Application of anthracene fluorescent material in detection of pH value, preparation of lysosome and / or mitochondrion targeted reagent and diagnosis and treatment of tumor cell drugs

Abstract

The application provides application of anthracene fluorescent material in detection of pH value, preparation of lysosome and / or mitochondrion targeted reagent and diagnosis and treatment of tumor cells, and belongs to the technical field of fluorescent sensing. The anthracene fluorescent material has high fluorescence quantum yield and good light stability; the anthracene fluorescent material has protonation effect between amino groups and hydrogen ions, can be used as a combination site of protons, can realize quantitative detection of pH value, and can be used as a targeting group of lysosomes in cells; the amino groups combine with hydrogen ions to generate quaternary ammonium salt, which can increase hydrophilicity, and the generated positive charge can be used as a mitochondrion targeting group, so that the anthracene fluorescent material realizes dual-color visualization targeting lysosomes and mitochondria in cells; and based on the transformation between amino group protonation and non-protonation, the anthracene fluorescent material can be used as an acid triggered probe for tumor diagnosis, realizes high selectivity visualization tumor detection and tumor treatment in a living body.

Description

Technical Field

[0001] This invention relates to the field of fluorescence sensing technology, and in particular to the application of anthracene fluorescent materials in pH detection, preparation of lysosome and / or mitochondrial targeting reagents, and drugs for the diagnosis and treatment of tumor cells. Background Technology

[0002] Cancer is a malignant disease caused by disordered cell division and growth following mutations. The transformation of normal cells into tumor cells is a complex process, involving not only disordered proliferation, division, and metastasis, but also mutations in tumor cells such as alterations in the intracellular microenvironment (polarity, hypoxia, pH, and viscosity). Therefore, intracellular microenvironment parameters have been used as effective indicators for cancer diagnosis. Among these intracellular microenvironment-related parameters, pH has attracted widespread attention from researchers because it plays a role in many important biological processes, such as metabolism, signal transduction, electron transport, and apoptosis.

[0003] Eukaryotic cells contain numerous organelles, such as the Golgi apparatus, lysosomes, and mitochondria. Lysosomes are not only the "stomach" of the cell but also participate in many important physiological processes, including secretion, migration signaling, apoptosis, and autophagy. Due to their unique role in the cell, lysosomes have become one of the most attractive targeting sites for tumor cells. Furthermore, the pH of tumor cell lysosomes is 3.8–4.7, lower than the pH of normal cells (4.5–6). This lower pH could potentially serve as an effective marker for distinguishing tumor cells from normal cells. However, current techniques typically employ probes containing localization groups such as morpholine for lysosome targeting, resulting in relatively limited functionality.

[0004] Mitochondria are the main sites of oxidative phosphorylation and adenosine triphosphate (ATP) biosynthesis, providing most of the energy required for aerobic cells to maintain their physiological activities. Mitochondria also participate in cell growth, differentiation, signal transduction, and apoptosis. Various mitochondrial diseases caused by internal or external factors, including oxidative stress, drastic fluctuations in ion concentration, aberrant oxidative phosphorylation, changes in electron transport chain complex enzymes, and mitochondrial DNA mutations, can occur during mitochondrial energy metabolism. Therefore, the development of accurate, visualized, and specific mitochondrial-targeting agents is of great significance. Summary of the Invention

[0005] The purpose of this invention is to provide the application of anthracene fluorescent materials in pH detection, preparation of lysosome and / or mitochondrial targeting reagents, and diagnosis and treatment of tumor cells. The anthracene fluorescent materials described in this invention can accurately detect pH values, thereby achieving intracellular two-color visualization of lysosomes and mitochondrial targeting, as well as tumor cell diagnosis and treatment.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides an application of anthracene fluorescent material in pH detection, wherein the anthracene fluorescent material has the structure shown in Formula I:

[0008]

[0009] Preferably, the anthracene fluorescent material is suitable for a pH range of 4.0 to 6.0.

[0010] This invention provides the application of anthracene fluorescent material in the preparation of lysosome-targeting reagents and / or mitochondrial-targeting reagents, wherein the anthracene fluorescent material has the structure shown in Formula I:

[0011]

[0012] Preferably, the lysosome targeting reagent is a lysosome targeting reagent for both normal cells and tumor cells.

[0013] Preferably, the mitochondrial targeting reagent is a mitochondrial targeting reagent for tumor cells.

[0014] Preferably, the tumor cells include breast tumor cells.

[0015] Preferably, the lysosome-targeting reagent and the mitochondrial-targeting reagent are visual targeting reagents.

[0016] This invention provides an application of anthracene fluorescent material in the preparation of diagnostic and therapeutic tumor drugs, wherein the anthracene fluorescent material has the structure shown in Formula I:

[0017]

[0018] Preferably, the tumor-treating drug is a visual tumor-treating drug.

[0019] Preferably, the tumor includes a breast tumor.

[0020] This invention provides the application of anthracene fluorescent materials in pH detection, wherein the anthracene fluorescent material has the structure shown in Formula I. In this invention, the anthracene fluorescent material with the structure shown in Formula I includes anthracene groups, boronic acid ester groups, and a basic aniline group. Anthracene consists of three linearly fused benzene rings, extending the aromatic and conjugated π-system, exhibiting superior blue light emission performance and high photoluminescence efficiency. The modification of the boronic acid ester group and the aniline group weakens the strong intermolecular π-π interactions caused by the planar and rigid structure of anthracene, thereby avoiding fluorescence quenching and redshift of solid-state emission wavelength. Therefore, the anthracene fluorescent material has a high fluorescence quantum yield and good photostability. Furthermore, the amino group in the anthracene fluorescent material exhibits a protonation effect with hydrogen ions, which can serve as a proton binding site, enabling quantitative detection. It measures pH value and can serve as a targeting group for lysosomes in cells; the amino group combines with hydrogen ions to form a quaternary ammonium salt, which can increase hydrophilicity, and the generated positive charge can also serve as a mitochondrial targeting group, thereby realizing the dual-color visualization of anthracene fluorescent materials targeting lysosomes and mitochondria in cells; and based on the protonation and deprotonation transition of amino groups, the anthracene fluorescent material can be used as an acid-triggered probe for tumor diagnosis, realizing highly selective visualization of tumor detection and tumor treatment in vivo. Biotoxicity experiments show that the anthracene fluorescent material has significant toxicity to tumor cells but almost no toxicity to normal cells, thus its use as a probe can realize the integrated diagnosis and treatment of tumor cells in vivo. Attached Figure Description

[0021] Figure 1 The UV absorption and fluorescence emission spectra of the normalized compound T1 in tetrahydrofuran from Example 1 are shown.

[0022] Figure 2 The fluorescence spectra and fluorescence ratios (I1) of compound T1 in Example 1 under different pH conditions are shown. 430 / I 480 ) Change over time;

[0023] Figure 3 This is a graph showing the reversibility of fluorescence intensity of compound T1 in Example 1 under pH 2 and pH 8 conditions;

[0024] Figure 4 The fluorescence emission spectrum of compound T1 in Example 1 at pH values ​​of 4–6 is shown.

[0025] Figure 5 This is a graph showing the fluorescence intensity ratios of compound T1 in Example 1 when it contains different metal ions and amino acids at pH 4.

[0026] Figure 6 This is a graph showing the photostability of compound T1 in Example 1 at pH 4.

[0027] Figure 7 These are confocal micrographs of compound T1 in Example 2, showing the colocalization of lysosomes and mitochondria in normal and tumor cells.

[0028] Figure 8 These are confocal fluorescence microscopy images of compound T1 in different channels in normal cells and cancer cells in Example 3;

[0029] Figure 9 This is a bar chart showing the biotoxicity of compound T1 in Example 3 to normal cells and tumor cells;

[0030] Figure 10 This is a diagram showing the antitumor effect of compound T1 in mice in Example 3. Detailed Implementation

[0031] This invention provides an application of anthracene fluorescent material in pH detection, wherein the anthracene fluorescent material has the structure shown in Formula I:

[0032]

[0033] In this invention, the pH range suitable for the anthracene fluorescent material is preferably 4.0 to 6.0, specifically 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 or 6.0.

[0034] In this invention, the amino groups and hydrogen ions in the anthracene fluorescent material exhibit a protonation effect, which can serve as proton binding sites to achieve quantitative detection of pH values. This invention does not specifically limit the method for pH detection using the anthracene fluorescent material; any detection method well-known to those skilled in the art can be used. Specifically, the fluorescence properties of the anthracene fluorescent material can be utilized for detection.

[0035] In an embodiment of the present invention, the method for testing pH value specifically involves mixing anthracene fluorescent material solution, tetrahydrofuran, and the pH value to be tested solution, performing fluorescence intensity detection, and obtaining the fluorescence intensity of the pH value to be tested solution; based on the standard curve and the fluorescence intensity of the pH value to be tested solution, the pH value of the pH value to be tested solution is obtained. In the present invention, the volume ratio of the anthracene fluorescent material solution, tetrahydrofuran, and the pH value to be tested solution is preferably 1:50:950; the solvent of the anthracene fluorescent material solution is preferably tetrahydrofuran, and the concentration of the anthracene fluorescent material solution is preferably 3 μmol / L. The present invention does not impose any special limitation on the fluorescence intensity detection method, and any method well known to those skilled in the art can be used. In the present invention, the standard curve is preferably a linear curve of the fluorescence intensity of the standard pH value solution versus the pH value, and the standard pH value solution is preferably a Britton-Robinson buffer solvent with a pH value of 4.0 to 6.0; the detection method of the fluorescence intensity of the standard pH value solution is preferably consistent with the detection method of the fluorescence intensity of the pH value to be tested solution in the above technical solution, and will not be repeated here.

[0036] This invention provides the application of anthracene fluorescent material in the preparation of lysosome-targeting reagents and / or mitochondrial-targeting reagents, wherein the anthracene fluorescent material has the structure shown in Formula I:

[0037]

[0038] In this invention, the lysosome-targeting reagent can be a lysosome-targeting reagent for both normal cells and tumor cells, and the color channels that target lysosomes differ between normal cells and tumor cells; the mitochondrial-targeting reagent can be a mitochondrial-targeting reagent for tumor cells (it does not target mitochondria in normal cells). In this invention, the tumor cells preferably include breast tumor cells; in the embodiments of this invention, mouse breast tumor cells 4T1 are specifically used as an example. In this invention, the lysosome-targeting reagent and the mitochondrial-targeting reagent are specifically visual targeting reagents.

[0039] In this invention, the amino groups in the anthracene fluorescent material can serve as proton binding sites, acting as targeting groups for lysosomes in cells. The amino groups combine with hydrogen ions to form quaternary ammonium salts, which increase hydrophilicity, and the generated positive charge can also serve as mitochondrial targeting groups, thereby enabling two-color visualization of the anthracene fluorescent material targeting lysosomes and mitochondria in cells. This invention does not specifically limit the method for targeting lysosomes and mitochondria using anthracene fluorescent materials; methods well-known to those skilled in the art can be used. Specifically, based on the fact that the amino groups in the anthracene fluorescent material can first target lysosomes, and then be activated in the acidic environment of tumor cell lysosomes to generate quaternary ammonium salts, which then target mitochondria.

[0040] In an embodiment of the present invention, anthracene fluorescent material solution is added to normal cells and tumor cells for incubation, respectively. Then, lysosomal and mitochondrial targeting dyes are added to the normal and tumor cells for co-incubation, followed by laser confocal imaging. In this invention, the solvent for the anthracene fluorescent material solution is preferably a PBS solution containing dimethyl sulfoxide (DMSO), with a preferred volume fraction of 1‰. The concentration of the anthracene fluorescent material during incubation is preferably 10 μg / mL. The preferred incubation temperature is 37°C, and the preferred incubation time is 2 hours. The preferred co-incubation temperature is 37°C, and the preferred incubation time is 30 minutes. Results show that in normal cells, the green channel fluorescence of the anthracene fluorescent material can precisely target lysosomes. Simultaneously, in tumor cells, the blue channel fluorescence overlaps with the lysosomal dye, while the green channel fluorescence overlaps with the mitochondrial dye fluorescence. This indicates that the anthracene fluorescent material, as a probe, can provide dual-color visualization targeting of lysosomes and mitochondria.

[0041] This invention provides an application of anthracene fluorescent material in the preparation of diagnostic and therapeutic tumor drugs, wherein the anthracene fluorescent material has the structure shown in Formula I:

[0042]

[0043] In this invention, the tumor preferably includes a breast tumor; in specific embodiments of this invention, mouse breast tumor cells 4T1 and human breast tumor cells MDA-MB-231 are used as examples. In this invention, the tumor-treating drug is specifically a visually-guided tumor-treating drug.

[0044] In this invention, based on the protonation and deprotonation transition of the anthracene fluorescent material, it can serve as an acid-triggered probe for tumor diagnosis, enabling highly selective and visualized tumor detection and treatment in vivo, thereby achieving integrated diagnosis and treatment of tumor cells in vivo. In this invention, the drug can specifically be a diagnostic drug for tumors and a therapeutic drug for tumors. This invention does not impose any particular limitation on the method of using the diagnostic drug for tumors and the therapeutic drug for tumors; any method well-known to those skilled in the art can be used.

[0045] In an embodiment of the present invention, anthracene fluorescent material solution is added to normal cells and tumor cells respectively for incubation, followed by laser confocal imaging. In this invention, the solvent of the anthracene fluorescent material solution is preferably a PBS solution containing dimethyl sulfoxide (DMSO), with a preferred volume fraction of 1‰. The concentration of the anthracene fluorescent material during incubation is preferably 10 μg / mL. The incubation temperature is preferably 37°C, and the incubation time is preferably 2 hours. Results show that the fluorescence of the green channel is stronger in normal cells, while the fluorescence of the blue channel is stronger in tumor cells, thus aiding in tumor diagnosis.

[0046] Furthermore, in the embodiments of the present invention, anthracene fluorescent material solution was added to the normal cells and tumor cells respectively. The results showed that when the concentration of anthracene fluorescent material was 10 μg / mL, the survival rate of normal cells was over 90%, while the mortality rate of tumor cells was as high as 50%, indicating that the anthracene fluorescent material can specifically kill tumor cells. Anthracene fluorescent material was introduced into mice by intratumoral injection. The results showed that when the injection dose of anthracene fluorescent material was 50 mg / kg, the weight change of the mice was very small, indicating that the systemic cytotoxicity of anthracene fluorescent material during anti-tumor process was negligible. The tumor volume of the mice hardly increased after administration, indicating that the anthracene fluorescent material effectively inhibited tumor growth.

[0047] The present invention does not specifically limit the source of the anthracene fluorescent material having the structure shown in Formula I, and it can be prepared using methods well known to those skilled in the art. In the present invention, the preparation method of the anthracene fluorescent material preferably includes the following steps:

[0048] 9,10-Dibromoanthracene, pinacol 4-pyridineborate, tetrakis(triphenylphosphine)palladium, potassium carbonate aqueous solution were mixed with the first organic solvent and the Suzuki reaction was carried out under a protective atmosphere to give the intermediate product.

[0049] The intermediate product, pinacol diborate, [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride, potassium acetate, and a second organic solvent were mixed and subjected to a Miyaura borate esterification reaction under a protective atmosphere to obtain anthracene fluorescent material.

[0050] This invention involves mixing 9,10-dibromoanthracene, 4-pyridineboronic acid pinacol ester, tetrakis(triphenylphosphine)palladium, and an aqueous solution of potassium carbonate with a first organic solvent, and conducting a Suzuki reaction under a protective atmosphere to obtain an intermediate product. In this invention, the molar ratio of 9,10-dibromoanthracene, 4-pyridineboronic acid pinacol ester, tetrakis(triphenylphosphine)palladium, and potassium carbonate in the aqueous solution is preferably 1:1.2:0.05:1; the concentration of the potassium carbonate aqueous solution is preferably 2 mol / L. In this invention, the first organic solvent is preferably a mixture of toluene and ethanol, with a volume ratio of toluene to ethanol preferably 3:1; the amount of the first organic solvent is sufficient to ensure the smooth progress of the Suzuki reaction, and this invention does not impose any special limitations on this. In this invention, the temperature of the Suzuki reaction is preferably 80–100°C, more preferably 85–90°C; the time is preferably 12–24 h, more preferably 20–24 h. After the Suzuki reaction is completed, the present invention preferably pours the obtained product system into water and extracts it with dichloromethane. The resulting organic phase is then rotary evaporated to remove the organic solvent. The residue is purified by silica gel chromatography to obtain the intermediate product. In the present invention, the eluent used for purification is preferably petroleum ether:ethyl acetate = 1:3 by volume.

[0051] After obtaining the intermediate product, the present invention mixes the intermediate product, pinacol diborate, [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride, potassium acetate, and a second organic solvent, and carries out a Miyaura borate esterification reaction under a protective atmosphere to obtain anthracene fluorescent material. In the present invention, the molar ratio of the intermediate product, pinacol diborate, [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride, and potassium acetate is preferably 1:1.2:0.1:2; the second organic solvent is preferably 1,4-dioxane, and its amount is sufficient to ensure the smooth progress of the Miyaura borate esterification reaction; the present invention does not impose any special limitations on this. In the present invention, the temperature of the Miyaura borate esterification reaction is preferably 100–130°C, more preferably 100–110°C; the time is preferably 12–24 h, more preferably 20–24 h. Following the Miyaura borate esterification reaction, the present invention preferably cools the resulting product system, mixes it with water, extracts it with dichloromethane, dries it with anhydrous magnesium sulfate, filters it, and rotary evaporates the resulting filtrate to remove the organic solvent. The residue is then purified using a silica gel chromatography column to obtain anthracene fluorescent material. In the present invention, the eluent used for purification is preferably petroleum ether:ethyl acetate = 3:1 by volume.

[0052] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0053] The preparation method of the anthracene fluorescent material (i.e., compound T1) used in the following examples includes the following steps:

[0054] (1) Preparation of compound 1, the reaction route is as follows:

[0055]

[0056] In a 100 mL two-necked flask, 9,10-dibromoanthracene (2 mmol), 4-pyridineborate pinacol ester (2.4 mmol), tetrakis(triphenylphosphine)palladium (0.1 mmol), toluene (15 mL), ethanol (5 mL), and potassium carbonate aqueous solution (2 mol / L, 10 mL) were added. Under argon protection, the resulting mixture was reacted at 85 °C for 24 h. After the reaction was completed, the resulting product system was poured into water and extracted with dichloromethane. The resulting organic phase was rotary evaporated to remove the organic solvent. The residue was purified by silica gel chromatography. The eluent used for purification was petroleum ether:ethyl acetate = 1:3 by volume, to give compound 1 with a yield of 45%. The molecular ion peak of the mass spectrometer was 347.03, and the actual molecular weight was 349.03.

[0057] (2) Preparation of compound T1, the reaction route is as follows:

[0058]

[0059] Compound 1 (5 mmol), pinacol diborate (6 mmol), [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (0.5 mmol), potassium acetate (10 mmol), and 1,4-dioxane (50 mL) were mixed and stirred at 100 °C for 24 h under argon protection. After the reaction was completed, the resulting product system was cooled and mixed with water, extracted with dichloromethane, dried with anhydrous magnesium sulfate for 12 h, filtered, and the filtrate was rotary evaporated to remove the organic solvent. The residue was purified by silica gel chromatography with petroleum ether:ethyl acetate in a volume ratio of 3:1 to give compound T1 in a yield of 69%.

[0060] 1¹H NMR (500MHz, DMSO) δ 8.27 (d, J = 8.7Hz, 2H), 7.71 (d, J = 8.8Hz, 2H), 7.58–7.47 (m, 2H), 7.44–7.34 (m, 2H), 7.01 (d, J = 8.3Hz, 2H), 6.80 (d, J = 8.3Hz, 2H), 5.32 (s, 2H), 1.53 (s, 12H). Molecular ion peak: 395.21, actual molecular weight: 395.08.

[0061] Example 1

[0062] The compound T1 was used as a pH testing reagent, and different pH environments were provided using Britton-Robinson buffer solvent. The specific steps are as follows:

[0063] Using tetrahydrofuran as a solvent, a solution with a concentration of 3×10⁻⁶ was prepared. -3 A mol / L solution of compound T1 (referred to as T1 solution).

[0064] Take 3 μL of the solution and place it in a cuvette. Add 3 mL of tetrahydrofuran and record the UV absorption wavelength and fluorescence intensity at this point. The normalized fluorescence emission spectrum and UV absorption wavelength are shown below. Figure 1 As shown. By Figure 1 It can be seen that the absorption wavelength and emission wavelength of compound T1 in tetrahydrofuran are 375 nm and 495 nm, respectively.

[0065] Take 3 μL of the L1 solution and place it in a cuvette. Add 150 μL of tetrahydrofuran and 2850 μL of buffer solvents with different pH values ​​(1–12). Record the fluorescence intensity ratio (IL) at 0 h, 12 h, 24 h, and 48 h. 430 / I 480 ) changes, results as Figure 2 As shown. By Figure 2 It can be seen that the fluorescence of compound T1 is relatively stable under different pH conditions.

[0066] Take 3 μL of the solution and place it in a cuvette. Add 150 μL of tetrahydrofuran and 2850 μL of a buffer solvent with a pH of 2. Record the fluorescence intensity. Then, adjust the pH of the system to 8 with sodium hydroxide and record the fluorescence intensity. After that, adjust the pH of the system to 2 with hydrochloric acid and record the fluorescence intensity. Repeat this reversible experiment. Figure 3 This is a graph showing the reversibility of fluorescence intensity of compound T1 in Example 1 at pH 2 and pH 8. Figure 3In the diagram, "2-1, 2-2, 2-3, 2-4" represent the fluorescence intensity curves of compound T1 at pH 2, and "8-1, 8-2, 8-3, 8-4" represent the fluorescence intensity curves of compound T1 at pH 8. Figure 3 It is known that compound T1 can undergo reversible reactions in acidic and weakly alkaline environments, exhibiting good reversibility. Using it as a probe can help increase its sensitivity in organisms.

[0067] Take 3 μL of the LT1 solution and place it in a cuvette. Add 150 μL of tetrahydrofuran, then add 2850 μL of buffer solvent with a pH of 4.0–6.0. ​​Record the fluorescence intensity for each step. The results are as follows: Figure 4 As shown. By Figure 4 It can be seen that compound T1 can accurately detect the pH value of a solution in the pH range of 4.0 to 6.0.

[0068] Take 3 μL of the solution and place it in a cuvette. Add 150 μL of tetrahydrofuran and 2850 μL of a pH 4 buffer solution. Then add 10 μM Na+ solution. + NH4 + Ca 2+ Mg 2+ Mn 2+ Cu 2+ Zn 2+ Al 3+ Hcy, GSH, Gly, Pro, and His were used as interfering agents, and their fluorescence intensity ratios (IL, I ... 430 / I 480 A control group without added interfering substances was set up, and the results were as follows: Figure 5 As shown in Figure 5, bar number 1 corresponds to the control group, and bars numbered 2 to 14 correspond to the interfering substance Na. + NH4 + Ca 2+ Mg 2+ Mn 2+ Cu 2+ Zn 2+ Al 3+ The experimental groups of Hcy, GSH, Gly, Pro, and His. (By...) Figure 5 It can be seen that the fluorescence intensity ratio of compound T1 hardly changed in the presence of the above-mentioned interfering substances, indicating that compound T1 can still detect pH value in the presence of interfering substances.

[0069] Take 3 μL of the solution and place it in a cuvette. Add 150 μL of tetrahydrofuran and 2850 μL of pH 4 buffer solution. Irradiate the solution with a xenon lamp for 80 min and record the fluorescence intensity ratio (IL). 430 / I480 The result is as follows Figure 6 As shown. By Figure 6 It can be seen that the fluorescence intensity ratio of compound T1 remains almost unchanged, indicating that compound T1 has good photostability.

[0070] Example 2

[0071] In this embodiment, cells were cultured at 37°C in DMEM medium (Dulbecco modified Eagle medium) containing 10% FBS (fetal bovine serum) and an atmosphere of 5% CO2 and 95% air. Prior to the experiment, cells were cultured at 2 × 10⁶ cells per tray. 5 Cells were seeded at a density of 2 mL of culture medium into 35 mm glass-bottomed culture dishes and incubated at 37 °C for 24 h in an incubator containing 5% CO2 and 95% air. During this period, the cells adhered to the glass surface, and cell experiments were then performed.

[0072] Compound T1 solution (PBS solution containing dimethyl sulfoxide, volume fraction of 1‰) was added to normal cells (human umbilical vein endothelial cells HUVEC) and tumor cells (mouse mammary tumor cells 4T1), respectively, to achieve a concentration of 10 μg / mL of compound T1 in the resulting mixture. The mixture was incubated at 37°C for 2 h. Then, lysosomal localization dye and mitochondrial localization dye were added to the normal cell system and tumor cell system, respectively, and the mixtures were co-incubated at 37°C for 30 min. Laser confocal imaging was then performed, and the results are as follows: Figure 7 As shown. By Figure 7 It was observed that in normal cells, the green channel fluorescence of compound T1 can precisely target lysosomes. Simultaneously, in tumor cells, the blue channel fluorescence overlaps with lysosomal dyes, while the green channel fluorescence overlaps with mitochondrial dye fluorescence. This indicates that compound T1, as a probe, can be used for dual-color visualization targeting of lysosomes and mitochondria.

[0073] Example 3

[0074] In this embodiment, cells were cultured at 37°C in DMEM medium (Dulbecco modified Eagle medium) containing 10% FBS (fetal bovine serum) and an atmosphere of 5% CO2 and 95% air. Prior to the experiment, cells were cultured at 2 × 10⁶ cells per tray. 5 Cells were seeded at a density of 2 mL of culture medium into 35 mm glass-bottomed culture dishes and incubated at 37 °C for 24 h in an incubator containing 5% CO2 and 95% air. During this period, the cells adhered to the glass surface, and cell experiments were then performed.

[0075] Compound T1 solution (using PBS solution containing dimethyl sulfoxide, with a volume fraction of 1‰) was added to normal cells (human umbilical vein endothelial cells HUVEC) and tumor cells (mouse breast tumor cells 4T1 and human breast tumor cells MDA-MB-231) to achieve a concentration of 10 μg / mL in the resulting mixture. The mixture was incubated at 37°C for 2 h, followed by laser confocal imaging. The results are as follows: Figure 8 As shown. By Figure 8 It is known that the green channel exhibits stronger fluorescence in normal cells, while the blue channel exhibits stronger fluorescence in tumor cells. This difference can serve as a simple and effective strategy for diagnosing tumor cells.

[0076] Different concentrations of compound T1 were added to normal cells (human umbilical vein endothelial cells HUVEC) and tumor cells (mouse breast tumor cells 4T1 and human breast tumor cells MDA-MB-231), and incubated at 37°C for 24 h. The results are as follows: Figure 9 As shown. By Figure 9 It is known that normal cells have a survival rate of over 90% when the concentration of compound T1 is 10 μg / mL, while the mortality rate of tumor cells is as high as 50%, which indicates that compound T1 can specifically kill tumor cells.

[0077] This embodiment also investigated the antitumor efficacy of compound T1 in breast cancer mice (primary tumor volume 100 mm). 3 Specifically, compound T1 solution (a PBS solution containing dimethyl sulfoxide with a volume fraction of 1‰) was introduced into mice via intratumoral injection at a dose of 50 mg / kg. The results showed that compound T1 had a significant anticancer effect. The body weight and tumor volume changes of the mice that received intratumoral injection were recorded over the next 6 days, as shown in Figure 10. Figure 10 In the graph, 'a' represents the change in mouse body weight, and 'b' represents the change in tumor volume; from Figure 10 It can be seen that the weight change of mice is very small, indicating that the systemic cytotoxicity of compound T1 is negligible during the anti-cancer process; the tumor volume of mice hardly increased after administration, indicating that compound T1 effectively inhibited tumor growth.

[0078] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An application of anthracene fluorescent material in pH detection, wherein the anthracene fluorescent material has the structure shown in Formula I:

2. The application according to claim 1, characterized in that, The applicable pH range for the anthracene fluorescent material is 4.0 to 6.

0.

3. The application of an anthracene fluorescent material in the preparation of lysosomal targeting reagents and / or mitochondrial targeting reagents, wherein the anthracene fluorescent material has the structure shown in Formula I:

4. The application according to claim 3, characterized in that, The lysosome-targeting reagent is a lysosome-targeting reagent for both normal cells and tumor cells.

5. The application according to claim 3, characterized in that, The mitochondrial targeting reagent is a mitochondrial targeting reagent for tumor cells.

6. The application according to claim 4 or 5, characterized in that, The tumor cells include breast tumor cells.

7. The application according to claim 3, characterized in that, The lysosome-targeting reagent and mitochondrial-targeting reagent are visual targeting reagents.

8. The application of an anthracene fluorescent material in the preparation of diagnostic and therapeutic tumor drugs, wherein the anthracene fluorescent material has the structure shown in Formula I:

9. The application according to claim 8, characterized in that, The tumor-treating drugs mentioned are those that can be visualized for diagnosis and treatment.

10. The application according to claim 8, characterized in that, The tumors include breast tumors.

Images