Reversible near-infrared small-molecule probe, preparation method thereof and application of reversible near-infrared small-molecule probe in monitoring of hemangioma redox steady state

Abstract

The invention discloses a reversible near-infrared small-molecule probe and a preparation method and application thereof in the aspect of monitoring the redox steady state of hemangioma, and the probe is prepared by selecting boron dipyrromethene (BODIPY) of a donor-acceptor (D-A) structure, introducing 3, 4-dihydroxy benzaldehyde to the 3-site of a BODIPY skeleton through a vinyl bridge by virtue of a Knoevenagel condensation reaction, and then introducing 3, 4-dihydroxy benzaldehyde to the 3-site of the BODIPY skeleton by virtue of a vinyl bridge, so as to obtain the reversible near-infrared small-molecule probe. And a small molecule probe with reversible response is designed and synthesized. The probe images the redox state in hemangioma by monitoring the dynamic change of superoxide anion (O2 <.->) and glutathione (GSH). The redox probe provided by the invention has the characteristics of specificity and sensitive response, and provides a potential promising tool for researching the treatment mechanism of anti-cancer drugs and illuminating various redox-related pathological processes.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescence probe cell imaging, and particularly relates to a reversible near-infrared small molecule probe, a preparation method thereof, and an application in monitoring the redox homeostasis of hemangioma. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the understanding of the overall background of the present application, and is not necessarily to be regarded as an admission or any form of implication that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] According to the research and understanding of the inventors, cancer cells are in an oxidative stress state due to the disruption of ROS homeostasis leading to excessive production of ROS, and activate the antioxidant system by upregulating glutathione (GSH) to adapt to oxidative stress. Therefore, the levels of intracellular O2 ·- or GSH can directly reflect the degree of oxidative stress in living cells. Dynamically and sensitively evaluating the redox cycle of local O2 ·- and GSH is very important.

[0004] The etiology and specific pathogenesis of hemangioma are not very clear, and its formation may be caused by various reasons. As infants and young children are the high-incidence group of hemangioma, combined with the significant role played by redox regulation in cancer progression, the study of the redox homeostasis of hemangioma has attracted attention.

[0005] So far, many fluorescent probes for separately detecting O2 ·- or GSH have been developed. However, the O2 ·- / GSH redox pair is fluctuating and variable, and physically combining organic probes with the ability to detect a single O2 ·- or GSH usually cannot simultaneously monitor O2 ·- and GSH. This is attributed to the heterogeneity of different probes in cell uptake and cell distribution. Therefore, studying the changes in the O2 ·- / GSH redox homeostasis in tumor cells requires reversible probes that can simultaneously detect these two substances. It is reported that near-infrared (NIR) fluorescent probes have excellent deep tissue penetration ability, low background interference, and good biocompatibility, and reversible organic NIR fluorescent probes provide good sensitivity and controllability, enabling them to be used for real-time monitoring of biological processes, especially in cell imaging and dynamic monitoring, and can provide more information.

[0006] Therefore, if a near-infrared fluorescent probe that can simultaneously detect these two substances can be provided, it may provide effective monitoring of the changes in the redox state of hemangioma. Summary of the Invention

[0007] To address the current deficiencies in the research on hemangiomas and the problems existing in the above-mentioned prior art, the present invention provides a reversible near-infrared small molecule probe, its preparation method, and its application in the monitoring of the redox homeostasis of hemangiomas. This project provides a boron dipyrromethene (BODIPY) chromophore with excellent photostability, high molar extinction coefficient, good biocompatibility, and easy synthesis and modification. By introducing 3,4-dihydroxybenzaldehyde with reversible responsiveness into the 3-position of the BODIPY skeleton through a vinyl bridge, the redox monitoring of O2 ·- / GSH can be achieved, with high sensitivity and specificity.

[0008] One of the objectives of the present invention is to provide a near-infrared fluorescent probe that can dynamically monitor the redox homeostasis of hemangiomas.

[0009] Another objective of the present invention is to provide a preparation method for the above-mentioned near-infrared fluorescent probe that can dynamically monitor the redox homeostasis of hemangiomas.

[0010] A third objective of the present invention is to provide an imaging method for the above-mentioned near-infrared fluorescent probe that can dynamically monitor the redox homeostasis of hemangiomas.

[0011] A fourth objective of the present invention is to provide the application of the above-mentioned near-infrared fluorescent probe that can dynamically monitor the redox homeostasis of hemangiomas in hemangioma cell imaging.

[0012] To achieve the above objectives, the technical solution of the present invention is as follows:

[0013] In the first aspect, the present invention provides a reversible near-infrared small molecule probe, and its structural formula is:

[0014]

[0015] Its structure includes: a donor-acceptor (D-A) structure boron dipyrromethene (BODIPY), catechol; among them, BODIPY has excellent photostability, high molar extinction coefficient, excellent biocompatibility, and properties that are easy to synthesize and modify; through the Knoevenagel condensation reaction, 3,4-dihydroxybenzaldehyde is introduced into the 3-position of the BODIPY skeleton through a vinyl bridge, and a reversibly responsive small molecule probe is designed and synthesized. Catechol is easily oxidized by O2 ·- and then reduced by GSH.

[0016] As can be seen in the examples of the present invention, the BDPos provided by the present invention has excellent reversibility and stability after multiple O2 ·- / GSH redox reactions; has high specificity for O2 ·- / GSH; and has stability under physiological pH conditions.

[0017] A near-infrared small molecule fluorescence for reversibly monitoring cellular redox homeostasis, whose structural formula is:

[0018]

[0019] The reaction equation for the preparation method of the above fluorescence probe is:

[0020]

[0021] It includes the following steps: Dissolve compound 1 in a solvent, add 3,4-dihydroxybenzaldehyde, and add piperidine and acetic acid as reaction catalysts, and react at a temperature of 60 - 100 °C for 10 - 15 h to generate the reversible near-infrared small molecule probe.

[0022] Preferably, the molar ratio of compound 1 to 3,4-dihydroxybenzaldehyde is 1:0.9 - 1.2.

[0023] In the examples of the present invention, the solvent is toluene, and other conventional solvents in the chemical field can also be used.

[0024] More preferably, the reaction temperature is 60 - 80 °C and the reaction time is 10 - 12 h.

[0025] More preferably, after the reaction, it further includes the steps of rotary evaporation under reduced pressure and purification of the reaction product solution.

[0026] More preferably, silica gel column chromatography is used to purify the reaction product.

[0027] Even more preferably, the eluent for the silica gel column chromatography is a mixed solution of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol used for purifying the reaction product is 120 - 90:1 - 2; preferably 100:1.5.

[0028] In the examples of the present invention, the application of the reversible near-infrared small molecule probe system in the field of biological detection is provided, preferably in the monitoring of redox homeostasis of hemangiomas.

[0029] In the examples of the present invention, the reversible near-infrared small molecule probe system exhibits obvious absorption shift, reversible reactivity, and the ability to visualize the redox state between 708 nm and 620 nm.

[0030] In the third aspect, the present invention provides an application of a reversible near-infrared small molecule probe in vitro for monitoring O2 ·- / GSH. It includes the following steps: Add the probe to the sample to be tested, and then add the prepared O2 ·-The stock solution is directly subjected to fluorescence intensity measurement after incubation for a set time; then GSH is added, and after incubation for a set time, fluorescence spectrum measurement is performed again. The probe can cycle and monitor the above operations at least three times.

[0031] Preferably, the incubation time of O2 ·- and GSH is 5 min.

[0032] Preferably, the incubation temperature of O2 ·- and GSH is 37 °C.

[0033] In a fourth aspect, the present invention provides an application of a reversible near-infrared small molecule probe in monitoring the redox homeostasis of hemangioma, which is an application in monitoring the redox homeostasis at the hemangioma cell level. The method includes the following steps: The cultured HemEC cells are stimulated with 2-Me and then the probe is added for incubation. After incubation for a set time, fluorescence confocal imaging is directly performed; then GSH is added, and after incubation for a set time, fluorescence confocal imaging is performed again; the cycle is monitored twice.

[0034] Preferably, the stimulation time of 2-Me and GSH is 30 min.

[0035] The present invention also provides an application of a reversible near-infrared small molecule probe in hemangioma cell imaging.

[0036] In a fifth aspect, the present invention provides a method for monitoring the endogenous O2 ·- / GSH-mediated redox homeostasis in hemangioma. The method includes the following steps: The cultured HemEC cells are stimulated with phorbol myristate acetate (PMA) and then the probe is added for incubation. After incubation for a set time, fluorescence confocal imaging is directly performed; then α-lipoic acid (LPA) is added, and after incubation for a set time, fluorescence confocal imaging is performed again; the cycle is monitored twice.

[0037] Preferably, the stimulation time of PMA and LPA is 30 min.

[0038] In a sixth aspect, the present invention provides an application of the reversible near-infrared small molecule probe in monitoring the intracellular redox homeostasis during the treatment of hemangioma with therapeutic drugs. The method includes the following steps: HemEC cells are stimulated with a hemangioma therapeutic drug, and then the reversible near-infrared small molecule probe is added. Fluorescence confocal imaging is performed at different incubation time points to detect the fluorescence intensity. If the fluorescence intensity shows a gradually decreasing trend with the increase of the incubation time, it is determined that the hemangioma therapeutic drug destroys the redox homeostasis of HemEC, resulting in O2 ·-Gradual accumulation. If the introduction of GSH after incubation leads to an increase in the NIR fluorescence signal in the treated cells, it is determined that the hemangioma treatment drug exerts its therapeutic effect by disrupting the redox balance within tumor cells; otherwise, the hemangioma treatment drug has no significant effect on the redox state of hemangioma.

[0039] In the embodiments of the present invention, there is provided an application of a reversible near-infrared small molecule probe in monitoring the changes in intracellular redox homeostasis during the treatment of hemangioma with propranolol and timolol maleate. The steps are as follows: A group of cultured HemEC cells are stimulated with propranolol and then incubated with the probe. After the set incubation time, fluorescence confocal imaging is performed at different time points; another group of HemEC cells are incubated for the set time, and fluorescence confocal imaging is performed at different time points to monitor the changes in the fluorescence signals of the two groups of tumor cells.

[0040] It can be seen that the present invention provides a research tool for the therapeutic mechanism of a hemangioma treatment drug based on a reversible near-infrared small molecule probe. By monitoring the changes in the intracellular redox state during the treatment of hemangioma, it is possible to explore whether the drug treats hemangioma by disrupting the mechanism of hemangioma redox homeostasis.

[0041] The reversible near-infrared small molecule probe provided by the present invention can image the redox state in hemangioma by monitoring the dynamic changes of superoxide anion (O2 ·- ) and glutathione (GSH). During the selective oxidation with O2 ·- and the reduction process of GSH, a significant change in the absorption of the probe occurs between 708 and 620 nm, thereby showing a reversible responsiveness and successfully demonstrating the redox state. Importantly, it is found that the disruption of hemangioma redox homeostasis may be one of the mechanisms of propranolol in treating hemangioma.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] (1) The reversible near-infrared small molecule probe provided by the present invention is the near-infrared fluorescence probe BDPos for monitoring redox homeostasis. This probe selects the boron dipyrromethene (BODIPY) with an excellent donor-acceptor (D-A) structure that has excellent photostability, good biocompatibility, and is easy to synthesize and modify as the chromophore. 3,4-Dihydroxybenzaldehyde is introduced into the 3-position of the BODIPY skeleton through a vinyl bridge, and it has a reversible responsiveness and high sensitivity and specificity to the redox cycle of O2 ·- / GSH.

[0044] (2) This probe can indicate the changes in O2 ·-The increase in concentration enables the observation of O2 during the treatment with anti-tumor drugs. ·- / GSH redox state changes, providing a potentially promising tool for exploring the treatment mechanisms of anti-tumor drugs and various redox-related pathological processes.

[0045] (3) The synthesis steps of the near-infrared fluorescent probe of the present invention are simple, with high yield, easy purification, strong practicability, easy to promote, and little environmental pollution. Description of the Drawings

[0046] The specification drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention.

[0047] Figure 1 Absorption spectra of the near-infrared fluorescent probe in Example 2, after being oxidized by O2 ·- and after adding GSH.

[0048] Figure 2 Fluorescence response spectra of the near-infrared fluorescent probe in Example 2 to different concentrations of O2 ·- and GSH; the abscissa is the wavelength (nm), and the ordinate is the fluorescence emission intensity; among them, (a) is the fluorescence response spectrum to different concentrations of O2 ·- and (b) is the fluorescence response spectrum to different concentrations of GSH.

[0049] Figure 3 Linear relationships of the near-infrared fluorescent probe in Example 2 with different concentrations of O2 ·- and GSH; the abscissa is the concentration (μM), and the ordinate is the fluorescence emission intensity of the probe at the corresponding wavelength; among them, (a) is the linear relationship diagram for different concentrations of O2 ·- and (b) is the linear relationship diagram for different concentrations of GSH.

[0050] Figure 4 Fluorescence emission intensity of the near-infrared fluorescent probe in Example 2 at different pH values.

[0051] Figure 5 In Example 2, in the presence of different substances, the fluorescence intensity of the near-infrared fluorescent probe of Example 1. The abscissa is different substances, and the ordinate is the fluorescence emission intensity of the probe.

[0052] Figure 6 Fluorescence imaging diagrams of the near-infrared fluorescent probe in hemangioma cells in Example 3; among them, (a) is the experimental flow chart of the fluorescence imaging of the probe on hemangioma cells in Example 3; (b) is the monitoring of O2 in hemangioma cells simulated by 2-ME using BDPos·- Fluctuation; (c) is the change graph of the relative fluorescence intensity at different times in the 2-ME simulation test; (d) is to evaluate the ability of the probe to sense endogenous O2 ·- / GSH in cells using phorbol myristate acetate (PMA) and α-lipoic acid (LPA); (e) is the change graph of the relative fluorescence intensity in the PMA and LPA tests; the abscissa in Figures (c) and (e) is time (min), and the ordinate is the relative fluorescence intensity.

[0053] Figure 7 Schematic diagram for monitoring O2 ·- and GSH provided by Example 1. Detailed implementation mode

[0054] It should be noted that the following detailed description is exemplary and is intended to provide further illustration of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0055] It should be noted that the terms used herein are only for describing the specific implementation mode and are not intended to limit the exemplary implementation mode according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or their combinations.

[0056] As introduced in the background technology, currently, the etiology and specific pathogenesis of hemangioma are not very clear and the monitoring of the redox homeostasis of O2 ·- and GSH is very important. For this reason, the present invention proposes a method for dynamically monitoring the redox homeostasis of hemangioma using a near-infrared fluorescent probe. The present invention will be further described below in conjunction with the drawings and specific implementation modes.

[0057] Example 1, a preparation method of a near-infrared small molecule probe for dynamically monitoring the redox homeostasis of hemangioma

[0058] The reaction equation is:

[0059]

[0060] The specific preparation steps are as follows:

[0061] The present invention utilizes the previously existing raw material intermediate compound 1, which is simply and efficiently synthesized by a one-step reaction, and finally the product probe BDPos is obtained through silica gel column chromatography separation. Compound 1 is obtained by reacting 2-methylpyrrole, 2-trifluoroacetyl-4-methylpyrrole, and POCl3 in dichloromethane at 0 °C for 4 hours. Then, triethylamine and BF3·OEt2 are added and reacted at room temperature for 2 hours.

[0062] Synthesis of the probe: Dissolve compound 1 in toluene (1 equivalent), add 3,4-dihydroxybenzaldehyde (1 equivalent), piperidine (9.7 mM / ml, 0.2 ml), and acetic acid (17.5 mM / ml, 0.2 ml). Stir the reaction mixture at 80 °C for 12 hours. After the reaction is completed, add the obtained mixed solution to saturated sodium bicarbonate solution and extract it 3 times with DCM. Collect the organic phase solution, concentrate and rotary evaporate the solvent, and further purify the obtained solid. The reaction product is purified by silica gel column chromatography. The eluent for the silica gel column chromatography is a mixed solution of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol used for purifying the reaction product is 120 - 90:1 - 2; preferably 100:1.5. After detection, the yield of compound 1 obtained in this example is 31.7%.

[0063] Mass spectrometry and NMR characterization:

[0064] Compound 1. 1 H NMR (400 MHz, DMSO-d6) δ 7.40 (s, 2H), 6.66 (d, J = 7.8 Hz, 2H), 2.59 (s, 6H) ppm. 13 C NMR (100 MHz, DMSO-d6) δ 162.58, 130.99, 130.75, 124.26, 123.14, 121.82, 15.43 ppm. 19 F-NMR (376 MHz, DMSO-d6): δ = -55.10 (t, 3F), -143.31 (q, 2F) ppm. HRMS (ESI - ): calculated for C 12 H 10 BF5N2: [M - H] - = 287.0775, found: m / z [M - H] - = 287.0717.

[0065] Probe BDPos. 11H NMR (400 MHz, DMSO-d6) δ 9.83 (s, 1H), 9.42 (s, 1H), 7.72 (d, J = 8.7 Hz, 1H), 7.54 (d, J = 8.2 Hz, 2H), 7.35 (m, 3H), 6.82 (d, J = 8.4 Hz, 2H), 6.39 (s, 1H), 3.04 (s, 3H) ppm. 13 13C NMR (100 MHz, DMSO-d6) δ 161.98, 157.28, 152.53, 142.18, 135.34, 129.33, 124.51, 124.08, 122.73, 118.57, 116.66, 114.40, 113.45, 112.86, 105.13, 103.64, 101.74, 15.03 ppm. HRMS (ESI - ): calculated for C 19 H 14 BF5N2O2: [M-H] - = 407.0988, found: m / z [M-H] - = 407.0941.

[0066] Effect experiment:

[0067] At physiological pH values, the catechol unit is easily oxidized by O2 ·- to benzoquinone, thus reconstructing the π-conjugated structure of the whole molecule. This structural change leads to the loss of fluorescence properties but causes a red shift of the main absorption spectral band. Subsequently, the oxidized form of BDPosO is easily reduced by GSH, restoring the NIR fluorescence signal. The mechanism of the O2 ·- / GSH-mediated redox homeostasis change process is as Figure 7 shown.

[0068] Example 2,

[0069] 1. Fluorescence response experiment of the probe to O2 ·- and GSH:

[0070] Detect O2 ·- / GSH in the physiological environment simulated by PBS buffer solution: Use PBS buffer solution as the sample to be measured. After the probe is added to PBS (pH 7.4) buffer and diluted with water, different concentrations of O2 ·- are added to make the final concentration of the probe 5.0 μM and the PBS concentration 50 mM. The results are as Figure 1 shown. Free BDPos exhibits a strong absorption peak at 620 nm. It is oxidized by O2 at pH 7.4 ·-After oxidation, the absorption peak redshifts to 708 nm. Subsequently, after adding GSH to the mixture of BDPos and O2 ·- , the absorption peak returns to 620 nm. As shown in (a) and (b) of Figure 2 , after incubation with different concentrations of O2 ·- (0 - 45 μM), the fluorescence decreases significantly; and then after incubation with different concentrations of GSH (0 - 67.5 μM), the fluorescence increases significantly. As shown in (a) and (b) of Figure 3 , the detection limits of BDPos for O2 ·- and GSH (calculated according to the formula 3σ / k) are 68 nM and 92 nM respectively, and their linear formulas and correlation coefficients are: F = 790.8356 - 16.7651[O2 ·- , R 2 = 0.9937; F = 16.21685 + 10.9306[GSH], R 2 = 0.9924. It shows that the fluorescent probe has the ability to detect endogenous O2 ·- and GSH in biological systems.

[0071] Similarly, the sample to be detected can also be cells.

[0072] 2. The specificity of the fluorescence response of BDPos to O2 ·- was evaluated

[0073] The fluorescence change of BDPos with O2 ·- was compared with a series of biologically relevant substances. As shown in Figure 5 , label 1 represents 100 μM ONOO - , 2 represents 100 μM 1 O2, 3 represents 100 μM H2O2, 4 represents 100 μM ClO - , 5 represents 100 μM · OH, 6 represents 100 μM NO, 7 represents 5 mM GSH, 8 represents 5 mM Cys, 9 represents 5 mM Vc, 10 represents 100 μM Hcy, 11 represents 100 μMH2S, 12 represents 100 μM Ca 2+ , 13 represents 100 μM Fe 2+ , 14 represents 100 μM Fe 3+ , 15 represents 100 μM Zn 2+ , 16 represents 100 μM Na + , 17 represents 100 μM Cu 2+ , 18 represents 100 μM K + , 19 represents 100 μM Mg 2+, 20 represents 100 μM Co 2+ , 21 represents 100 μM HSO3 - , 22 represents 100 μM HCO3 - , 23 represents 100 μM NO2 - , 24 represents 100 μM SO4 2- , 25 represents 100 μM CO3 2- , 26 represents 100 μM H2PO4 - , 27 represents 100 μM NO3 - , 28 represents the blank sample, 29 represents 50 μM O2 ·- , 30 represents adding 50 μM O2 first ·- , and then adding 100 μM GSH. The results show that only the addition of O2 ·- will cause a significant decrease in fluorescence at 654 nm, while in the presence of other ROS (ONOO - , H2O2, 1 O2, ClO - , ·OH and NO), reducing substances (GSH, Cys, Vc, Hcy and H2S), metal ions (Ca 2+ , Fe 2+ , Zn 2+ , Fe 3+ , Na + , Cu 2+ , K + , Mg 2+ and Co 2+ ) and acid radical ions (HSO3 - , HCO3 - , NO2 - , SO4 2- , CO3 2- , H2PO4 - and NO3 - ), negligible fluorescence changes are detected. In summary, the probe BDPos has high selectivity for O2 ·- .

[0074] 3. The effect of pH on the fluorescence response was studied in the pH range of 4.0 - 11.0

[0075] It was Figure 4 observed that BDPos maintained a weak and stable fluorescence emission throughout the tested pH range, showed an almost constant fluorescence decrease after treatment with O2 ·- , and the addition of GSH to BDPos and O2 ·-After the mixed solution, the recovery and stability of the fluorescence intensity were observed, indicating the stability of the probe under physiological pH conditions. In addition, the redox cycle of superoxide anion and glutathione was monitored by the chemical system probe, and the results were good.

[0076] In summary, these results indicate that BDPos has high specificity for O2 ·- / GSH and can be used for fluorescence visualization research and redox monitoring of O2 ·- in physiological environment.

[0077] Example 3, Fluorescent imaging experiment of the probe on hemangioma cells

[0078] The viability of hemangioma cells was evaluated by standard MTT

[0079] After treatment with 50 μM BDPos for 24 h, the viability of hemangioma cells exceeded 83%, indicating that the probe has good biocompatibility and low cytotoxicity.

[0080] 2-Methoxyestradiol (2-ME) is a known experimental anti-cancer agent that generates O2 ·- . As shown in (a) - (e) of Figure 6 , BDPos was used to monitor the O2 ·- fluctuations in hemangioma cells simulated by 2-ME. Compared with the control group without 2-ME treatment, the BDPos-pretreated hemangioma cells showed fluorescence quenching when stimulated with 1.0 μg / mL 2-ME, indicating an increase in intracellular O2 ·- concentration. After adding 1 mM GSH to these cells, the fluorescence immediately increased, indicating that GSH reduced BDPosO and converted it into BDPos. Under the same conditions, another reversible fluorescence cycle for imaging was observed. Meanwhile, the ability of our probe to sense intracellular endogenous O2 ·- / GSH was evaluated. The hemangioma cells were treated with phorbol 12-myristate 13-acetate (PMA) and α-lipoic acid (LPA), which are stimulants for intracellular O2 ·- and GSH respectively, and significant reversible fluorescence response changes occurred in the fluorescence signal of the probe. This indicates that the probe successfully monitored the dynamic cellular redox changes controlled by PMA and LPA. The above results indicate that the changes in the NIR fluorescence signal can be attributed to the dynamic changes in intracellular and GSH levels, thus inferring the feasibility of the reversible probe BDPos in visualizing the redox state of the living system.

[0081] Example 4, Effects of propranolol and timolol maleate on the redox homeostasis of hemangioma cells

[0082] To gain an in-depth understanding of the effects of hemangioma drugs on intracellular redox homeostasis in hemangioma cells, BDPos was used to monitor intracellular redox homeostasis during the treatment of HemEC with propranolol or timolol maleate. HemEC cells were stimulated with propranolol and timolol maleate respectively, and then the probe was added. Fluorescence confocal imaging was performed at different incubation time points. As the incubation time increased, the fluorescence intensity of HemEC cells stimulated by propranolol showed a gradually decreasing trend, while the change in the fluorescence signal of HemEC treated with timolol maleate was negligible. These observations indicate that propranolol disrupts the redox homeostasis of HemEC, leading to the gradual accumulation of O2 ·- . It is worth noting that the introduction of GSH after 12 hours of propranolol incubation resulted in an increase in the NIR fluorescence signal in the treated cells. This finding suggests that propranolol may exert its therapeutic effect by disturbing the redox balance within tumor cells, while timolol maleate seems to have no significant effect on the redox state of hemangiomas.

[0083] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A reversible near-infrared small molecule probe, characterized in that, Its structural formula is:

2. The preparation method of a reversible near-infrared small molecule probe according to claim 1, characterized in that The reaction equation is: It includes the following steps: Dissolve compound 1 in a solvent, add 3,4-dihydroxybenzaldehyde, and add piperidine and acetic acid as reaction catalysts, and react at a temperature of 60-100 °C for 10-15 h to generate the reversible near-infrared small molecule probe.

3. Use of a reversible near-infrared small molecule probe according to claim 1 for in vitro monitoring of O2 ·- / GSH.

4. The application of a reversible near-infrared small molecule probe according to claim 1 in hemangioma cell imaging.

5. The application of a reversible near-infrared small molecule probe according to claim 1 in monitoring the redox homeostasis of hemangioma.

6. The application of the reversible near-infrared small molecule probe according to claim 1 in monitoring the intracellular redox homeostasis during the treatment of hemangioma with therapeutic drugs.

7. The application according to claim 6, wherein Stimulate HemEC cells with a hemangioma therapeutic drug, and then add the reversible near-infrared small molecule probe according to claim 1. At different incubation time points, perform fluorescence confocal imaging and detect the fluorescence intensity.

8. The application according to claim 7, wherein If the fluorescence intensity shows a gradually decreasing trend with the increase of the incubation time, it is determined that the hemangioma treatment drug has disrupted the redox homeostasis of HemEC, resulting in the gradual accumulation of O2 ·- ; If the introduction of GSH after incubation results in an increase in the NIR fluorescence signal in the treated cells, it is determined that the hemangioma therapeutic drug exerts its therapeutic effect by disrupting the intracellular redox balance of tumor cells; otherwise, the hemangioma therapeutic drug has no significant effect on the redox state of hemangioma.

9. A method for monitoring the endogenous O2 ·- / GSH-mediated redox homeostasis in hemangiomas, characterized in that, Based on a reversible near-infrared small molecule probe with the following structural formula:

10. The method for monitoring the endogenous O2 ·- / GSH-mediated redox homeostasis in hemangiomas, characterized in that, It includes the following steps: Stimulate the cultured HemEC cells with phorbol 12-myristate 13-acetate and then add the reversible near-infrared small molecule probe for incubation. After incubating for the set time, directly perform fluorescence confocal imaging; then add α-lipoic acid, and after incubating for the set time, perform fluorescence confocal imaging again; monitor twice in a cycle.

Images