Glutathione probe as well as preparation method and application thereof

Abstract

The invention relates to a glutathione probe, a preparation method and application thereof, and a bifunctional glutathione probe, which consumes bimolecular glutathione through a sequential activation strategy, so that high fluorescence enhancement is realized, and the accuracy of glutathione response imaging is realized. The Cy-ResNPs shows selective accumulation in mitochondria, the level of reactive oxygen species (ROS) is effectively improved by consuming glutathione, including induction of superoxide anions, and efficient photodynamic therapy is achieved. Intracellular oxidative stress storms induced by a variety of pathways promote iron apoptosis, cell apoptosis, and lethal toxicity in cancer cells. Finally, the Cy-ResNPs realizes accurate diagnosis and effective treatment in transplantable and rapid proliferative melanoma tumors. The research has important significance for tumor therapeutic research by consuming the intelligent small molecular fluorescent agent of glutathione in the future.

Description

Technical Field

[0001] The present invention relates to the field of fluorescent dyes, and in particular to a novel cyanine glutathione-responsive diagnostic and therapeutic fluorescent probe, as well as a preparation method and application thereof. Background Art

[0002] In the tumor microenvironment (TME), tumor cells produce high levels of reactive oxygen species (ROS) to support their rapid proliferation, but excessive levels of reactive oxygen species can induce cell death; therefore, tumor cells upregulate the antioxidant system to prevent apoptosis. Glutathione is the main antioxidant that can effectively scavenge free radicals and react with toxic ROS in mitochondria. The concentration of glutathione in tumor cells can reach 1-10mM, which is about 100-1000 times that of the extracellular environment. Compared with healthy tissue, the glutathione concentration in tumor tissue is about four times higher. Therefore, these relatively high glutathione levels can be used to develop redox-responsive therapeutic agents that can specifically release active drugs or fluorophores in tumor cells. In addition, the high nucleophilicity of the thiol group in glutathione is also the basis for designing glutathione-specific fluorescent probes and glutathione-triggered therapeutics. A variety of probes that utilize unique recognition mechanisms (such as Michael addition and aromatic nucleophilic substitution) have been developed. These probes can not only improve diagnostic efficacy but also enhance the precision of treatment, highlighting the important role of glutathione-responsive small molecule fluorescent probes in combining imaging and treatment.

[0003] Although many glutathione-responsive fluorescent probes have been developed, there are few reports on small-molecule near-infrared (NIR) fluorescent probes that can combine diagnosis with therapy, especially with low-laser-intensity photodynamic therapy. Summary of the invention

[0004] The purpose of the present invention is to design a novel near-infrared cyano fluorescent probe (Cy-Res), which has high selectivity for glutathione through a dual-site sequential reaction strategy, and realizes selective imaging of tumor cells. It also effectively consumes glutathione, thereby inducing the massive generation of multiple reactive oxygen species (ROS), aggravating oxidative stress, and combining the potent photodynamic therapy (PDT) effect of cyano dyes, ultimately achieving the purpose of glutathione-responsive tumor diagnosis and treatment.

[0005] The above purpose is achieved through the following technical solutions:

[0006]

[0007] In formula I, R1 and R2 are selected from hydrogen, C1-C16 chain alkyl, polyethylene glycol group, C1-C4 alkyl carboxylic acid and its derivatives, C1-C5 alkyl sulfonic acid and its salt, C1-C4 alcohol; R3 and R4 are selected from hydrogen and halogen.

[0008] In this technical solution, a glutathione probe is synthesized by replacing Cl in IR780 with resorufin. The probe uses the photoinduced electron transfer (PeT) effect to cause fluorescence quenching, so that it has very weak fluorescence before reacting with glutathione.

[0009] In this technical solution, when the probe targets the mitochondria in the cell, the resorufin molecule and glutathione undergo Michael addition rapidly, followed by a slow cleavage of the linker between Cyanine 7 (Cy7) and resorufin, ultimately releasing compounds 3 and 4 with strong fluorescence emission, achieving a significant enhancement of fluorescence, and presenting a concentration and time gradient. Due to the twofold consumption of glutathione in the process, the intracellular ROS level increases, leading to cellular oxidative stress, achieving an anti-tumor effect. Further photodynamic therapy generates more ROS, exacerbating the oxidative stress, and ultimately achieving an excellent anti-tumor effect.

[0010] Taking the glutathione probe Cy-Res in Example 1 as an example, after reacting with glutathione, compounds 3 and 4 are obtained, and the reaction pathway is:

[0011]

[0012] In this technical solution, the glutathione probe not only labels glutathione in cells, but also produces a continuous fluorescence response to glutathione, and the fluorescence intensity is positively correlated with the concentration of glutathione, thus realizing glutathione monitoring in vivo and in vitro. It can also label mitochondria in cells, consume intracellular glutathione to increase the level of intracellular reactive oxygen species (ROS), and cause oxidative stress to produce tumor-suppressing effects in vivo and in vitro. After interacting with glutathione, it can cooperate with photodynamic therapy to achieve an anti-tumor effect.

[0013] In some preferred embodiments, the glutathione probe is selected from the following compounds:

[0014]

[0015] The present invention also provides a method for preparing a glutathione probe, which is used to prepare any of the above-mentioned glutathione probes, and the preparation method comprises the following steps:

[0016] The compound represented by formula II is mixed with the compound represented by formula III to react, thereby obtaining a glutathione probe represented by formula I;

[0017] Formula II: Formula III:

[0018] In formula II, R1 and R2 are selected from hydrogen, C1 to C 16Chain alkyl, polyethylene glycol group, C1-C4 alkyl carboxylic acid and its derivatives, C1-C5 alkyl sulfonic acid and its salts, C1-C4 alcohol; R3 and R4 are selected from hydrogen and halogen.

[0019] In the technical scheme, the compound shown in formula II is a cyanine fluorophore, and the compound shown in formula III is resorufin. Both compounds can be synthesized in an existing manner or purchased commercially.

[0020] In an embodiment, after being dissolved in a first solvent, a condensing agent is added, and the reaction system is reacted at 40 to 70° C. to obtain a glutathione probe. The first solvent may be DMF. The condensing agent may be DIPEA. The reaction mixture is stirred at 40 to 70° C. for a certain period of time to obtain an intermediate, preferably, the reaction temperature is 60° C. and the reaction time is 2 hours.

[0021] The present invention also provides an application of a glutathione probe, which not only labels glutathione in cells, but also produces a continuous fluorescence response to glutathione, and the fluorescence intensity is positively correlated with the concentration of glutathione, thereby realizing in vivo and in vitro glutathione monitoring. The glutathione probe can also label mitochondria in cells, consume intracellular glutathione to increase the level of intracellular reactive oxygen species (ROS), and cause oxidative stress to produce tumor-inhibiting effects in vivo and in vitro. After acting with glutathione, it can cooperate with photodynamic therapy to achieve an anti-tumor effect.

[0022] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0023] 1. The glutathione probe provided by the present invention causes fluorescence quenching through the action of PeT, consumes two molecules of glutathione to generate a compound with a high fluorescence quantum yield, and realizes the monitoring of glutathione in the tumor microenvironment;

[0024] 2. The glutathione probe provided by the present invention can increase ROS by consuming intracellular glutathione, causing intracellular oxidative stress and achieving anti-tumor effects. It can also achieve a strong photodynamic effect under the action of low-power near-infrared laser, enhancing the compliance of clinical use;

[0025] 3. The glutathione probe provided by the present invention can generate superoxide free radicals by consuming glutathione, which can make up for the defect of oxygen requirement in most photodynamic therapy mechanisms and explore the treatment of hypoxic tumors;

[0026] 4. The preparation process of the present invention is simple, the reaction conditions are mild, the synthesis path is short, and it is conducive to industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The drawings described herein are used to provide a further understanding of the embodiments of the present invention, constitute a part of this application, and do not constitute a limitation of the embodiments of the present invention. In the drawings:

[0028] Figure 1 The absorption spectra of Cy-Res after reacting with different concentrations of glutathione in a specific embodiment of the present invention (A) are shown. The absorption spectra of Cy-Res after reacting with glutathione (5 mM) for different time periods (B) are shown.

[0029] Figure 2 The fluorescence intensity changes after the Cy-Res reacted with different concentrations of glutathione for different time periods in a specific embodiment of the present invention are shown (C), reaction time 90 min / ex = 750 nm (A), reaction time 90 min / ex = 591 nm (B).

[0030] Figure 3 The fluorescence intensity ex=750nm (A) and ex=591nm (B) of Cy-Res after reacting with different interferents (1mM) for 90min in a specific embodiment of the present invention are shown.

[0031] Figure 4 It shows the monitoring of intracellular glutathione level by flow cytometry after cells were treated with Cy-ResNPs in a specific embodiment of the present invention.

[0032] Figure 5 The figure shows the Cy-ResNPs imaging of B16F10 tumor mice by intratumoral injection in a specific embodiment of the present invention.

[0033] Figure 6 The fluorescence imaging of B16F10 cells co-stained with Cy-ResNPs and the commercial mitochondrial dye Mito-TrackerGreen in a specific embodiment of the present invention is shown.

[0034] Figure 7 The graph shows the absorption change of DPBF at a wavelength of 414 nm under irradiation with a wavelength of 808 nm before and after the reaction of Cy-Res with glutathione at different time points in a specific embodiment of the present invention.

[0035] Figure 8 The figures show the effect of (A) Cy-ResNPs (5 μM) on intracellular ROS level in B16F10 cells before and after laser irradiation (L). (B) The effect of Cy-ResNPs (3 μM) on intracellular O2- level before and after laser irradiation in a specific embodiment of the present invention.

[0036] Fig. 9 The cell viability of A549 (A) and B16F10 (B) cells treated with Cy-ResNPs for 48 hours in a specific embodiment of the present invention is shown. The phototoxicity and dark toxicity (CD) of Cy-ResNPs on A549 (C) and B16F10 (D) cells were evaluated by CCK-8 method.

[0037] Fig.10 It shows that in a specific embodiment of the present invention, flow cytometry analysis of B16F10 cells treated with Cy-ResNPs was performed using Annexin V-FITC / PI double staining method.

[0038] Fig.11 The Western blot analysis of the expression of GPX4, Bcl-2, Bax, full-length Caspase-3 and cleaved Caspase-3 in a specific embodiment of the present invention is shown.

[0039] Fig.12 The changes in tumor volume of mice bearing subcutaneous B16F10 tumors after 12 days of treatment with PBS, Cy-ResNPs, and Cy-ResNPs combined with PDT in a specific embodiment of the present invention are shown. DETAILED DESCRIPTION

[0040] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments and drawings. The exemplary embodiments of the present invention and their description are only used to explain the present invention and are not intended to limit the present invention.

[0041] All raw materials of the present invention are not particularly limited to their sources, and can be prepared by commercial purchase or conventional methods well known to those skilled in the art. All raw materials of the present invention are not particularly limited to their purity, and the present invention preferably adopts analytically pure or conventional purity requirements in the field of fluorescent dyes. All raw materials of the present invention, their brands and abbreviations all belong to conventional brands and abbreviations in this area, and each brand and abbreviation is clear and definite in the field of its related use, and those skilled in the art can purchase from commercial or prepare by conventional methods according to the brands, abbreviations and corresponding uses.

[0042] The present invention has no particular limitation on the expression of the substituents, and all expressions familiar to those skilled in the art are adopted. Based on common sense, those skilled in the art can correctly understand the meanings according to the expressions.

[0043] 1. Preparation of glutathione probe

[0044] [Example 1]

[0045]

[0046] Preparation of Cy-Res: Resorcinol (25.6 mg, 0.12 mmol) and IR-780 iodide (66.7 mg, 0.1 mmol) were added to 2 mL DMF, followed by N,N-diisopropylethylamine (25 μL, 0.15 mmol) and stirred at 60° C. for 2 hours. The product was precipitated with ether (30 mL) and purified by reverse phase silica gel column chromatography to obtain the final solid product.

[0047] 1 H NMR(400MHz, DMSO-d6)δ:7.94(d,J=8.8Hz,1H),7.76(d,J=14.1Hz,2H),7.57-7.48(m, 3H),7.44-7.31(m,6H),7.21(t,J=7.3Hz,2H),6.81(d,J=7.7Hz,1H),6.32(d,J=2.1Hz ,1H),6.26(d,J=14.2Hz,2H),4.15(t,J=7.3Hz,4H),2.77(t,J=6.1Hz,4H),2.57-2.45 (m,4H),2.05-1.92(m,2H),1.74(q,J=7.3Hz,4H),1.32(s,12H),0.94(t,J=7.3Hz,6H).

[0048] 13 C NMR(101MHz, CDCl3)δ:186.20,172.11,162.53,161.84,149.36,146.88,145.67,142.17,140.86,134.83,134.65,132.68,1 28.80,125.34,122.16,121.77,113.31,111.00,107.21,101.94,100.91,49.08,46.36,28.02,24.71,20.98,20.91,11.69.

[0049] 2. Photophysical properties of the probe and glutathione-responsive imaging

[0050] [Example 2]

[0051] In this example, the glutathione probe Cy-Res was subjected to UV absorption experiments before and after the reaction with glutathione. Cy-Res was incubated with different concentrations of glutathione (0, 0.2, 1, 5 mM) at 37°C in the dark, and the UV-visible absorption spectra were recorded at 0 min, 30 min and 90 min in a CH3CN-PB (10 mM) buffer solution (3:7 v / v, pH 7.4).

[0052] The results are shown in the figure ( Figure 1 ), Cy-Res showed a maximum absorption peak in the near-infrared region at 770 nm. The absorption spectrum showed that with the increase of glutathione concentration or the extension of reaction time, the maximum absorption peak was slightly red-shifted, indicating that glutathione was added to Cy7, and a new absorption peak appeared in the range of 575-620 nm. This shows that the addition of glutathione gradually destroyed the skeleton of Resorufin, and the coupling between Cy7 and Resorufin was also destroyed.

[0053] [Example 3]

[0054] In this example, an experiment was conducted on the fluorescence response change of the glutathione probe Cy-Res before and after the reaction with glutathione.

[0055] Cy-Res was incubated with different concentrations of glutathione (0, 0.2, 1, 5 mM) at 37°C in the dark, and the fluorescence spectra were sampled and recorded at 0 min, 30 min and 90 min in CH3CN-PB (10 mM) buffer solution (3:7 v / v, pH 7.4) at 591 and 750 nm, respectively.

[0056] As shown in the figure ( Figure 2 ), the results showed that the fluorescence intensity of Cy-Res increased significantly with the increase of glutathione concentration and reaction time.

[0057] [Example 4]

[0058] In this example, the selectivity of the glutathione probe Cy-Res to glutathione was verified.

[0059] In CH3CN-PB (10mM) buffer solution (3:7v / v, pH 7.4), the concentration of Cy-Res was kept at 10μM. Hcy (homocysteine), glutathione (GSH), Pro (proline), His (histidine), Lys (lysine), Gly (glycine), Trp (tryptophan), Arg (arginine), H2O2 (hydrogen peroxide), Ca2+ (calcium ion), Cu2+ (copper ion) were incubated at 37°C in the dark for 90min, and then fluorescence measurement (λex=750nm / 591nm) was performed.

[0060] As shown in the figure ( Figure 3 ), the results of the selectivity experiment confirmed that Cy-Res has good selectivity for glutathione and can selectively detect glutathione.

[0061] [Example 5]

[0062] In this example, in order to detect the selective imaging ability of Cy-Res on glutathione at the cellular level, B16F10 cells were treated with 5 μM Cy-ResNPs for 0.5 h and then illuminated with 808 nm laser (80 mW / cm 2 After irradiation for 5 minutes, the cells were cultured for another 24 hours, collected and washed with PBS, and CMFDA fluorescence was detected by flow cytometry to evaluate glutathione consumption.

[0063] As shown in the figure ( Figure 4 ), as the concentration of glutathione increased, the fluorescence of CMFDA gradually decreased, indicating that Cy-Res can consume glutathione in the body and present a concentration gradient.

[0064] [Example 6]

[0065] In vivo glutathione responsive imaging

[0066] In this example, in order to detect the selective imaging ability of Cy-Res on glutathione in vivo, a tumor-bearing mouse model was established by subcutaneously injecting 3×105 B16F10 cells (75 μL) into the right hind limb. When the tumor volume reached about 60 mm 3 The mice were randomly divided into four groups (n=4) and injected intratumorally as follows: 100 μL of NEM (1 mM) or 100 μL of PBS (100 μL) was injected intratumorally before the Cy-ResNPs (20 μM) 50 μL was injected intratumorally and imaged using IVIS 1 h later.

[0067] As shown in the figure ( Figure 5 ), the fluorescence intensity of Cy-ResNPs-treated tumors was significantly higher than that of NEM-pretreated tumors, confirming the glutathione-targeted imaging ability of Cy-ResNPs in vivo.

[0068] Conclusion: The above results show that Cy-Res can achieve a significant enhancement of fluorescence intensity by consuming glutathione, and the fluorescence intensity is positively correlated with the concentration of glutathione, thus realizing glutathione monitoring in vivo and in vitro.

[0069] 3. Experiments on the application of probes in glutathione-activated anti-tumor therapy

[0070] [Example 7]

[0071] In this example, in order to detect the colocalization of Cy-ResNPs with granules, B16F10 cells were stained with MitoTracker Green FM (λex=488nm, λem=510nm) and Hoechst33342 for 30 minutes. After replacing the medium with fresh 1640 medium (10% FBS), the cells were treated with Cy-ResNPs (1.2 μM) for 60 minutes and then imaged with CLSM.

[0072] As shown in the figure ( Figure 6 ), and co-staining with the mitochondrial dye MitoTracker Green (MTG) showed good colocalization with a Pearson correlation coefficient (PCC) of 0.74, indicating mitochondrial targeting. Notably, we observed obvious mitochondrial damage, such as shrinkage and fragmentation, suggesting that Cy-ResNPs may induce mitochondrial oxidative stress damage by depleting intracellular glutathione.

[0073] [Example 8]

[0074] In this example, to evaluate the ROS generation capacity in vitro, a commercial reactive oxygen species probe was used in this study. 1,3-diphenylisobenzofuran (DPBF) was used to indicate the generation of 1O2 in DMF. Its attenuation at a wavelength of 410 nm represented the generation of 1O2. After Cy-Res or compound 4 was fully mixed with DPBF in DMF, an 808 nm laser (80 mW / cm 2 ) was used to irradiate the mixture every 15 seconds for a total of 2 minutes, and then the changes in the UV-visible absorption spectrum of the solution were recorded.

[0075] As shown in the figure ( Figure 7 ), Cy-Res has a weak singlet oxygen production capacity, while compound 4 has a significantly improved singlet oxygen production efficiency. This highlights the potential of Cy-Res as an activatable photosensitizer that can enhance the therapeutic effect in the glutathione-rich tumor microenvironment.

[0076] [Example 9]

[0077] In this example, in order to detect the ability of intracellular ROS generation, B16F10 cells were first cultured with 5 μM Cy-ResNPs for 0.5 h and then irradiated with 808 nm laser (80 mW / cm 2 5min), and then cultured for 24 hours, the cells were collected and washed with PBS, and the ROS level was detected using a DCFH-DA detection kit and flow cytometry. Alternatively, B16F10 cells were cultured in a confocal dish and cultured with 3μM Cy-ResNPs for 3 hours, and then cultured with 5μM DCFH-DA for 30 minutes. 808nm laser irradiation (80mW / cm 2 After 30 minutes of incubation, the generation of ROS was monitored using confocal laser scanning microscopy (CLSM) (ex / em=488nm / 525nm). To evaluate the type of ROS generated, 3 μM Cy-ResNPs were incubated with 5 μM dihydroethidine (DHE) for 30 minutes and then illuminated with an 808 nm laser (80 mW / cm 25 min) and the superoxide anion level was detected by CLSM (ex=488 nm, em=570-630 nm).

[0078] As shown in the figure ( Figure 8 A), Cy-ResNPs deplete intracellular glutathione, which normally acts to neutralize ROS, resulting in a significant increase in DCF fluorescence intensity compared to the control group. After irradiation, a stronger fluorescence signal was observed, indicating that the product compound 4 of Cy-ResNPs significantly increased the generation of ROS through the photodynamic effect. As shown in the figure ( Figure 8 B), DHE staining showed that Cy-ResNPs induced the formation of superoxide anions. Cy-ResNPs treatment directly induced the production of superoxide anions.

[0079] [Example 10]

[0080] In this example, in order to evaluate the in vitro antitumor effect of Cy-ResNPs, A549 or B16F10 cells were seeded into 96-well plates and cultured for 24 hours. After culturing for 2 hours, the cells were illuminated with 808 nm laser (0 or 120 mW / cm 2 The cells were irradiated for 5 minutes and then cultured for 48 hours. After 48 hours of culture, cell viability was assessed using a standard CCK-8 cytotoxicity assay and the IC 50 value.

[0081] As shown in the figure ( Fig. 9 ), when glutathione was detected, the efficient ROS induction ability due to glutathione consumption indicated that Cy-ResNPs had potential anticancer activity. Therefore, we first evaluated the cytotoxicity of Cy-ResNPs using the CCK-8 assay. The results showed that after 48 hours of treatment of B16F10 cells, the half-maximal inhibitory concentration (HIC) of Cy-ResNPs was 0.6 μM and that of A549 cells was 1.6 μM. The half-inhibitory concentration value of IR780 of A549 cells was approximately 10 μM. These results indicate that Cy-ResNPs can enhance the oxidative stress of tumor cells by consuming intracellular glutathione, thereby exhibiting anticancer effects. After the Cy-ResNPs concentration for B16F10 and A549 cell lines was around 50%, the inhibition rate increased to around 70% and 80%, respectively.

[0082] [Example 11]:

[0083] In this example, in order to verify the mechanism of ferroptosis and apoptosis, Western blot analysis was performed. B16F10 cells were first cultured with different concentrations of Cy-ResNPs for 2 hours, and then illuminated with 808 nm laser (0 or 80 mW / cm 25min) and then cultured for another 12 hours. Cells were lysed in RIPA buffer containing 1mM PMSF. After centrifugation at 12,000g for 10 minutes, the protein lysate was quantified using the BCA assay. Proteins were separated by SDS-PAGE, transferred to a PVDF membrane, and blocked with 5% BSA. Primary antibody incubation was performed overnight, followed by secondary antibody incubation. Bands were observed using the Bio-Rad Chemi Doc XRS system. Cell apoptosis was assessed using Annexin V-FITC / PI double staining. Cy-ResNPs (1.2μM) were incubated with cells for 4 hours and then irradiated with 808nm laser (0 or 80mW / cm 2 The cells were then incubated with Annexin V-FITC (5 μL) and PI (10 μL) for 15 min and apoptosis was assessed by flow cytometry.

[0084] The results are shown in the figure ( Fig.10 ), after Cy-ResNPs treatment, the apoptosis rate reached 20.7%, which was about 11 times higher than that of the negative control group (1.9%). After 808 nm laser irradiation, the apoptosis rate further increased to 32.9%, which was 17 times higher than that of the control group. Western blot analysis showed that ( Fig.11 ), Cy-ResNPs treatment significantly downregulated the expression of Bcl-2 and FLCaspase-3, while upregulated the expression of pro-apoptotic proteins Bax and cleaved Caspase-3. PDT treatment amplified these effects, indicating that Cy-ResNPs induced apoptosis by increasing ROS levels through mitochondrial targeting, and ROS generated by PDT further enhanced this effect.

[0085] [Example 7]

[0086] In this example, to evaluate the anti-tumor effect in vivo, 3×10 5 B16F10 cells (75 μL) were used to establish a tumor mouse model. When the tumor volume reached about 60 cubic millimeters, the mice were randomly divided into four groups (n=4) and injected intratumorally as follows: 1. PBS (50 μL); 2. PBS (50 μL) + laser (L); 3. Cy-ResNPs (50 μM / 50 μL); 4. Cy-ResNPs (50 μM / 50 μL) + laser (L). Mice in groups 2 and 4 were irradiated with 808 nm laser (80 mW / cm2 for 5 minutes) 2 hours after injection. The tumor volume was measured every two days, and the second treatment was performed on the 6th day. The experiment was terminated on the 12th day because the tumor volume exceeded 1500 cubic millimeters.

[0087] The results are shown in the figure ( Fig.12), the anti-tumor efficacy evaluation showed that Cy-ResNPs significantly increased oxidative stress in tumors by consuming glutathione, effectively inhibiting tumor growth. When used in combination with photodynamic therapy (PDT), the inhibitory effect of Cy-ResNPs on tumor cells was even more obvious.

[0088] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A glutathione probe, characterized in that It has the structural formula shown in Formula I: In Formula I, R1 and R2 are independently selected from hydrogen, C1 to C 16 Chain alkyl, polyethylene glycol group, C1-C4 alkyl carboxylic acid and its derivatives, C1-C5 alkyl sulfonic acid and its salts, C1-C4 alcohol; R3 and R4 are independently selected from hydrogen and halogen.

2. A glutathione probe according to claim 1, characterized in that R1 and R2 are independently selected from hydrogen, C1-C 16 Chain alkyl, polyethylene glycol group, C1~C 16 Alkyl carboxylic acids, C1-C5 alkyl sulfonic acids and salts thereof, C1-C4 alcohols; R3 and R4 are independently selected from hydrogen and halogen.

3. A glutathione probe according to claim 1 or 2, characterized in that: The glutathione probe is selected from the following compounds: 。 4. A method for preparing a glutathione probe, characterized in that: For preparing the glutathione probe according to any one of claims 1 to 3, the preparation method comprises the following steps: The compound represented by formula II is mixed with the compound represented by formula III to react, thereby obtaining a glutathione probe represented by formula I; Formula II: Formula III: In Formula II, R1 and R2 are independently selected from hydrogen, C1 to C 16 Chain alkyl, polyethylene glycol group, C1-C4 alkyl carboxylic acid and its derivatives, C1-C5 alkyl sulfonic acid and its salts, C1-C4 alcohol; R3 and R4 are independently selected from hydrogen and halogen.

5. The method for preparing a glutathione probe according to claim 4, characterized in that: After the compound represented by formula II and the compound represented by formula III are dissolved in a first solvent, the reaction system is reacted at 25-70° C. under the action of a base to obtain a glutathione probe.

6. An application of a glutathione probe, using a glutathione probe according to claim 1 or 2, characterized in that: The glutathione probe is used to label glutathione in cells, produces a continuous fluorescence response to glutathione, and the fluorescence intensity is positively correlated with the concentration of glutathione, thereby realizing in vivo and in vitro glutathione monitoring.

7. The use according to claim 6, characterized in that: The glutathione probe can target mitochondria in cells, consume intracellular glutathione to increase the level of intracellular reactive oxygen species (ROS), and achieve anti-tumor effects through synergistic photodynamic therapy with glutathione.

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