Mitochondria-targeted calix [4] arene supramolecular photosensitizer as well as preparation method and application thereof

Abstract

The invention discloses a mitochondria-targeted calix [4] arene supramolecular photosensitizer as well as a preparation method and application thereof. The calix [4] arene supramolecular photosensitizer is shown as a formula (I). According to the invention, a calix [4] arene rigid skeleton is taken as a core, a mitochondrial targeting fluorophore is covalently modified, and a novel calix [4] arene supramolecular photosensitizer integrating real-time fluorescence tracing, mitochondrial precise targeting and efficient I-type reactive oxygen species (ROS) generation is constructed, so that normal tissues and tumor tissues can be accurately distinguished, and accurate diagnosis and treatment are provided clinically. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a mitochondrion-targeted calix[4]arene supramolecular photosensitizer, a preparation method thereof and an application thereof. Background Art

[0002] As one of the major public health problems globally, the incidence and mortality of cancer have been continuously rising, posing a serious threat to human health. Traditional treatment and diagnosis processes are often disjointed, lacking real-time visualization guidance, which limits the accuracy and effectiveness of treatment. Therefore, developing a diagnostic and therapeutic integrated platform that can simultaneously achieve diagnosis and treatment has important clinical significance.

[0003] Among various diagnostic and therapeutic integrated technologies, the strategy based on fluorescence imaging and photodynamic therapy (PDT) has attracted much attention due to its unique advantages. Fluorescence imaging technology has the advantages of high sensitivity, high resolution, good real-time performance, no ionizing radiation, etc., and can achieve efficient labeling and visualization of tumor cells and tissues. Photodynamic therapy is a new non-invasive treatment method that uses photosensitizers to generate cytotoxic substances such as reactive oxygen species (ROS) under specific wavelength light irradiation to kill tumor cells. Combining fluorescence imaging with PDT can not only achieve accurate diagnosis and localization of tumors, but also perform precise treatment under image guidance, which is an ideal strategy for realizing tumor diagnostic and therapeutic integration. The supramolecular photodynamic therapy based on nanocarriers has significant advantages in cancer diagnostic and therapeutic integration. Nanocarriers can improve the water solubility and stability of photosensitizers, enhance tumor targeting and reduce side effects, while realizing intelligent response release and improving the PDT treatment efficiency. In addition, the nanoplatform can integrate imaging technologies such as fluorescence and photoacoustics to achieve precise diagnosis and treatment and enhance the anti-cancer effect. However, the application of nanocarriers is restricted by three key limitations: (1) The average tumor accumulation rate of nano-drugs is only 0.7% of the injection dose, significantly affecting the curative effect; (2) The drug loading efficiency of the carrier is low, and it is difficult to achieve high-dose precise delivery; (3) Drug leakage during the preparation and storage processes leads to batch-to-batch differences, hindering standardized production. Obviously, there is an urgent need to develop a single-molecule diagnostic and therapeutic integrated supramolecular photosensitizer to construct a new supramolecular photosensitizer that integrates real-time fluorescence tracing, precise mitochondrion targeting and efficient type I reactive oxygen species (ROS) generation, aiming to break through the inherent defects of traditional nanoplatforms, so as to provide accurate diagnosis and treatment for clinical practice. Summary of the Invention

[0004] One of the objectives of the present invention is to provide a single-molecule diagnosis and treatment integrated supramolecular photosensitizer. With a calix[4]arene rigid framework as the core, covalently modified with a mitochondrial-targeted fluorescent group, a novel calix[4]arene supramolecular photosensitizer integrating real-time fluorescence tracing, precise mitochondrial targeting, and efficient type I reactive oxygen species (ROS) generation is constructed, enabling it to accurately distinguish normal tissues from tumor tissues, thereby providing accurate diagnosis and treatment for clinical applications.

[0005] Another objective of the present invention is to provide a preparation method for the above-mentioned calix[4]arene supramolecular photosensitizer.

[0006] A third objective of the present invention is to provide the pharmaceutical use of the above-mentioned calix[4]arene supramolecular photosensitizer in the preparation of fluorescence imaging targeting tumor cell mitochondria and exerting photodynamic therapy effects, especially fluorescence imaging and / or treatment of tumor cells and tissues in vitro and in vivo.

[0007] The objectives of the present invention are achieved by the following technical means:

[0008] A mitochondrial-targeted calix[4]arene supramolecular photosensitizer, as shown in formula (I):

[0009]

[0010] Among them, R is 1,2,3,3-tetramethyl-3H-indolium iodide, 1,1,2,3-tetramethyl-1H-benzo[e]indolium iodide, 3-ethyl-1,1,2-trimethyl-3H-indolium iodide, or 3-ethyl-1,1,2-trimethyl-1H-benzo[e]indolium iodide.

[0011] In some specific examples, R is selected from

[0012] In some specific examples, the mitochondrial-targeted calix[4]arene supramolecular photosensitizer is as shown in any of the following:

[0013]

[0014] The present invention also provides a preparation method for the mitochondrial-targeted calix[4]arene supramolecular photosensitizer shown in formula (I),

[0015]

[0016] wherein the definition of R is as described above.

[0017] In the present invention, for the preparation method of the calix[4]arene supramolecular photosensitizers C4AZ1 and C4AZ2 with mitochondrial targeting function, it includes the following steps:

[0018] (1) Under the conditions of trifluoroacetic acid or sulfuric acid, the compound tetraester removed reacts with hexamethylenetetramine at 60-90 °C to prepare compound 1;

[0019] (2) In the presence of sodium acetate or sodium ethoxide, compound 1 and 1,2,3,3-tetramethyl-3H-indole iodide react at 60-90 °C to prepare compound C4AZ1;

[0020] (3) In the presence of sodium acetate or sodium ethoxide, compound 1 and 3-ethyl-1,1,2-trimethyl-1H-benzo[e]indole iodide react at 60-90 °C to prepare compound C4AZ2.

[0021] For the present invention, in step (1), the reaction temperature is preferably 70 °C; the molar ratio of the compound tetraester removed to hexamethylenetetramine is 1:10.0-12.0, preferably 1:10.0.

[0022] For the present invention, in step (2), the reaction temperature is preferably 70 °C; the molar ratio of compound 1 to 1,2,3,3-tetramethyl-3H-indole iodide is 1:2.0-4.0, preferably 1:3.0; the molar ratio of compound 1 to sodium acetate is 1:2.0-4.0, preferably 1:3.0.

[0023] For the present invention, in step (3), the reaction temperature is preferably 70 °C; the molar ratio of compound 1 to 1,1,2,3-tetramethyl-1H-benzo[e]indole iodide is 1:2.0-4.0, preferably 1:3.0; the molar ratio of compound 1 to sodium acetate is 1:2.0-4.0, preferably 1:3.0.

[0024] The present invention also provides the application of the mitochondrion-targeted calix[4]arene supramolecular photosensitizer in the preparation of tumor detection reagents. Specifically, it is used in the preparation of fluorescence imaging diagnostic drugs targeting tumor cells; more specifically, it is used in the preparation of fluorescence imaging diagnostic drugs targeting the mitochondria of tumor cells.

[0025] The present invention also provides the application of the mitochondrion-targeted calix[4]arene supramolecular photosensitizer in the preparation of photodynamic tumor treatment drugs.

[0026] The present invention also provides the application of the combination of the mitochondrion-targeted calix[4]arene supramolecular photosensitizer and light irradiation in the preparation of drugs for treating tumors.

[0027] The tumors of the present invention can be human hepatocellular carcinoma cells (HepG2); human colorectal cancer cells (SW480); mouse mammary tumor cells (4T1), preferably HepG2.

[0028] Adopting the technical solution of the present invention, the mitochondrion-targeted calix[4]arene supramolecular photosensitizer has the following advantages:

[0029] (1) Molecular-level diagnosis and treatment integration: Fluorescence imaging, mitochondrion targeting, and type I ROS photodynamic therapy are all integrated into a single molecule, avoiding the preparation complexity brought by traditional nanocarriers and the biosafety problems caused by potential drug leakage.

[0030] (2) Advantages of the calixarene core skeleton: The unique structure of calixarene not only provides an ideal platform for covalent modification but also can effectively fix functional groups, ensuring the stability and high efficiency of the overall structure.

[0031] (3) Precise mitochondrion targeting: By introducing a mitochondrion-targeting fluorescent group, accurate localization of mitochondria can be achieved, enhancing the enrichment effect of the photosensitizer in tumor cells.

[0032] (4) Real-time fluorescence imaging ability: The fluorescent group enables the supramolecular photosensitizer to perform real-time imaging in vivo, contributing to the dynamic monitoring of drug distribution and the treatment process.

[0033] (5) Efficient generation of type I ROS: It does not rely on high concentrations of oxygen and can continuously generate a large amount of ROS even in the hypoxic environment of tumors, thereby overcoming the problem of poor treatment effects caused by tumor hypoxia.

[0034] (6) Significant anti-tumor effect: The calix[4]arene supramolecular photosensitizer can target the mitochondria of tumor cells and generate ROS to kill tumor cells after irradiation with excitation light. Description of the Drawings

[0035] Figure 1 are the (A) ultraviolet-visible absorption spectra and (B) fluorescence emission spectra of compounds C4AZ1 and C4AZ2 in DMSO;

[0036] Figure 2 are the normalized absorbance (A / A0) and integral area ratio (Area / Area0) of (A) C4AZ1 and (B) C4AZ2 after white light (25 mW / cm 2 ) irradiation at different times;

[0037] Figure 3 are the changes in the fluorescence intensity (I / I0 - 1) of (A) DCFH-DA at 525 nm; the change in the absorbance (A / A0) of (B) ABDA at 378 nm; the change in the fluorescence intensity (I / I0 - 1) of (C) DHR123 at 525 nm; the change in the fluorescence intensity (I / I0 - 1) of (D) HPF at 515 nm;

[0038] Figure 4Construction of a tumor cell hypoxia model. (A) Intracellular hypoxia confirmation was performed in HepG2 cells using ROS-ID as an anaerobic probe. (B) Quantitative analysis of the mean fluorescence intensity;

[0039] Figure 5 (A) Flow cytometry analysis of the uptake of C4AZ1 (2.5 μM) by HepG2 cells incubated for different times. (B) Quantitative analysis of the fluorescence intensity. (C) CLSM images of HepG2 cells after incubation with C4AZ1 (2.5 μM) for different times;

[0040] Figure 6 (A) Flow cytometry analysis of the uptake of C4AZ1 (2.5 μM) by HUVEC cells incubated for different times. (B) Quantitative analysis of the fluorescence intensity. (C) CLSM images of HUVEC cells after incubation with C4AZ1 (2.5 μM) for different times;

[0041] Figure 7 Effects of different concentrations of C4AZ1 and C4AZ2 with or without light treatment on the viability of (A) NIH-3T3, (B) HUVEC, and (C) LO2 cells;

[0042] Figure 8 Effects of different concentrations of C4AZ1 and C4AZ2 with or without light treatment on the viability of (A) HepG2, (B) 4T1, and (C) SW480 cells under normoxic conditions;

[0043] Figure 9 Effects of different concentrations of C4AZ1 and C4AZ2 with or without light treatment on the viability of (A) HepG2, (B) 4T1, and (C) SW480 cells under hypoxic conditions;

[0044] Figure 10 Cellular mitochondrial co-localization images. Laser confocal microscopy imaging of HepG2 and 4T1 cells and the fluorescence intensity correlation coefficient map of C4AZ1 and Mito Tracker Green in the presence of C4AZ1 and Mito Tracker Green;

[0045] Figure 11 ROS imaging and fluorescence intensity of HepG2 cells pretreated with C4AZ1 after white light irradiation under normoxic and hypoxic environments;

[0046] Figure 12 Laser confocal microscopy imaging and fluorescence intensity ratio of HepG2 cells treated with C4AZ1 under normoxic or hypoxic environments with or without white light irradiation after staining with JC-1;

[0047] Figure 13 Images of HepG2 cells treated with C4AZ1 under normoxic or hypoxic conditions, with or without white light irradiation, after staining with Calcein-AM / PI and observed under an inverted fluorescence microscope;

[0048] Figure 14 Analysis of apoptosis of HepG2 cells treated with C4AZ1 under normoxic or hypoxic conditions, with or without white light irradiation, by flow cytometry after staining with Annexin V-FITC / PI;

[0049] Figure 15 Effect of different concentrations of C4AZ1 on the cell cycle of HepG2 cells under white light irradiation;

[0050] Figure 16 Fluorescence distribution imaging of C4AZ1 in HepG2 tumor-bearing mice. (A) In vivo fluorescence imaging of HepG2 tumor-bearing mice at different time points after tail vein injection of C4AZ1 (200 μM, 100 μL). (B) Fluorescence intensity change curve in the tumor area. (C) Ex vivo fluorescence imaging of major organs and tumors 24 h after tail vein injection. (D) Quantitative analysis of ex vivo tissue fluorescence intensity;

[0051] Figure 17 Antitumor efficacy of each group after 14 days of different treatments. (A) Tumor volume change curve during 14 days, (B) Representative tumor pictures, (C) Tumor weight pictures after 14 days of treatment.

[0052] Figure 18 H&E, TUNEL, and Ki67 staining images of tumors;

[0053] Figure 19 (A) Erythrocyte hemolysis test of different concentrations of C4AZ1. (B) Body weight change curve of mice in each group during 14 days after different treatments;

[0054] Figure 20 H&E staining pictures of major organs (heart, liver, spleen, lung, and kidney) of mice in different groups after 14 days of treatment;

[0055] Figure 21 Blood biochemical indexes 14 days after tail vein injection of C4AZ1.

[0056] Figure 22 Blood routine indexes 14 days after tail vein injection of C4AZ1. Specific implementation manners

[0057] The following further describes the content of the present invention in conjunction with embodiments, but the protection scope of the present invention is not limited to the following embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the protection scope of the present invention.

[0058] In the following experimental methods of the embodiments, unless otherwise specified, they are all conventional methods. Unless otherwise specified, the test materials used in the following embodiments are all obtained from regular biochemical reagent stores.

[0059] Example 1 Synthesis of Compounds C4AZ1 and C4AZ2

[0060]

[0061] Synthesis of Compound 1

[0062] Dissolve de-tetraester (596 mg, 1 mmol) and hexamine (1.4 g, 10 mmol) in 10 mL of trifluoroacetic acid. Under nitrogen protection, stir and heat to 70 °C, and reflux for 4 h. After monitoring the reaction by TLC and the reaction is completed, quench the reaction with water to form a white precipitate. Ultrasonic to form a suspension, filter by suction. After the filter cake is dissolved in dichloromethane, it is dried with anhydrous sodium sulfate and concentrated under reduced pressure. The crude product is purified by flash column chromatography (eluent: V DCM / V MeOH = 200:1) to obtain a light white solid powder of Compound 1 (300 mg), yield: 70%. The specific structural characterization is as follows:

[0063] 1 H NMR (400 MHz, Chloroform-d) δ 9.77 (s, 2H), 8.69 (s, 2H), 7.61 (s, 4H), 6.97 (d, J = 8.0 Hz, 4H), 6.81 (t, J = 8.0 Hz, 2H), 4.72 (s, 4H), 4.46 (d, J = 12.0 Hz, 4H), 4.35 (q, J = 8.0 Hz, 4H), 3.50 (d, J = 12.0 Hz, 4H), 1.36 (t, J = 4.0 Hz, 6H).

[0064] 13 C NMR (101 MHz, DMSO-D6) δ 191.0, 168.8, 158.7, 152.3, 132.7, 131.0, 129.4, 128.5, 125.7, 72.4, 61.1, 48.6, 30.3, 14.0.

[0065] Synthesis of Compound C4AZ1

[0066] Compound 1 (217 mg, 0.33 mmol), 1,2,3,3-tetramethyl-3H-indolium iodide (301 mg, 1 mmol), and sodium acetate (82 mg, 1 mmol) were dissolved in 10 mL of anhydrous ethanol. Under a nitrogen atmosphere, the mixture was heated to 70 °C and refluxed with stirring overnight. After monitoring the reaction by TLC until completion, the solvent was removed under reduced pressure. The crude product was purified by flash column chromatography (dichloromethane:methanol = 100:1, v / v) to give 682 mg of a red-gold solid with a yield of 56%. The specific structural characterization is as follows:

[0067] 1 H NMR (400 MHz, Chloroform-d) δ 7.86 (s, 2H), 7.62 (d, J = 12.0 Hz, 2H), 7.37 - 7.27 (m, 6H), 7.21 (s, 2H), 7.11 (t, J = 8.0 Hz, 3H), 7.00 (d, J = 8.0 Hz, 2H), 6.94 (d, J = 8.0 Hz, 2H), 6.86 (d, J = 8.0 Hz, 2H), 6.73 (t, J = 8.0 Hz, 2H), 6.31 (d, J = 16.0 Hz, 2H), 4.57 (t, J = 4.0 Hz, 4H), 4.48 (q, J = 8.0 Hz, 5H), 4.16 (t, J = 12.0 Hz, 5H), 3.58 (s, 6H), 3.23 (d, J = 12.0 Hz, 2H), 3.06 (d, J = 12.0 Hz, 2H), 1.71 (s, 12H), 1.40 (t, J = 8.0 Hz, 6H).

[0068] 13 C NMR (101 MHz, Chloroform-d) δ 180.8, 170.2, 167.9, 149.8, 144.7, 141.9, 138.5, 137.1, 135.9, 133.8, 131.3, 128.0, 127.7, 127.0, 126.6, 123.5, 122.0, 120.5, 120.2, 107.7, 94.3, 69.5, 60.9, 52.1, 46.6, 29.8, 29.6, 29.2, 27.0, 26.9, 12.8.

[0069] HRMS m / z calcd for C 62 H 64 N2O8 2+ [M + K]+ 501.7145, found 501.7078.

[0070] Synthesis of Compound C4AZ2

[0071] Compound 1 (217 mg, 0.33 mmol), 3-ethyl-1,1,2-trimethyl-1H-benzo[e]indolium iodide (365 mg, 1 mmol), and sodium acetate (82 mg, 1 mmol) were dissolved in 10 mL of anhydrous ethanol. Under a nitrogen atmosphere, the mixture was heated to 70 °C and refluxed with stirring overnight. After monitoring the reaction by TLC until completion, the solvent was removed under reduced pressure. The crude product was purified by flash column chromatography (dichloromethane:methanol = 100:1, v / v) to obtain 700 mg of a purple-gold solid with a yield of 51%. The specific structural characterization is as follows:

[0072] 1 H NMR (400 MHz, DMSO-d6) δ 8.16 (d, J = 8.0 Hz, 4H), 8.01 - 7.94 (m, 4H), 7.88 (d, J = 16.0 Hz, 2H), 7.58 (t, J = 8.0 Hz, 7H), 7.41 (t, J = 4.0 Hz, 2H), 6.98 (d, J = 8.0 Hz, 2H), 6.90 (d, J = 8.0 Hz, 2H), 6.71 (d, J = 8.0 Hz, 2H), 6.57 (d, J = 12.0 Hz, 2H), 4.58 (d, J = 12.0 Hz, 4H), 4.35 (d, J = 8.0 Hz, 4H), 4.31 - 4.23 (m, 4H), 4.14 (t, J = 14.2 Hz, 4H), 3.14 (d, J = 12.0 Hz, 2H), 3.02 (d, J = 12.0 Hz, 2H), 1.91 (s, 12H), 1.31 (t, J = 4.0 Hz, 12H).

[0073] 13 C NMR (126 MHz, CDCl3) δ 181.7, 179.9, 173.9, 168.6, 161.0, 155.7, 152.3, 151.7, 150.5, 139.1, 138.1, 137.7, 133.6, 133.4, 133.3, 131.8, 131.5, 130.7, 130.3, 130.1, 130.1, 128.6, 128.2, 127.7, 127.5, 127.1, 126.1, 125.9, 125.5, 124.9, 122.9, 122.0, 120.9, 111.5, 110.5, 107.8, 97.4, 72.9, 72.4, 64.0, 62.3, 61.5, 53.5, 53.5, 51.0, 43.7, 40.0, 31.8, 30.2, 27.8, 27.3, 14.5, 14.2, 14.1, 14.1, 13.1.

[0074] HRMS m / z calcd for C 72 H73 N2O8 2+ [M + Na]+558.2627, found 558.2302.

[0075] Photophysical Property Tests of Compounds C4AZ1 and C4AZ2 in Example 2

[0076] Ultraviolet-Visible Absorption Spectra and Fluorescence Absorption Spectra: DMSO solutions of 10 μM C4AZ1 and C4AZ2 were prepared respectively, and their ultraviolet absorption spectra and fluorescence spectra were measured. The results are as Figure 1 , in DMSO solution, the absorption spectra of compounds C4AZ1 and C4AZ2 are similar, having a considerable absorption band in the wavelength range of 500 - 600 nm. However, the absorption peak wavelength of C4AZ2 is slightly longer than that of C4AZ1. These remarkable light absorption characteristics are beneficial to enhancing their ROS generation ability. In the DMSO solutions of the two compounds, significant fluorescence is exhibited at wavelengths of 590 nm and 620 nm.

[0077] Determination of Stability: The photostability of C4AZ1 and C4AZ2 in DMSO solution under continuous white light (25 mW / cm 2 ) irradiation was evaluated. By combining ultraviolet-visible absorption spectra and fluorescence emission spectra, their photodegradation kinetics and changes in luminescence performance were analyzed. The normalized absorbance A / A0 and the integral area ratio (Area / Area0) were calculated. The results are as Figure 2 shown. Within 30 min, there were no obvious changes in the UV-VIS absorption spectra and fluorescence intensities of the two compounds, indicating that the compounds described in the present invention have good photostability and can maintain the chemical structure and functional integrity under continuous light irradiation, thereby ensuring the efficient and stable generation of reactive oxygen species in photodynamic therapy to improve the anti-cancer efficacy.

[0078] Reactive Oxygen Species (ROS) Test in Example 3

[0079] Total ROS test: In this study, 2',7'-dichlorodihydrofluorescein (DCFH-DA) was used to detect the ROS generation ability of two molecules. The method is as follows: Weigh DCFH-DA (4.87 mg, 0.01 mmol) and dissolve it in 1.0 mL DMSO to prepare a 10 mM stock solution. Take an appropriate amount of the DCFH-DA stock solution and add a certain volume of 0.01 mol / L sodium hydroxide solution. Incubate the above mixed solution at room temperature for 30 min. After incubation, add an appropriate amount of PBS buffer to dilute the solution to obtain a DCFH solution with a working concentration of 10 μM. Considering that both of these compounds show strong absorption in the visible light region, we used an easily available and harmless white light source to stimulate ROS generation. The experimental groups were: blank control group (PBS + DCFH); experimental group (test molecule + DCFH). Pipette 1 μL of the compound stock solution into 1 mL of the DCFH working solution, mix, and irradiate with a white light source (25 mW / cm2). Detect the fluorescence intensity of the test solution at different time intervals. The excitation wavelength of DCFH is 488 nm, and the emission wavelength is 510 - 550 nm. In this study, the ROS generation abilities of C4AZ1, C4AZ2, and RB were tested.

[0080] Singlet oxygen test: 9,10-Anthracenediyl-bis(methylene)dicarboxylic acid (ABDA) is a commonly used singlet oxygen ( 1 1O2) indicator. After the photosensitizer is irradiated with light, it will release 1 1O2, and ABDA reacts with 1 1O2 and is gradually decomposed, and its absorbance decreases with the increase of 1 1O2. The experimental method is as follows: The preparation and storage methods of the ABDA stock solution are the same as those of DCFH-DA. The experimental groups were: blank control group (PBS + ABDA); experimental group (test molecule + ABDA). Pipette 1 μL of the compound stock solution into 1 mL of the ABDA working solution, mix, and irradiate with a white light source (25 mW / cm 2 2). Detect the ultraviolet-visible absorption spectrum of the test solution at different time intervals. In this study, the 1 1O2 generation abilities of C4AZ1, C4AZ2, and RB were tested.

[0081] Oxygen free radical test: DHR123 was used as an indicator to verify whether a specific molecule can generate superoxide anion (O2 ·- •−), and L-ascorbic acid was used as a scavenger to confirm O2 ·-The existence is as follows. The specific experimental methods are as follows: The preparation and preservation methods of the DHR123 stock solution and working solution are the same as those of DCFH-DA. The experiments were divided into three groups: blank control group (PBS + DHR123); experimental group (test molecule + DHR123); scavenger group (test molecule + DHR123 + L-ascorbic acid). Pipette 1 μL of the compound stock solution into 1 mL of the DHR123 working solution, mix and irradiate with a white light source (25 mW / cm2), and detect the fluorescence intensity of the test solution at different time intervals. The excitation wavelength of DHR123 is 488 nm, and the emission wavelength is 510 - 550 nm. This study tested the O2 ·- generating ability.

[0082] Hydroxyl radical test: Use HPF as an indicator to study whether the compound can generate ·OH. The method is as follows: Weigh HPF (4.10 mg, 0.01 mmol) powder and dissolve it in 1.0 mL of N,N-dimethylformamide (DMF) to prepare a 10 mM stock solution. The HPF stock solution is aliquoted and stored in the dark at -20 °C or -80 °C. Dilute the stock solution with PBS to prepare a 10 μM HPF working solution. The experimental groups are: blank control group (PBS + HPF); experimental group (test molecule + HPF). Pipette 1 μL of the compound stock solution into 1 mL of the HPF working solution, mix and irradiate with a white light source (25 mW / cm2), and detect the fluorescence intensity of the test solution at different time intervals. The excitation wavelength of HPF is 488 nm, and the emission wavelength is 510 - 550 nm. This study tested the ·OH generating ability of C4AZ1 and C4AZ.

[0083] The results are as Figure 3 shown in Figure A. The fluorescence emission of DCFH alone did not change significantly. On the contrary, when DCFH was co-incubated with C4AZ1 and C4AZ2 respectively, its fluorescence signal showed a rapid increase, which was about 5.1 times and 4.2 times higher than that of the positive control rose bengal (RB). This finding emphasizes the excellent ROS generating ability of the test compounds, indicating that the compounds described in the present invention can directly enhance the killing efficiency of PDT on cancer cells, more efficiently induce DNA damage, mitochondrial function collapse and programmed death of tumor cells, and especially achieve a more thorough clearance effect on deep solid tumors.

[0084] The results are as Figure 3 shown in Figure B. In the blank group, under white light irradiation, the absorption peak of ABDA at 378 nm did not change significantly. Subsequently, we compared the singlet oxygen yields of the two compounds with that of the commercial photosensitizer rose bengal (RB). It was observed that when the C4AZ1 or C4AZ2 compound was added to the ABDA probe and exposed to white light, the absorbance signal changed little. This indicates that these compounds hardly generated1 The ability of O2.

[0085] The results are as Figure 3 shown in C. The fluorescence intensity of the DHR123 solution in the blank control group changed little. However, after adding C4AZ1 and C4AZ2, the fluorescence intensity increased significantly and then tended to be stable. The enhancement of the DHR123 fluorescence intensity was confirmed to be caused by reactive oxygen species (ROS), as demonstrated by the radical scavenger Vc. After introducing Vc, the emission intensities of C4AZ1 and C4AZ2 both decreased, indicating that they have the ability to generate O2 ·- The ability.

[0086] The results are as Figure 3 shown in D. The fluorescence emission of HPF in the blank control group maintained at the baseline level under white light irradiation. In sharp contrast, the fluorescence intensity increased significantly after co-incubating C4AZ1 and C4AZ2 with HPF, indicating that the compounds can generate ·OH, and more ·OH is generated by C4AZ1. Therefore, we speculate that the PDT effect of C4AZ1 is better.

[0087] The above results indicate that the compounds of the present invention do not rely on molecular oxygen and will not lead to a deterioration in the treatment effect due to the hypoxic environment of solid tumors.

[0088] Example 4: Cell culture and construction of tumor cell hypoxia model

[0089] The culture media used for human hepatoma cells (HepG2), mouse mammary tumor cells (4T1), human hepatocytes (LO2), and human umbilical vein endothelial cells (HUVEC) were: DMEM high-glucose medium containing 10% fetal bovine serum (FBS); the culture medium used for human colorectal cancer cells (SW480) was: RPMI-1640 medium containing 10% FBS; the culture medium used for mouse embryonic cells (3T3) was: DMEM high-glucose medium containing 10% newborn calf serum (NCS). The cell culture conditions were all in a 37 °C constant temperature incubator containing 5% CO2.

[0090] To simulate the hypoxic tumor environment and detect intracellular ROS under hypoxic conditions, DFO induces hypoxia by chelating iron and stabilizes the proteolysis of HIF-1α by inhibiting the activity of iron-dependent prolyl hydroxylase, thereby reducing the potential of oxygen transport. For hypoxia, 1 mL of fresh medium was replaced, which contained 4 μL of DFO (stock concentration: 50 mM, working concentration: 200 μM). Then the hypoxic cells were placed back in the incubator (5% CO2, 20% O2, 37 °C) and incubated for 24 h. Next, the cells were washed thoroughly 3 times with PBS. According to the product manual, ROS-ID was used TMThe hypoxia / oxidative stress detection kit (stock concentration: 1 mM, working concentration: 0.5 μM) was incubated in an incubator for 30 min to indicate the hypoxia situation within cells. When live cells are in an environment with normal oxygen concentration, it is non-fluorescent. When the oxygen level decreases, it turns into red fluorescence. Hypoxia detection: Red fluorescence (E x / E m : 575 / 600 nm).

[0091] The results were as Figure 4 shown. The red fluorescence intensity within HepG2 cells in the DFO treatment group reached 20.4 times that of the normoxia control group (p < 0.01), indicating that a hypoxic microenvironment was successfully established and could be used for subsequent experiments under hypoxic conditions (such as Example 6).

[0092] Example 5: Cell imaging and flow cytometry quantitative analysis

[0093] HepG2 and HUVEC cells were seeded at a density of 5×10 3 cells per dish in a confocal laser scanning microscopy (CLSM)-specific culture dish (diameter 20 mm), and 1 mL of complete medium was added to each dish. The culture dishes were placed in a humidified incubator at 37 °C and 5% CO2 and incubated overnight until the cells were completely adherent. The adherent cells were incubated with C4AZ1 (2.5 μM) for 0 h, 0.5 h, 1 h, 2 h, 4 h, and 8 h respectively. After the drug incubation was completed, the original medium was discarded, and the cells were gently washed 3 times with sterile PBS buffer or DMEM medium to remove unbound drugs and residual medium. 1 mL of Hoechst33342 staining solution (1 μg / mL) was added to each dish, and the cells were incubated in the dark for 10 min to stain the cell nuclei. Fluorescence images were obtained using a confocal laser scanning microscope (CLSM).

[0094] Cell collection for flow cytometry: After the drug incubation was completed, the cells were digested with trypsin and collected into a centrifuge tube, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The cells were resuspended with PBS. The fluorescence intensity of C4AZ1 was detected using a flow cytometer, and at least 10,000 cells were detected for each sample group.

[0095] Data analysis: FlowJo VX software (BD Biosciences, USA) was used to perform quantitative analysis on the flow cytometry data. The mean fluorescence intensity (MFI) of cells in each group was statistically analyzed and compared.

[0096] The results were as Figure 5As shown, after co-incubating C4AZ1 with HepG2 cells for 30 min, faint red fluorescence began to appear in the cytoplasm, indicating that the compound had started to enter the cells. As the co-incubation time was extended to 4 h, the intensity of the red fluorescence gradually increased and reached a peak, proving that C4AZ1 could effectively enter HepG2 cells and accumulate in the cytoplasm. Flow cytometry (FCM) for intracellular fluorescence quantitative analysis of C4AZ1 at different time points further supported these observations. As Figure 6 shown, there was no obvious change in the fluorescence intensity of HUVEC cells, and it was significantly lower than that of tumor cells. This indicated that C4AZ1 had a certain uptake specificity for cancer cells.

[0097] Example 6: Cell safety

[0098] Solution preparation: Appropriate amounts of C4AZ1 and C4AZ2 stock solutions were pipetted and diluted with incomplete medium into working solutions with concentration gradients of 0.1, 0.25, 0.5, 1, 2.5, 5, and 10 μM for standby.

[0099] HUVEC cells were evenly seeded in 96-well plates at a density of 5×10 3 cells / well, and cultured in an incubator for 24 h. Then, the medium in the wells was discarded. 100 μL of the above sample medium was added to each group, and 100 μL of incomplete DMEM medium was added to each well in the control group. They were placed in a cell culture incubator at 37 °C and 5% CO2 in the dark for 24 h for dark toxicity study. For phototoxicity study, after incubating in the dark for 8 h, the cells were irradiated with white light (25 mW / cm 2 , 12 min), and then incubated under the corresponding conditions until 24 h. The medium was removed, and the cells were washed once with PBS solution. Then, the cells were incubated with fresh serum-free medium containing 10% CCK-8 in the dark for 1 h. Finally, the OD value at 450 nm was measured by an enzyme-labeled instrument. 4-6 replicates were set for each group to ensure the reliability of the experimental results. The cell viability was calculated according to the following formula:

[0100] Cell viability (%) = [OD treated - OD blank] / [OD control - OD blank] × 100%

[0101] where OD treated is the OD value of each experimental group; OD control is the OD value of the control group; OD blank is the OD value of the blank group.

[0102] The measurement methods for LO2 cells and NIH-3T3 cells were the same as those for HUVEC cells.

[0103] The results are as Figure 7As shown, under light and dark conditions, the cytotoxicity of these two compounds (with the highest concentration gradient of 10 μM) to three normal cell lines, LO2, NIH-3T3, and HUVEC, is extremely low, and the cell survival rate remains above 80%.

[0104] Example 6: Cell light / dark toxicity

[0105] Solution preparation: Appropriate amounts of C4AZ1 and C4AZ2 stock solutions were aspirated and diluted with incomplete medium into working solutions with concentration gradients of 0.1, 0.25, 0.5, 1, 2.5, 5, and 10 μM for standby.

[0106] HepG2 cells were evenly seeded in 96-well plates at a density of 5×10 3 cells / well and cultured in an incubator for 24 h. Then, the medium in the wells was discarded. 100 μL of the above sample medium was added to each group, and 100 μL of incomplete DMEM medium was added to each well in the control group. The plates were placed in a cell incubator at 37 °C and 5% CO2 and cultured in the dark for 24 h for dark toxicity study. For light toxicity study, after incubating in the dark for 8 h, the cells were irradiated with white light (25 mW / cm 2 , 12 min), and then incubated for another 16 h under the corresponding conditions until 24 h. The medium was removed, and the cells were washed once with PBS solution. Then, the cells were incubated with fresh serum-free medium containing 10% CCK-8 in the dark for 1 h. Finally, the OD value at 450 nm was measured using a microplate reader. 4-6 replicates were set for each group to ensure the reliability of the experimental results, and the cell survival rate was calculated.

[0107] For the cytotoxicity test under hypoxic conditions, the original medium in each well was replaced with 100 μL of fresh medium containing 0.4 μL of DFO (stock concentration: 50 mM, working concentration: 200 μM) and different concentrations of C4AZ1 and C4AZ2 solutions.

[0108] The measurement methods for SW480 cells and 4T1 cells were the same as those for HepG2 cells.

[0109] The results are as Figure 8 - 9 shown. Both compounds showed significant dose-dependent cytotoxicity under photoactivation conditions, and the inhibitory effect on HepG2 cells was the most prominent. The half-inhibitory concentration (IC 50 ) was calculated through the dose-effect curve. The IC 50The values were 0.46 ± 0.07 μM and 0.61 ± 0.14 μM respectively, which were significantly lower than those of 4T1 (1.09 ± 0.03 μM and 1.65 ± 0.16 μM) and SW480 cells (1.14 ± 0.10 μM and 1.63 ± 0.05 μM). Even under hypoxic conditions, C4AZ1 and C4AZ2 could still maintain significant cytotoxicity and had good therapeutic effects on the above three types of tumor cells. In both normoxic and hypoxic environments, the dark toxicity of the two compounds at a dosing concentration of 10 μM to the three cancer cells was relatively low under dark conditions, and the cell viability was maintained at about 70%, indicating that the compounds described in the present invention could effectively avoid the tumor cells entering the stress state, thus significantly reducing the killing efficiency during illumination.

[0110] Example 7: Fluorescence colocalization

[0111] In this experiment, two cell lines, 4T1 and HepG2, were selected for research. They were inoculated into a special culture dish for laser confocal microscopy (diameter 20 mm) at a density of 5×10 3 cells per dish, and 1 mL of complete medium was added to each dish. The culture dishes were placed in a humidified incubator at 37 °C and 5% CO2 and incubated overnight until the cells were completely adherent. The stock solution of C4AZ1 was diluted to the working concentration (2.5 μM) with complete medium, and the adherent cells were incubated with C4AZ1 (2.5 μM) for 4 h. After the drug incubation was completed, the medium was discarded, and the cells were gently washed 3 times with sterile PBS buffer to remove unbound drugs and residual medium. 1 mL of MitoTracker staining solution (prepared according to the instructions) was added to each dish, and the cells were incubated in the dark for 30 min to stain the mitochondria. After discarding the MitoTracker staining solution, the cells were washed 2 times with PBS, and then 1 mL of Hoechst 33342 staining solution (1 μg / mL) was added, and the cells were incubated in the dark for 10 min to stain the nuclei. Fluorescence images were obtained using a laser confocal microscope (CLSM).

[0112] The results were as Figure 10 shown. The red fluorescence signal of C4AZ1 in HepG2 cells had good overlap with the green fluorescence of Mito-tracker Green (Pearson correlation coefficient R = 0.92), proving that C4AZ1 could selectively accumulate in mitochondria. A similar phenomenon was also observed in 4T1 cells (R = 0.91), confirming that C4AZ1 had broad-spectrum mitochondrial targeting ability to cancer cells.

[0113] Example 8: Detection of intracellular ROS

[0114] HepG2 cells were selected as the research object, and the adherent cells were incubated with C4AZ1 (2.5 μM) for 4 h. The medium was discarded, and the cells were gently washed 3 times with PBS buffer. Then, 10 μM DCFH-DA, DHE, and HPF staining solutions were added respectively, and the cells were incubated in the dark for 30 min. Subsequently, the cells were gently washed 3 times with PBS buffer, and then irradiated with white light (25 mW / cm 2 ) for 3 min, and fluorescence images were obtained using CLSM.

[0115] ROS detection under hypoxic conditions: The medium in each dish was replaced with 1 mL of fresh medium (containing 200 μM DFO and 2.5 μM C4AZ1), and the cells were incubated in an incubator at 37 °C, 5% CO2, and 20% O2 for 4 hours. ROS probe incubation: 10 μM DCFH-DA, DHE, and HPF staining solutions were added respectively, and the cells were incubated in the dark for 30 minutes. The cells were gently washed 3 times with PBS buffer, and then irradiated with white light (25 mW / cm 2 ) for 3 minutes, and fluorescence images were obtained using CLSM.

[0116] The results are as Figure 11 shown. Cells treated with C4AZ1 and DCFH-DA probes showed bright green fluorescence under both normoxic and hypoxic conditions, while control cells showed no obvious fluorescence signal. Under normoxic conditions, incubation of cells with C4AZ1 and DHE probes led to a significant increase in red fluorescence. Notably, even under hypoxic conditions (where the production of O ·- is usually expected to be inhibited), a significant increase in red fluorescence was also observed. For cells co-cultured with C4AZ1 and HPF probes, a significant increase in green fluorescence was detected after white light irradiation under both normoxic and hypoxic conditions. This indicates that ·OH can be generated regardless of the oxygen level, breaking through the absolute dependence of traditional type II photosensitizers on oxygen.

[0117] Example 9: Detection of mitochondrial membrane potential

[0118] In this experiment, HepG2 cells were selected as the research object.

[0119] The experimental groups were as follows: control group, normoxic dark group: C4AZ1-Light, normoxic light group: C4AZ1+Light, hypoxic dark group: C4AZ1-Light, hypoxic light group: C4AZ1+Light.

[0120] Drug treatment was as follows: normoxic dark group: C4AZ1 (1.0 μM) was incubated in the dark for 24 h; normoxic light group: C4AZ1 (1.0 μM) was incubated in the dark for 8 h and then irradiated with white light (25 mW / cm 2, 12 min), continue to culture until 24 h; Hypoxic dark group: C4AZ1 (1.0 μM) + DFO (200 μM), incubate in the dark for 24 h; Hypoxic light group: C4AZ1 (1.0 μM) + DFO (200 μM), incubate in the dark for 8 h, then irradiate with white light (25 mW / cm 2 , 12 min), and then continue to culture until 24 h; Control group: Only add an equal volume of serum-free medium without drug treatment.

[0121] Cell staining and fluorescence imaging: After the drug treatment is completed, gently wash the cells 3 times with pre-warmed PBS (pH 7.4). Prepare the staining solution according to the instructions: Take 2 μL of JC-1 (500×), add it to 900 μL of sterilized deionized water, and vigorously vortex to fully dissolve and mix JC-1. Then add 100 μL of 10× Incubation Buffer, and after mixing, it is 1 mL of JC-1 working solution. Incubate at 37 °C in the dark for 20 min. After discarding the staining solution, wash 3 times with PBS to remove free dye. Use a laser confocal microscope (CLSM) to obtain fluorescence images.

[0122] The results are as Figure 12 shown. Under normoxic and hypoxic conditions, in the experimental group treated with C4AZ1 photoactivation, compared with the control group without photoactivation and the PBS blank control group, significantly enhanced green fluorescence was emitted in the cells, indicating that after C4AZ1 photoactivation, the mitochondrial membrane potential was significantly reduced or lost, which could lead to apoptosis of tumor cells.

[0123] Example 10: Live / dead cell staining experiment

[0124] Cell seeding: Seed HepG2 cells in a 6-well cell culture plate at a density of 5 × 10 4 cells per well, add 2 mL of complete medium, and incubate in a 37 °C, 5% CO2 incubator for 24 h until the cells adhere to the wall.

[0125] The experimental grouping and drug treatment are the same as in Example 9.

[0126] Cell staining and fluorescence microscopy imaging: Discard the medium, gently wash the cells 3 times with pre-cooled PBS. Add 1 mL of PBS containing calcein-AM (1 μM) and PI (1 μM) to each well, and incubate in the dark for 30 minutes. Discard the staining solution, wash 3 times with PBS to remove residual dye, and perform fluorescence microscopy imaging.

[0127] As shown in Figure 13, after light irradiation, strong red fluorescence signals due to PI uptake were observed in C4AZ1-treated HepG2 cells under both hypoxic and normoxic conditions, indicating that a large number of cells were in a state of necrosis or late apoptosis due to the loss of membrane integrity. In contrast, the PBS control group and the group without light treatment showed bright green fluorescence, indicating that C4AZ1 has excellent ability to eliminate tumor cells.

[0128] Example 11: Detection of apoptosis

[0129] Cell seeding: Seed HepG2 cells in a 6-well cell culture plate at a density of 5×10 4 cells per dish, add 2 mL of complete medium, and incubate in a 37°C, 5% CO2 incubator for 24 h until the cells adhere to the wall.

[0130] The experimental grouping and drug treatment are the same as in Example 9.

[0131] Cell staining and cell collection: Discard the medium, wash the cells twice with PBS, add 0.25% trypsin to digest the cells, collect the cell suspension into a centrifuge tube, centrifuge at 1000 rpm for 5 min, and discard the supernatant. Resuspend the cells with 1×Binding Buffer and adjust the cell density to 1×10 5 cells / mL. Add 5 μL of Annexin V-FITC and 5 μL of PI staining solution, mix gently, and incubate in the dark for 15 min. Add 500 μL of 1×Binding Buffer, mix well and immediately perform detection. Use a flow cytometer to detect the fluorescence signals of cells in each group, and at least 10,000 cells in each sample are detected.

[0132] Data analysis: Use FlowJo VX software to analyze the data and statistically calculate the apoptosis rate (early apoptosis + late apoptosis) and necrosis rate of cells in each group.

[0133] The results are as Figure 14 shown. For cells treated only with C4AZ1, the cell populations of early apoptosis, late apoptosis, and necrotic cells were 15.3%, 1.34%, and 0.53% respectively, close to the blank control group. For white light irradiation at 25 mW / cm 2 for 12 min, the percentages of cells in early and late apoptosis increased to 33.0% and 36.3% respectively. Excitingly, the hypoxic environment did not significantly weaken the PDT efficacy, and the percentages of cells in early and late apoptosis were 43.0% and 10.1% respectively. This further verified the PDT efficacy of the compound and overcame the obstacle of tumor hypoxia.

[0134] Example 12: Cell cycle experiment

[0135] Cell seeding: Seed HepG2 cells into a 6-well cell culture plate at a density of 5×10 4 cells per dish, add 2 mL of complete medium, and incubate in a 37°C, 5% CO2 incubator for 24 h until the cells adhere to the wall.

[0136] Drug treatment: After culturing in the incubator for 24 h, discard the medium and wash once with PBS. Add 1 mL of incomplete medium to the control group and add incomplete medium with different concentrations of C4AZ1 (0.1, 0.5, 1 μM) to the experimental groups. Incubate in a 37°C, 5% CO2 cell culture incubator for 8 h, and continue to culture for 24 h after white light irradiation (25 mW / cm 2 , 12 min).

[0137] Cell fixation and staining: Discard the medium, wash twice with PBS, digest with 0.25% trypsin (37°C, 1 - 2 min), add complete medium to terminate digestion, and centrifuge to collect cells (800 rpm, 5 min). Slowly add pre-cooled 70% ethanol (dropwise while shaking), and fix at -20°C overnight (at least 12 h). Centrifuge to remove ethanol (800 rpm, 5 min), and wash twice with PBS to remove residual ethanol. Prepare the staining solution according to the kit instructions: staining buffer (0.5 mL) + PI staining solution (25 μL) + RNase A (10 μL), and stain for 30 min in the dark (operate on ice). Use a flow cytometer to detect the fluorescence signals of cells in each group. Each sample should be detected for at least 10,000 effective cell samples, excluding aggregates.

[0138] Data analysis (ModFit LT): Import the flow cytometer data, and Modfit automatically fits the DNA distribution curve to calculate the proportions of each stage of the cell cycle.

[0139] The results are as Figure 15 shown. With the change of the compound concentration, the cell cycle distribution of HepG2 cells showed a dose-dependent G0 / G1 phase arrest. Compared with the control group, the proportions of G0 / G1 phase cells in the 0.1, 0.5, and 1.0 μM treatment groups increased significantly from 48.7% to 54.6%, 64.1%, and 69.3% respectively. At the same time, the total proportion of S phase and G2 / M phase cells decreased correspondingly. This phenomenon indicates that the compound can effectively inhibit the transition of cells from the G0 / G1 phase to the S phase, and may hinder DNA synthesis and subsequent cell division processes, resulting in cell cycle arrest at the G0 / G1 phase.

[0140] Example 13: In vivo live imaging experiment

[0141] (1) Female BALB / c Nude mice (4 weeks old, 11 - 15 g) in good health were selected and raised under standard specific pathogen - free (SPF) conditions. After being raised in the SPF experimental animal center for one week, 100 μL of cell suspension containing 5×10 6 hepatocellular carcinoma cell line (HepG2) was implanted subcutaneously into the right axilla of the mice to establish a subcutaneous HepG2 tumor model. New disposable syringes were used for tumor implantation in different mice. Normal food and water were provided, and leisure devices were placed in the cages to ensure that the mice were in a comfortable growth environment. During the tumor formation period of the mice, the mice were observed and weighed every 2 days. Special attention was paid to the site of cancer cell inoculation, and the growth of the tumor was observed. The tumor volume was recorded with a vernier caliper. The tumor volume calculation formula was: V = a 2 ×b / 2, where a and b represent the shortest and longest diameters of the tumor, respectively. After the tumor volume reached approximately 100 mm 3 , in - vivo fluorescence imaging and photodynamic therapy experiments were carried out.

[0142] (2) To systematically evaluate the in - vivo distribution and tumor - targeting ability of C4AZ1, this study was analyzed by combining in - vivo fluorescence imaging with ex - vivo organ imaging. The specific experiments were as follows: 12 h before the experiment, the mice were fasted and allowed free access to water to reduce background fluorescence interference. The HepG2 - tumor - bearing mice were deeply anesthetized with isoflurane, and then C4AZ1 (0.2 mM, 100 μL) was injected via the tail vein. At different time points (0 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, and 48 h) after injection, the mice were placed in a small animal in - vivo fluorescence imaging system for photographing to collect in - vivo fluorescence imaging pictures. After the imaging was completed, the mice were euthanized with CO2, dissected, and the main organs (heart, liver, spleen, lung, and kidney) and tumor tissues were collected. They were rinsed 3 times in pre - cooled PBS to remove the surface blood. The tissue samples were laid flat on a black background plate and placed in a small animal in - vivo fluorescence imaging system for photographing to study the biodistribution of C4AZ1 in the main organs.

[0143] Data processing and quantitative analysis: Fluorescence signal calibration: Living 4.5 software was used for fluorescence intensity quantification. The ROI (Region of Interest) tool was used to delineate the regions of each organ and tumor for calculation.

[0144] The results are as Figure 16As shown, the fluorescence intensity at the tumor site increased with time and reached its maximum at 8 h. Moreover, compared with other tissues, an abnormally strong signal was visible in the tumor area for more than 24 h. This result suggests that the 8th h after drug injection is the optimal treatment time, and light irradiation at this time can maximize the ROS killing effect. The in vitro fluorescence signal of C4AZ1 mainly accumulates in tumors. In addition, fluorescence signals were mainly detected in the liver and kidneys, indicating that the compound is mainly cleared through the hepatobiliary metabolic system and the renal excretion system.

[0145] Example 14: Photodynamic Therapy for Tumors in Vivo

[0146] In this experiment, mice models with subcutaneous HepG2 tumors were randomly divided into 4 groups, with 5 mice in each group. The specific grouping is as follows: PBS group; PBS + Light group; C4AZ1 group (0.2 mM, 100 μL); C4AZ1 + Light group (0.2 mM, 100 μL). 8 h after intravenous injection of the drug, the tumor site was irradiated with white light for 15 min (50 mW / cm 2 ). The body weight and tumor volume of the mice were monitored every 2 days for 14 days, and the corresponding growth curves were plotted. After the treatment, the mice were sacrificed and the tumors were dissected for measurement, weighing, and photographing, and the tumor volume (Tumor Volume, mm 3 ) and the tumor inhibition rate (Tumor inhibition rate, TIR) of each experimental group were calculated.

[0147] The formula for calculating the tumor inhibition rate of each experimental group is: TIR = (1 - T t / T0) × 100%, where T t is the average TV of the experimental group and T0 is the average TV of the PBS blank control group.

[0148] To further evaluate the inhibitory effect of the PDT group on tumor growth, 14 days after the treatment, the mice in each group were euthanized and the tumor tissues were collected. Hematoxylin and eosin (H&E), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, and Ki67 immunohistochemistry were used to analyze the pathological changes to evaluate the treatment effects of different treatments.

[0149] The results are as Figure 17 shown. Compared with "PBS", the inhibitory effects of "PBS + Light" and "C4AZ1" treatments on tumor growth were negligible, indicating that neither single white light irradiation nor drug treatment could produce a significant anti-tumor effect. The C4AZ1 + Light group (PDT treatment group) showed a significant tumor inhibition effect within 14 days (tumor inhibition rate 94.8 ± 1.8%, p < 0.001), verifying the necessity of photoactivation and the synergistic effect.

[0150] The results were as follows Figure 18 As shown, H&E staining showed that compared with the PBS, PBS+L, and C4AZ1 groups, the tumor tissues in the C4AZ1+L group presented more obvious nuclear fragmentation and cytoplasmic reduction. The TUNEL immunofluorescence staining assay also showed that significant apoptosis and necrosis occurred in the tumor tissues after C4AZ1+L treatment, while similar damage was not observed in other groups. The results of Ki67 immunohistochemical staining also showed the same trend, indicating that the proliferation ability of tumor cells in the C4AZ1+Light group was significantly reduced. These results suggest that C4AZ1 effectively achieved fluorescence imaging-guided type I PDT and had a significant inhibitory effect on tumor growth.

[0151] Example 15: In vivo biosafety evaluation

[0152] In vivo biosafety was mainly evaluated through hemolysis experiments, whether there were obvious abnormal changes in the body weights of mice in each group during the 2-week cycle of mouse tumor photodynamic therapy, histological examinations of the main organs of mice after treatment, and routine blood and biochemical experiments.

[0153] (1) Hemolysis experiment: Fresh anticoagulated whole blood from healthy mice was centrifuged at 3000 rpm for 10 min, the plasma and white blood cell layer were discarded, and the red blood cell precipitate was washed 3 times with physiological saline and then prepared into a 2% red blood cell suspension. Gradient concentrations of C4AZ1 (20 - 200 μM) were prepared with physiological saline. Positive control: ultrapure water (complete hemolysis); negative control: physiological saline (0% hemolysis). Incubate in the dark at 37 °C for 4 h, and then centrifuge at 3000 rpm for 10 min. Take pictures of the samples on the same horizontal line. Use a pipette to aspirate the supernatant of the samples into a 96-well plate and measure the absorbance value at 540 nm to calculate the hemolysis rate. Hemolysis rate calculation formula:

[0154] Hemolysis rate (%) = (OD 实验组 - OD 阴性对照 ) / (OD 阳性对照 - OD 阴性对照 ) × 100%

[0155] (2) During the photodynamic therapy of mouse living tumors, record the body weights of mice in each group (every other day) to obtain a curve graph of the body weights of mice in each group changing with time.

[0156] (3) After the treatment cycle ended, euthanize the test mice, dissect and collect the main organs of the mice (heart, liver, spleen, lung, kidney), and fix and preserve the main organs of the mice with 4% formalin. Then each tissue was paraffin-embedded and sectioned (5 μm), and H&E staining was used for imaging and histological analysis.

[0157] (4) Blood biochemistry and blood routine tests: Before blood collection from tumor-bearing mice at the end of the treatment cycle, isoflurane inhalation anesthesia was used. Blood was collected from the orbital venous plexus and directly collected into blood collection tubes (first the yellow tube and then the purple tube). The single blood collection volume was approximately 0.8 - 1.0 mL (yellow tube: 0.5 mL, purple tube: 0.3 mL). Yellow coagulant + separator gel tube (for blood biochemistry detection): After blood collection, it was allowed to stand for 30 min, centrifuged at 3000×g for 15 min, and the upper-layer serum was separated into an EP tube and stored at -80°C or immediately analyzed on the machine. Purple EDTAK2 anticoagulant tube (for blood routine detection): After blood collection, it was gently inverted and mixed 8 - 10 times immediately, stored at room temperature, and analyzed within 2 h. The detected indicators included alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total protein (TP), albumin (ALB), blood urea nitrogen (BUN), creatinine (CREA), hemoglobin (HGB), red blood cells (RBC), white blood cells (WBC), platelets (PLT), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC).

[0158] The results were as Figure 19 shown. After incubation of C4AZ1 (concentration 200 μM) with 2% blood cells at 37°C for 4 h, the hemolysis rate of red blood cells was still less than 5%. The low hemolysis rate indicated that C4AZ1 had no obvious destructive effect on the red blood cell membrane structure at a concentration of 200 μM. Compared with the PBS group, during the 14-day treatment period, no obvious weight changes occurred in all other treatment groups, and the body weight remained stable, indicating that light irradiation and the photodynamic therapy with the photosensitizer C4AZ1 did not cause obvious physiological toxicity to the mice.

[0159] The results were as Figure 20 shown. As shown by H&E staining, there were no obvious signs of tissue morphology or pathological abnormalities in the main organs of all experimental mice in different groups, indicating that light irradiation, C4AZ1, and its PDT treatment did not cause physiological toxicity to the mice, demonstrating that the photosensitizer C4AZ1 had good biocompatibility.

[0160] The results were as Figure 21 - 22As shown, the key indicators of liver and kidney function (ALT, AST, ALP, TP, ALB, BUN, CREA) and hematological parameters (HGB, RBC, WBC, PLT, HCT, MCV, MCH, MCHC) in all groups were within the normal physiological range, indicating that each experimental treatment did not cause significant hepatotoxicity, nephrotoxicity or hematopoietic system abnormalities. It is worth noting that there was no statistical difference in the levels of transaminases (ALT / AST) between the C4AZ1 combined with L treatment group and the simple PBS control group (p>0.05), suggesting that C4AZ1 did not produce detectable hepatocyte damage at the experimental dose. At the same time, the consistency of blood urea nitrogen (BUN) and creatinine (CREA) levels among groups further confirmed that the experimental treatment had no significant effect on renal function. In terms of the safety of the hematological system, the erythrocyte parameters (HGB, RBC, HCT) and platelets (PLT) in all groups were maintained within the normal fluctuation range. In particular, the stability of the WBC count indicated that the experimental treatment did not trigger a significant inflammatory response or immune system activation.

Claims

1. A mitochondrion-targeted calix[4]arene supramolecular photosensitizer, as shown in formula (I): Among them, R is 1,2,3,3-tetramethyl-3H-indolium iodide, 1,1,2,3-tetramethyl-1H-benzo[e]indolium iodide, 3-ethyl-1,1,2-trimethyl-3H-indolium iodide or 3-ethyl-1,1,2-trimethyl-1H-benzo[e]indolium iodide; preferably, R is selected from 2. A mitochondrion-targeted calix[4]arene supramolecular photosensitizer, as shown in any of the following:

3. A preparation method of the mitochondrion-targeted calix[4]arene supramolecular photosensitizer shown in claim 1 or 2, wherein the definition of R is as shown in claim 1 or 2.

4. The preparation method according to claim 3, wherein comprising the following steps: (1) Under the conditions of trifluoroacetic acid or sulfuric acid, a tetraester is reacted with hexamethylenetetramine at 60-90 °C to prepare compound 1; (2) In the presence of sodium acetate or sodium ethoxide, compound 1 and 1,2,3,3-tetramethyl-3H-indole iodide are reacted at 60-90 °C to prepare compound C4AZ1; (3) In the presence of sodium acetate or sodium ethoxide, compound 1 and 3-ethyl-1,1,2-trimethyl-1H-benzo[e]indole iodide are reacted at 60-90 °C to prepare compound C4AZ2.

5. The preparation method according to claim 4, characterized in that, In step (1), the reaction temperature is preferably 70 °C; the molar ratio of the tetraester to hexamethylenetetramine is 1:10.0-12.0, preferably 1:10.0; preferably, in step (2), the reaction temperature is preferably 70 °C; the molar ratio of compound 1 to 1,2,3,3-tetramethyl-3H-indole iodide is 1:2.0-4.0, preferably 1:3.0; the molar ratio of compound 1 to sodium acetate is 1:2.0-4.0, preferably 1:3.

0.

6. The preparation method according to claim 4, characterized in that, The reaction temperature in step (3) is preferably 70 °C; the molar ratio of compound 1 to 1,1,2,3-tetramethyl-1H-benzo[e]indole iodide is 1:2.0-4.0, preferably 1:3.0; the molar ratio of compound 1 to sodium acetate is 1:2.0-4.0, preferably 1:3.

0.

7. Use of the mitochondrion-targeted calix[4]arene supramolecular photosensitizer shown in claim 1 or 2 in the preparation of a tumor detection reagent, preferably in the preparation of a fluorescence imaging diagnostic drug targeting tumor cells; more preferably in the preparation of a fluorescence imaging diagnostic drug targeting the mitochondria of tumor cells.

8. Use of the mitochondrion-targeted calix[4]arene supramolecular photosensitizer shown in claim 1 or 2 in the preparation of a photodynamic tumor treatment drug.

9. Use of the mitochondrion-targeted calix[4]arene supramolecular photosensitizer shown in claim 1 or 2 in combination with light irradiation in the preparation of a tumor treatment drug.

10. The application according to any one of claims 7 to 9, characterized in that, The tumor is human liver cancer cells, human colorectal cancer cells or mouse breast tumor cells; preferably human liver cancer cells.

Images