Near-infrared fluorescent probe for detecting circulating hypoxia as well as preparation method and application of near-infrared fluorescent probe

Abstract

The invention discloses a near-infrared fluorescent probe for detecting circulating hypoxia as well as a preparation method and application thereof, and belongs to the technical field of biological analysis and detection.The novel near-infrared fluorescent probe for detecting circulating hypoxia is constructed by taking indolo-thioxanthene as a fluorophore and 2, 5-bis (trifluoromethyl) azobenzene as a hypoxia recognition group through conjugate connection of the indolo-thioxanthene and the 2, 5-bis (trifluoromethyl) azobenzene. The material can rapidly and reversibly respond to hypoxia, has high selectivity and high sensitivity, and realizes reversible hypoxia cycle detection in living cells and mice.

Description

Technical Field

[0001] The invention belongs to the technical field of biological analysis and detection, and specifically relates to a near-infrared fluorescent probe for detecting circulatory hypoxia and a preparation method and application thereof. Background Art

[0002] Circulatory hypoxia, i.e., the hypoxia-reoxygenation cycle, exists in a variety of pathological processes, including solid tumors, tissue ischemia-reperfusion, etc. It has an important impact on tumor metastasis, drug resistance and radiotherapy inhibition, tissue ischemia-reperfusion injury, etc. Therefore, monitoring the circulatory hypoxia process in living bodies is of great significance. At present, the means of detecting oxygen content in living bodies include magnetic resonance imaging, positron emission tomography, oxygen electrode imaging, and optical imaging. Among them, optical imaging has the characteristics of high sensitivity, high temporal and spatial resolution, and non-invasive in situ imaging, which is suitable for tracking and detecting oxygen content in living bodies. Phosphorescent probes containing heavy metal ions are sensitive to oxygen, but have potential metal toxicity risks and are expensive. Organic small molecule fluorescent probes have low biological toxicity and rapid metabolism. The development of such probes to track the dynamic changes of oxygen content in living bodies has important practical significance.

[0003] Under physiological hypoxic conditions, the levels of reductive enzymes, including nitroreductase, azoreductase, and CYP450 reductase, are abnormally increased. Based on this, people use nitrobenzene, azobenzene, aromatic nitrogen oxides, quinoline, etc. as hypoxic recognition groups to design fluorescent probes that specifically recognize hypoxic environments. However, the recognition reactions of these probes are almost irreversible, and it is impossible to dynamically track the cyclic changes in oxygen content. At present, only two reversible hypoxic fluorescent probes based on 3,5-bis(trifluoromethyl)azobenzene as recognition groups have been reported in the prior art, and no other new recognition groups or probes have been reported. The development of reversible hypoxia-responsive recognition groups and fluorescent probes based on organic small molecules is a difficult problem that needs to be solved urgently but is difficult. Summary of the invention

[0004] Technical issues solved:

[0005] The present application aims at the deficiencies of the prior art and solves the technical problems that the current phosphorescent probes for heavy metal ions have potential metal toxicity risks and are expensive, the recognition reactions of the probes are almost irreversible, and the cyclic changes of oxygen content cannot be dynamically tracked. A near-infrared fluorescent probe for detecting cyclic hypoxia and its preparation method and application are provided.

[0006] Technical solution:

[0007] To achieve the above objectives, this application is implemented through the following technical solutions:

[0008] A method for preparing a near-infrared fluorescent probe for detecting circulatory hypoxia specifically comprises the following steps:

[0009] Step 1: Preparation of Compound 1: Take 0.513 g of 2,5-di(trifluoromethyl)aniline and dissolve it in 15 mL of CH2Cl2 to obtain solution A, take 2.697 g of oxone and dissolve it in 30 mL of pure water to obtain solution B, pour solution B into solution A, stir vigorously in a N2 atmosphere at room temperature in the dark, track the reaction progress on a chromatographic plate until the raw material point disappears, stop the reaction, separate the liquids, extract the aqueous layer with CH2Cl2, wash with saturated brine, dry with anhydrous Na2SO4, remove the solvent, and obtain a green liquid substance, namely compound 1, which is directly used in the next step reaction;

[0010] Step 2: Preparation of near-infrared fluorescent probe for detecting circulatory hypoxia: 150 mg of SHD was added to the compound 1 prepared in the first step, dissolved in 30 mL of glacial acetic acid, stirred at 45 °C in the dark, and the reaction was tracked on the chromatographic plate until the raw material point disappeared. After the reaction was completed, 30

[0011] mLCH2Cl2 and 30mL water, separate the liquids, extract the aqueous phase with CH2Cl2, wash with saturated brine, and dry with anhydrous Na2SO4. After desolvation, purify the chromatographic column and elute with dichloromethane and methanol gradient to obtain a blue solid 2,5-CF3-SHD probe, which is a near-infrared fluorescent probe for detecting circulatory hypoxia.

[0012] Furthermore, in the first step, the aqueous layer was extracted with CH2Cl2 (30 mL x 3), washed with saturated brine (30 mL x 3), dried over anhydrous Na2SO4, and the solvent was evaporated under reduced pressure to obtain a green liquid substance, namely compound 1.

[0013] Furthermore, in the second step, the aqueous phase was extracted with CH2Cl2 (30 mL x 3), washed with saturated brine (30 mL x 3), dried over anhydrous Na2SO4, and the solvent was evaporated under reduced pressure and then purified by chromatographic column.

[0014] Furthermore, in the second step, dichloromethane and methanol are gradient eluted, and the gradient is 40:1, 30:1, and 20:1.

[0015] Furthermore, the yield in the second step is 7-15%.

[0016] The present application also discloses a near-infrared fluorescent probe for detecting cyclic hypoxia prepared by any of the above preparation methods. The near-infrared fluorescent probe for detecting cyclic hypoxia uses 2,5-bis(trifluoromethyl)azobenzene as the hypoxia recognition group and indolethioanthene as the fluorophore. The two are conjugated to construct a small molecule near-infrared fluorescent probe 2,5-CF3-SHD for detecting cyclic hypoxia. The chemical structure of 2,5-CF3-SHD is shown in formula (I):

[0017]

[0018] Application of a near-infrared fluorescent probe for detecting circulatory hypoxia in detecting circulatory hypoxia, wherein the near-infrared fluorescent probe for detecting circulatory hypoxia is encapsulated with an amphiphilic polymer into a nanoprobe for detecting circulatory hypoxia, wherein the amphiphilic polymer is DSPE-PEG n The nanoprobe is 2,5-CF3-SHD@DP, and the chemical structure of the amphiphilic polymer is shown in formula (II):

[0019]

[0020] Where n is 23, 43 or 113.

[0021] Further, 24.8 mg DSPE-PEG 1000 Dissolved in 44 mL THF, added 0.5 mL THF solution containing 0.68 mg 2,5-CF3-SHD, and after 40 kHz ultrasound for 10 min, quickly poured into 44 mL pure water and 40 kHz ultrasound for 10-15 min; stirred at room temperature for 3 h, THF was removed by N2 gas flow at 45 °C, centrifuged and washed with a 100 kDa ultrafiltration tube at 4500 r / min for 10 min, dispersed in PBS and stored at 4 °C to obtain 2,5-CF3-SHD@DP nanoprobes.

[0022] Furthermore, the fluorophore indolothioxanthene can be combined with an anion, the anion being I - Br - PF4 - One or more of the .

[0023] The present application also discloses the application of a near-infrared fluorescent probe for detecting circulatory hypoxia in imaging, biomarkers or sensing of circulatory hypoxia in cells or mice. The near-infrared fluorescent probe for detecting circulatory hypoxia can respond rapidly to a hypoxic environment, and the fluorescence signal reaches a peak value within 10-15 minutes.

[0024] Principle explanation: The near-infrared fluorescent probe for detecting circulatory hypoxia in the present application uses azobenzene containing two trifluoromethyl substituents as the hypoxia recognition group, and the substituents are located at the ortho or meta position of the azo bond, as a circulatory hypoxia probe; the near-infrared fluorescent probe for detecting circulatory hypoxia can detect hypoxia with an intracellular oxygen content of 10% or less, and is widely used in circulatory hypoxia imaging, biomarkers or sensing fields in cells / mice.

[0025] Beneficial effects:

[0026] The present application provides a method for preparing a near-infrared fluorescent probe for detecting circulatory hypoxia, which has the following advantages compared with the prior art:

[0027] Beneficial effects:

[0028] 1. The near-infrared fluorescent probe for detecting cyclic hypoxia in the present application can quickly respond to the hypoxic environment, and the fluorescence signal reaches a peak in about 10 minutes;

[0029] 2. The near-infrared fluorescent probe for detecting cyclic hypoxia of the present application has a weak fluorescence signal itself. When liver microsomes and NADPH are present in a hypoxic environment, the fluorescence signal is significantly enhanced; when the environmental oxygen content returns to normal, the probe fluorescence signal recovers; when the environment becomes hypoxic again, the probe fluorescence signal increases accordingly; when the environmental oxygen content returns to normal again, the probe fluorescence signal recovers again, and the fluorescence signal intensity is positively correlated with the degree of hypoxia. The fluorescence signal is weak in a normoxic environment, and increases when the oxygen content is 10%, and is strongest when the oxygen content is 0.1%. When a hypoxia-reoxygenation cycle occurs in the environment, the probe fluorescence signal increases-weakens in a corresponding manner;

[0030] 3. The near-infrared fluorescent probe for detecting circulatory hypoxia of the present application effectively indicates the cyclic changes in intracellular oxygen content and the dynamic changes in oxygen content during ischemia-reperfusion of the mouse hind legs;

[0031] 4. The near-infrared fluorescent probe for detecting circulatory hypoxia of the present application has high selectivity, good sensitivity, rapid response and dynamic reversibility in the recognition of hypoxia, and realizes circulatory hypoxia imaging in cells and living mice. The probe has broad application prospects in biology and medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Response diagram of different degrees of hypoxia of the near-infrared fluorescent probe for detecting circulatory hypoxia prepared in this application;

[0033] Figure 2 The response kinetic diagram of the near-infrared fluorescent probe for detecting circulatory hypoxia prepared in this application to hypoxia, wherein a is a fluorescence emission diagram; b is a diagram showing the change of fluorescence intensity at 765 nm over time;

[0034] Figure 3 This is a graph showing the response of the near-infrared fluorescent probe for detecting cyclic hypoxia prepared in the present application to the hypoxia-reoxygenation cycle, wherein a is a fluorescence emission graph; b is a graph showing the change in fluorescence intensity at 765 nm;

[0035] Figure 4 This is a selectivity diagram of the near-infrared fluorescent probe for detecting circulatory hypoxia prepared in the present application in response to hypoxia, wherein a is a fluorescence emission diagram; b is a fluorescence intensity diagram at 775 nm;

[0036] Figure 5The response diagram of the near-infrared fluorescent probe for detecting circulatory hypoxia prepared in this application to different oxygen contents in cells, wherein a is a fluorescence microscopic imaging diagram of H9C2 cells incubated with 2,5-CF3-SHD@DP probe (2 μM) under different hypoxia levels; b is a bar graph of the average fluorescence intensity in cells under various oxygen contents;

[0037] Figure 6 The response diagram of the near-infrared fluorescent probe for detecting cyclic hypoxia prepared in this application to the hypoxia-reoxygenation cycle in cells, wherein a is a fluorescence microscopic imaging diagram of H9C2 cells incubated with 2,5-CF3-SHD@DP probe (2 μM) under normoxic-hypoxic cycle; b is a diagram of the change of the average fluorescence intensity in the cells under normoxic-hypoxic cycle;

[0038] Figure 7 The imaging diagram of the ischemia-reperfusion process of the mouse hind leg by the near-infrared fluorescent probe for detecting circulatory hypoxia prepared in this application, wherein af is the imaging during which equal amounts of probe solution are injected into the left and right hind leg muscles of the mouse, the tourniquet is tied to the left leg for 30 minutes, and the imaging is tracked during this period; gk is the imaging during which the tourniquet is released after 30 minutes and the imaging is tracked; the control group is the imaging diagram of the mouse before the probe injection; ex =745nm,λ em =820nm. DETAILED DESCRIPTION

[0039] In order to further illustrate the present invention, the present invention is further described in detail below in conjunction with embodiments.

[0040] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection scope of the present invention is not limited to the following examples.

[0041] Preparation of SHD: Compound SHD was synthesized by a method known in the literature (S. Yao, Y. Chen, H. Xu, et al., Hypoxia-responsive near infrared thioxanthene-hemicyanine nanoparticle for multimodal imaging-guided photothermal / photodynamic therapy, Dyes and Pigments, 2022, 206, 110583.). 1HNMR (400MHz, CD3OD, δ, ppm) 8.31 (d, J = 12Hz, 1H), 7.59 (d, J = 8Hz, 1H), 7.50-7.40 (m, 4H), 7.35 (t, J = 8Hz, 1H), 6.91 (s, 1H), 6.85 (d, J = 12Hz, 1H),6.38(d,J=12Hz,1H),4.20(t,J=8Hz,2H),2.80(t,J=8Hz,2H),2.70(t,J=8Hz,2H),1.96-1.88(m,4H),1,78(s,6H),1.06(t,J=8Hz,3H).

[0042] Embodiment 1:

[0043] A method for preparing a near-infrared fluorescent probe for detecting circulatory hypoxia specifically comprises the following steps:

[0044] Step 1, preparation of compound 1: 0.513 g of 2,5-di(trifluoromethyl)aniline was dissolved in 15 mL of CH2Cl2 to obtain solution A, 2.697 g of oxone was dissolved in 30 mL of pure water to obtain solution B, solution B was poured into solution A, stirred vigorously at room temperature in a N2 atmosphere and protected from light, the reaction progress was tracked by the chromatographic plate until the raw material point disappeared, the reaction was stopped, the liquid was separated, and the aqueous layer was

[0045] After extraction with CH2Cl2, washing with saturated brine, drying with anhydrous Na2SO4, and removing the solvent, a green liquid substance, namely compound 1, was directly used for the next step reaction; the aqueous layer was extracted with CH2Cl2 (30mlⅹ3), washed with saturated brine (30mlⅹ3), dried with anhydrous Na2SO4, and evaporated to remove the solvent under reduced pressure;

[0046] Step 2: Preparation of near-infrared fluorescent probe for detecting circulatory hypoxia: 150 mg of SHD was added to the compound 1 prepared in the first step, dissolved in 30 mL of glacial acetic acid, stirred at 45 °C in the dark, and the reaction was tracked on the chromatographic plate until the raw material point disappeared. After the reaction was completed, 30

[0047] mLCH2Cl2 and 30 mL water, separate the liquids, extract the aqueous phase with CH2Cl2 (30 ml x 3), wash with saturated brine (30 ml x 3),

[0048] 3) Drying with anhydrous Na2SO4, removing the solvent by vacuum rotary evaporation, and purifying by chromatographic column, eluting with dichloromethane and methanol in a gradient ratio of 40:1, 30:1, and 20:1, to obtain a blue solid 2,5-CF3-SHD probe, i.e., a near-infrared fluorescent probe for detecting cyclic hypoxia, with a yield of 7-15%.

[0049] The near-infrared fluorescent probe for detecting cyclic hypoxia uses 2,5-bis(trifluoromethyl)azobenzene as the hypoxia recognition group and indolethioanthene as the fluorophore, and the two are conjugated to construct a small molecule near-infrared fluorescent probe 2,5-CF3-SHD for detecting cyclic hypoxia. The chemical structure of 2,5-CF3-SHD is shown in formula (I):

[0050]

[0051] 1 HNMR(400MHz,CD3OD,δ,ppm)8.46(d,J=14.8Hz,1H),8.17-8.13(m,2H),8.80(s,1H) ,8.04(d,J=8.4Hz,1H),7.95(d,J=8.4Hz,1H),7.77-7.68(m,3H),7.62-7.55(m,2H), 7.22(s,1H),6.90(d,J=14.8Hz,1H),4.49(t,J=16Hz,2H),2.84(t,J=4Hz,J=8Hz,2H ), 2.74 (t, J = 4Hz, J = 8Hz, 2H), 2.02-1.98 (m, 4H), 1.96 (s, 6H), 1.09 (t, J = 7.2Hz, 3H). 13 CNMR(101MHz,CD3OD,δ,ppm)179.73,151.35,151.02,149.46,149.36,146.01,143.04,141.26,137.30,133.46,133.10,132.35,131.14,129.1 6,128.78,128.56,127.99,127.94,127.73,122.68,122.47,119.75,11 3.89,113.13,108.79,51.56,32.27,26.69,26.38,21.40,20.09,10.11.

[0052]

[0053] Embodiment 2:

[0054] An application of the near-infrared fluorescent probe for detecting circulatory hypoxia prepared in Example 1 in detecting circulatory hypoxia, wherein the near-infrared fluorescent probe for detecting circulatory hypoxia is wrapped with an amphiphilic polymer to form a nanoprobe for detecting circulatory hypoxia, wherein the amphiphilic polymer is DSPE-PEG n The nanoprobe is 2,5-CF3-SHD@DP, and the chemical structure of the amphiphilic polymer is shown in formula (II):

[0055]

[0056] Where n is 23, 43 or 113;

[0057] Take 24.8mg DSPE-PEG 1000 Dissolved in 44 mL THF, added 0.5 mL THF solution containing 0.68 mg 2,5-CF3-SHD, and after 40 kHz ultrasound for 10 min, quickly poured into 44 mL pure water and 40 kHz ultrasound for 10-15 min; stirred at room temperature for 3 h, THF was removed by N2 gas flow at 45 °C, centrifuged and washed with a 100 kDa ultrafiltration tube at 4500 r / min for 10 min, dispersed in PBS and stored at 4 °C to obtain 2,5-CF3-SHD@DP nanoprobes.

[0058] The fluorophore indolothioxanthene can bind to an anion, the anion is I - Br - PF4 - One or more of the .

[0059] Embodiment 3:

[0060] Fluorescence spectra of the probe 2,5-CF3-SHD in response to different degrees of hypoxia:

[0061] Rat liver microsomes (RLM) are widely used in the evaluation of drug metabolism and in the study of hypoxia-responsive probes and hypoxia-controlled-release prodrugs. The rat liver microsomes used in the present invention are from BD Gentest, derived from male SD rats, item number 452501.

[0062] 21% oxygen: incubation in normoxic air;

[0063] 10% oxygen: adjust the oxygen content in the glove box to 10%, and place all solutions in the glove box until the oxygen content is balanced before use. Scan the fluorescence after the samples are incubated in the glove box;

[0064] 0.1% oxygen: adjust the oxygen content in the glove box to 0.1%, and place all solutions in the glove box until the oxygen content is balanced before use. Scan the fluorescence after the samples are incubated in the glove box.

[0065] PBS solution containing 2,5-CF3-SHD probe (20 μM, pH 7.40, 3% DMSO, v / v) was incubated at 37°C for 20 min under various oxygen concentrations ([RLM] = 250 μg / mL, [NADPH] = 100 μM), and the fluorescence spectrum was tested. The results are shown in Figure 1 The fluorescence signal of the probe 2,5-CF3-SHD increases with the decrease of the oxygen content in the environment. The fluorescence signal of the probe begins to increase in the 10% O2 environment, indicating that the probe can respond to moderate hypoxia and has high sensitivity.

[0066] Embodiment 4:

[0067] Response time of probe 2,5-CF3-SHD to hypoxia:

[0068] Before the hypoxia test, all solutions were placed in a glove box with 0.1% O2 until the oxygen content was balanced. During the reoxygenation test, the probe solution was exposed to air away from light until normoxia was restored and the probe fluorescence signal was stable;

[0069] The probe 2,5-CF3-SHD@DP (20μM), mouse liver microsomes (250μg / ml), and NADPH (100μM) were added to PBS buffer (0.1M, pH7.40) and the fluorescence spectrum was scanned; the solution was transferred to an anoxic glove box to remove oxygen, incubated at 37°C for 15 minutes, and the fluorescence spectrum was scanned; the above solution was placed in the air to return to normoxia, and the fluorescence spectrum was scanned; the solution was transferred to an anoxic glove box for a second time to remove oxygen, and mouse liver microsomes (250μg / ml) and NADPH (100μM) were added, incubated at 37°C for 15 minutes, and the fluorescence spectrum was scanned. This completes the normoxia-hypoxia-reoxygenation-hypoxia cycle of the solution, and the rest is analogous, and the results are shown in Figure 3 The probe fluorescence signal is weak under normoxia, and the fluorescence is significantly enhanced near 765nm after hypoxia; the fluorescence signal almost disappears after reoxygenation. This cycle can be repeated many times, indicating that the probe 2,5-CF3-SHD@DP is a reversible hypoxic fluorescence probe that can indicate solution cyclic hypoxia.

[0070] Embodiment 5:

[0071] Selectivity of probe response to hypoxia:

[0072] The selectivity test was carried out in PBS buffer (0.1 M, pH 7.40) containing 2,5-CF3-SHD@DP (20 μM). Figure 4 All active oxygen, active sulfur, active nitrogen species and metal ions were prepared in milliQ aqueous solution. Under normoxic conditions, various metal ions (2 mM Ca 2+ Mg 2+ , 50 μM Mn 2+ ,Fe 3+ ,Fe 2+ ,Co 2+ ,Ni 2+ ,Cu 2+ ,Zn 2+ ), reactive oxygen species (100 μM ℃l - ,H2O2,20μM ONO2 - ), reactive nitrogen species (100 μM SNP, NO2 - ) and reduced sulfur species (100 μM HS- ,Hcy,GSH,Cys), oxalic acid (100μM), ascorbic acid (100μM) and incubated at 37℃ for 2h, the fluorescence intensity of the probe did not change significantly. 2+ , NO donor SNP, high concentration of HS - When present, the fluorescence intensity of the probe appears weak or fluctuates slightly, and the fluorescence signal is greatly enhanced only under hypoxic conditions. Therefore, the probe is highly selective in recognizing hypoxic environments and is not interfered by common species of living organisms.

[0073] Embodiment 6:

[0074] The probe responds to different levels of intracellular hypoxia:

[0075] The cell experiment was conducted in H9C2 rat cardiomyocytes. Cell hypoxia incubation: The cell culture dish containing the 2,5-CF3-SHD@DP (2μM) probe and the oxygen content indicator were placed in a hypoxic gas production bag (0.1% O2) or a microaerobic gas production bag (6-10% O2) produced by Mitsubishi Corporation of Japan, sealed and placed in a 37°C incubator for 4 hours to control the concentration of reductase and the degree of hypoxia in the cells, simulating the reducing environment under pathological conditions. Cell normoxic incubation: The cell culture dish containing the 2,5-CF3-SHD@DP (2μM) probe was placed in a 5% CO2 incubator and incubated at 37°C for 4 hours. The excitation filter was 690 / 50nm, the emission filter was 792 / 64nm, the exposure time was 1s, and the fluorescence microscopy imaging results of the cells are shown in the figure. Figure 5 As shown. Under normoxic conditions (21% O2), the intracellular fluorescence signal is weak. Under oxygen concentrations of 6-10%, obvious fluorescence signals appear in the cells. When the oxygen concentration is 0.1%, the intracellular fluorescence signal is significantly enhanced. This result is consistent with the test results in the solution, indicating that the probe can respond to various levels of hypoxia and has high sensitivity.

[0076] Embodiment 7:

[0077] Probe response to cellular cyclic hypoxia:

[0078] The cell culture dish containing 2,5-CF3-SHD@DP (2μM) probe was placed in a hypoxic gas-generating bag (0.1% O2), incubated in a 37°C incubator for 2 hours, and imaged under a fluorescence microscope after washing with PBS; the above cells were placed in a 5% CO2 incubator, reoxygenated and incubated at 37°C for 2 hours, and imaged under a fluorescence microscope; the cells were placed in a hypoxic gas-generating bag (0.1% O2) again, incubated in a 37°C incubator for 2 hours, and imaged under a fluorescence microscope; the reoxygenation-hypoxia cycle was performed according to the same steps, and the results are shown in FIG. Figure 6As shown. Under hypoxic conditions, there is an obvious fluorescence signal in the cells, which disappears after reoxygenation, is re-enhanced after the second hypoxia, and disappears again after the third reoxygenation. That is, in the hypoxia-reoxygenation cycle of the incubation environment, the probe fluorescence signal shows a cycle of enhancement-weakening. This phenomenon is consistent with the cyclic change of the probe fluorescence signal under the hypoxia-reoxygenation cycle in the solution, confirming that the probe can reversibly respond to hypoxia and is an example of a reversible hypoxia fluorescent probe.

[0079] Embodiment 8:

[0080] Probe imaging of ischemia-reperfusion in mouse hind legs:

[0081] All animal experiments were performed in accordance with the guidelines of the Animal Care and Use Committee. Nude mice were provided by Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. Isoflurane was used as an anesthetic, and the fluorescence signals were collected by the Bolu Teng AniView600 in vivo imaging system at 745 nm excitation and 820 nm band.

[0082] The hind legs of mice were tied with a tourniquet to cause ischemia, and the tourniquet was cut and reperfusion was performed. The ischemia-reperfusion process has been shown to be accompanied by a decrease and increase in oxygen content (Y. Zhang, W. Zhao, Y. Chen, et al., Rational construction of a versatile arylazo-based NIR probe for cycling hypoxia imaging in vivo, Nat. Commun. 2021, 12, 2772.).

[0083] Physiological saline (12.5 μM, 50 μL) containing 2,5-CF3-SHD@DP probe was injected intramuscularly on the outside of the two hind legs of mice. A tourniquet was tied around the left leg to cause ischemia and hypoxia. The image was taken every 5 minutes. After 30 minutes, the tourniquet was removed to allow reperfusion and reoxygenation. The image was taken every 10 minutes. The fluorescence signals of the left and right hind legs were basically the same. The tracking imaging results are shown in the figure. Figure 7 The right hind leg was always in a normoxic state, and the fluorescence signal remained almost unchanged; the fluorescence signal of the left hind leg continued to increase within 30 minutes after ligation; after the tourniquet was removed, the fluorescence signal rapidly weakened, and after 30 minutes, the fluorescence signals of the left and right legs were almost the same. This shows that the 2,5-CF3-SHD@DP probe can quickly respond to the changes in the hypoxia-reoxygenation cycle of living mice.

[0084] Implementation results:

[0085] like Figure 1 As shown, Figure 1The response diagrams of different degrees of hypoxia of the near-infrared fluorescent probe for detecting circulatory hypoxia prepared in this application, specifically, the fluorescence emission diagrams of PBS buffer (0.1M, pH7.40) containing 2,5-CF3-SHD@DP probe (20μM), rat liver microsomes (RLM, 250μg / ml) and NADPH (100μM) under different oxygen contents, Hypoxic treatment: adjust the oxygen content in the glove box to the target value, and bubble into the solution until the oxygen content reaches the corresponding value.

[0086] like Figure 2 Middle a, Figure 2 As shown in b, Figure 2 The response kinetic diagram of the near-infrared fluorescent probe for detecting circulatory hypoxia prepared in this application to hypoxia, wherein a is a fluorescence emission diagram; b is a diagram showing the change of fluorescence intensity at 765 nm over time, specifically under complete hypoxic conditions, PBS buffer (0.1 M, pH 7.40) containing 2,5-CF3-SHD@DP probe (20 μM), rat liver microsomes (RLM, 250 μg / ml) and NADPH (100 μM), incubated at 37°C for 0-30 min, fluorescence emission diagram and a diagram showing the change of fluorescence intensity at 765 nm over time; Hypoxic treatment: in a completely hypoxic glove box, aerate the solution until the oxygen content is <0.1%.

[0087] like Figure 3 As shown, Figure 3 The response diagram of the near-infrared fluorescent probe for detecting cyclic hypoxia prepared in the present application to the hypoxia-reoxygenation cycle, wherein a is a fluorescence emission diagram; b is a fluorescence intensity change diagram at 765nm, specifically a PBS buffer (0.1M, pH 7.4) containing 2,5-CF3-SHD@DP probe (20μM), rat liver microsomes (RLM, 250μg / ml) and NADPH (100μM), a fluorescence emission diagram under normoxia-hypoxia-reoxygenation cycle and a fluorescence intensity change diagram at 765nm; hypoxic conditions: in a completely hypoxic glove box, aerate the solution until the oxygen content is <0.1%; normoxic conditions: open the reaction system to expose it to air until the oxygen content returns to normal.

[0088] like Figure 4 As shown, Figure 4 The selectivity diagram of the near-infrared fluorescent probe for detecting circulatory hypoxia prepared in this application for hypoxia response, wherein a is a fluorescence emission diagram; b is a fluorescence intensity diagram at 775 nm, specifically a PBS buffer (0.1 M, pH 7.4) containing 2,5-CF3-SHD@DP probe (20 μM), in hypoxia (containing 250 μg / ml mouse liver microsomes, 100 μM NADPH), or in the presence of various competitive species under normoxic conditions, and the fluorescence emission diagram and fluorescence intensity at 775 nm scanned after incubation at 37°C for 1 to 2 hours; the metal ion Ca2+ Mg 2+ :2mM, other metal ions 50μM; ONO2 - :20μM, other ROS / RNS / RSS, oxalic acid, ascorbic acid: 100μM; hypoxic conditions: in a completely hypoxic glove box, aerate the solution until the oxygen content is <0.1%.

[0089] like Figure 5 As shown, Figure 5 The response diagram of the near-infrared fluorescent probe for detecting circulatory hypoxia prepared in this application to different oxygen contents in cells, wherein a is a fluorescence microscopic imaging diagram of H9C2 cells incubated with 2,5-CF3-SHD@DP probe (2 μM) under different hypoxia levels; b is a bar graph of the average fluorescence intensity in cells under various oxygen contents in a, n=5, and the hypoxic and microaerobic environments are provided by cell hypoxic gas production bags or microaerobic gas production bags produced by Mitsubishi Corporation of Japan.

[0090] like Figure 6 As shown in Figure 6 The response diagram of the near-infrared fluorescent probe for detecting cyclic hypoxia prepared in this application to the hypoxia-reoxygenation cycle in the cell, wherein a is the fluorescence microscopic imaging of H9C2 cells incubated with 2,5-CF3-SHD@DP probe (2 μM) under normoxic-hypoxic cycle; b is the change diagram of the average fluorescence intensity in the cell under normoxic-hypoxic cycle in figure a, n=5; the hypoxic environment is provided by a hypoxic gas production bag; normoxic conditions: the cell culture dish is placed in an incubator containing 5% CO2 until the oxygen content in the culture medium returns to normal and the fluorescence intensity in the cell remains stable.

[0091] Figure 7 The imaging diagram of the near-infrared fluorescent probe for detecting circulatory hypoxia prepared in this application on the ischemia-reperfusion process of the mouse hind leg, wherein af is the same amount of probe solution injected into the left and right hind leg muscles of the mouse, the tourniquet was tied on the left leg for 30 minutes, and the imaging was tracked, gk is the tourniquet was released after 30 minutes, and the imaging was tracked, and the control group is the imaging diagram of the mouse before the probe injection; ex =745nm,λ em =820nm.

[0092] The present invention is described above by way of example. It should be noted that, without departing from the core of the present invention, any simple deformation, modification or other equivalent replacement that can be made by those skilled in the art without inventive effort falls within the protection scope of the present invention.

Claims

1. A method for preparing a near-infrared fluorescent probe for detecting circulatory hypoxia, characterized in that: The specific steps include: Step 1, preparation of compound 1: 0.513 g of 2,5-di(trifluoromethyl)aniline was dissolved in 15 mL of CH2Cl2 to obtain solution A, 2.697 g of oxone was dissolved in 30 mL of pure water to obtain solution B, solution B was poured into solution A, stirred vigorously at room temperature in a N2 atmosphere and away from light, the reaction progress was tracked by a chromatographic plate until the raw material point disappeared and the reaction was stopped, the liquid was separated, and the aqueous layer was concentrated with CH2Cl2 After extraction, washing with saturated brine, drying with anhydrous Na2SO4, and desolvation, a green liquid substance, namely compound 1, was obtained, which was directly used in the next step reaction; Step 2: Preparation of near-infrared fluorescent probe for detecting circulatory hypoxia: 150 mg of SHD was added to the compound 1 prepared in the first step. Dissolve in 30 mL of glacial acetic acid, stir at 45 °C in the dark, and track the reaction on the chromatographic plate until the raw material point disappears. After the reaction is complete, add 30 mL CH2Cl2 and 30 mL water, separate the liquids, extract the aqueous phase with CH2Cl2, wash with saturated brine, dry with anhydrous Na2SO4, remove the solvent and purify with a chromatographic column, elute with dichloromethane and methanol gradient to obtain a blue solid 2,5-CF3-SHD probe, which is a near-infrared fluorescent probe for detecting circulatory hypoxia.

2. The method for preparing a near-infrared fluorescent probe for detecting circulatory hypoxia according to claim 1, characterized in that: In the first step, the aqueous layer was extracted with CH2Cl2 (30 mL x 3), washed with saturated brine (30 mL x 3), dried over anhydrous Na2SO4, and evaporated to remove the solvent under reduced pressure to obtain a green liquid substance, namely compound 1.

3. The method for preparing a near-infrared fluorescent probe for detecting circulatory hypoxia according to claim 1, characterized in that: In the second step, the aqueous phase was extracted with CH2Cl2 (30 mL x 3), washed with saturated brine (30 mL x 3), dried over anhydrous Na2SO4 The product was dried, the solvent was evaporated under reduced pressure and then purified by chromatography.

4. The method for preparing a near-infrared fluorescent probe for detecting circulatory hypoxia according to claim 1, characterized in that: In the second step, dichloromethane and methanol are used for gradient elution, and the gradient is 40:1, 30:1, and 20:

1.

5. The method for preparing a near-infrared fluorescent probe for detecting circulatory hypoxia according to claim 1, characterized in that: The yield in the second step is 7-15%.

6. A near-infrared fluorescent probe for detecting cyclic hypoxia prepared by the preparation method according to any one of claims 1 to 5, characterized in that: The near-infrared fluorescent probe for detecting cyclic hypoxia uses 2,5-bis(trifluoromethyl)azobenzene as the hypoxia recognition group and indolethioanthene as the fluorophore, and the two are conjugated to construct a small molecule near-infrared fluorescent probe 2,5-CF3-SHD for detecting cyclic hypoxia. The chemical structure of 2,5-CF3-SHD is shown in formula (I):

7. Use of the near-infrared fluorescent probe for detecting circulatory hypoxia according to claim 6 in detecting circulatory hypoxia, characterized in that: The near-infrared fluorescent probe for detecting circulatory hypoxia is encapsulated with an amphiphilic polymer into a nanoprobe for detecting circulatory hypoxia. The amphiphilic polymer is DSPE-PEG. n The nanoprobe is 2,5-CF3-SHD@DP, and the chemical structure of the amphiphilic polymer is shown in formula (II): Where n is 23, 43 or 113.

8. The use according to claim 7, characterized in that: Take 24.8 mg DSPE-PEG 1000 Dissolve in 44 mL THF, add 0.5 mL THF solution containing 0.68 mg 2,5-CF3-SHD, sonicate at 40 kHz for 10 min, quickly pour into 44 mL pure water, sonicate at 40 kHz for 10-15 min; stir at room temperature for 3 h, remove THF with N2 flow at 45 °C, and filter with 100 kDa ultrafiltration tube 4500 The mixture was washed by centrifugation at r / min for 10 min, and then dispersed in PBS and stored at 4°C to obtain 2,5-CF3-SHD@DP nanoprobes.

9. The use according to claim 7, characterized in that: The fluorophore indolothioxanthene can bind to an anion, the anion is I - Br - PF4 - One or more of the .

10. Use of the near-infrared fluorescent probe for detecting circulatory hypoxia according to claim 6 in imaging, biomarking or sensing of circulatory hypoxia in cells or mice, characterized in that: The near-infrared fluorescent probe for detecting circulatory hypoxia can respond rapidly to the hypoxic environment, and the fluorescence signal reaches a peak within 10-15 minutes.

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