 / TAX cells at 37°C for 24 h. After incubation, the cells were washed three times with PBS and digested with trypsin to obtain cell suspension.

[0137] (3) Treat the cells in (2) with the compound ITPM ​​for 2 minutes;

[0138] (4) Rapid and wash-free fluorescent screening of cell viability was performed by flow cytometry (BD FACS AriaⅢ).

[0139] The results are as follows Figure 11 As shown, the cell death rate of normal A549 cells after treatment with cisplatin or paclitaxel is significantly higher than that of drug-resistant cell lines. ITPM ​​can precisely quantify the live / dead cell ratio of corresponding tumor cells after cisplatin and paclitaxel treatment, thus rapidly and accurately reporting the efficacy of cisplatin and paclitaxel treatment on tumor cells with different levels of resistance / sensitivity. This method provides a novel fluorescence detection approach for clinically assessing the sensitivity of tumors to chemotherapeutic drugs.

[0140] Example 9: ITPM ​​possesses two-photon properties, enabling two-photon imaging of tissues.

[0141] The compound ITPM ​​has excellent AIE properties, exhibiting outstanding fluorescence imaging capabilities not only at the cellular level but also at the tissue level.

[0142] ITPM tissue two-photon imaging steps:

[0143] BALB / c nude mice (4 weeks old, 12-15g) were purchased from Nanfang Hospital, Southern Medical University, China. The mice were then packed with pre-selective nitrogen at a density of 2×10⁻⁶. 6 150 μL of A549 or A549 / DDP cells were subcutaneously injected into each nude mouse, causing subcutaneous tumors to grow to approximately 500 mm. 3 Mice were anesthetized and euthanized; mouse tissues (including tumor tissue, heart, liver, spleen, lungs, and kidneys) were carefully separated; after washing twice with PBS, the tissues were fixed overnight with 4% paraformaldehyde. The corresponding tissues were cut into small pieces of approximately 2 mm, stained with 10 μM ITPM ​​for 3 minutes, and imaged using a two-photon microscope (Olympus FV1200MPE). Two-photon excitation wavelength: 900 nm. Emission filter: 575-630 nm. 3D two-photon images (Z-stack) were reconstructed using Imaris 9.0.1.

[0144] The results are as follows Figure 12As shown, 900nm near-infrared pulsed laser was used for ex vivo two-photon imaging of tumor tissue. (Compared to...) Figure 12 Compared to single-photon imaging in A and B, the two-photon imaging results of IPTM ( Figure 12 C and D) exhibit higher resolution, signal-to-noise ratio, and deeper tissue penetration; in addition, as Figure 12 As shown in E-12H, ITPM ​​can also display ultra-clear two-photon tissue structures and local details of different tissues such as the liver, kidneys, heart, and spleen through reconstructed 3D images. These results demonstrate that ITPM ​​possesses excellent two-photon imaging capabilities for deep tissues.

[0145] Example 10: ITPM ​​exhibits excellent in vivo fluorescence imaging capabilities.

[0146] In addition to its outstanding two-photon imaging capabilities in deep tissues, ITPM's unique AIE luminescence advantage enables long-term in vivo imaging in living animals.

[0147] ITPM small animal in vivo imaging procedure: BALB / c nude mice were purchased from Nanfang Hospital of Southern Medical University, China (4 weeks old, 12-15g). A density of 2×10⁻⁶ was used for in vivo imaging. 6 150 μL of A549 or A549 / DDP cells were subcutaneously injected into each nude mouse to induce subcutaneous tumor growth to a certain size. Fluorescence imaging of the tumor site in the tumor-bearing nude mice at different time points after intratumoral injection of ITPM ​​(20 μM) was tested using a multispectral small animal imaging system (FX Pro, Bruker). Excitation wavelength was 450 nm, and fluorescence was collected at 600 nm.

[0148] The results are as follows Figure 13 As shown, a strong fluorescence signal with a high signal-to-noise ratio can be detected at the tumor site 10 minutes after intratumoral injection in a tumor-bearing mouse model, and the fluorescence signal can last for up to 144 hours, indicating that ITPM ​​has significant long-term in vivo imaging capabilities. ITPM ​​can be applied to in vivo imaging-guided PDT for the treatment of various tumors (including drug-resistant tumors).

[0149] Example 11: Compound ITPM ​​exhibits excellent PDT therapeutic effects.

[0150] (1) ITPM-labeled tumor cells can produce a large amount of ROS after light exposure:

[0151] A549 cells and drug-resistant A549 / DDP cells were incubated with 10 μM ITPM ​​for 2 minutes; washed three times with PBS; and then incubated under an LED white light (4.2 mW / cm²). 2Irradiate the cells for 30 minutes; then incubate them in DMEM complete medium containing 5 μM CFH-DA for 30 minutes (37°C); wash twice with pre-cooled PBS, digest the cells with trypsin, and measure the cell fluorescence intensity by flow cytometry.

[0152] The results are as follows Figure 14-1 As shown, ITPM ​​can effectively generate a large amount of ROS in both normal A549 tumor cells and drug-resistant A549 / DDP (cisplatin-resistant) tumor cells after light irradiation. Due to ITPM's dual targeting capability, it can not only generate ROS at the cell membrane to damage the cell membrane, but also severely damage the cell nuclear structure through photoinduced reactive oxygen species (ROS).

[0153] (2) The MTT trial showed that ITPM ​​has excellent PDT therapeutic effects:

[0154] Cell viability was assessed using the MTT assay to evaluate the efficacy of ITPM ​​in PDT treatment of different tumor cell types (including drug-resistant cells). A549 cells and A549 / DDP (cisplatin-resistant) cells were used as examples.

[0155] Cells were seeded at a density of 8000 cells per well in two 96-well plates and cultured overnight (in complete DMEM medium). One group was the Dark group (control) without light treatment, and the other was the +L light treatment group. The medium was then replaced with 200 μL of fresh DMEM medium containing different concentrations of ITPM ​​(0, 5, 10, 20, and 30 μM). After incubation for 2 minutes, the light-treated groups were exposed to white light (4.2 mW / cm²). 2 Irradiate the plates under light for 30 minutes, while a separate Dark group plate is placed in the dark as a control. After treatment, incubate the plates in a cell culture incubator. After 24 hours, incubate with DMEM medium containing 10% MTT solution for 2 hours, then discard the medium. Add 100 μL of DMSO solution, shake and incubate for 15 minutes, then analyze the results using a microplate reader (Varioskan™ LUX multimode microplate reader). The absorbance is 595 nm.

[0156] MTT experiment ( Figure 14-2 The results showed that ITPM ​​exhibited highly efficient photodynamic therapy (PDT) killing effects on A549 tumor cells and drug-resistant A549 / DDP tumor cells under white light irradiation. Specifically, after light irradiation, ITPM ​​effectively generated a large amount of reactive oxygen species (ROS). Due to its dual targeting capability, ITPM ​​not only generated ROS at the cell membrane to disrupt the cell membrane, but also severely damaged the nuclear structure through photoinduced reactive oxygen species (ROS), causing the death of a large number of tumor cells, demonstrating excellent PDT therapeutic efficacy. ITPM ​​can serve as an excellent photosensitizer for PDT therapy.

[0157] Example 12: Compound ITPM ​​can achieve self-reporting of cell death levels during self-PDT.

[0158] ITPM possesses a unique dual-targeting capability, specifically targeting the cell membrane of living cells while simultaneously anchoring to the nucleus of dead cells. This enables precise analysis of tumor cell viability after drug treatment, thereby determining the efficacy of chemotherapy drugs. Furthermore, ITPM ​​exhibits rapid and efficient ROS generation, making it an excellent photosensitizer for PDT (photodynamic therapy). The dual-targeting characteristics of ITPM ​​allow it to directly report the degree of cancer cell death during its own photodynamic therapy, achieving self-reported PDT.

[0159] ITPM Self-Reporting PDT:

[0160] (1) A549 cells were labeled with 10 μM ITPM ​​for 2 minutes. After washing three times with PBS, time-series images were continuously captured using a laser scanning confocal microscope (FV3000, Olympus). The cells were continuously irradiated with a 405 nm laser with an emission wavelength of 550-650 nm.

[0161] (2) Seed cells at a density of 20,000 cells per well in 6-well plates or confocal dishes and culture overnight (in DMEM complete medium); replace the medium with DMEM containing 10 μM ITPM ​​and incubate for 2 minutes; expose the cells to white light (4.2 mW / cm²). 2 Cells were continuously irradiated for 0, 15, or 30 minutes, respectively. After irradiation, the cells were washed twice with PBS. Cells were stained with 10 μM PI for 10 minutes and then washed twice with PBS. Cells in confocal dishes were imaged under a confocal microscope, while cells in 6-well plates were digested with trypsin and analyzed for fluorescence by flow cytometry.

[0162] In this method, normal A549 cells stained with ITPM ​​were used as a schematic template. Real-time confocal imaging of single cells was performed under continuous 405nm laser irradiation. The results are as follows: Figure 15-1 As shown, the dynamic migration process of ITPM ​​during PDT treatment is clearly presented. At the beginning of PDT, ITPM ​​is located on the cell membrane (living cell). With prolonged light exposure, ITPM ​​continuously generates ROS to exert its PDT effect, leading to gradual disruption of the cell membrane and the formation of numerous vesicles. Subsequently, ITPM ​​enters the cell and gradually targets the cell nucleus, where it exhibits progressively increasing fluorescence. Finally, when most of the ITPM ​​targets the cell nucleus, the fluorescence intensity in the nucleus reaches its maximum. Simultaneously, ITPM ​​continues to exert its PDT effect within the cell nucleus, powerfully destroying tumor cells.

[0163] Furthermore, the results were quantitatively evaluated using a self-reported PDT process combining co-localization fluorescence imaging and flow cytometry ITPM, such as... Figure 15-2As shown, before white light irradiation, ITPM ​​anchored to the cell membranes of live cells. Flow cytometry analysis yielded consistent results: the proportion of live cells was almost 100%. After 15 minutes of irradiation, fluorescence images showed that both the cell membrane and nucleus were stained, consistent with the increased proportion of live and dead cells observed in flow cytometry analysis, indicating that ROS production led to the death of some cancer cells. After 30 minutes of irradiation, ITPM ​​was almost entirely fixed to the cell nuclei, and the PI staining and flow cytometry results were highly consistent.

[0164] In summary, ITPM ​​can achieve highly efficient PDT for cancer cells, and ITPM ​​can self-report the entire PDT process in situ through spatiotemporally resolved fluorescence migration from the cell membrane to the cell nucleus.

[0165] Example 13: ITPM ​​demonstrates highly effective tumor PDT treatment.

[0166] The phototherapy effect of ITPM ​​on tumors (including drug-resistant tumors) was evaluated using mouse tumor-bearing models. Two mouse models were used: one bearing A549 tumor cells and the other bearing A549 / DDP cisplatin-resistant tumor cells. BALB / c nude mice (4 weeks old, 12-15g) were purchased from Nanfang Hospital, Southern Medical University, China. A density of 2×10⁻⁶ cells was used. 6 150 μL of A549 or A549 / DDP cells were subcutaneously injected into each nude mouse, causing subcutaneous tumors to grow to approximately 200 mm. 3 BALB / c tumor-bearing nude mice were randomly divided into four groups, and either PBS or ITPM ​​(10mM, 100μL / 200mm) was injected into the tumor, respectively. 3 (Tumors) In the light-treated group, mice were exposed to light for 30 minutes every two days. Tumor volume and body weight were monitored every three days. Tumor volume was defined as follows: Volume = (Tumor length) × (Tumor width)² / 2.

[0167] The results are as follows Figure 16 As shown, neither cisplatin-sensitive A549 tumors nor cisplatin-resistant A549 / DDP tumors showed any significant inhibitory effect on tumor growth from white light or ITPM ​​alone. However, white light irradiation in the ITPM ​​group (ITPM+L group) exhibited a significant inhibitory effect on tumor growth. ITPM ​​demonstrates highly effective tumor phototherapy (PDT).

Claims

1. A fluorescent compound exhibiting aggregation-induced emission properties, characterized in that, Its chemical structural formula is shown in formula (II): (II)。 2. The use of the fluorescent compound with aggregation-induced emission properties as described in claim 1 in the preparation of cell staining agents.

3. The application according to claim 2, characterized in that, The cell staining agent is used to distinguish between live and dead cells.

4. The application according to claim 2, characterized in that, The cell staining agent is used to evaluate the drug's efficacy on cells.

5. The use of the fluorescent compound with aggregation-induced emission properties as described in claim 1 in the preparation of two-photon tissue imaging agents.

6. The use of the fluorescent compound with aggregation-induced emission properties as described in claim 1 in the preparation of photosensitizers with dual targeting functions of cell membrane and cell nucleus.

7. The use of the fluorescent compound with aggregation-induced emission properties as described in claim 1 in the preparation of antitumor drugs.

Images