Anoxic and endogenous formaldehyde dual-response supramolecular fluorescent probe and a preparation method and application thereof

Abstract

The application provides a hypoxia and endogenous formaldehyde dual-response supramolecular fluorescent probe and a preparation method and application thereof, and belongs to the technical field of fluorescent probes.The supramolecular fluorescent probe provided by the application is composed of an azobenzene-based calixarene and a formaldehyde response fluorescent probe encapsulated in the azobenzene-based calixarene.The supramolecular fluorescent probe provided by the application can simultaneously respond to hypoxia and high endogenous formaldehyde microenvironment characteristics, generate a strong near-infrared fluorescent signal for analysis and detection, thereby reducing the interference of a 'false positive' signal, and can realize high-specificity imaging under the condition of a tumor microenvironment, thereby providing a new mode for precise imaging of tumors.Compared with the existing fluorescent probes, the supramolecular fluorescent probe provided by the application combines the detection of two important biomarkers in the process of tumor occurrence and development, does not need to introduce a large volume of a targeting group into the molecule, and has the advantages of simple preparation method, novel structure, excellent imaging performance, precise near-infrared fluorescent imaging of tumors and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of fluorescent probes, and particularly relates to a hypoxia and endogenous formaldehyde dual-responsive supramolecular fluorescent probe and a preparation method and application thereof. BACKGROUND

[0002] Tumor is a major public health problem in China. At present, the incidence and mortality of malignant tumors in China continue to rise, and the annual medical cost caused by malignant tumors exceeds 220 billion. Precise diagnosis and imaging recognition of tumors are one of the keys to improve the cure rate of tumors. Fluorescence imaging technology has become one of the most promising methods for precise diagnosis of cancer due to its advantages of convenience, real-time imaging, good spatial resolution and high sensitivity. However, the currently developed fluorescent probes usually only respond to a single microenvironment feature in tumors, which is easy to cause insufficient specificity and interference of "false positive" signals (Wu, L. et al., Nat. Rev. Chem. 2021, 5, 406-421). Through the detection of two important biomarkers in the process of tumor occurrence and development, the problem of false positive signals existing in most fluorescent probes at present will be effectively improved.

[0003] Endogenous formaldehyde plays an important role in physiological and pathological processes of human body, and is widely involved in folate cycle in vivo, involved in amino acid, neurotransmitter, protein synthesis, DNA demethylation and gene expression regulation and other physiological processes. For example, in breast cancer cells, methylation synthesis enzymes such as serine hydroxymethyltransferase (SHMT) and histone demethylase (LSD) are highly expressed, resulting in much higher endogenous formaldehyde content in tumor tissues than in normal tissues. Through the determination of endogenous formaldehyde in tumor tissues of breast cancer patients, it is found that the endogenous formaldehyde content in tumor tissues is as high as 0.75 ± 0.12 mM, and the maximum can reach 2.35 mM; while the endogenous formaldehyde concentration of normal tissues is only 0.1-0.2 mM. In addition, the endogenous formaldehyde content may be related to the degree of tumor progression. Therefore, high endogenous formaldehyde is an important microenvironment feature of tumor. In addition, hypoxia is another important microenvironment feature of tumor, which is mainly caused by excessive oxygen consumption and insufficient oxygen supply of tumor. More importantly, hypoxia and endogenous formaldehyde level have certain correlation in tumor metastasis, and the level of endogenous formaldehyde is up-regulated in the process of hypoxia-induced cancer cell metastasis. The development of near-infrared fluorescent probes activated by tumor-related hypoxia and high endogenous formaldehyde is expected to realize high specificity imaging of tumor to meet the needs of precise diagnosis and treatment of tumor. However, there are few fluorescent probes that can simultaneously respond to tumor hypoxia and high endogenous formaldehyde microenvironment, and most of such probes are combined by covalent bond, which has complex synthesis, purification and separation process, limiting its further wide application. SUMMARY

[0004] In view of the above technical problems, the present application provides a hypoxia and endogenous formaldehyde dual-responsive supramolecular fluorescent probe and a preparation method and application thereof.

[0005] The present application adopts the following technical solutions:

[0006] I. Hypoxia and endogenous formaldehyde dual-responsive supramolecular fluorescent probe

[0007] The hypoxia and endogenous formaldehyde dual-responsive supramolecular fluorescent probe provided by the present application is composed of an azobenzene-based calixarene and a formaldehyde-responsive fluorescent probe encapsulated in the azobenzene-based calixarene, and the chemical structural formula of the azobenzene-based calixarene is as follows:

[0008]

[0009] wherein R is a methyl group, a fluorine atom or a bromine atom

[0010] The chemical structural formula of the formaldehyde-responsive fluorescent probe is as follows:

[0011]

[0012] The hypoxia and endogenous formaldehyde dual-responsive supramolecular fluorescent probe provided by the present application is formed into a stable supramolecular complex through non-covalent interaction between the azobenzene-based calixarene and the formaldehyde-responsive fluorescent probe encapsulated in the azobenzene-based calixarene. By utilizing the azobenzene-based calixarene's characteristic of azo bond cleavage under the hypoxic microenvironment of a tumor and the formaldehyde-responsive fluorescent probe's characteristic of activation under the action of high endogenous formaldehyde in the tumor, the hypoxia and endogenous formaldehyde dual-responsive supramolecular fluorescent probe can simultaneously respond to the characteristics of the hypoxic and high endogenous formaldehyde microenvironment of a tumor and generate a strong near-infrared fluorescent signal for the analysis and detection of the tumor (as shown in the following formula). Figure 1

[0013] II. Preparation method of hypoxia and endogenous formaldehyde dual-responsive supramolecular fluorescent probe

[0014] The azobenzene-based calixarene and the formaldehyde-responsive fluorescent probe are mixed in proportion and then dissolved in a buffer solution to obtain a supramolecular fluorescent probe solution. The molar ratio of the azobenzene-based calixarene and the formaldehyde-responsive fluorescent probe is 1:1 to 1:2 (preferably 1:1). The buffer solution is a PBS buffer solution with a pH of 6.0 to 8.0 (preferably pH 7.4).

[0015] The preparation method of the hypoxia and endogenous formaldehyde dual-responsive supramolecular fluorescent probe provided by the present application is simple, and only needs to mix the azobenzene-based calixarene and the formaldehyde-responsive fluorescent probe in proportion and then dissolve them in a buffer solution. Alternatively, the two raw materials can be pre-dissolved in a solvent such as DMSO and then added to a buffer solution to configure a target concentration, which is easy for industrial production.

[0016] III. Application of hypoxia and endogenous formaldehyde dual-responsive supramolecular fluorescent probe ​

[0017] The hypoxia and endogenous formaldehyde dual-response supramolecular fluorescent probe of the application can simultaneously respond to the characteristics of tumor hypoxia and high endogenous formaldehyde microenvironment, produce strong near-infrared fluorescent signals for analysis and detection, thereby reducing the interference of "false positive" signals, realizing high-specificity imaging of tumors, and having a good application prospect in the precise diagnosis and imaging research of tumors. The application of the prepared supramolecular fluorescent probe A in fluorescence imaging is described below as an example.

[0018] MCF-7 cells of human breast cancer were selected as the research object, and the imaging performance of the probe in breast cancer MCF-7 cells was studied by laser scanning confocal microscopy (CLSM). After being cultured in a cell incubator at 37°C, 21% oxygen (volume percentage), and 5% carbon dioxide (volume percentage) for 24 hours, the cells were trypsinized and transferred to a cell imaging dish, and then the cells in the imaging dish were divided into three groups: (i) the cells in the first group were cultured in a cell incubator at 37°C, 21% oxygen (volume percentage), and 5% carbon dioxide (volume percentage) for 24 hours, and then the supramolecular fluorescent probe A (the molar ratio of the azobenzene-based calixarene and the formaldehyde-responsive fluorescent probe in the fluorescent probe A was 1:1, and the final concentration of both was 5 μmol / L) was added, and the incubation was continued for 2 hours; (ii) the cells in the second group were cultured in a cell incubator at 37°C, 1% oxygen (volume percentage), and 5% carbon dioxide (volume percentage) for 24 hours, and then the supramolecular fluorescent probe A (the molar ratio of the azobenzene-based calixarene and the formaldehyde-responsive fluorescent probe in the fluorescent probe A was 1:1, and the final concentration of both was 5 μmol / L) was added, and the incubation was continued for 2 hours; (iii) the cells in the third group were cultured in a cell incubator at 37°C, 1% oxygen (volume percentage), and 5% carbon dioxide (volume percentage) for 24 hours, and then the MCF-7 cells were pretreated with the formaldehyde scavenger sodium bisulfite (NaHSO3), and then the supramolecular fluorescent probe A (the molar ratio of the azobenzene-based calixarene and the formaldehyde-responsive fluorescent probe in the fluorescent probe A was 1:1, and the final concentration of both was 5 μmol / L) was added, and the incubation was continued for 2 hours. Finally, the culture medium in the imaging dish was washed with PBS buffer, and laser scanning confocal microscopy was used to select 633 nm excitation light source and waveband at 650-750 nm as the output signal channel for cell imaging research. The results are shown in Figure 5 Figure 5 ​It can be seen that only the second group of cells shows strong fluorescence signal, and the first group and the third group of cells are not observed near-infrared fluorescence signal. This is because, in the experimental cells, the first group of cells is not hypoxic, the third group of cells has low endogenous formaldehyde content due to sodium bisulfite, and only the second group of cells has the characteristics of hypoxia and high endogenous formaldehyde microenvironment. The above results show that the supramolecular fluorescent probe can enter the tumor cells and react with the endogenous formaldehyde in the cells, and produce strong fluorescence signal under the stimulation of hypoxia, which is used for specific fluorescence imaging of hypoxic tumor cells.

[0019] In summary, the beneficial effects of the present application are:

[0020] 1. The hypoxia and endogenous formaldehyde dual-responsive supramolecular fluorescent probe provided by the present application can simultaneously detect two biomarkers-hypoxia and high endogenous formaldehyde associated with the occurrence and development of tumors, turn on the near-infrared fluorescence signal, realize high specificity imaging of tumors, thereby avoiding the interference of "false positive" signals, and the probe does not need to introduce a large volume of targeting group in the molecule, has the advantages of novel structure, high targeting and good imaging performance.

[0021] 2. The preparation method of the hypoxia and endogenous formaldehyde dual-responsive supramolecular fluorescent probe provided by the present application is simple and effective, and can quickly and efficiently obtain the fluorescent probe.

[0022] 3. In view of the good imaging performance, the hypoxia and endogenous formaldehyde dual-responsive supramolecular fluorescent probe provided by the present application has important significance for the accurate diagnosis of tumors. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is a schematic diagram of the application of the hypoxia and endogenous formaldehyde dual-responsive supramolecular fluorescent probe in tumor imaging.

[0024] Figure 2 (a) is a fluorescence spectrum of non-covalent mutual bonding between the azobenzene-based calixarene and the formaldehyde-responsive fluorescent probe host-guest; (b) is a MALDI-TOF mass spectrum of the supramolecular fluorescent probe A.

[0025] Figure 3 is the stability of the supramolecular fluorescent probe A.

[0026] Figure 4 is a fluorescence response performance diagram of the supramolecular fluorescent probe A to formaldehyde and hypoxia.

[0027] Figure 5 is a tumor cell imaging diagram of the supramolecular fluorescent probe A. DETAILED DESCRIPTION

[0028] The technical solutions of the present application will be further described in detail below in combination with several preferred embodiments and the accompanying drawings. The embodiments are implemented on the premise of the technical solutions of the present application, and detailed implementation modes and specific operation processes are given, but the protection scope of the present application is not limited to the following embodiments.

[0029] The experimental materials used in the following examples are commercially available from conventional biochemical reagent companies, unless otherwise specified.

[0030] Example 1: Preparation of supramolecular fluorescent probe A

[0031] This embodiment provides a supramolecular fluorescent probe, and the preparation method thereof is as follows: 0.5 μmol of azobenzenyl calixarene 1 and 0.5 μmol of formaldehyde responsive fluorescent probe (in a molar ratio of 1:1) are mixed and dissolved in 10 μL of DMSO, and then added to 100 mL of PBS buffer (10 mM, pH=7.4) to obtain supramolecular fluorescent probe A (the final concentration of both azobenzenyl calixarene and formaldehyde responsive fluorescent probe is 5 μmol / L).

[0032] The chemical structural formula of azobenzenyl calixarene 1 is as follows:

[0033]

[0034] The synthesis method of azobenzenyl calixarene 1 is as follows:

[0035]

[0036] In a 25 mL round-bottom flask, p-fluoroaniline (110 mg) is dissolved in 2 mL of water, and 0.5 mL of hydrochloric acid is added. After the reaction solution is cooled to 0°C by adding an ice bath, 69 mg of 2 mL NaNO2 solution is slowly added. After 1 hour of reaction, the obtained reaction solution is slowly added to a reaction solution containing calixarene 1 (50 mg) and sodium acetate (245 mg) dissolved in MeOH-DMF (5:8, v:v). After the mixture is stirred at room temperature for 24 hours, azobenzenyl calixarene 1 is obtained. 1 H NMR, 13 C NMR and high-resolution mass spectrometry data are as follows: 1 H NMR (400 MHz, DMSO- d6 ): δ 9.97 (s, 4H), 7.82 (t, J =4.0, 8H), 7.78(d, J =12.0, 8H), 7.31 (t, J =8.0, 8H), 4.11(s, 8H). 13C NMR (100 MHz, DMSO- d6 ) δ 164.5, 162.1, 157.4, 149.2, 145.2, 130.1, 124.5, 124.4, 124.1, 116.5, 116.3, 31.6. High resolution mass spectrometry calculated for C 52 H 36 F4N8O4: 911.2718 [M-H] - ; found, 911.2710 [M-H] - .

[0037] The chemical structure of the formaldehyde-responsive fluorescent probe is as follows:

[0038]

[0039] The synthesis method of the formaldehyde-responsive fluorescent probe is as follows:

[0040]

[0041] Specifically, the compound hemicyanine A (0.2 mmol) and 2-methylamino benzoic acid (0.3 mmol) are dissolved in 10 mL of dichloromethane. Then, dicyclohexyl carbodiimide (61 mg, 0.4 mmol) and 4-dimethylaminopyridine (37 mg, 0.3 mmol) are added to the above reaction solution. The mixture is stirred at room temperature for 12 hours. After the reaction is completed, the solvent is rotary evaporated, and the crude product is purified by silica gel column chromatography (CH2Cl2 / MeOH = 30:1, v / v) to obtain a dark blue formaldehyde-responsive fluorescent probe. The obtained 1 H NMR, 13 C NMR and high resolution mass spectrometry data are as follows 1 H NMR (400 MHz, DMSO- d6 ): delta 8.57 (d, J = 16.0, 1H), 8.05 (d, J = 8.0, 1H), 7.73 (d, J = 8.0, 1H), 7.62 (d, J = 8.0, 1H), 7.44-7.58 (m, 6H), 7.24 (d, J = 8.0, 1H), 6.82 (d, J = 8.0, 1H), 6.64-6.71 (m, 2H), 3.93 (s, 3H), 2.89 (d, J= 4.0, 3H), 2.67-2.76 (m, 4H), 1.84 (m, 2H), 1.73 (s, 6H). 13 C NMR (100 MHz, DMSO- d6 ) delta 179.3, 166.3, 159.1, 153.1, 153.0, 152.7, 145.5, 142.8, 142.7, 136.5, 132.2, 131.1, 130.0, 129.3, 128.6, 128.1, 123.1, 120.1, 119.9, 115.0, 114.6, 114.2, 111.9, 110.8, 107.9, 107.1, 51.2, 33.5, 29.9, 29.2, 27.5, 27.5, 24.0, 20.3. High resolution mass spec. calcd for C 34 H 33 N2O3: 517.2486 [M-I] + found, 517.2469

[0042] The successful preparation of supramolecular fluorescent probe was studied by fluorescence titration method and high resolution mass spectrometry. Specifically, at room temperature, different concentrations of azobenzene calixarene were added to the formaldehyde responsive fluorescent probe (5 μmol / L) solution, and the fluorescence spectrum changes of the solution at λ ex = 679 nm and the fluorescence intensity changes at λ ex / em = 690 / 750 nm were tested. Further, the mass spectrum signal of the obtained solution was studied by MALDI-TOF mass spectrometry. From Figure 2 (a) and Figure 2 (b), it can be seen that the azobenzene calixarene and the formaldehyde responsive fluorescent probe can specifically produce host-guest interaction, and the azobenzene calixarene and the formaldehyde responsive fluorescent probe can specifically produce characteristic molecular weight addition signals at m / z = 1429.5449, indicating the successful preparation of supramolecular fluorescent probe A.

[0043] Example 2: Stability of supramolecular fluorescent probe

[0044] The supramolecular fluorescent probe provided in the present application has good in-vitro stability. The stability of the supramolecular fluorescent probe A prepared in Example 1 in vitro was studied. The supramolecular fluorescent probe A (the final concentration of both the azobenzene-based calixarene and the formaldehyde-responsive fluorescent probe was 5 μmol / L) configured in Example 1 was stored at room temperature, and the stability of the supramolecular fluorescent probe was tested by using ultraviolet absorption spectrum. Quartz colorimetric cells were selected as sample cells, and the supramolecular fluorescent probe was placed at room temperature for 12 hours. The ultraviolet absorption spectrum changes of the sample solution at 0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 8.0, 10.0 and 12 hours were tested, respectively. The results are shown in Figure 3 From Figure 3 It can be seen that the ultraviolet absorption spectrum of the supramolecular fluorescent probe has no obvious change within 12 hours, which proves that the supramolecular fluorescent probe has good stability.

[0045] Example 3: Optical properties of the supramolecular fluorescent probe

[0046] The supramolecular fluorescent probe provided in the present application can respond to two microenvironment characteristics of tumor hypoxia and high endogenous formaldehyde with high selectivity and light up the near-infrared fluorescent signal. Therefore, the optical properties of the supramolecular fluorescent probe A prepared in Example 1 under the conditions of hypoxic microenvironment and endogenous formaldehyde were studied. Specifically, to the solution of the supramolecular fluorescent probe A (the final concentration of both the azobenzene-based calixarene and the formaldehyde-responsive fluorescent probe was 5 μmol / L) configured in Example 1, (i) PBS; (ii) formaldehyde; (iii) sodium dithionite (chemical mimic of azo reductase) and (iv) formaldehyde and sodium dithionite were added, respectively. After incubation of the above solutions, the fluorescence spectrum changes of the solutions at λ ex = 679 nm and the fluorescence intensity changes at λ ex / em = 690 / 750 nm were tested by using a fluorescence spectrometer. From Figure 4 It can be seen that the fluorescence signal of the solution of the supramolecular fluorescent probe A is weak under the condition of only single sodium dithionite or single formaldehyde; and when sodium dithionite and formaldehyde exist simultaneously, the supramolecular fluorescent probe A observes a strong near-infrared fluorescent signal at λ em = 710 nm. This result shows that the supramolecular fluorescent probe A can only be lighted up under the condition of hypoxic microenvironment and endogenous formaldehyde together. This reduces the "false positive" signal in the detection process, improves the reliability of the detection result, and is conducive to the application of biological imaging.

[0047] Example 4: Application of the supramolecular fluorescent probe in tumor cell imaging

[0048] The supermolecular fluorescent probe provided in the application can respond to two microenvironment characteristics of tumor hypoxia and high endogenous formaldehyde with high selectivity, and light up the near-infrared fluorescent signal. Therefore, the application of the supermolecular fluorescent probe A prepared in Example 1 in tumor cell fluorescent imaging is studied.

[0049] MCF-7 cells of human breast cancer were selected as the research object, and the imaging performance of the probe in breast cancer MCF-7 cells was studied by laser scanning confocal microscope (CLSM). After the breast cancer cells were cultured in a cell incubator at 37°C, 21 % oxygen (volume percentage), and 5 % carbon dioxide (volume percentage) for 24 hours, the cells were trypsinized and transferred to a cell imaging dish (cell concentration 1 * 10 6 ) 4. The cells in the imaging dish were divided into three groups: (i) the first group of cells were cultured in a cell incubator at 37°C, 21 % oxygen (volume percentage), and 5 % carbon dioxide (volume percentage) for 24 hours, and then supermolecular fluorescent probe A (the molar ratio of azobenzene-based calixarene and formaldehyde-responsive fluorescent probe in fluorescent probe A was 1:1, and the final concentration of both was 5 μmol / L) was added, and the incubation was continued for 2 hours; (ii) the second group of cells were cultured in a cell incubator at 37°C, 1 % oxygen (volume percentage), and 5 % carbon dioxide (volume percentage) for 24 hours, and then supermolecular fluorescent probe A (the molar ratio of azobenzene-based calixarene and formaldehyde-responsive fluorescent probe in fluorescent probe A was 1:1, and the final concentration of both was 5 μmol / L) was added, and the incubation was continued for 2 hours; (iii) the third group of cells were cultured in a cell incubator at 37°C, 1 % oxygen (volume percentage), and 5 % carbon dioxide (volume percentage) for 24 hours, and then the MCF-7 cells were pretreated with formaldehyde scavenger sodium bisulfite (NaHSO3), and then supermolecular fluorescent probe A (the molar ratio of azobenzene-based calixarene and formaldehyde-responsive fluorescent probe in fluorescent probe A was 1:1, and the final concentration of both was 5 μmol / L) was added, and the incubation was continued for 2 hours. Finally, the culture medium in the imaging dish was washed with PBS buffer, and laser scanning confocal microscope was used to select 633 nm excitation light source and waveband at 650-750 nm as the output signal channel for cell imaging research. The results are shown in Figure 5 Figure 5 ​It can be seen that only the second group of cells showed strong fluorescence signal, and no near-infrared fluorescence signal was observed in the first and third groups of cells. This is because, in the experimental cells, the first group of cells is not hypoxic, the third group of cells has low endogenous formaldehyde content due to sodium bisulfite, and only the second group of cells has both hypoxia and high endogenous formaldehyde microenvironment characteristics. The above results show that the supramolecular fluorescent probe can enter breast cancer cells and react with endogenous formaldehyde in the cells, and produce strong fluorescence signal under hypoxic stimulation, which can be used for specific fluorescence imaging of hypoxic breast cancer cells.

Claims

1. A hypoxia and endogenous formaldehyde dual-responsive supramolecular fluorescent probe, characterized in that: The formaldehyde-responsive fluorescent probe is composed of an azophenyl calixarene and formaldehyde occluded in the azophenyl calixarene, and the chemical structural formula of the azophenyl calixarene is as follows: wherein R is methyl, fluorine or bromine; The chemical structural formula of the formaldehyde-responsive fluorescent probe is as follows: ； The azophenyl calixarene and the formaldehyde-responsive fluorescent probe form a stable supramolecular complex through non-covalent interaction between host and guest. 2.The method for preparing the hypoxia and endogenous formaldehyde dual-responsive supramolecular fluorescent probe according to claim 1, characterized in that: The azophenyl calixarene and the formaldehyde-responsive fluorescent probe are mixed sufficiently and then dissolved in a buffer to obtain a supramolecular fluorescent probe solution; The molar ratio of the azophenyl calixarene and the formaldehyde-responsive fluorescent probe is 1:1-1:2; The buffer is a PBS buffer with a pH of 6.0-8.

0.

3. The use of the hypoxia and endogenous formaldehyde dual-responsive supramolecular fluorescent probe according to claim 1 in the preparation of a tumor fluorescent imaging detection reagent. 4.The use of the hypoxia and endogenous formaldehyde dual-responsive supramolecular fluorescent probe according to claim 3 in the preparation of a tumor fluorescent imaging detection reagent, characterized in that: The hypoxia and endogenous formaldehyde dual-responsive supramolecular fluorescent probe is used as a tumor fluorescent imaging detection reagent for tumors and tumor microenvironments.

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