A two-photon fluorescent probe for detecting hypoxic level and its in-situ imaging tumor tissue application

Abstract

This invention provides a two-photon fluorescent probe for detecting hypoxia levels. The probe comprises a core fluorophore composed of a 2-dimethylamino-7-hydroxynaphthalene two-photon fluorophore and a probe composed of a sulfonate methylene group. This invention also provides a method for preparing and applying the two-photon fluorescent probe for detecting hypoxia levels. The fluorescent probe of this invention enables the monitoring of hypoxia levels in tumor and normal tissues by in-situ detection of nitroreductase (NTR) content within tissues. It features fast response, high sensitivity, high optical stability, good biocompatibility, high selectivity, and high depth, high resolution, and low biological background in two-photon imaging. In cell imaging applications, this probe exhibits good cell permeability and can detect NTR content at the cellular level, indicating cellular hypoxia.

Description

Technical Field

[0001] This invention relates to the field of fluorescent chemical sensor technology, and more specifically to a two-photon fluorescent probe for detecting hypoxia levels, its preparation method, and its application. Background Technology

[0002] Hypoxia is a pathological state caused by a decrease in the partial pressure of oxygen within tissues, commonly seen in highly aggressive malignant tumors. Hypoxia also plays an important role in various diseases such as stroke, ischemia, and inflammatory diseases. Similarly, many research groups have reported that reduced tissue blood supply leads to hypoxia at the tissue level. Hypoxia is also associated with the adverse effects of metabolic anticancer drugs on treatment and is an indicator of certain types of cancer. In tumor disease research, a positive correlation has also been observed between hypoxia levels and cell growth in tumor regions. Therefore, it is necessary to detect hypoxia levels in cells and tumors, which is becoming increasingly important for examining tumor status and anticancer drug formulation.

[0003] Currently, several methods exist for detecting hypoxia levels in tissues, such as electrochemical methods, immunohistochemistry, nuclear magnetic resonance (MRI) molecular imaging, and PET molecular imaging. However, these techniques all have drawbacks, including invasiveness or high cost. In contrast, biomarker-based fluorescence detection methods are attracting increasing attention due to their advantages such as high sensitivity, good specificity, ease of operation, and low background interference.

[0004] Under hypoxic conditions, several reductases, including nitroreductase (NTR), quinone reductase, and azoreductase, are overexpressed. As one of the most representative hypoxic enzymes, NTR serves as an indicative biomarker of transcriptional responses under hypoxic stress and is directly related to the degree of hypoxia in solid tumors, thus attracting considerable attention. Therefore, the detection of NTR levels can be used to assess the degree of hypoxia in biological systems.

[0005] NTR sensors utilize a specific strategy employed by nitroreductases to reduce nitro groups to amino groups in the presence of electron donors such as NADH / NADPH, and are designed to monitor hypoxia levels in tumors. NADH, a reduced nicotinamide adenine dinucleotide, is a known reducing agent that can act as an electron donor in biological systems, providing electrons for biological processes. Several fluorescent, bioluminescent, and chemiluminescent sensors for NTR have been developed. Among the various reported NTR detection methods, fluorescence-based methods are of great significance due to their high sensitivity, simplicity of operation, fast response, and applicability for high-throughput screening of live cells.

[0006] Intracellular biological process imaging based on small-molecule fluorescent probes has gained increasing attention due to its high sensitivity and large spatiotemporal resolution, becoming a powerful imaging tool. However, the number of known fluorescent probes that can specifically detect hypoxia levels is currently limited, and most are single-photon (OP) excitation or have short excitation and emission wavelengths (<500 nm), which can easily lead to photodamage to biological tissues. Furthermore, the organism's own fluorescence can cause significant background interference to the analytical results, affecting the accuracy of detection. In particular, OP imaging, due to its short excitation wavelength, struggles to achieve high-resolution, high-depth tissue imaging.

[0007] Two-photon (TP) imaging is a nonlinear optical imaging technique that combines continuous two-photon absorption and long-wavelength fluorescence emission. The excitation and emission wavelengths can be longer, resulting in greater tissue penetration, less photodamage and photobleaching, and higher resolution. It has become an important tool for detecting active proteases and imaging in vivo tissues. However, current techniques for detecting NTR based on two-photon fluorescent probes still suffer from problems such as long response times (>100 min), low detection sensitivity (>2 μg / mL), shallow penetration depth, and inability to be applied to in vivo tissue testing.

[0008] Therefore, there is an urgent need to develop new two-photon fluorescent probes to achieve rapid, sensitive, and efficient monitoring of NTR, and ultimately apply them to imaging hypoxia levels in living tissues. This is of great significance for tumor diagnosis, tumor treatment, and new drug development. Summary of the Invention

[0009] The purpose of this invention is to address the shortcomings of existing technologies by providing a two-photon fluorescent probe for detecting hypoxia levels and its preparation method. The recognition group of this probe can be rapidly inserted into the active cavity of the NTR, and it has the characteristics of rapid response, high sensitivity, good selectivity, good biosafety, and the ability to perform in vivo tissue imaging.

[0010] According to a first aspect of the present invention, a two-photon fluorescent probe for detecting hypoxia levels is provided, the fluorescent probe comprising a core fluorophore composed of a 2-dimethylamino-7-hydroxynaphthalene two-photon fluorophore and a probe composed of a sulfonate methylene group.

[0011] As an optional implementation, the fluorescent probe has the structural formula shown in Formula I:

[0012] Formula I.

[0013] As an optional implementation, the response time of the fluorescent probe is ≤15 min.

[0014] As an optional implementation, the detection limit of the fluorescent probe is 0.059 μg / mL.

[0015] According to a second aspect of the present invention, a method for preparing the aforementioned two-photon fluorescent probe capable of detecting hypoxia levels is provided, comprising the following steps:

[0016] 7-(dimethylamino)-3-formylnaphthalene-2-yl(4-nitrophenyl)methanesulfonate was prepared by reacting 2-dimethylamino-7-hydroxynaphthalene with (4-nitrophenyl)methanesulfonyl chloride;

[0017] The desired fluorescent probe can be obtained by preparing 3-(2,2-dicyanovinyl)-7-(dimethylamino)naphth-2-yl(4-nitrophenyl)methanesulfonate from 7-(dimethylamino)-3-formylnaphth-2-yl(4-nitrophenyl)methanesulfonate.

[0018] As an optional implementation method, the specific process for preparing 7-(dimethylamino)-3-formylnaphthalene-2-yl(4-nitrophenyl)methanesulfonate is as follows:

[0019] (4-Nitrophenyl)methanesulfonyl chloride was added dropwise to a DMF solution containing triethylamine of 2-dimethylamino-7-hydroxynaphthalene under a nitrogen atmosphere at 0°C, and the mixture was stirred at room temperature to obtain a first mixed solution.

[0020] The first mixed solution was diluted with DCM and washed with deionized water. The resulting organic phase was dried with anhydrous Na2SO4 and concentrated under vacuum. Finally, it was purified by silica gel chromatography to obtain a yellow solid 7-(dimethylamino)-3-formylnaphthalene-2-yl(4-nitrophenyl)methanesulfonate.

[0021] As an optional implementation, in the first mixed solution, the molar ratio of 2-dimethylamino-7-hydroxynaphthalene and (4-nitrophenyl)methanesulfonyl chloride is 0.39:(0.54~0.64), the concentration of 2-dimethylamino-7-hydroxynaphthalene is 0.10~0.16 mmol / mL, the concentration of (4-nitrophenyl)methanesulfonyl chloride is 0.14~0.24 mmol / mL, and the volume percentage concentration of the triethylamine DMF solution is 0.031~0.043 v / v.

[0022] As an optional implementation, the specific process for preparing 3-(2,2-dicyanovinyl)-7-(dimethylamino)naphth-2-yl(4-nitrophenyl)methanesulfonate is as follows:

[0023] 7-(dimethylamino)-3-formylnaphthalene-2-yl(4-nitrophenyl)methanesulfonate and malononitrile were added to ethanol, followed by piperidine. The mixture was then stirred at 0°C for 20 min under argon atmosphere and then at room temperature for 3 h to obtain a second mixed solution.

[0024] After removing the organic solvent from the second mixed solution by rotary evaporation, the solution was dissolved in dichloromethane and washed twice with deionized water. The organic phase was collected, dried on anhydrous Na2SO4, and then concentrated under vacuum to obtain a red solid 3-(2,2-dicyanovinyl)-7-(dimethylamino)naphth-2-yl(4-nitrophenyl)methanesulfonate.

[0025] As an optional implementation, in the second mixed solution, the molar ratio of 7-(dimethylamino)-3-formylnaphthalene-2-yl(4-nitrophenyl)methanesulfonate to malononitrile is 0.19:(0.19~0.38), and the concentration of piperidine is 0.40~0.55 mmol / mL.

[0026] In a third aspect of the present invention, the application of the aforementioned two-photon fluorescent probe capable of detecting hypoxia levels in the preparation of a reagent for detecting hypoxia levels in tumor tissue is provided.

[0027] As can be seen from the above technical solutions of the present invention, the two-photon fluorescent probe for detecting hypoxia levels proposed in this invention has the following characteristics:

[0028] High specificity: The fluorescent probe of this invention is activated only when NTR and coenzyme NADH are present. After being activated by coenzyme NADH, the nitro group is reduced to amino group, followed by 1,6-elimination, which spontaneously releases an electron-rich naphthol fluorophore, accompanied by an increase in fluorescence, thereby achieving high specificity.

[0029] Rapid response: The fluorescent probe of this invention can react completely with NTR within 15 min, which can greatly shorten the reaction time compared with the reported probe response time (>100 min);

[0030] High sensitivity: The detection limit of the fluorescent probe of this invention reaches 0.059 μg / mL, which greatly improves the detection sensitivity;

[0031] High selectivity: The fluorescent probe of this invention will only show significant fluorescence enhancement when NTR and coenzyme NADH are added simultaneously;

[0032] High biocompatibility: The fluorescent probe molecules of this invention have high biocompatibility with cells and organisms;

[0033] Vivo tissue imaging: The fluorescent probe of this invention can successfully distinguish the NTR and hypoxia level inside tumor tissue, and under two-photon laser irradiation, the penetration depth can reach 224 μm;

[0034] High time stability: The fluorescent probe of this invention has good stability and can image NTR activity for a long time. Attached Figure Description

[0035] Figure 1 This is a synthesis route diagram of XN3 according to the present invention.

[0036] Figure 2 shows the 1H NMR spectrum (i), 1C NMR spectrum (ii), and high-resolution mass spectrum (iii) of the XN3 of the present invention.

[0037] Figure 3 This is a docking simulation of the XN3 and NTR active cavity of the present invention and its mechanism of action.

[0038] Figure 4 This is the fluorescence emission spectrum of XN3 and NTR of the present invention.

[0039] Figure 5 This invention relates to the kinetic performance test of the XN3-NTR reaction.

[0040] Figure 6 The fluorescence spectra (A) and linear relationship (B) between fluorescence intensity and NTR concentration after the reaction of XN3 with different concentrations of NTR are shown in the figure.

[0041] Figure 7 This invention relates to the XN3's performance test against 26 common interfering substances.

[0042] Figure 8 This is a cytotoxicity assay using different concentrations of the NTR probe XN3.

[0043] Figure 9 This is an imaging image of XN3 in cancer cells HepG2 and HeLa under different hypoxic conditions according to the present invention.

[0044] Figure 10 This is a comparison of cell imaging of the XN3 of the present invention in cancer cells HepG2 and HeLa and normal cells L929 and LO2.

[0045] Figure 11 The XN3 of this invention is at a two-photon excitation wavelength (λ) ex At 810 nm, the imaging images of normal and tumor tissue sections of mice are shown in (A), the corresponding fluorescence intensity map (B), and the imaging images of different penetration depths corresponding to two-photon excitation (C).

[0046] Figure 12These are fluorescence imaging images of mouse tumor tissue sections incubated with XN3 for 40 min to 24 h according to the present invention.

[0047] Figure 13 The XN3 of this invention is at a two-photon excitation wavelength (λ) ex = 810 nm (red channel) or single-photon excitation wavelength (λ) ex = 488 nm (green channel) Imaging images of human transverse colon cancer tissue and adjacent tissue sections (A), corresponding fluorescence intensity maps (BC), and corresponding imaging images at different penetration depths (D).

[0048] Figure 14 The images are TP & OP fluorescence images, DAPI and H & E staining images of human transverse colon cancer tissue obtained by XN3 in situ imaging according to the present invention. Detailed Implementation

[0049] To better understand the technical content of the present invention, specific embodiments are described below in conjunction with the accompanying drawings.

[0050] Various aspects of the invention are described in this disclosure with reference to the accompanying drawings, in which numerous illustrative embodiments are shown. The embodiments of this disclosure are not necessarily intended to encompass all aspects of the invention. It should be understood that the various concepts and embodiments described above, as well as those described below in more detail, can be implemented in any of a number of ways.

[0051] This invention designs a two-photon near-infrared fluorescent probe for detecting NTR, i.e., hypoxia levels. Compared with previously reported two-photon NTR probes, the probe of this invention, through docking simulation calculations, allows the recognition group to rapidly insert into the NTR active cavity, improving response efficiency. Test results show that the probe can fully respond to the target protease NTR within 15 minutes, significantly shortening the detection time. The detection limit of this probe for NTR is 0.059 μg / mL, greatly improving detection sensitivity. In addition, the probe has high biosafety and excellent two-photon imaging performance, enabling deeper and higher resolution imaging detection in human tumor tissues.

[0052] In an exemplary embodiment of the present invention, a two-photon fluorescent probe for detecting hypoxia levels is provided. The fluorescent probe comprises a core fluorophore composed of a 2-dimethylamino-7-hydroxynaphthalene two-photon fluorophore and a probe composed of a sulfonate methylene group, namely XN3.

[0053] As an optional implementation, the fluorescent probe has the structural formula shown in Formula I:

[0054] Formula I.

[0055] As an optional implementation, the response time of the fluorescent probe is ≤15 min.

[0056] As an optional implementation, the detection limit of the fluorescent probe is 0.059 μg / mL.

[0057] Combination Figure 1 As shown, in another exemplary embodiment of the present invention, a method for preparing the aforementioned two-photon fluorescent probe capable of detecting hypoxia levels is provided, comprising the following steps:

[0058] 7-(dimethylamino)-3-formylnaphthalene-2-yl(4-nitrophenyl)methanesulfonate (denoted as XP4) was prepared by using 2-dimethylamino-7-hydroxynaphthalene (denoted as X1C) and (4-nitrophenyl)methanesulfonyl chloride.

[0059] The desired fluorescent probe XN3 was obtained by preparing 3-(2,2-dicyanovinyl)-7-(dimethylamino)naphth-2-yl(4-nitrophenyl)methanesulfonate using XP4.

[0060] As an optional implementation method, the specific process for preparing 7-(dimethylamino)-3-formylnaphthalene-2-yl(4-nitrophenyl)methanesulfonate is as follows:

[0061] (4-Nitrophenyl)methanesulfonyl chloride was added dropwise to a DMF solution containing triethylamine (X1C) at 0°C and stirred at room temperature to obtain a first mixed solution.

[0062] The first mixed solution was diluted with DCM and washed with deionized water. The resulting organic phase was dried with anhydrous Na2SO4 and concentrated under vacuum. Finally, it was purified by silica gel chromatography to obtain yellow solid XP4.

[0063] As an optional embodiment, in the first mixed solution, the molar ratio of 2-dimethylamino-7-hydroxynaphthalene and (4-nitrophenyl)methanesulfonyl chloride is 0.39:(0.54~0.64), particularly preferably 0.39:0.59; the concentration of 2-dimethylamino-7-hydroxynaphthalene is 0.10~0.16 mmol / mL, particularly preferably 0.13 mmol / mL; the concentration of (4-nitrophenyl)methanesulfonyl chloride is 0.14~0.24 mmol / mL, particularly preferably 0.19 mmol / mL; and the volume percentage concentration of the triethylamine DMF solution is 0.031~0.043 v / v%, particularly preferably 0.037 v / v.

[0064] As an optional implementation method, the specific process for preparing XN3 is as follows:

[0065] XP4 and malononitrile were added to an ethanol solution, followed by piperidine. The mixture was then stirred at 0°C for 20 min under argon atmosphere and then at room temperature for 3 h to obtain a second mixed solution.

[0066] After removing the organic solvent from the second mixed solution by rotary evaporation, the solution was dissolved in dichloromethane and washed twice with deionized water. The organic phase was collected, dried on anhydrous Na2SO4, and then concentrated under vacuum to obtain a red solid 3-(2,2-dicyanovinyl)-7-(dimethylamino)naphth-2-yl(4-nitrophenyl)methanesulfonate XN3.

[0067] As an optional embodiment, in the second mixed solution, the molar ratio of XP4 to malononitrile is 0.19:(0.19~0.38), and the concentration of piperidine is 0.40~0.55 mmol / mL, particularly preferably 0.475 mmol / mL.

[0068] In another exemplary embodiment of the present invention, an application of the aforementioned two-photon fluorescent probe capable of detecting hypoxia levels is provided in detecting hypoxia levels in tumor tissue. Tumor tissue has a lower hypoxia level than normal tissue and can be specifically imaged. Therefore, by detecting the hypoxia level in tumor tissue, its application in in situ imaging of tumor tissue is realized.

[0069] The aforementioned two-photon fluorescent probe can be used for deep imaging of NTR levels in mouse tumor tissues or for deep imaging of NTR levels in clinical human tumor tissues. According to docking simulation calculations, the recognition group of the probe can be rapidly inserted into the NTR active cavity, improving the response efficiency and clearly indicating the hypoxia status in the tissue, i.e., nitroreductase (NTR) activity. At the same time, it can accurately and clearly distinguish the boundary between tumor tissue and normal tissue, providing a new detection scheme for the application research of small molecules with the potential of image-assisted clinical tumor surgical resection.

[0070] To facilitate better understanding, the present invention will be further illustrated below with several specific examples, but the preparation process is not limited to these examples, and the content of the present invention is not limited to these examples.

[0071] Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0072] Example 1

[0073] [Reference Figure 1 The synthetic route shown illustrates the preparation of the XN3 fluorescent probe.

[0074] Step 1: Under a nitrogen atmosphere at 0°C, add (4-nitrophenyl)methanesulfonyl chloride (137.9 mg, 0.59 mmol) dropwise to a DMF (3 mL) solution of 2-dimethylamino-7-hydroxynaphthalene (X1C, 83.9 mg, 0.39 mmol) and triethylamine (0.11 mL).

[0075] The mixture was stirred at room temperature for 8 hours, diluted with DCM (20 mL), and washed with water (3 × 10 mL). The organic phase was dried over Na2SO4 and concentrated by air separation. It was then purified by silica gel chromatography (using ethyl acetate / petroleum ether as eluent, v / v = 1 / 4) to give a yellow solid XP4 (85.7 mg, yield: 53%).

[0076] Step 2: Add XP4 and malononitrile (12.55 mg, 0.19 mmol) obtained in Step 1 to a solution of ethanol (4 mL), then add piperidine (0.19 mL, 1.9 mmol), stir at 0°C for 20 minutes under argon atmosphere, and then stir at room temperature for 3 hours.

[0077] After removing the organic solvent by rotary evaporation, the solution was dissolved in dichloromethane and washed twice with deionized water. The organic phase was collected, dried on anhydrous Na2SO4, and then concentrated under vacuum to give red solid XN3 (61.6 mg, yield: 70%).

[0078] Example 2

[0079] [XN3 Characterization and Mechanism]

[0080] The XN3 obtained in Example 1 was characterized as shown in Figure 2. The H spectrum positions and proton numbers of the probe were found to be consistent with each other through the proton NMR spectrum, carbon NMR spectrum and high-resolution mass spectrometry. The calculated value of the mass spectrometry data was 461.0919 and the measured value was 461.0930. This confirms that the XN3 fluorescent probe was successfully synthesized in this invention.

[0081] Combination Figure 3 As shown, the XN3 two-photon fluorescent probe molecule of the present invention is initially in a "off" state, wherein the intrinsic fluorescence of N,N-dimethylaminonaphthalene is significantly quenched by the adjacent electron-deficient nitro aromatic group; under NTR catalysis, the nitro group is reduced to amino, and then 1,6-elimination spontaneously releases an electron-rich naphthol fluorophore. With the increase of fluorescence, the corresponding fluorescent group is generated, thereby achieving the effect of specific detection of NTR.

[0082] Example 3

[0083] [In vitro testing of XN3]

[0084] The in vitro reaction system consisted of a 384-well plate with a total volume of 100 μL per well. This included 5 μL of a 400 μM fluorescent probe XN3 stock solution, 10 μL of a 100 μg / mL NTR stock solution, 10 μL of a 5 mM NADH stock solution, and 85 μL of PBS solution at pH 7.4. The probe stock solution was prepared from DMSO, and the final test system contained 5% DMSO at a final concentration of 20 μM. The NTR concentration was 10 μg / mL, and the NADH concentration was 500 μM. The reaction system was incubated at 37°C and 1000 rpm for 40 min in the dark. The specific experimental results are as follows:

[0085] (1) such as Figure 4 As shown, the response performance of XN3 to NTR was tested. Specifically, under the condition of NTR concentration of 10 μg / mL, using a 485 nm wavelength laser as the excitation wavelength, a significant enhancement of fluorescence was observed after the reaction, while the fluorescence decreased significantly after adding the inhibitor dicoumarin. These changes in photophysical properties confirm that XN3 can be activated by NTR and release a fluorophore, causing fluorescence enhancement. These data indicate that XN3 can effectively detect changes in NTR activity.

[0086] (2) For example Figure 5 As shown, the time-kinetic response of XN3 to NTR was tested. Specifically, in a PBS test system (0.1M, 5% DMSO) at pH = 7.4, the concentrations of XN3 and NTR were 20 μM and 10 μg / mL, respectively. The reaction time-kinetic test results showed that the reaction between XN3 and NTR reached equilibrium at 15 min (λex = 485nm).

[0087] (3) such as Figure 6 As shown, the concentration-response performance of XN3 to NTR was tested. Specifically, in a PBS test system (0.1 M, 5% DMSO) at pH = 7.4, with an XN3 concentration of 20 μM, different concentrations of NTR (0-10 μg / mL) were added, and the reaction was carried out at 37℃ for 40 min to obtain the final concentration gradient (λex = 485 nm). The results showed that the fluorescence intensity of XN3 after reacting with NTR gradually increased with increasing NTR concentration.

[0088] (4) such as Figure 7 As shown, the interference performance of probe XN3 was tested. Specifically, 26 common substances were selected as interfering substances, of which 1-26 were Na.+ S 2- HS - Fe 2+ Cu 2+ Zn 2+ K + Mg 2+ Fe 3+ Cr 3+ Ca 2+ Na + Al 3+ SO3 2- SO4 2- CO3 2- V C V E The interfering substances were H2O2, Glu, Cys, Tyr, Arg, Trp, GSH, and BSA. 27 was NTR+NADH (10 μg / mL), and 28 was the blank control group. The test solution system was PBS (0.1 M, 5% DMSO) at pH 7.4, with an XN3 concentration of 20 μM. Solutions of various interfering substances were added sequentially, and the fluorescence emission spectra (λex = 485 nm) were measured after 40 min of incubation.

[0089] The results showed that only when NTR and coenzyme NADH were present was there a significant fluorescence enhancement, while other interfering ions had no effect. This proves that XN3 has high specificity for the detection of NTR.

[0090] Example 4

[0091] [XN3 Intracellular Assay]

[0092] (1) First, the cytotoxicity of probe XN3 was tested. Specifically, the standard MTT assay was used for analysis. HeLa cells were digested with 0.25% trypsin and then the cells were prepared into a cell suspension with cell culture medium and seeded into 96-well plates. The cells were cultured at 37°C for 24 hours. Probes of different concentrations (0 μM, 1 μM, 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 80 μM) were added to the well plates and cultured for 24 hours. MTT was added and cultured for 4 hours. Finally, triple lysis buffer was added and incubated for another 2 hours. The absorbance of each well was measured using a microplate reader. Control wells and zeroing wells were also set up.

[0093] The results are as follows Figure 8 As shown, the results indicate that after 24 h of incubation, the probe had virtually no effect on cell viability at 0-80 μM, demonstrating that the fluorescent probe molecules of this invention have high biosafety for cells and organisms.

[0094] (2) XN3 was used to perform fluorescence imaging of NTRs in HepG2 and HeLa cancer cells. Tumors express NTRs in a hypoxic environment. The changes in NTR activity under different hypoxic conditions were observed by using OPFM to evaluate the feasibility of XN3 for tumor hypoxia imaging. HepG2 or HeLa cells were cultured at 37°C for 4 hours under normoxic (20% O2) and different hypoxic conditions (10%, 5% and 1% O2), and then incubated with XN3 (20 μM) at 37°C for 40 minutes. A group pretreated with In (50 μM) for 1 hour (1% O2) was used as an additional control. Before imaging with a confocal laser microscope, the cells were washed three times with 1 mL of PBS solution at pH = 7.4 to remove the culture medium, and finally, images were taken under a two-photon confocal microscope.

[0095] The results are as follows Figure 9 As shown, HepG2 cells incubated with XN3 under normoxic conditions showed almost no fluorescence after TPFM imaging. Furthermore, as oxygen concentration decreased (from 20% to 1%), NTR activity and / or expression levels increased, and the signal displayed by the probe gradually strengthened. The same phenomenon was observed in another cancer cell line, HeLa cells, indicating that these cells produced more NTRs with increasing hypoxia. In addition, in both cancer cell lines, cells treated with inhibitors under hypoxic conditions (1% O2) showed significantly reduced fluorescence signals. This further demonstrates that XN3 has the ability to image changes in tumor-endogenous NTR activity, meaning that the XN3 probe can effectively image hypoxic conditions in cancer cells.

[0096] (3) To assess whether XN3 can selectively image cancer cells, imaging experiments were performed in cancer cells and normal cells. LO2 cells, L929 cells, HepG2 cells, and HeLa cells were co-incubated with XN3 (20 μM, DMEM containing 1% DMSO) at different oxygen concentrations (20% or 1%), with the control group being the image without XN3 (DMEM containing 1% DMSO). Before imaging with a confocal laser microscope, the cells were washed three times with 1 mL of PBS solution at pH = 7.4 to remove the culture medium, and finally, images were taken under a two-photon confocal microscope.

[0097] The results are as follows Figure 10 As shown, only cancer cells under hypoxic (1% O2) conditions emitted strong fluorescence, while normal cells treated with XN3 showed almost no fluorescence signal. These results further demonstrate that XN3 can selectively image cancer cells under hypoxic conditions.

[0098] In summary, the XN3 fluorescent probe of the present invention has the ability to image NTR activity in living cells.

[0099] Example 5

[0100] [XN3 Mouse Tumor Tissue Intrast Test]

[0101] (1) To verify the tissue penetration effect of the probe, tumor tissue section imaging tests were performed on mice with subcutaneous implantation of 4T1 tumors. Tissue sections of tumor tissue and normal tissue were imaged under different light sources. Three control experiments were conducted on the tumor tissue: blank control group (PBS), experimental group with probe XN3 added (XN3), and experimental group with NTR inhibitor (In, dicumarol) pretreated and then probe XN3 added (DIC+XN3). Specifically, tumor tissue sections with a thickness of 300 μm were placed in three 1.5 mL centrifuge tubes. The first group was the blank control group (PBS): 300 μL of PBS solution was added to the centrifuge tube; the second group was the XN3 group: 300 μL of 60 μM XN3 solution was added to the centrifuge tube; the third group was the In+XN3 group: dicumarol was added to the centrifuge tube for 1 h pretreatment to inhibit NTR activity, and then 300 μL of 60 μM XN3 solution was added. All experimental groups were incubated at 37°C and 1000 rpm for 40 min in an incubator. For normal tissues: 100 μM XN3 solution was added. Then, the tissues were incubated at 37°C and 1000 rpm for 40 min in an incubator.

[0102] After incubation, the tissue sections were placed on glass slides, covered with coverslips, and imaged under a laser confocal fluorescence microscope using single-photon (λex = 488 nm) and two-photon (λex = 810 nm) excitation, respectively.

[0103] The results are as follows Figure 11 As shown, normal tissue exhibited almost no fluorescence, while in tumor tissue, the PBS group (serving as a blank control) also showed extremely low fluorescence. The XN3 group showed significant fluorescence enhancement, and the In+XN3 group showed only weak fluorescence. Furthermore, when excited by two-photon excitation, the fluorescence penetration depth was 224 μm, which is significantly greater than the penetration depth under single-photon excitation. This result demonstrates that the XN3 of this invention possesses excellent two-photon performance and provides deeper tissue penetration imaging and lower background interference for tissue sections.

[0104] (2) To determine the fluorescence stability of tumor tissue stained with XN3, tumor sections were incubated with XN3 (60 μM, 10 mM PBS, pH 7.4, containing 1% DMSO) for 40 minutes. The inhibitor group was also pretreated with In (1 mM) for 1 h, followed by incubation with XN3 (60 μM, 10 mM PBS, pH 7.4, containing 1% DMSO) for 40 minutes. Samples were excited at intervals using an 810 nm laser over 24 h after incubation, and TP fluorescence images were recorded at wavelengths of 520-620 nm. Images at different times were acquired using the same method.

[0105] The results are as follows Figure 12 As shown, the fluorescence generated by the XN3 imaging tumor tissue of the present invention under two-photon (TP) excitation is very stable, and the fluorescence intensity can still be maintained at more than 86.5% after 24 h. The corresponding data under single-photon (OP) excitation also show the same.

[0106] Example 6

[0107] [XN3 Imaging Effect Detection on Clinical Human Tumor Tissue]

[0108] (1) The activity of NTRs in human tumor tissues was detected by two-photon fluorescence microscopy. All tissues were less than 5 mm × 5 mm in size. Fresh transverse colon cancer tissues were incubated with XN3 (80 μM, 10 mM PBS, pH 7.4, containing 1% DMSO) in centrifuge tubes for 40 minutes. At the same time, fresh adjacent tissues were incubated with XN3 (80 μM, 10 mM PBS, pH 7.4, containing 1% DMSO) for 40 minutes as a control group. In addition, an inhibitor group was studied by pretreating fresh transverse colon cancer tissues with In (1 mM) for 1 hour, and then incubating them with XN3 (80 μM, 10 mM PBS, pH 7.4, containing 1% DMSO). Fresh transverse colon cancer tissues were incubated with PBS buffer (1% DMSO) as a control group.

[0109] After incubation, the tissue sections were placed on glass slides, covered with coverslips, and imaged under a laser confocal fluorescence microscope using single-photon (λex = 488 nm) and two-photon (λex = 810 nm) excitation, respectively.

[0110] The results are as follows Figure 13As shown, adjacent normal tissue exhibited almost no fluorescence. In transverse colon cancer tissue, the PBS group (as a blank control) also showed extremely low fluorescence, while the XN3 group showed significant fluorescence enhancement, and the In+XN3 group showed only weak fluorescence. Furthermore, the image clarity obtained using two-photon excitation was significantly higher than that obtained using single-photon excitation. This result demonstrates that XN3 possesses excellent two-photon performance and provides higher resolution, deeper tissue penetration imaging, and lower background interference for tissue sections.

[0111] (2) To assess the effectiveness of XN3 in detecting clinical human tumor tissues, histological examination of clinical specimens was performed. Fresh transverse colon cancer tissue was incubated with XN3 (80 μM, 10 mM PBS, pH 7.4, containing 1% DMSO) in centrifuge tubes for 40 minutes, then fixed with formalin for histopathological analysis. Sections were stained with H&E and then examined histologically using a BZ-X800 microscope under bright field. XN3 fluorescence was measured at λex = 488 nm (OP) or 810 nm (TP); λem = 520-620 nm, while DAPI fluorescence was detected under the DAPI filter group (λex = 340-370 nm; λem = 420-70 nm).

[0112] The results are as follows Figure 14 As shown, images of the resected tumor tissue stained with XN3, DAPI, and H&E were finally obtained. The fluorescence of the two-photon channel (red) and the single-photon channel (green) clearly showed the boundary between the tumor tissue and normal tissue, which coincided with the tumor boundary identified by H&E staining. This result verifies the accuracy of the fluorescent probe of the present invention in diagnosing tumor tissue.

[0113] While the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the invention. Those skilled in the art can make various modifications and refinements without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention shall be determined by the claims.

Claims

1. A two-photon fluorescent probe capable of detecting hypoxic level, characterized in that, The structural formula of the fluorescent probe is shown as formula I. Formula I.

2. The two-photon fluorescent probe for detecting hypoxic level according to claim 1, wherein, The response time of the fluorescent probe is ≤15 min.

3. The two-photon fluorescent probe for detecting hypoxia levels according to claim 1, characterized in that, The lower limit of detection of the fluorescent probe is 0.059 μg / mL.

4. A method for preparing the two-photon fluorescent probe capable of detecting the level of hypoxia according to any one of claims 1 to 3, characterized in that, The method comprises the following steps: 7-(dimethylamino)-3-formylnaphthalen-2-yl (4-nitrophenyl)methyl sulfonate is prepared from 2-dimethylamino-7-hydroxynaphthalene and (4-nitrophenyl)methylsulfonyl chloride; 3-(2,2-dicyanoethenyl)-7-(dimethylamino)naphthalen-2-yl (4-nitrophenyl)methyl sulfonate is prepared from 7-(dimethylamino)-3-formylnaphthalen-2-yl (4-nitrophenyl)methyl sulfonate, and the desired fluorescent probe is obtained.

5. The preparation method according to claim 4, characterized in that, The specific process for preparing 7-(dimethylamino)-3-formylnaphthalen-2-yl (4-nitrophenyl)methyl sulfonate is as follows: (4-nitrophenyl)methylsulfonyl chloride is added dropwise into a DMF solution containing 2-dimethylamino-7-hydroxynaphthalene and triethylamine under a nitrogen atmosphere at 0°C, and a first mixed solution is obtained by stirring at room temperature; The first mixed solution is diluted with DCM and washed with deionized water, and the obtained organic phase is dried over anhydrous Na2SO4 and concentrated under vacuum, and finally purified by silica gel chromatography to obtain 7-(dimethylamino)-3-formylnaphthalen-2-yl (4-nitrophenyl)methyl sulfonate in the form of a yellow solid.

6. The production method according to claim 5, wherein In the first mixed solution, the molar ratio of 2-dimethylamino-7-hydroxynaphthalene to (4-nitrophenyl)methylsulfonyl chloride is 0.39:(0.54-0.64), the concentration of 2-dimethylamino-7-hydroxynaphthalene is 0.10-0.16 mmol / mL, the concentration of (4-nitrophenyl)methylsulfonyl chloride is 0.14-0.24 mmol / mL, and the volume percentage concentration of the DMF solution of triethylamine is 0.031-0.043 v / v%.

7. The preparation method according to claim 4, characterized in that, The specific process for preparing 3-(2,2-dicyanoethenyl)-7-(dimethylamino)naphthalen-2-yl (4-nitrophenyl)methyl sulfonate is as follows: 7-(dimethylamino)-3-formylnaphthalen-2-yl (4-nitrophenyl)methyl sulfonate and malononitrile are added into ethanol, piperidine is added, and then the second mixed solution is obtained by stirring under an argon atmosphere at 0°C for 20 min and at room temperature for 3 h. After the organic solvent of the second mixed solution is removed by rotary evaporation, it is dissolved in dichloromethane and washed with deionized water twice, and the organic phase is collected, dried over anhydrous Na2SO4, and then concentrated under vacuum to obtain 3-(2,2-dicyanoethenyl)-7-(dimethylamino)naphthalen-2-yl (4-nitrophenyl)methyl sulfonate in the form of a red solid.

8. The preparation method according to claim 7, characterized in that, In the second mixed solution, the molar ratio of 7-(dimethylamino)-3-formylnaphthalen-2-yl (4-nitrophenyl)methyl sulfonate to malononitrile is 0.19:(0.19-0.38), and the concentration of piperidine is 0.40-0.55 mmol / mL.

9. Use of the two-photon fluorescent probe for detecting hypoxic level according to any one of claims 1-3 in the preparation of a reagent for detecting the hypoxic level in tumor tissue.

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