Hypoxia and endogenous formaldehyde dual-response supramolecular fluorescent probe as well as preparation method and application thereof

Abstract

The invention provides an anoxic and endogenous formaldehyde dual-response supramolecular fluorescent probe as well as a preparation method and application thereof, and belongs to the technical field of fluorescent probes. The supramolecular fluorescent probe provided by the invention is composed of azobenzene calixarene and a formaldehyde response fluorescent probe included in the azobenzene calixarene. The supramolecular fluorescent probe provided by the invention can simultaneously respond to oxygen deficit and high endogenous formaldehyde microenvironment characteristics and generate a stronger near-infrared fluorescent signal for analysis and detection, so that false positive signal interference is reduced, high-specificity imaging under tumor microenvironment conditions can be realized, and a new mode is provided for precise imaging of tumors. Compared with the existing fluorescent probe, the supramolecular fluorescent probe provided by the invention combines the detection of two important biomarkers in the tumor generation and development processes, and does not need to introduce a large-volume targeting group into the molecule; the preparation method is simple, the structure is novel, the imaging performance is excellent, and the tumor is accurately subjected to near-infrared fluorescence imaging tracing.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent probes, and in particular relates to a hypoxia and endogenous formaldehyde dual-response supramolecular fluorescent probe and a preparation method and application thereof. Background Art

[0002] Cancer is a major public health problem in China. At present, the incidence and mortality of malignant tumors in my country continue to rise, and the medical expenses caused by malignant tumors exceed 220 billion yuan each year. Accurate diagnosis and imaging identification of tumors is one of the keys to improving the cure rate of tumors. Fluorescence imaging technology has become one of the most promising methods for accurate diagnosis of cancer due to its advantages such as convenience, real-time imaging, good spatial resolution imaging rate and high sensitivity. However, the fluorescent probes currently developed usually only respond to a single microenvironment feature in the tumor, which is prone to interference from "false positive" signals caused by insufficient specificity (Wu, L. et al., Nat. Rev. Chem. 2021, 5, 406-421). By combining the detection of two important biomarkers in the process of tumor occurrence and development, the false positive signal problem existing in most current fluorescent probes will be effectively improved.

[0003] Endogenous formaldehyde plays an important role in the physiological and pathological processes of the human body. It is widely involved in the folic acid cycle in the body, and participates in a variety of physiological processes such as amino acid, neurotransmitter, protein synthesis, DNA demethylation and gene expression regulation. For example, in breast cancer cells, methyl synthases such as serine hydroxymethyltransferase (SHMT) and histone demethylase (LSD) are highly expressed, resulting in endogenous formaldehyde content in tumor tissues being much higher than that in normal tissues. By measuring endogenous formaldehyde in tumor tissues of breast cancer patients, it was found that the endogenous formaldehyde content in tumor tissues was as high as 0.75 ± 0.12mM, and the maximum could reach 2.35mM; while the endogenous formaldehyde concentration in normal tissues was only 0.1~0.2mM. In addition, the endogenous formaldehyde content may be related to the degree of tumor progression. Therefore, high endogenous formaldehyde is an important microenvironmental feature of tumors. In addition, hypoxia is another important microenvironmental feature of tumors, which is mainly caused by excessive oxygen consumption and insufficient oxygen supply in tumors. More importantly, hypoxia and endogenous formaldehyde levels have a certain correlation in tumor metastasis. In the process of hypoxia-induced cancer cell metastasis, endogenous formaldehyde levels are upregulated. The development of near-infrared fluorescent probes activated by hypoxia and high endogenous formaldehyde, which are interrelated in tumors, is expected to achieve highly specific tumor imaging to meet the needs of precise diagnosis and treatment of tumors. However, there are currently very few fluorescent probes that can simultaneously respond to tumor hypoxia and high endogenous formaldehyde microenvironments, and most of these probes are bound by covalent bonds, and the synthesis, purification and separation processes are complicated, which limits their further widespread application. Summary of the invention

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

[0005] The present invention adopts the following technical scheme: 1. Dual-responsive supramolecular fluorescent probe for hypoxia and endogenous formaldehyde The hypoxia and endogenous formaldehyde dual-responsive supramolecular fluorescent probe of the present invention is composed of azophenylcalixarene and a formaldehyde-responsive fluorescent probe included in the azophenylcalixarene. The chemical structural formula of the azophenylcalixarene is as follows: Where R is a methyl group, a fluorine atom or a bromine atom The chemical structural formula of the formaldehyde-responsive fluorescent probe is as follows: The hypoxia and endogenous formaldehyde dual-responsive supramolecular fluorescent probe provided by the present invention is a stable supramolecular complex formed by azophenylcalixarene and a formaldehyde-responsive fluorescent probe included in the azophenylcalixarene through non-covalent interactions between the host and the guest. By utilizing the azo bond breaking characteristics of azophenylcalixarene in the hypoxic microenvironment of tumors and the activation characteristics of the formaldehyde-responsive fluorescent probe under the action of high endogenous formaldehyde in tumors, it can simultaneously respond to the characteristics of the microenvironment of tumor hypoxia and high endogenous formaldehyde, and generate a strong near-infrared fluorescent signal for tumor analysis and detection (such as Figure 1 as shown).

[0006] 2. Preparation of supramolecular fluorescent probes with dual response to hypoxia and endogenous formaldehyde Azophenylcalixarene and formaldehyde-responsive fluorescent probe are fully mixed in proportion and dissolved in a buffer to obtain a supramolecular fluorescent probe solution. The molar ratio of azophenylcalixarene and formaldehyde-responsive fluorescent probe group is 1:1-1:2 (preferably 1:1). The buffer is a PBS buffer with a pH of 6.0-8.0 (preferably pH 7.4).

[0007] The preparation method of the hypoxia and endogenous formaldehyde dual-responsive supramolecular fluorescent probe provided by the present invention has simple steps, and only requires mixing azophenylcalixarene and the formaldehyde-responsive fluorescent probe in proportion and then dissolving them in a buffer solution. The two raw materials can also be pre-dissolved in a solvent such as DMSO in proportion and then added to the buffer solution to be configured to a target concentration, which is easy for industrial production.

[0008] 3. Application of supramolecular fluorescent probes with dual response to hypoxia and endogenous formaldehyde The present invention is a hypoxia and endogenous formaldehyde dual-responsive supramolecular fluorescent probe that can simultaneously respond to tumor hypoxia and high endogenous formaldehyde microenvironment characteristics, generate strong near-infrared fluorescence signals for analysis and detection, thereby reducing "false positive" signal interference, achieving high-specific imaging of tumors, and having good application prospects in accurate diagnosis and imaging research of tumors. The following uses the prepared supramolecular fluorescent probe A as an example to illustrate the application of the probe of the present invention in fluorescence imaging.

[0009] Human breast cancer cells MCF-7 were selected as the research object, and the imaging performance of the probe in breast cancer MCF-7 cells was studied by confocal laser scanning microscopy (CLSM). After culturing in a cell culture incubator at 37°C, 21% oxygen (volume percentage), 5% carbon dioxide (volume percentage) for 24 hours, the cells were digested with trypsin and transferred to a cell imaging dish. The cells in the imaging dish were then divided into three groups: (i) After the cells in the first group were cultured in a cell culture incubator at 37°C, 21% oxygen (volume percentage), 5% carbon dioxide (volume percentage) for 24 hours, supramolecular fluorescent probe A (the molar ratio of azophenylcalixarene 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 continued for 2 hours; (ii) After the cells in the second group were cultured in a cell culture incubator at 37°C, 1% oxygen (volume percentage), 5% carbon dioxide (volume percentage) for 24 hours, supramolecular fluorescent probe A (the molar ratio of azophenylcalixarene 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 continued for 2 hours; (iii) After the cells in the third group were cultured in a cell culture incubator at 37°C, 1 After culturing in a cell culture incubator with 5% oxygen (volume percentage) and 5% carbon dioxide (volume percentage) for 24 hours, MCF-7 cells were pretreated with formaldehyde scavenger sodium bisulfite (NaHSO3), and then supramolecular fluorescent probe A was added (the molar ratio of azophenylcalixarene and formaldehyde-responsive fluorescent probe in fluorescent probe A was 1:1, and the final concentration of both was 5 μmol / L), and incubation was continued for 2 hours. Finally, the culture medium in the imaging dish was washed away with PBS buffer, and a laser scanning confocal microscope was used to select a 633 nm excitation light source and use the 650-750 nm band as the output signal channel for cell imaging research. The results are shown in Figure 5 As shown. Figure 5It can be seen that only the second group of cells showed a 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 were not hypoxic, the third group of cells had a low endogenous formaldehyde content due to sodium bisulfite, and only the second group of cells had both hypoxia and high endogenous formaldehyde microenvironment characteristics. The above results show that the supramolecular fluorescent probe can enter tumor cells and react with endogenous formaldehyde in cells, producing a strong fluorescence signal under hypoxia stimulation, which can be used for specific fluorescence imaging of hypoxic tumor cells.

[0010] In summary, the beneficial effects of this application are: 1. The present application provides a supramolecular fluorescent probe that responds to both hypoxia and endogenous formaldehyde, which can simultaneously detect two interrelated biomarkers in the occurrence and development of tumors - hypoxia and high endogenous formaldehyde, turn on near-infrared fluorescence signals, and achieve highly specific tumor imaging, thereby avoiding the interference of "false positive" signals. At the same time, the probe does not require the introduction of large-volume targeting groups in the molecule, and has the advantages of novel structure, high targeting and good imaging performance.

[0011] 2. The present application provides a method for preparing a supramolecular fluorescent probe that responds to both hypoxia and endogenous formaldehyde. The simple host-guest non-covalent construction strategy is simple and effective, and the fluorescent probe can be obtained quickly and efficiently.

[0012] 3. In view of the good imaging performance, the hypoxia and endogenous formaldehyde dual-responsive supramolecular fluorescent probe provided in this application is of great significance for the accurate diagnosis of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Schematic diagram of the application of hypoxia and endogenous formaldehyde dual-responsive supramolecular fluorescent probe in tumor imaging.

[0014] Figure 2 (a) is the fluorescence spectrum of the non-covalent bonding between azophenylcalixarene and formaldehyde-responsive fluorescent probe host and guest; (b) is the MALDI-TOF mass spectrum of supramolecular fluorescent probe A.

[0015] Figure 3 is the stability of supramolecular fluorescent probe A.

[0016] Figure 4 This is the fluorescence response performance diagram of supramolecular fluorescent probe A to formaldehyde and hypoxia.

[0017] Figure 5 This is the image of tumor cells imaged by supramolecular fluorescent probe A. DETAILED DESCRIPTION

[0018] The technical solution of the present invention is further described in detail below in conjunction with several preferred embodiments and drawings. This embodiment is implemented on the premise of the technical solution of the invention, and a detailed implementation method and specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments.

[0019] Unless otherwise specified, the experimental materials used in the following examples can be purchased from conventional biochemical reagent companies.

[0020] Example 1: Preparation of supramolecular fluorescent probe A The present embodiment provides a supramolecular fluorescent probe, the preparation method of which is as follows: 0.5 μmol of azophenylcalixarene 1 and 0.5 μmol of formaldehyde-responsive fluorescent probe (according to a molar ratio of 1:1) are mixed and dissolved in 10 μL of DMSO, and then added into 100 mL of PBS buffer (10 mM, pH = 7.4) to obtain supramolecular fluorescent probe A (the final concentrations of both azophenylcalixarene and formaldehyde-responsive fluorescent probe are 5 μmol / L).

[0021] The chemical structure of azophenylcalixarene 1 is: The synthesis method of azophenylcalixarene 1 is as follows: In a 25 mL round-bottom flask, p-fluoroaniline (110 mg) was dissolved in 2 mL of water and 0.5 mL of hydrochloric acid was added. After cooling the reaction solution to 0°C in an ice bath, 69 mg of 2 mL of NaNO2 solution was slowly added. After reacting for 1 hour, the reaction solution obtained above was slowly added to the reaction solution containing calixarene 1 (50 mg) and sodium acetate (245 mg) dissolved in MeOH-DMF (5:8, v:v). After the mixture was stirred at room temperature for 24 hours, azophenylcalixarene 1 was 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). 13 C NMR (100 MHz, DMSO- d6) δ164.5, 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 theoretical calculated value C 52 H 36 F4N8O4: 911.2718 [MH] - ; Measured value, 911.2710 [MH] - . The chemical structure of the formaldehyde-responsive fluorescent probe is as follows: The synthesis method of formaldehyde-responsive fluorescent probe is as follows: Specifically, compound hemicyanine A (0.2 mmol) and 2-methylaminobenzoic acid (0.3 mmol) were dissolved in 10 mL of dichloromethane. Then, dicyclohexylcarbodiimide (61 mg, 0.4 mmol) and 4-dimethylaminopyridine (37 mg, 0.3 mmol) were added to the above reaction solution. The mixture was stirred at room temperature for 12 hours. After the reaction, the solvent was dried and the crude product was purified by silica gel column chromatography (CH2Cl2 / MeOH = 30:1, v / v) to obtain a dark blue formaldehyde-responsive fluorescent probe. 1 H NMR, 13 C NMR and high-resolution mass spectrometry data are as follows 1 H NMR (400 MHz, DMSO- d6 ): δ 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 ) δ179.3, 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 spectrometry theoretical calculated value C 34 H 33 N2O3: 517.2486 [MI] + ; Measured value, 517.2469 The successful preparation of supramolecular fluorescent probe was studied by fluorescence titration and high-resolution mass spectrometry. Specifically, different concentrations of azophenylcalixarene were added to the formaldehyde-responsive fluorescent probe (5 μmol / L) solution at room temperature, and the test solution was tested at λ ex = Fluorescence spectrum changes at 679 nm and at λ ex / em = 690 / 750 nm fluorescence intensity change. Further, the mass spectrometry signal of the obtained solution was studied by MALDI-TOF mass spectrometry. Figure 2 (a) and Figure 2 (b) It can be seen that azophenylcalixarene and the formaldehyde-responsive fluorescent probe can specifically produce host-guest interactions. Azophenylcalixarene and the formaldehyde-responsive fluorescent probe can specifically produce a characteristic molecular weight addition signal at a charge-to-mass ratio of 1429.5449, indicating the successful preparation of supramolecular fluorescent probe A.

[0022] Example 2: Stability of supramolecular fluorescent probe A supramolecular fluorescent probe provided in the present application has good in vitro stability. This example studies the in vitro stability of the supramolecular fluorescent probe A prepared in Example 1. The supramolecular fluorescent probe A configured in Example 1 (the final concentrations of both azophenylcalixarene and formaldehyde-responsive fluorescent probe are 5 μmol / L) is stored at room temperature, and the stability of the supramolecular fluorescent probe is tested using ultraviolet absorption spectroscopy. The test uses a quartz cuvette as the sample pool, and the supramolecular fluorescent probe is placed at room temperature for 12 hours. The changes in the ultraviolet absorption spectrum of the sample solution are tested after 0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 8.0, 10.0 and 12 hours. The results are shown in the figure. Figure 3 As shown, from Figure 3It can be seen that the ultraviolet absorption spectrum of the supramolecular fluorescent probe has no obvious change within 12 hours, proving that the supramolecular fluorescent probe has good stability.

[0023] Example 3: Optical properties of supramolecular fluorescent probes The supramolecular fluorescent probe provided in the present application can highly selectively respond to the two microenvironmental characteristics of tumor hypoxia and high endogenous formaldehyde, and light up near-infrared fluorescence signals. Therefore, this embodiment studies the optical properties of the supramolecular fluorescent probe A prepared in Example 1 under hypoxic microenvironment and endogenous formaldehyde conditions. Specifically, to the solution of supramolecular fluorescent probe A (the final concentration of both azophenylcalixarene and formaldehyde-responsive fluorescent probe is 5 μmol / L) configured in Example 1, (i) PBS; (ii) formaldehyde; (iii) insurance powder (azoreductase chemical mimetic) and (iv) formaldehyde and insurance powder were added respectively. After incubating the above solution, the solution was tested at λ using a fluorescence spectrometer. ex = Fluorescence spectrum changes at 679 nm and at λ ex / em = Fluorescence intensity change at 690 / 750 nm. Figure 4 It can be seen that the supramolecular fluorescent probe A solution has a weak fluorescence signal when there is only single insurance powder or single formaldehyde; while when there is both insurance powder and formaldehyde, the supramolecular fluorescent probe A has a strong fluorescence signal at λ em = A strong near-infrared fluorescence signal was observed at 710 nm. This result shows that the supramolecular fluorescent probe A can only be lit up when stimulated by the hypoxic microenvironment and endogenous formaldehyde. This reduces the "false positive" signal in the detection process, improves the reliability of the detection results, and is conducive to its application in biological imaging.

[0024] Example 4: Application of supramolecular fluorescent probe in tumor cell imaging The supramolecular fluorescent probe provided in the present application can highly selectively respond to two microenvironmental characteristics of tumor hypoxia and high endogenous formaldehyde to light up near-infrared fluorescence signals. Therefore, this example studies the application of the supramolecular fluorescent probe A prepared in Example 1 in tumor cell fluorescence imaging.

[0025] Human breast cancer cells MCF-7 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 the breast cancer cells were cultured in a cell culture incubator at 37°C, 21% oxygen (volume percentage), and 5% carbon dioxide (volume percentage) for 24 hours, the cells were digested with trypsin and transferred to a cell imaging dish (cell concentration 1 * 10 6), and then the cells in the imaging dish were divided into three groups: (i) After the first group of cells were cultured in a cell culture incubator at 37°C, 21% oxygen (volume percentage), 5% carbon dioxide (volume percentage) for 24 hours, supramolecular fluorescent probe A (the molar ratio of azophenylcalixarene 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 incubation continued for 2 hours; (ii) After the second group of cells were cultured in a cell culture incubator at 37°C, 1% oxygen (volume percentage), 5% carbon dioxide (volume percentage) for 24 hours, supramolecular fluorescent probe A (the molar ratio of azophenylcalixarene 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 incubation continued for 2 hours; (iii) After the third group of cells were cultured in a cell culture incubator at 37°C, 1 After culturing in a cell culture incubator with 5% oxygen (volume percentage) and 5% carbon dioxide (volume percentage) for 24 hours, MCF-7 cells were pretreated with formaldehyde scavenger sodium bisulfite (NaHSO3), and then supramolecular fluorescent probe A was added (the molar ratio of azophenylcalixarene and formaldehyde-responsive fluorescent probe in fluorescent probe A was 1:1, and the final concentration of both was 5 μmol / L), and incubation was continued for 2 hours. Finally, the culture medium in the imaging dish was washed away with PBS buffer, and a laser scanning confocal microscope was used to select a 633 nm excitation light source and use the 650-750 nm band as the output signal channel for cell imaging research. The results are shown in Figure 5 As shown. Figure 5 It can be seen that only the second group of cells showed a 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 were not hypoxic, the third group of cells had a low endogenous formaldehyde content due to sodium bisulfite, and only the second group of cells had 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, producing a strong fluorescence signal under hypoxia stimulation, which can be used for specific fluorescence imaging of hypoxic breast cancer cells.

Claims

1. A supramolecular fluorescent probe that responds to hypoxia and endogenous formaldehyde, characterized in that: It is composed of azophenylcalixarene and a formaldehyde-responsive fluorescent probe included in the azophenylcalixarene. The chemical structure of the azophenylcalixarene is as follows: Wherein R is methyl, fluorine or bromine; The chemical structural formula of the formaldehyde-responsive fluorescent probe is as follows: 。 2. The hypoxia and endogenous formaldehyde dual-responsive supramolecular fluorescent probe according to claim 1, characterized in that: The azophenylcalixarene and the formaldehyde-responsive fluorescent probe form a stable supramolecular complex through non-covalent interaction between the host and the guest.

3. The method for preparing the hypoxia and endogenous formaldehyde dual-responsive supramolecular fluorescent probe according to claim 1, characterized in that: The azophenylcalixarene and the formaldehyde-responsive fluorescent probe are fully mixed and dissolved in a buffer solution to obtain a supramolecular fluorescent probe solution.

4. The method for preparing the hypoxia and endogenous formaldehyde dual-responsive supramolecular fluorescent probe according to claim 3, characterized in that: The molar ratio of the azophenylcalixarene to the formaldehyde-responsive fluorescent probe is 1:1-1:

2.

5. The method for preparing the hypoxia and endogenous formaldehyde dual-responsive supramolecular fluorescent probe according to claim 3, characterized in that: The buffer solution is a PBS buffer solution with a pH value of 6.0 to 8.

0.

6. Use of the hypoxia and endogenous formaldehyde dual-responsive supramolecular fluorescent probe as claimed in claim 1 in the preparation of tumor fluorescence imaging detection reagents.

7. Use of the hypoxia and endogenous formaldehyde dual-responsive supramolecular fluorescent probe according to claim 6 in the preparation of tumor fluorescence imaging detection reagents, characterized in that: Hypoxia and endogenous formaldehyde dual-responsive supramolecular fluorescent probes are used as detection agents for tumor fluorescence imaging in tumors and their tumor microenvironments.

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