AIE type fluorescent probe with tumor targeting property as well as preparation and application of AIE type fluorescent probe

Abstract

The invention discloses an AIE type fluorescent probe with tumor targeting as well as preparation and application thereof, and belongs to the technical field of medicines, the probe can accurately target HDAC6 high-expression tumor cells, the specific binding capacity of the probe reduces interference to normal cells, and the detection accuracy is improved. The probe is fused with AIE performance dye molecules and emits strong fluorescence during aggregation, so that the imaging sensitivity and definition are remarkably enhanced. By optimizing the structure and synthesis conditions, the fluorescence intensity and stability of the probe are improved, the sensitivity of early tumor detection is ensured, and the survival rate and life quality of patients are improved. The preparation method is simple, convenient, feasible, low in cost and beneficial to practical application. The probe can be used for preparing tumor detection products, assisting in tumor diagnosis and classification, monitoring treatment changes and evaluating the curative effect. High-precision imaging can be realized in the aspect of tumor imaging, the method can be used for evaluating curative effect and guiding formulation of subsequent treatment schemes, and the application prospect is wide.

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 the preparation and application thereof. Background Art

[0002] Breast cancer has become the most common cancer in women and one of the main causes of cancer death, which has seriously threatened women's life and health. Among breast cancer patients, triple-negative breast cancer (TNBC), a subtype of breast cancer with special biological and clinical pathological characteristics, is characterized by strong invasiveness, rapid progression, low survival rate and poor prognosis compared with 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 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 the diagnosis and treatment of breast cancer, and is related to the occurrence, development and evolution of breast cancer: in hormone-dependent breast cancer, HDAC6 is upregulated to varying degrees in breast cancer, affecting ER gene transcription, resulting in high expression of ER and HDAC6 levels 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 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: 116236.), which can be metabolized in vivo to generate hydroxylamine derivatives with toxic or side effects (ChemMedChem, 2016, 11(1): 15-21), limiting 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 concentrated in target tissues, they are prone to fluorescence quenching, limiting their application in complex biological environments.

[0005] Therefore, developing a reagent with AIE performance that can be used for accurate imaging and detection of HDAC6 expression in TNBC has positive significance for tumor detection. This probe can specifically recognize and bind to HDAC6, thereby achieving high-sensitivity and high-specificity imaging of HDAC6 at the cellular and tissue levels. By monitoring the expression changes of HDAC6, the progression and treatment effect of TNBC can be evaluated, providing an important basis for the formulation and adjustment of personalized treatment plans, which has positive significance for tumor detection. Summary of the invention

[0006] In view of the problem of lack of fluorescent probes for accurate imaging of triple-negative breast cancer (TNBC) in clinical practice, HDAC6 is a potential target for TNBC treatment, and the development of AIE-type fluorescent probes targeting HDAC6 tumors has positive significance for tumor detection. The present invention aims to provide an AIE-type fluorescent probe with tumor targeting, and 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 method for preparing the above-mentioned tumor-targeting AIE type fluorescent probe comprises:

[0014] S1, preparing 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 a tumor-targeting AIE fluorescent probe.

[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 10h-14h.

[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] 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 tumors and performing in vitro pathological staining of tumor tissues.

[0027] The above-mentioned tumor-targeting AIE-type fluorescent probe is used for non-diagnostic and therapeutic purposes in tumor imaging. 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 AIE type fluorescent probe with tumor targeting provided by the present invention has the following characteristics: 1) tumor targeting and specificity: the probe targets tumor cells with high expression of HDAC6. As an important target in tumor treatment, the high expression of HDAC6 is often related to the malignancy and drug resistance of the tumor. The targeting 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 fluorescence signal in an aggregated state, thereby improving the sensitivity and clarity of imaging, thereby achieving imaging of tumor cells; 3) near-infrared dyes are used for labeling, so that 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 improve its fluorescence intensity and stability, thereby enhancing the sensitivity of detection, which is helpful for accurate detection in the early stages of tumors and improving the survival rate and quality of life of patients.

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

[0031] The application of the tumor-targeting AIE fluorescent probe provided by the present invention can be used to prepare tumor detection products, such as chips, kits or nucleic acid membrane strips, etc. These products can be used to detect the expression level of tumor HDAC6, thereby assisting the diagnosis and classification of tumors. They can also be used to monitor changes in the process of tumor treatment and evaluate the treatment effect; in terms of tumor imaging, the probe can specifically target tumor cells with high expression of HDAC6 to achieve high-precision imaging, which helps doctors to formulate more accurate surgical plans before surgery and reduce surgical risks and complications. At the same time, it can also be used to evaluate the efficacy after tumor treatment and guide the formulation of subsequent treatment plans. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0033] Figure 2 The carbon nuclear magnetic resonance spectrum of the tumor-targeting AIE type 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 an ultraviolet absorption spectrum diagram, and b is a 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 The present invention is a photostability analysis of the tumor-targeting AIE-type fluorescent probe; wherein a is a fluorescence imaging diagram of the fluorescent probe molecule at different times before and after illumination, and b is a quantitative analysis of the fluorescence intensity of the fluorescent probe molecule at different times before and after illumination;

[0037] Figure 6 The effect of the tumor-targeting AIE fluorescent probe of the present invention on the survival rate of normal breast 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] Fig. 9 These are the results of in vivo fluorescence imaging of mice bearing breast MDA-MB-231 tumors before and after intravenous injection of the AIE type fluorescent probe of the present invention, wherein a is the imaging diagram at different times after the fluorescent probe molecule is injected into the mice bearing breast MDA-MB-231 tumors through the tail vein, b is a quantitative analysis of the fluorescence intensity signal-to-noise ratio of the tumor and the surrounding normal tissues at different times, and c is the result of fluorescence imaging of the main organs taken out 24 hours after the fluorescent probe molecule was injected into the mice bearing breast MDA-MB-231 tumors. DETAILED DESCRIPTION

[0041] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme 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 described embodiments 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 creative work 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 the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[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 was 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] An optical imaging system with high detection sensitivity (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-naphthalene dicarboxylic 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), and then Pd(PPh3)4 (40.0 mg, 0.04 mmol) was added, and the mixture was refluxed for 2 hours. After the reaction was completed, ethanol was distilled off under reduced pressure, and the mixture was extracted with 20 mL of dichloromethane, washed with 50 mL of saturated brine, dried over anhydrous Na2SO4, and dichloromethane was distilled off under reduced pressure 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 5:1) as the eluent to obtain 143.5 mg of compound 3 with 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, and the mixture was heated to 75°C 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 N,N-dimethylformamide (DMF), K2CO3 (82.9 mg, 0.60 mmol) and 1,5-dibromopentane (55.2 mg, 0.24 mmol) were added, and 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, washed with saturated brine for 3 times, dried over anhydrous Na2SO4, and 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 the 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 N,N-dimethylformamide (DMF), heated to 70°C for reaction for 12 hours; after the reaction, the reaction solution was poured into water, a yellow solid was precipitated, and suction filtration was performed. The filter cake was washed with water 3 times and vacuum dried to obtain a crude product, which was chromatographed on a 200-300 mesh silica gel column with an eluent of dichloromethane and methanol (volume ratio of 12:1) to obtain 44.1 mg of fluorescent probe compound 6 after purification, 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 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, and its absorption spectrum was measured using an ultraviolet spectrophotometer; its fluorescence emission spectrum was measured using a fluorescence spectrometer, and the spectrum diagram is shown in the attached 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 fluorescent imaging.

[0079] Example 3

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

[0081] The AIE-type fluorescent probe molecule in Example 1 was prepared into a 0.01 mol / L mother solution using DMSO and deionized water H2O, respectively, and recorded as solution A and solution B. Take 100 μL of solution A and place it in a 0.2 mL centrifuge tube, which is a fluorescent probe molecule solution with a water content of 0%; take 90 μL of solution A and 10 μL of solution B and place them in a 0.2 mL centrifuge tube, which is a fluorescent probe molecule solution with a water content of 10%. Similarly, prepare probe molecule solutions with water content of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90%, respectively. Then, arrange the prepared solutions neatly 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 4 shown.

[0082] By the attached Figure 4 From the data, it can be seen 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 the 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 show 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 (Mol Imaging 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 is based on Example 1 and detects the photostability of the tumor-targeting AIE type fluorescent probe molecule of the present invention.

[0085] The tumor-targeting AIE fluorescent probe molecules of Example 1 were prepared into working solutions of different concentrations using DMSO. The fluorescent probe molecules in these test tubes 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) to observe the changes in fluorescence intensity with illumination time. For specific results, see the attached Figure 5 shown.

[0086] By the attached Figure 5 It can be seen from the data that before illumination, the fluorescent probe molecule has already shown a certain fluorescence intensity; as the illumination time increases (5min to 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, which 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-targeted fluorescent probe, providing strong support for the diagnosis and treatment of tumors.

[0088] Example 5

[0089] Based on Example 1, this example uses the MTT method 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] The MCF-10A cells were revived and cultured until the cell density in the T25 cell culture flask reached 80% to 90%, digested with trypsin, centrifuged, the supernatant was discarded, and the cells were resuspended and mixed with 2 mL of complete culture medium, and the cells were counted; the cells were inoculated in 96-well plates at a number of 10,000 cells per well, the edge wells were filled with sterile PBS, and 3 96-well plates were inoculated for each cell type, and 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 to the wall. The original culture medium was discarded, and the culture medium was used to dilute the 0.01 mol / L tumor-targeting AIE 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), and 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 of 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 an enzyme 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, see the attached Figure 6 shown.

[0099] By the attached Figure 6 From the data, we can see that when the concentration of the fluorescent probe molecules 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 molecules are less toxic to cells; even when the concentration of the fluorescent probe molecules 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 molecules have high biocompatibility and low toxicity and can be used for biological imaging.

[0100] Example 6

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

[0102] 1. Cell Culture

[0103] (1) Prepare 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 generation number 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 use a sterile pipette to aspirate the PBS waste solution.

[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 surface of the cells and 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. 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 that were 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. The solution was gently mixed to cover the surface of the cells and observed under a microscope. When the cells were completely detached from the bottom of the 12-well culture plate and the cell connections were loose, 1 mL of complete culture medium was added and pipetted to form a cell suspension.

[0116] (4) The above 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 cell uptake of the fluorescent probe and compound 3. The specific results are shown in the attached Figure 7 shown.

[0117] By the attached Figure 7 From the data, we can see 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, and 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 good 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] Based on Example 1, this example measures the uptake of the fluorescent probe of the present invention by human breast cancer MDA-MB-231 cells, and observes the uptake of the fluorescent probe to be measured by human breast cancer MDA-MB-231 cells using a laser confocal microscope. The specific steps are as follows:

[0121] (1) MDA-MB-231 cells were cultured 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 to the wall, 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 is not 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 min, wash gently three times with PBS.

[0125] (5) Add 1 mL of PBS to each dish and observe the uptake of different concentrations of fluorescent probes by cells 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 could successfully enter the cells; comparing the fluorescence intensities of different concentrations of fluorescent probes (0.25 μM and 1 μM), 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] Based on Example 1, this example conducts animal experiments to detect 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] Take MDA-MB-231 tumor cells, centrifuge at 1000 rpm for 5 min, wash the tumor cells three times with sterile PBS, and make appropriate dilutions. Count the cells with a hemocytometer to make a concentration of 1×10 7 Each mouse was subcutaneously inoculated with 100 μL of tumor cell suspension 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 at .

[0132] (2) Targeted verification

[0133] 5 to 10 days after tumor inoculation, 10 nmol of fluorescent probe was injected through the tail vein, and then a small animal in vivo optical imaging system with high detection sensitivity was used to monitor the fluorescent signal in the mouse 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 mouse were removed for imaging. For specific results, see the attached Fig. 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 tissue and organ in vitro imaging results showed that the tumor 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 to MDA-MB-231 tumors and can be enriched in tumor tissues. Its fluorescence signal intensity is high enough to be used for imaging of 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 effects.

[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 shows good targeting at both the cellular level and the animal level, and can perform dynamic non-invasive in vivo evaluation of HDAC6 molecules expressed by tumors, solving the problem of tumor-specific imaging. The probe complex has no toxic side effects, is safe and reliable, and has a simple preparation process.

[0138] The above contents are 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 fluorescent probe, characterized in that: The structure of the AIE type fluorescent probe is as follows:

2. The tumor-targeting AIE 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, preparing 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 a tumor-targeting AIE fluorescent probe.

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

6. The method for preparing a tumor-targeting AIE type fluorescent probe according to claim 3, characterized in that: 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 10h-14h.

7. The method for preparing a tumor-targeting AIE type fluorescent probe according to claim 3, characterized in that: 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 fluorescent probe according to any one of claims 1 to 2 in the preparation of tumor detection products.

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 tumors and performing in vitro pathological staining of tumor tissues.

10. The 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 expression of HDAC6.

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