A tumor-targeting AIE fluorescent probe and its preparation and application

Abstract

The present invention discloses an AIE-type fluorescent probe with tumor targeting, its preparation and application, and belongs to the field of medical technology. The probe can accurately target tumor cells with high expression of HDAC6, and its specific binding ability reduces interference with normal cells and improves the accuracy of detection. The probe is integrated with AIE performance dye molecules, which emit strong fluorescence when aggregated, significantly enhancing the sensitivity and clarity of imaging. By optimizing the structure and synthesis conditions, the fluorescence intensity and stability of the probe are improved, ensuring the sensitivity of early tumor detection, and helping to improve patient survival rate and quality of life. The preparation method is simple and easy, low in cost, and conducive to practical application. The probe can be used to prepare tumor detection products, assist in tumor diagnosis and classification, monitor treatment changes, and evaluate efficacy. In terms of tumor imaging, it can achieve high-precision imaging, can be used for efficacy evaluation and to guide the formulation of subsequent treatment plans, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical applications, and in particular relates to an AIE-type fluorescent probe with tumor targeting and its preparation and application. Background Art

[0002] Breast cancer has become the most common cancer in women and one of the leading causes of cancer death, posing a serious threat to women's lives and health. Among breast cancer patients, triple-negative breast cancer (TNBC), a subtype of breast cancer with unique biological and clinical pathological characteristics, is characterized by strong invasiveness, rapid progression, low survival rate, and poorer prognosis than other types of breast cancer. However, if early diagnosis and timely and correct intervention treatment can be achieved, the survival rate of breast cancer patients can be significantly improved. However, since TNBC patients lack traditional breast cancer endocrine therapy targets, such as estrogen receptor (ER), progesterone receptor (PR), and its human epidermal growth factor receptor 2 (HER-2), there is a lack of fluorescent probes that can be used for precise imaging of TNBC in clinical practice. Therefore, the development of targeted fluorescent probes for TNBC has become a problem that needs to be solved urgently.

[0003] Histone deacetylase 6 (HDAC6) is an effective target for breast cancer diagnosis and treatment, and is associated with the occurrence, development, and progression of breast cancer. In hormone-dependent breast cancer, HDAC6 is upregulated to varying degrees, affecting ER gene transcription, resulting in high expression of ER and HDAC6 in cancer cells. In TNBC, HDAC6 silences the ER promoter, leading to the loss of estrogen receptor expression in TNBC. Although ERα is not expressed in TNBC, HDAC6 remains highly expressed, making HDAC6 a potential target for TNBC diagnosis.

[0004] Although some fluorescent probes targeting HDAC6 have been developed, they still have some shortcomings in their application. 1) Most of the HDAC6 ligands of fluorescent probes are based on the zinc ion binding group of hydroxamic acid (-CONHOH-) (Eur J Med Chem, 2017, 141: 596-602; Org Biomol Chem, 2018, 16, 7820-7832; Mol Imaging Biol, 2020, 22(3): 476-485; Sensors and Actuators B: Chemical, 2022, 364: 131882; Chem Commun, 2023, 59, 10109-10112; E ... Chem, 2024, 268: 116-236.), which can be metabolized in the body to produce hydroxylamine derivatives with toxic or side effects (ChemMedChem, 2016, 11(1): 15-21), which limits their clinical application; 2) Most of the existing HDAC6 fluorescent probes do not have AIE (aggregation-induced emission) properties (J MaterChem B, 2019, 7: 3528-3536; EBioMedicine, 2023, 98: 104880.), and are usually prepared by coupling HDAC6 ligands with near-infrared fluorescent dyes (such as IRDye800CW). When they are highly enriched in target tissues, they are prone to fluorescence quenching, which limits their application in complex biological environments.

[0005] Therefore, developing a reagent with AIE properties for precise imaging and detection of HDAC6 expression in TNBC has significant implications for tumor detection. This probe can specifically recognize and bind to HDAC6, enabling highly sensitive and specific imaging of HDAC6 at the cellular and tissue levels. By monitoring changes in HDAC6 expression, the progression and treatment efficacy of TNBC can be assessed, providing important evidence for the development and adjustment of personalized treatment plans, and thus has positive implications for tumor detection. Summary of the Invention

[0006] In response to the clinical problem of lack of fluorescent probes for accurate imaging of triple-negative breast cancer (TNBC), HDAC6 is a potential target for TNBC treatment. The development of AIE-type fluorescent probes targeting HDAC6-containing tumors has positive significance for tumor detection. The present invention aims to provide an AIE-type fluorescent probe with tumor targeting, as well as its preparation and application.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention provides an AIE-type fluorescent probe with tumor targeting, and the structure of the AIE-type fluorescent probe is as follows:

[0009]

[0010] The tumor targeting is targeting tumor cells with high expression of HDAC6, and the tumor is any one of breast tumor, lung cancer, liver cancer, and gastric cancer.

[0011] Furthermore, the tumor is a breast tumor.

[0012] Furthermore, the breast tumor is triple-negative breast cancer.

[0013] The preparation method of the above-mentioned tumor-targeting AIE-type fluorescent probe comprises:

[0014] S1, prepare compound 3; the structural formula of compound 3 is:

[0015] S2, preparing compound 4 by heating compound 3 with ammonia water; the structural formula of compound 4 is:

[0016] S3, preparing compound 5 by reacting compound 4 with potassium carbonate and 1,5-dibromopentane; the structural formula of compound 5 is:

[0017] S4, compound 5 was reacted with selenocyanate to prepare an AIE-type fluorescent probe with tumor targeting.

[0018] In S1, the preparation of compound 3 is as follows: 4-(diphenylamino)phenylboronic acid, 4-bromo-1,8-naphthalene dicarboxylic anhydride, and potassium carbonate are mixed for reaction, extracted, washed, dried, and purified by column chromatography, wherein the molar ratio of 4-(diphenylamino)phenylboronic acid, 4-bromo-1,8-naphthalene dicarboxylic anhydride and K2CO3 is 1-3:1:1-2

[0019] Furthermore, the solvent is a mixed solvent of toluene, ethanol and water in a volume ratio of 2:2:1.

[0020] Furthermore, the reaction catalyst is any one of Pd(PPh3)4 and Pd(OAc)2.

[0021] Furthermore, the extraction agent used in the extraction is any one of dichloromethane and ethyl acetate.

[0022] In S2, the molar ratio of the compound 3 to aqueous ammonia is 1:2-3, the reaction temperature is 70° C.-80° C., and the reaction time is 1 h-3 h.

[0023] In S3, the molar ratio of the compound 4, K2CO3 and 1,5-dibromopentane is 1:3-4:1-2, and the reaction time at room temperature is 10 h to 14 h.

[0024] In S4, the molar ratio of the compound 5 to selenocyanate is 1:2-4, the reaction temperature is 60° C.-80° C., and the reaction time is 10 h-14 h.

[0025] The application of the above-mentioned tumor-targeting AIE-type fluorescent probe in the preparation of tumor detection products.

[0026] The tumor detection product is a chip, a kit or a nucleic acid membrane strip; the tumor detection includes detecting the expression of tumor HDAC6, detecting the presence of tumor and performing in vitro pathological staining of tumor tissue.

[0027] The above-mentioned tumor-targeting AIE-type fluorescent probe is used in tumor imaging for non-diagnostic and therapeutic purposes. The AIE-type fluorescent probe can specifically target tumor cells with high expression of HDAC6.

[0028] Compared with the prior art, the present invention achieves the following technical effects:

[0029] The present invention provides an AIE-type fluorescent probe with tumor targeting, which has 1) tumor targeting and specificity: the probe targets tumor cells with high expression of HDAC6. HDAC6 is an important target in tumor treatment, and its high expression is often associated with the malignancy and drug resistance of the tumor. The targeting property helps to achieve accurate detection of specific tumor cells; it can specifically bind to tumor cells with high expression of HDAC6, thereby reducing interference with normal cells and improving the accuracy and reliability of detection; 2) AIE performance: by introducing dye molecules with AIE performance, the probe can emit a stronger fluorescent signal in an aggregated state, improving the sensitivity and clarity of imaging, thereby achieving imaging of tumor cells; 3) using near-infrared dyes for labeling, the probe has excellent tissue penetration ability and low background interference, and can achieve high-definition imaging in vivo; 4) high detection sensitivity: by optimizing the structure and synthesis conditions of the probe, the probe can increase its fluorescence intensity and stability, thereby enhancing the sensitivity of detection, facilitating accurate detection of tumors in the early stages, and improving the survival rate and quality of life of patients.

[0030] The preparation method of the tumor-targeting AIE-type fluorescent probe provided by the present invention has a simple preparation process and is easy to operate and control; the raw materials are readily available and the cost is low, which is conducive to practical application. Through the carefully designed molecular structure, the fluorescent probe can specifically recognize and bind to the HDAC6 receptor, ensuring the accuracy and reliability of the probe in complex biological environments.

[0031] The present invention provides applications for tumor-targeting AIE-type fluorescent probes. The probes can be used to prepare tumor detection products, such as chips, kits, or nucleic acid membrane strips. These products can be used to detect HDAC6 expression levels in tumors, thereby assisting in tumor diagnosis and classification. They can also be used to monitor changes during tumor treatment and assess treatment efficacy. In terms of tumor imaging, the probe can specifically target tumor cells with high HDAC6 expression, achieving high-precision imaging, helping doctors develop more precise surgical plans before surgery and reducing surgical risks and complications. Furthermore, the probes can be used to evaluate the efficacy of tumor treatment after treatment, guiding the development of subsequent treatment plans. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a hydrogen nuclear magnetic resonance spectrum of the tumor-targeting AIE-type fluorescent probe of the present invention;

[0033] Figure 2 The carbon nuclear magnetic resonance spectrum of the tumor-targeting AIE fluorescent probe of the present invention;

[0034] Figure 3 Spectral characterization diagram of the tumor-targeting AIE-type fluorescent probe of the present invention; wherein a is the ultraviolet absorption spectrum diagram, and b is the fluorescence emission spectrum diagram;

[0035] Figure 4 : is an AIE effect diagram of the tumor-targeting AIE-type fluorescent probe of the present invention; wherein a is a fluorescence imaging diagram of the fluorescent probe molecule in water with different volume ratios, and b is a quantitative analysis of the fluorescence intensity of the fluorescent probe molecule in water with different volume ratios;

[0036] Figure 5 This is a photostability analysis of the tumor-targeting AIE-type fluorescent probe of the present invention; wherein a is the fluorescence imaging of the fluorescent probe molecule before and after illumination at different times, and b is the quantitative analysis of the fluorescence intensity of the fluorescent probe molecule before and after illumination at different times;

[0037] Figure 6 The effect of the tumor-targeting AIE fluorescent probe of the present invention on the survival rate of normal mammary epithelial cells MCF-10A;

[0038] Figure 7The flow cytometer is used to analyze the binding of tumor cell lines to the tumor-targeting AIE-type fluorescent probe and non-targeted compounds of the present invention; wherein, a is the flow cytometer test of the uptake of different concentrations of fluorescent probe molecules by MDA-MB-231 cells, b is the flow cytometer test of the uptake of different concentrations of compound 3 by MDA-MB-231 cells, c is the quantitative analysis of the uptake rate of different concentrations of fluorescent probe molecules and compound 3 by MDA-MB-231 cells, and d is the quantitative analysis of the average fluorescence intensity percentage uptake rate after the MDA-MB-231 cells uptake different concentrations of fluorescent probe molecules and compound 3;

[0039] Figure 8 The binding of breast cancer MDA-MB-231 cells to the AIE type fluorescent probe molecules of the present invention at different concentrations was observed using a laser confocal microscope;

[0040] Figure 9 These are the in vivo fluorescence imaging results of mice bearing breast MDA-MB-231 tumors before and after intravenous injection of the AIE-type fluorescent probe of the present invention. Among them, a is the imaging image at different times after the fluorescent probe molecule was injected into the tail vein of the mouse bearing breast MDA-MB-231 tumor, b is the quantitative analysis of the fluorescence intensity signal-to-noise ratio of the tumor and surrounding normal tissues at different times, and c is the result of fluorescence imaging of the main organs of the mouse bearing breast MDA-MB-231 tumor 24 hours after the fluorescent probe molecule was injected. DETAILED DESCRIPTION

[0041] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0042] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0043] In the present invention, unless otherwise specified, all experimental raw materials used are commercially available products well known to those skilled in the art.

[0044] MDA-MB-231 cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences; BALB / c nude mice were purchased from the Xi'an Branch of Chongqing Tengxin Biotechnology Co., Ltd.

[0045] 12-well white bottom permeable cell culture plate (Costar product, product number No. 3513);

[0046] Fetal Bovine Serum (Gibco product, product number 10099141);

[0047] 100 mm culture dish (Corning product, product number 430167);

[0048] DMEM medium (Gibco product, product number 11965-084);

[0049] Flow cytometer (BD product, product model: Accuri C6, USA);

[0050] Fluorescence inverted microscope (Leica product, product model DMI3000B, Germany);

[0051] High-sensitivity optical imaging system (PerkinElmer product, product model IVIS Spectrum, USA).

[0052] Example 1

[0053] This embodiment provides an AIE-type fluorescent probe with tumor targeting, and the specific preparation steps are as follows:

[0054] (1) Preparation of Compound 3

[0055]

[0056] 4-(Diphenylamino)phenylboronic acid (260.2 mg, 0.90 mmol, compound 1), 4-bromo-1,8-naphthalenedicarboxylic acid (166.2 mg, 0.60 mmol, compound 2), and K2CO3 (165.8 mg, 1.20 mmol) were dissolved in 25 mL of a mixed solvent of toluene / ethanol / water (2 / 2 / 1, V / V / V). Pd(PPh3)4 (40.0 mg, 0.04 mmol) was then added, and the mixture was refluxed for 2 hours. After the reaction, ethanol was distilled off under reduced pressure, the product was extracted with 20 mL of dichloromethane, washed with 50 mL of saturated brine, dried over anhydrous Na2SO4, and dichloromethane was evaporated under reduced pressure to obtain a crude product. The crude product was purified by chromatography on a 200-300 mesh silica gel column with petroleum ether and ethyl acetate (5:1 by volume) as eluent to afford 143.5 mg of compound 3 in a yield of 54.2%.

[0057] The structural formula of compound 3 is:

[0058] (2) Preparation of Compound 4

[0059]

[0060] Compound 3 (110.4 mg, 0.25 mmol) was dissolved in 20 mL of ethanol (EtOH), and 77 μL of 25% aqueous ammonia was added. The mixture was heated to 75°C and reacted for 2 hours. After the reaction was completed, the ethanol was evaporated to obtain a crude product, which was purified by 200-300 mesh silica gel column chromatography using petroleum ether and ethyl acetate (volume ratio of 3:1) as eluent to obtain 93.4 mg of compound 4 with a yield of 84.7%. The NMR data of compound 4 are as follows:

[0061] 1 H NMR (600M, CDCl3) δ8.62-8.64(m,2H),8.59(s,1H),8.45(d,J=8.4Hz,1H),7.74(t,J=7.8Hz,2 H),7.37(d,J=8.4Hz,2H),7.33(t,J=7.8Hz,4H),7.20(d,J=8.4Hz,6H),7.10(t,J=7.8Hz,2H).

[0062] 13 C NMR (100M, CDCl3) δ164.09,163.86,148.49,147.60,147.31,133.61,131.70,131.06,130.81 ,130.49,130.18,129.51,127.73,126.66,125.24,125.11,123.71,122.73,122.42,121.03.

[0063] The structural formula of compound 4 is:

[0064] (3) Preparation of Compound 5

[0065]

[0066] Compound 4 (88.1 mg, 0.20 mmol) was dissolved in 10 mL of N,N-dimethylformamide (DMF), and K2CO3 (82.9 mg, 0.60 mmol) and 1,5-dibromopentane (55.2 mg, 0.24 mmol) were added. The mixture was reacted at room temperature for 12 hours. After the reaction, 25 mL of water was added, and the mixture was extracted with 20 mL of ethyl acetate. The mixture was washed three times with saturated brine and dried over anhydrous Na2SO4. The ethyl acetate was evaporated to obtain a crude product. The crude product was purified by chromatography on a 200-300 mesh silica gel column using petroleum ether and ethyl acetate (volume ratio of 2:1) as eluent to obtain 78.1 mg of compound 5 with a yield of 66.2%. The NMR data of compound 5 are as follows:

[0067] 1 H NMR (600M, CDCl3) δ8.62-8.63(m,2H),8.40(d,J=8.4Hz,1H),7.72(t,J=7.8Hz,2H),7.36(d,J=8.4Hz,2H),7.32(t,J=7.8Hz,4H),7.20(d,J =7.8Hz, 6H), 7.10 (t, J = 7.8Hz, 2H), 4.21 (t, J = 7.8Hz, 2H), 3.43 (t, J = 7.2Hz, 2H), 1.94-1.98 (m, 2H), 1.77-1.82 (m, 2H), 1.58-1.62 (m, 2H).

[0068] 13 C NMR(100M, CDCl3)δ164.37,164.17,148.36,147.33,146.80,132.87,131.89,131.19,130.96,130.79,130.02, 129.48,128.86,127.68,126.67,125.04,123.63,122.85,122.48,121.19,40.03,33.59,32.43,27.21,25.68.

[0069] The structural formula of compound 5 is:

[0070] (4) Preparation of fluorescent probe compound 6

[0071]

[0072] Compound 5 (59.0 mg, 0.10 mmol) and KSeCN (43.2 mg, 0.30 mmol) were dissolved in 10 mL of N,N-dimethylformamide (DMF) and heated to 70° C. for 12 hours. After the reaction, the reaction solution was poured into water to precipitate a yellow solid, which was filtered and the filter cake was washed three times with water and dried in vacuo to obtain a crude product. The crude product was purified by 200-300 mesh silica gel column chromatography using dichloromethane and methanol (volume ratio of 12:1) as eluent to obtain 44.1 mg of fluorescent probe compound 6, i.e., the tumor-targeting AIE-type fluorescent probe of the present invention, with a yield of 71.8%. The nuclear magnetic resonance spectrum of the fluorescent probe molecule is shown in the attached FIG. Figure 1-2 shown.

[0073] 1H NMR (600M, CDCl3) δ8.62-8.63(m,2H),8.41(d,J=8.4Hz,1H),7.72(t,J=7.8Hz,2H),7.36(d,J=8.4Hz,2H),7.32(t,J=7.2Hz,4H),7.20(d,J =7.2Hz,6H),7.10(t,J=7.2Hz,2H),4.23(t,J=7.2Hz,2H),3.09(t,J=7.2Hz,2H),2.00-2.03(m,2H),1.80-1.85(m,2H),1.59-1.63(m,2H).

[0074] 13 C NMR (100M, CDCl3) δ164.40,164.20,148.38,147.32,146.89,132.95,131.84,131.24,131.01,130.79,130.03,129 .48,128.86,127.69,126.68,125.05,123.65,122.78,122.46,121.11,101.47,39.85,30.40,29.35,27.24,26.53.

[0075] Example 2

[0076] Based on Example 1, this example performs spectral characterization on the tumor-targeting AIE-type fluorescent probe molecule obtained in Example 1.

[0077] Using dimethyl sulfoxide (DMSO) as a solvent, the tumor-targeting AIE-type fluorescent probe of Example 1 was prepared into a working solution with a concentration of 10 μM. Its absorption spectrum was measured using an ultraviolet spectrophotometer; its fluorescence emission spectrum was measured using a fluorescence spectrometer. The spectrum is shown in the attached figure. Figure 3 shown.

[0078] By the attached Figure 3 It can be seen from the spectrum that the maximum ultraviolet absorption wavelength of the tumor-targeting AIE-type fluorescent probe molecule of the present invention is 425 nm, and the maximum fluorescence emission wavelength is 512 nm, indicating that the AIE-type fluorescent probe molecule of the present invention has fluorescent properties and can perform fluorescence imaging.

[0079] Example 3

[0080] Based on Example 1, this example detects the AIE effect of the tumor-targeting AIE-type fluorescent probe molecule of Example 1.

[0081] The AIE-type fluorescent probe molecules in Example 1 were prepared into 0.01 mol / L mother liquors using DMSO and deionized water H2O, respectively, and recorded as solution A and solution B. 100 μL of solution A was placed in a 0.2 mL centrifuge tube, which is a fluorescent probe molecule solution with a water content of 0%; 90 μL of solution A and 10 μL of solution B were placed in a 0.2 mL centrifuge tube, which is a fluorescent probe molecule solution with a water content of 10%. Similarly, probe molecule solutions with water content of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90% were prepared. The prepared solutions were then neatly arranged in a small animal in vivo optical imaging system for imaging to analyze the change in the fluorescence of the fluorescent probe molecules with the volume of water in the solution. For specific results, see the attached figure. Figure 4 shown.

[0082] By the attached Figure 4 The data show that when the volume content of water in the fluorescent probe molecule solution increases from 0% to 30%, the fluorescent probe molecule produces fluorescence; when the volume content of water in the fluorescent probe molecule solution increases from 30% to 50%, the fluorescence intensity of the fluorescent probe molecule gradually increases and reaches a maximum value; when the volume content of water in the fluorescent probe molecule solution increases from 50% to 90%, the fluorescence intensity of the probe molecule does not increase but decreases. These results indicate that the fluorescent probe molecule of the present invention has an AIE effect, that is, the fluorescence intensity is enhanced in the aggregated state, and when the volume content of water in the prepared solution is 50%, the strongest fluorescence signal can be generated. However, the currently reported HDA6 fluorescent probes IRDye800CW-SelSA, IRDye800CW-SAHA, and SeCF3-IRDye800cw do not have AIE performance. Compared with other fluorescent probes reported so far, such as HDA6 fluorescent probe IRDye800CW-SelSA (J Mater Chem B, 2019, 7: 3528-3536.), IRDye800CW-SAHA (MolImaging Biol, 2020, 22(3): 476-485.) and SeCF3-IRDye800cw (EBioMedicine, 2023, 98: 104880.), the fluorescent probe molecule of the present invention has unique AIE properties, which makes it have potential application value in fields such as tumor targeted imaging.

[0083] Example 4

[0084] This example, based on Example 1, detects the photostability of the tumor-targeting AIE-type fluorescent probe molecule of the present invention.

[0085] The tumor-targeting AIE-type fluorescent probe molecules of Example 1 were prepared into working solutions of different concentrations using DMSO. The working solutions were imaged using a small animal in vivo optical imaging system before exposure to fluorescent light and after exposure to different illumination times (5 min, 10 min, 15 min, 30 min, 1 h, 2 h, 4 h, and 6 h). The changes in fluorescence intensity over illumination time were observed. The specific results are shown in the attached figure. Figure 5 shown.

[0086] By the attached Figure 5 The data show that before illumination, the fluorescent probe molecule has already shown a certain fluorescence intensity; with the extension of illumination time (5min~6h), the fluorescence intensity of the fluorescent probe molecule does not show a significant decrease or quenching phenomenon, which indicates that the fluorescent probe molecule of the present invention has excellent anti-fluorescence quenching ability, can maintain high fluorescence stability under illumination, and can be used for biological imaging.

[0087] The experimental results of this embodiment show that the AIE-type fluorescent probe molecule of the present invention has excellent photostability. This photostability makes the probe molecule have greater potential in applications such as biological imaging and can provide stable and reliable fluorescent signals in complex biological environments. Therefore, the AIE-type fluorescent probe molecule of the present invention is expected to become an efficient tumor-targeting fluorescent probe, providing strong support for the diagnosis and treatment of tumors.

[0088] Example 5

[0089] In this example, based on Example 1, the MTT method was used to detect the toxicity of the tumor-targeting AIE fluorescent probe in Example 1 to normal breast cancer epithelial cells MCF-10A.

[0090] The specific method is as follows:

[0091] (1) Cell culture and inoculation

[0092] MCF-10A cells were revived and cultured until the cell density in a T25 cell culture flask reached 80% to 90%. The cells were then trypsinized and centrifuged, the supernatant discarded, and the cells were resuspended and mixed with 2 mL of complete culture medium. The cells were counted. The cells were seeded into 96-well plates at a density of 10,000 cells per well. The edge wells were filled with sterile PBS, and three 96-well plates were seeded for each cell type. The complete culture medium consisted of 90% DMEM and 10% fetal bovine serum.

[0093] (2) Fluorescent probe molecule processing

[0094] The 96-well plate was returned to the carbon dioxide incubator and cultured until the cells adhered. The original culture medium was discarded, and the culture medium was used to dilute the 0.01 mol / L tumor-targeting AIE-type fluorescent probe molecule solution of Example 1 dissolved in DMSO to different concentrations (0 μM, 0.5 μM, 1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, 50 μM). The diluted fluorescent probe molecule solution was added to the 96-well plate, and the cells were incubated with the fluorescent probe molecules for 12 hours, 24 hours, and 48 hours, respectively.

[0095] (3) MTT assay

[0096] Prepare 5 mg / mL MTT solution, filter it and store it in the dark at 4°C; after incubation, discard the culture medium containing the fluorescent probe molecule, add 100 μL of the mixture according to the volume ratio of complete culture medium:MTT solution at 9:1, and continue to culture for 4 hours; after 4 hours, gently aspirate the liquid in the 96-well plate, add 150 μL DMSO to each well, shake on a shaker for 10 minutes, and use a microplate reader to detect the absorbance of each well at 490 nm.

[0097] (4) Data Analysis

[0098] The cell survival rate under incubation with different concentrations of fluorescent probe molecules was calculated based on the absorbance value. For specific results, please refer to the attached Figure 6 shown.

[0099] By the attached Figure 6 The data show that when the concentration of the fluorescent probe molecule is in the range of 0-10 μmol / L, the survival rate of MCF-10A cells exceeds 80%, which indicates that at low concentrations, the fluorescent probe molecule has less toxicity to cells; even when the concentration of the fluorescent probe molecule is increased to 20 μmol / L and 50 μmol / L, the survival rate of MCF-10A cells still reaches nearly 70%, which further proves that the fluorescent probe molecule has high biocompatibility and low toxicity and can be used for biological imaging.

[0100] Example 6

[0101] This example tests the in vitro targeting of the fluorescent probe based on the fluorescent probe in Example 1.

[0102] 1. Cell Culture

[0103] (1) Prepare the complete culture medium required for cells

[0104] The complete culture medium consisted of 90% DMEM and 10% fetal bovine serum, and double antibody storage solution (penicillin + streptomycin) was added at a volume fraction of 1% of the culture medium. The final concentrations of penicillin and streptomycin were 100 U / mL and 100 μg / mL, respectively.

[0105] (2) Before the experiment begins, confirm the name, culture medium, subculture time, and other information of the cell line MDA-MB-231 marked on the 100 mm culture dish to ensure that the experiment is correct.

[0106] (3) Use a pipette and a sterile pipette tip to aspirate the cell culture medium.

[0107] (4) Rinse the cell surface with 2 mL of sterile PBS solution, and then aspirate the PBS waste liquid with a sterile pipette tip.

[0108] (5) Add 1 mL of 0.25% (w / v) trypsin solution containing 0.04% (w / v) EDTA to the culture medium to digest the cells. Mix gently so that the solution covers the cell surface. Observe under a microscope. When the cells are completely detached from the bottom of the culture dish and the cell connections are loose, add 2 mL of complete culture medium and pipette.

[0109] (6) After the cells are separated into single cells, the cell suspension is transferred to a 15 mL sterile centrifuge tube and centrifuged at 1000 rpm for 5 min.

[0110] (7) Use a sterile pipette to aspirate the supernatant medium and add 2 mL of fresh complete medium to resuspend the cell pellet and gently pipette to mix.

[0111] (8) Count the cell suspension using a cell counting plate, adjust the concentration of the cell suspension and inoculate it into a 12-well culture plate for a plating experiment. The cell density is 2×10 5 cells / well and culture overnight to allow the cells to fully adhere.

[0112] 2. In vitro targeting assay

[0113] (1) 1 mL of fluorescent probe 6 solution with different concentration gradients (0 μM, 0.2 μM, 0.4 μM, 0.6 μM, 0.8 μM, 1 μM) and intermediate compound 3 of Example 1 with different concentration gradients (0 μM, 0.2 μM, 0.4 μM, 0.6 μM, 0.8 μM, 1 μM) were added to a 12-well culture plate.

[0114] (2) The cells in the above culture plate were incubated for 4 h and then washed three times with PBS to remove free substances not taken up by the cells.

[0115] (3) 0.2 mL of 0.25% (w / v) trypsin solution containing 0.04% (w / v) EDTA was used to digest the cells. Gently mix until the solution covers the cell surface and observe under a microscope. When the cells are completely detached from the bottom of the 12-well culture plate and the cell connections are loose, add 1 mL of complete culture medium and pipette to form a cell suspension.

[0116] (4) The cell suspensions were placed in 1.5 mL centrifuge tubes, centrifuged at 1000 rpm for 5 min to precipitate the cells, and washed three times with sterile PBS to further remove impurities and unbound substances. The washed cells were detected by flow cytometry to analyze the cellular uptake of the fluorescent probe and compound 3. The specific results are shown in the attached figure. Figure 7 shown.

[0117] By the attached Figure 7 The data show that the fluorescence intensity of the fluorescent probe increases with the increase of its concentration until it reaches a peak and then decreases slightly, indicating that the cell uptake of the fluorescent probe increases with the increase of concentration, but after reaching a certain level, it may no longer increase due to the saturation effect; while the cell uptake of compound 3 does not increase with the increase of concentration, which shows that the fluorescent probe has good tumor targeting and can specifically identify and bind to tumor cells. The fluorescence intensity of compound 3 also increases with the increase of concentration, but the increasing trend is not obvious and is far lower than the fluorescence intensity of the fluorescent probe, indicating that the cell uptake of compound 3 does not increase significantly with the increase of concentration.

[0118] In summary, the fluorescent probe of the present invention has excellent tumor targeting and can specifically identify and bind to tumor cells; compound 3 does not have this property, and its cellular uptake does not increase significantly with increasing concentration. The AIE-type fluorescent probe of the present invention has potential application value in tumor diagnosis and treatment, and provides new ideas and methods for the diagnosis and treatment of tumors.

[0119] Example 7

[0120] In this example, based on Example 1, the uptake of the fluorescent probe of the present invention by human breast cancer MDA-MB-231 cells was measured. The uptake of the fluorescent probe to be tested by human breast cancer MDA-MB-231 cells was observed using a laser confocal microscope. The specific steps are as follows:

[0121] (1) MDA-MB-231 cells were plated at 1×10 5 The cells were plated into confocal microplates at a density of 100 μg / cm2 and cultured in an incubator until the cells adhered to the wall.

[0122] (2) After the cells adhered, the culture medium in the dish was discarded, and different concentrations (0.25, 1 μM) of the fluorescent probe solution of Example 1 were added, and the cells were cultured in a 37° C. incubator for 4 h.

[0123] (3) Gently wash three times with PBS to remove the probe that has not been taken up by the cells, add Hoechst solution (1 μL Hoechst diluted with 1 mL culture medium) and incubate for 20 min. Hoechst solution is used to label the cell nucleus and produce blue fluorescence.

[0124] (4) After 20 minutes, gently wash three times with PBS.

[0125] (5) Add 1 mL of PBS to each dish and observe the cells' uptake of different concentrations of fluorescent probes under a laser confocal microscope.

[0126] Laser confocal microscopy was used to observe the uptake of the fluorescent probe of the present invention by MDA-MB-231. Figure 8 shown.

[0127] By the attached Figure 8 The data show that after incubation with the fluorescent probe for 4 hours, a strong fluorescence signal was observed in the cells, indicating that the fluorescent probe can successfully enter the cells; comparing the fluorescence intensity of different concentrations of fluorescent probes (0.25μM and 1μM), it was found that there was almost no difference, which means that within the selected concentration range, the cell's uptake of the fluorescent probe has reached saturation, further indicating that the fluorescent probe can successfully enter the cells. These results provide valuable information for further exploring the application of fluorescent probes in tumor diagnosis and treatment.

[0128] Example 8

[0129] This example is based on Example 1, and animal experiments are conducted to test the targeting and accuracy of the fluorescent probe of the present invention. The specific experimental process is as follows:

[0130] (1) Establishment of tumor-bearing mouse model

[0131] MDA-MB-231 tumor cells were taken and centrifuged at 1000 rpm for 5 min. The tumor cells were then washed three times with sterile PBS and diluted appropriately. The cells were counted using a hemocytometer to obtain a concentration of 1×10 7 Each mouse was subcutaneously inoculated with 100 μL of tumor cell suspension of cells / μL at the right hind limb of the back. The inoculation site was observed daily after inoculation to ensure that there was no infection and that the tumor did not regress naturally after growth. 3 Imaging experiments were started.

[0132] (2) Targeted verification

[0133] 5-10 days after tumor inoculation, 10 nmol of fluorescent probe was injected through the tail vein. Then, a high-sensitivity small animal in vivo optical imaging system was used to monitor the fluorescent signal in the mice before and 24 hours after the injection of the fluorescent probe. 24 hours after the injection of the fluorescent probe, the main organs and tumor tissues of the mice were removed for imaging. For specific results, see the attached Figure 9 shown.

[0134] In vivo fluorescence imaging: The fluorescent probe began to accumulate in the tumor tissue 2 hours after injection, indicating that the probe had begun to migrate to the tumor tissue; 10 hours after the injection of the fluorescent probe, the fluorescent probe showed good enrichment in the tumor tissue, which further confirmed that the fluorescent probe has the ability to aggregate to the tumor tissue; 24 hours after the injection of the fluorescent probe, further in vitro imaging of tissues and organs showed that the tumors injected with the fluorescent probe group had a higher fluorescence intensity, which proved that the accumulation of the fluorescent probe in the tumor tissue was significant and its fluorescence signal intensity was sufficient for imaging purposes.

[0135] In vitro targeting: The fluorescent probe has good targeting properties for MDA-MB-231 tumors and can be enriched in tumor tissue. Its fluorescence signal intensity is high enough to be used for imaging MDA-MB-231 tumors. This indicates that the fluorescent probe of the present invention has potential application value, such as in tumor imaging, monitoring tumor progression, or evaluating treatment efficacy.

[0136] Through the establishment of a tumor-bearing mouse model and targeting verification experiments, it was successfully demonstrated that the probe has good targeting to MDA-MB-231 tumors, providing an important basis for the further application of the fluorescent probe of the present invention in tumor imaging and treatment.

[0137] In summary, the fluorescent probe in the present invention exhibits excellent targeting at both the cellular and animal levels, enabling dynamic and non-invasive in vivo assessment of HDAC6 molecules expressed in tumors, solving the problem of tumor-specific imaging. Furthermore, the probe complex has no toxic side effects, is safe and reliable, and has a simple preparation process.

[0138] The above content is only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A tumor-targeting AIE-type fluorescent probe, characterized in that: The structure of the AIE type fluorescent probe is as follows:

2. The tumor-targeting AIE-type fluorescent probe according to claim 1, characterized in that: The tumor targeting is targeting tumor cells with high expression of HDAC6, and the tumor is any one of breast tumor, lung cancer, liver cancer, and gastric cancer.

3. The method for preparing the tumor-targeting AIE-type fluorescent probe according to any one of claims 1 to 2, characterized in that: include: S1, prepare compound 3; the structural formula of compound 3 is: S2, preparing compound 4 by heating compound 3 with ammonia water, wherein the structural formula of compound 4 is: S3, preparing compound 5 by reacting compound 4 with potassium carbonate and 1,5-dibromopentane; the structural formula of compound 5 is: S4, compound 5 was reacted with selenocyanate to prepare an AIE-type fluorescent probe with tumor targeting.

4. The method for preparing a tumor-targeting AIE-type fluorescent probe according to claim 3, wherein: In S1, the preparation of compound 3 includes: mixing 4-(diphenylamino)phenylboronic acid, 4-bromo-1,8-naphthalene dicarboxylic anhydride, and potassium carbonate, extracting, washing, drying, and purifying by column chromatography, wherein the molar ratio of the 4-(diphenylamino)phenylboronic acid, 4-bromo-1,8-naphthalene dicarboxylic anhydride, and K2CO3 is 1 to 3:1:1 to 2.

5. The method for preparing a tumor-targeting AIE-type fluorescent probe according to claim 3, wherein: In S2, the molar ratio of the compound 3 to aqueous ammonia is 1:2-3, the reaction temperature is 70° C.-80° C., and the reaction time is 1 h-3 h.

6. The method for preparing a tumor-targeting AIE-type fluorescent probe according to claim 3, wherein: In S3, the molar ratio of the compound 4, K2CO3 and 1,5-dibromopentane is 1:3-4:1-2, and the reaction is carried out at room temperature for 10 h to 14 h.

7. The method for preparing a tumor-targeting AIE-type fluorescent probe according to claim 3, wherein: In S4, the molar ratio of the compound 5 to selenocyanate is 1:2-4, the reaction temperature is 60° C.-80° C., and the reaction time is 10 h-14 h.

8. Use of the tumor-targeting AIE-type fluorescent probe according to any one of claims 1 to 2 in the preparation of a tumor detection product.

9. The use according to claim 8, characterized in that The tumor detection product is a chip, a kit or a nucleic acid membrane strip; the tumor detection includes detecting the expression of tumor HDAC6, detecting the presence of tumor and performing in vitro pathological staining of tumor tissue.

10. Use of the tumor-targeting AIE-type fluorescent probe according to any one of claims 1 to 2 for non-diagnostic and non-therapeutic purposes in tumor imaging, characterized in that: The AIE-type fluorescent probe can specifically target tumor cells with high HDAC6 expression.

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