Molecular probe as well as preparation method and application thereof

Abstract

The invention discloses a molecular probe as well as a preparation method and application thereof, and relates to the technical field of fluorescent probes. The structural formula of the molecular probe is # imgabs0 #, two identical cyanine units which are covalently connected are used as parent bodies, reduced glutathione response groups are introduced into the two cyanine units at the same time, and the molecular probe, namely the double-cyanine molecular probe, capable of clearing reduced glutathione and achieving NIR-II fluorescence signal opening at the same time is constructed. The molecular probe can induce cell ferroptosis by consuming reduced glutathione in tumor cells. In addition, the molecular probe has very high photothermal conversion efficiency, and can be used for photothermal therapy of tumor cells. Therefore, the molecular probe provided by the invention is an accompanying diagnostic reagent integrating NIR-II fluorescence response, ferroptosis induction and photothermal therapy, and has an excellent anti-tumor effect and a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent probes, and in particular to a molecular probe and a preparation method and application thereof. Background Art

[0002] Tumor heterogeneity and individual differences in the tumor microenvironment limit cancer treatment. Companion diagnostics (CDx) is a diagnostic technology that is usually developed together with specific therapeutic drugs to determine whether a patient is suitable for a certain drug treatment. CDx helps doctors choose the most effective treatment plan by detecting patients' biomarkers, improving the safety and effectiveness of treatment and reducing unnecessary medical expenses. However, most CDx reagents approved by the U.S. Food and Drug Administration (FDA) are only applicable to in vitro detection of biomarkers, and it is difficult to dynamically monitor changes in targets in vivo.

[0003] Fluorescence imaging is a non-invasive bioimaging technology with fast response, high sensitivity and high spatiotemporal resolution. Compared with visible light or near-infrared light, light in the near-infrared II (NIR-II) region has deeper tissue penetration, lower tissue absorption and scattering, and the tissue autofluorescence in the NIR-II band is negligible, resulting in better imaging effects. Therefore, the development of CDx agents based on NIR-II fluorescence imaging has great application prospects. In addition, the separation of imaging agents and therapeutic agents will reduce the accuracy of treatment evaluation. Therefore, it is necessary to develop CDx agents that integrate in vivo imaging and treatment. Summary of the invention

[0004] Based on the above-mentioned deficiencies of the prior art, the purpose of the present invention is to provide a molecular probe and a preparation method and application thereof, aiming to provide an integrated CDx reagent with NIR-II fluorescence response, ferroptosis induction and photothermal therapy (PTT), and then monitor and regulate the cancer treatment process through companion diagnosis, providing a new strategy for cancer treatment related research.

[0005] The technical solution of the present invention is as follows:

[0006] In a first aspect of the present invention, a molecular probe is provided, wherein the structural formula of the molecular probe is:

[0007]

[0008] In a second aspect, the present invention provides a method for preparing the molecular probe as described above, comprising the following steps:

[0009] Under an inert atmosphere, 2-dicyanomethylidene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran, sodium acetate and a first solvent are mixed and reacted at a first preset temperature for a first preset time to obtain an intermediate compound;

[0010] Under an inert atmosphere, adding the intermediate compound and 3,5-bis(trifluoromethyl)benzenethiol to a second solvent, stirring at a second preset temperature for a second preset time, to obtain the molecular probe;

[0011] Among them, the structural formula of the intermediate compound is:

[0012]

[0013] Optionally, the first solvent comprises at least one of acetic anhydride and an alcohol solvent; and / or,

[0014] The first preset temperature is 60-100° C., and the first preset time is 12-24 hours.

[0015] Optionally, the second solvent is N,N-dimethylformamide; and / or,

[0016] The second preset temperature is 10-50° C., and the second preset time is 5-15 hours.

[0017] The third aspect of the present invention provides a use of the molecular probe as described above in the detection of reduced glutathione for non-disease diagnosis purposes.

[0018] The fourth aspect of the present invention provides a use of the molecular probe as described above in detecting the concentration of reduced glutathione for non-disease diagnosis purposes.

[0019] The fifth aspect of the present invention provides a use of the molecular probe as described above in the present invention in the preparation of a companion diagnostic reagent.

[0020] A sixth aspect of the present invention provides a method for detecting the concentration of reduced glutathione, comprising the following steps:

[0021] Prepare reduced glutathione solutions of different concentrations;

[0022] The molecular probes described above are added to reduced glutathione solutions of different concentrations, and fluorescence intensity detection is performed to obtain fluorescence intensity values ​​corresponding to reduced glutathione solutions of different concentrations, and the reduced glutathione concentrations and fluorescence intensity values ​​are plotted to obtain a standard curve;

[0023] Provide the test fluid;

[0024] Adding the molecular probe into the test solution, performing fluorescence intensity detection, and obtaining a first fluorescence intensity value;

[0025] The concentration of reduced glutathione in the test solution is obtained according to the first fluorescence intensity value and the standard curve.

[0026] The seventh aspect of the present invention provides a nanoparticle, which includes the molecular probe of the present invention as described above, and a liposome encapsulating the molecular probe.

[0027] Optionally, the liposomes include distearate phosphatidylethanolamine-polyethylene glycol 2000 and folic acid-functionalized distearate phosphatidylethanolamine-polyethylene glycol 2000.

[0028] Beneficial effects: The present invention uses two covalently linked identical cyanine units as the matrix, and simultaneously introduces reduced glutathione (GSH) response groups into the two cyanine units to construct a molecular probe that can clear GSH while turning on the NIR-II fluorescence signal, namely, a bicyanine molecular probe. The molecular probe can induce cell ferroptosis by consuming GSH in tumor cells. In addition, the molecular probe itself has a very high photothermal conversion efficiency and can perform photothermal treatment on tumor cells. Therefore, the molecular probe provided by the present invention is a CDx reagent that integrates NIR-II fluorescence response, ferroptosis induction and photothermal therapy, has excellent anti-tumor effects, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1a is the ESI-MS mass spectrum of compound 2 in Example 1, Figure 1b is the hydrogen nuclear magnetic resonance spectrum of compound 2 in Example 1, Figure 1c It is the carbon NMR spectrum of compound 2 in Example 1.

[0030] Figure 2a is the ESI-MS mass spectrum of LET-13 in Example 1, Figure 2b is the hydrogen nuclear magnetic resonance spectrum of LET-13 in Example 1, Figure 2c is the carbon-13 NMR spectrum of LET-13 in Example 1, Figure 2d This is the nuclear magnetic resonance fluorine spectrum of LET-13 in Example 1.

[0031] Figure 3 This is a diagram showing the feasibility verification result of using LET-13 for fluorescence imaging detection of GSH in Example 2, wherein a is a fluorescence spectrum of the mixed solution changing with time, and b is a diagram showing the fluorescence intensity of the mixed solution at 928 nm changing with time.

[0032] Figure 4This is a diagram showing the feasibility verification result of using LET-13 in Example 3 to detect GSH concentration using fluorescence imaging, wherein a is a fluorescence spectrum of a mixed solution containing different concentrations of GSH, and b is a diagram showing the fluorescence intensity of the mixed solution at 928 nm changing with the GSH concentration.

[0033] Figure 5 4 is a graph showing the verification result of the detection specificity of LET-13 for GSH in Example 4, wherein a is a fluorescence intensity comparison graph of LET-13 reacting with different amino acids in vitro, and b is a NIR-II fluorescence imaging graph of LET-13 reacting with different amino acids in vitro.

[0034] Figure 6 These are the test results of the morphology, particle size and absorption spectrum of LET-13 nanoparticles in Example 5, where a is a transmission electron microscope image, b is a particle size distribution graph, and c is an absorption spectrum graph.

[0035] Figure 7 These are the photothermal performance test results of the LET-13 nanoparticles in Example 5, wherein a is a graph showing how the temperature of solutions containing different concentrations of LET-13 nanoparticles changes with illumination time, b is a graph showing how the temperature of a solution containing 20 μM LET-13 nanoparticles changes with illumination time at different laser powers, c is a graph showing the photothermal cycle of a solution containing 20 μM LET-13 nanoparticles, and d is a graph showing how the temperature of a solution containing 20 μM LET-13 nanoparticles changes with time after laser irradiation and a linear fitting graph of the negative logarithmic function of the time and temperature changes.

[0036] Figure 8 Figure 3 is a graph showing the results of the tumor cell killing test, where a is the cell survival rate of 4T1 cells treated with LET-13 nanoparticles at different concentrations of LET-13 nanoparticles, and b is the cell survival rate of 4T1 cells treated with LET-13 nanoparticles with and without laser irradiation.

[0037] Fig. 9 Figures 2 and 3 are the results of GSH scavenging ability and lipid peroxidation test, where a is the cell survival rate of 4T1 cells treated with LET-13 nanoparticles with and without laser irradiation, b is the intracellular lipid peroxidation staining image of 4T1 cells treated with LET-13 nanoparticles with and without laser irradiation, and c is the quantification image of intracellular lipid peroxidation concentration of 4T1 cells treated with LET-13 nanoparticles with and without laser irradiation.

[0038] Fig.10 Figure 2 is a graph showing the results of a GSH visualization test, where a is the NIR-II fluorescence imaging of tumor-bearing mice over time after tail vein administration of LET-13 nanoparticles, and b is a quantification graph of the NIR-II fluorescence signal at the tumor site.

[0039] Fig.11 These are the results of tumor treatment tests, where a is the change in tumor volume after LET-13 nanoparticle treatment, b is the tumor weight result after 15 days of treatment, and c is the change in mouse weight after treatment.

[0040] Fig.12 H&E staining images of the main organs of mice in each group after tumor treatment test. DETAILED DESCRIPTION

[0041] The present invention provides a molecular probe and a method for preparing the same. To make the purpose, technical solution and effect of the present invention clearer and more specific, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0042] If there are descriptions involving "first", "second", etc. in the embodiments of the present invention, the descriptions of "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or implying their relative importance or implicitly indicating the number of the indicated technical features.

[0043] The molecular probe provided in the embodiment of the present invention is the first biscyanine CDx reagent that can integrate NIR-II fluorescence response, ferroptosis induction and photothermal therapy. Specifically, the molecular probe has the structural formula:

[0044]

[0045] The present invention uses two identical covalently linked cyanine units as the parent, and simultaneously introduces a GSH response group into the two cyanine units, constructing a molecular probe that can remove GSH while realizing the opening of NIR-II fluorescence signal, namely a bicyanine molecular probe. The molecular probe can realize the specific detection of GSH, and can induce cell ferroptosis by consuming GSH in tumor cells. As shown in the following reaction formula, the molecular probe (LET-13) provided by the present invention can specifically bind to GSH, and while consuming two molecules of GSH, the generated product LET-13G has a very strong fluorescence signal, so that the specific detection of GSH can be realized, and cell ferroptosis can be induced by consuming GSH in tumor cells.

[0046]

[0047] In addition, the molecular probe itself has a high photothermal conversion efficiency and can perform photothermal therapy on tumor cells. In summary, the molecular probe provided by the present invention is a CDx reagent that integrates NIR-II fluorescence response, ferroptosis induction and photothermal therapy, has excellent anti-tumor effects, and has broad application prospects.

[0048] In the embodiment of the present invention, the fluorescence signal of the solution containing the molecular probe and GSH is significantly enhanced with the increase of GSH concentration, and the emission wavelength is at 928nm (excitation wavelength is 808nm). In a specific embodiment, when the concentration of the molecular probe in the solution is 10μM, its fluorescence intensity has a good linear relationship with the concentration of GSH in the solution in the range of 0-20μM.

[0049] In the embodiment of the present invention, the molecular probe is specific for GSH and will not be interfered by other amino acids such as L-cysteine ​​(Cys), L-homocysteine ​​(Hcy), L-methionine (Met), L-phenylalanine (Phe), glycine (Gly), L-lysine (Lys), L-glutamine (Gin), L-glutamic acid (Glu), L-histidine (His), L-serine (Ser), L-alanine (Ala), L-arginine (Arg), L-dithiothreitol (DTT), etc.

[0050] The present invention also provides a method for preparing the molecular probe according to the present invention, comprising the following steps:

[0051] S11. Under an inert atmosphere, 2-dicyanomethylidene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran (TCF), sodium acetate and a first solvent are mixed and reacted at a first preset temperature for a first preset time to obtain an intermediate compound;

[0052] S12, under an inert atmosphere, adding the intermediate compound and 3,5-bis(trifluoromethyl)benzenethiol (BTBT) to a second solvent, stirring at a second preset temperature for a second preset time, to obtain the molecular probe;

[0053] Among them, the structural formula of the intermediate compound is:

[0054]

[0055] The preparation method provided by the present invention is simple and safe, has high yield, cheap raw materials, and is easy to realize industrial production. In this embodiment, the intermediate compound is first prepared, and then the intermediate compound is reacted with BTBT to obtain a molecular probe. Wherein, the maximum absorption peak of the intermediate compound is located at 900nm, the maximum emission wavelength is located at 925nm and can be tailed to more than 1000nm, and the photostability is good, which is an ideal fluorescent parent structure. The fluorescence signal of the molecular probe is very weak. When GSH is present, the molecular probe can specifically bind to two molecules of GSH, and the generated product LET-13G has a very strong fluorescence signal. This allows the molecular probe to be used as a GSH probe for fluorescence imaging, while consuming two molecules of GSH, which can be used to induce cell ferroptosis. And the molecular probe itself has a very high photothermal conversion efficiency, and can be used to treat tumor cells with photothermal therapy. In other words, the molecular probe prepared by the embodiment of the present invention can consume intracellular GSH to induce ferroptosis, while realizing the fluorescence response of GSH, and can treat tumors with photothermal therapy.

[0056] In step S11, the inert atmosphere is one of a nitrogen atmosphere, a helium atmosphere, an argon atmosphere and a neon atmosphere.

[0057] In some embodiments, the first solvent includes at least one of acetic anhydride and an alcohol solvent (such as methanol, ethanol, etc.), but is not limited thereto.

[0058] In some embodiments, the first preset temperature is 60-100°C, for example, it can be 60°C, 70°C, 80°C, 90°C or 100°C, etc.; the first preset time is 12-24h, for example, it can be 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h or 24h, etc.

[0059] In step S12, the inert atmosphere is one of a nitrogen atmosphere, a helium atmosphere, an argon atmosphere and a neon atmosphere.

[0060] In some embodiments, the second solvent is N,N-dimethylformamide (DMF).

[0061] In some embodiments, the second preset temperature is 10-50°C, for example, it can be 10°C, 20°C, 30°C, 40°C or 50°C, etc.; the second preset time is 5-15h, for example, it can be 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h or 15h, etc.

[0062] The present invention also provides an application of the molecular probe described above in detecting GSH for non-disease diagnosis purposes. The molecular probe can specifically bind to GSH and generate LET-13G with a strong fluorescent signal, so GSH can be detected by the molecular probe.

[0063] At present, the method for determining the concentration or content of GSH mainly adopts high performance liquid chromatography, however, this method usually requires complex pre-treatment of the sample to be tested, which is time-consuming and leads to a long sample detection time. Based on this, an embodiment of the present invention also provides an application of a molecular probe as described above in an embodiment of the present invention in detecting the concentration of GSH for non-disease diagnosis purposes. In a system containing a molecular probe and GSH, as the concentration of GSH increases, the fluorescence intensity increases, and the GSH concentration is linearly related to the fluorescence intensity. Therefore, the molecular probe can be used to detect the concentration of GSH, and it is convenient and quick without the need for sample pre-treatment.

[0064] The present invention also provides a method for detecting GSH concentration, which comprises the following steps:

[0065] S21, preparing GSH solutions of different concentrations;

[0066] S22, adding the molecular probe described in the embodiment of the present invention to GSH solutions of different concentrations, respectively, performing fluorescence intensity detection, obtaining fluorescence intensity values ​​corresponding to GSH solutions of different concentrations, and plotting glutathione concentrations and fluorescence intensity values ​​to obtain a standard curve;

[0067] S23, providing a test liquid;

[0068] S24, adding the molecular probe to the test solution, performing fluorescence intensity detection, and obtaining a first fluorescence intensity value;

[0069] S25. Obtaining the concentration of GSH in the test solution according to the first fluorescence intensity value and the standard curve.

[0070] The detection method provided by the embodiment of the present invention is convenient, efficient, does not require sample pretreatment, and has a short detection time.

[0071] The present invention also provides an application of the molecular probe described above in the present invention in the preparation of CDx reagents. The molecular probe provided by the present invention has both an imaging agent and a therapeutic agent, and can be used to prepare CDx reagents that integrate in vivo imaging and treatment.

[0072] An embodiment of the present invention further provides a nanoparticle, which includes the molecular probe described above in the embodiment of the present invention, and a liposome encapsulating the molecular probe.

[0073] In some embodiments, the liposomes include distearate phosphatidylethanolamine-polyethylene glycol 2000 and folic acid-functionalized distearate phosphatidylethanolamine-polyethylene glycol 2000.

[0074] In some embodiments, the mass ratio of the molecular probe, distearate phosphatidylethanolamine-polyethylene glycol 2000 and folic acid functionalized distearate phosphatidylethanolamine-polyethylene glycol 2000 is 1:(1-20):(0.1-1), for example, the mass ratio of the molecular probe, distearate phosphatidylethanolamine-polyethylene glycol 2000 and folic acid functionalized distearate phosphatidylethanolamine-polyethylene glycol 2000 is 1:1:0.1, 1:10:0.1, 1:20:0.1, 1:1:0.5, 1:10:0.5, 1:20:0.5, 1:1:1, 1:10:1 or 1:20:1, etc.

[0075] In some embodiments, the method for preparing the nanoparticles comprises the following steps:

[0076] The molecular probe, distearate phosphatidylethanolamine-polyethylene glycol 2000, and folic acid-functionalized distearate phosphatidylethanolamine-polyethylene glycol 2000 were dissolved in tetrahydrofuran (THF), and after ultrasonication, deionized water was added to obtain a mixed solution;

[0077] Argon gas was introduced into the mixed solution to remove THF, and free molecules were washed away using an ultrafiltration tube to obtain nanoparticles.

[0078] The present invention will be further described below by means of specific examples.

[0079] Unless otherwise specified in the following examples, the raw materials, reagents, etc. used are all commercially available products.

[0080] Example 1

[0081] This embodiment provides a molecular probe (denoted as LET-13) and a preparation method thereof, wherein the synthesis route is:

[0082]

[0083] According to the above synthetic route, the preparation method of LET-13 comprises the following steps:

[0084] Under N2 atmosphere, compound 1 (717 mg, 1.0 mmol) and TCF (1 g, 5.0 mmol) were dispersed in anhydrous ethanol (20 mL), and then sodium acetate (420 mg, 5.0 mmol) was added. The mixture was refluxed at 80°C for 12 hours and then cooled to room temperature.

[0085] After ethanol was evaporated under vacuum, it was washed three times with dichloromethane (DCM). The crude product was further purified by column chromatography using DCM / methanol (10 / 1, v / v) as eluent to obtain an intermediate compound, namely compound 2 (708 mg, yield 64%).

[0086] Under N2 atmosphere, compound 2 (200 mg, 0.26 mmol) was dissolved in anhydrous DMF (5 mL), and then BTBT (76 mg, 0.26 mmol) was added and stirred at room temperature (20°C) for 5 h. After removing the solvent, the crude product was purified by column chromatography with DCM / methanol (20 / 1, v / v) as the eluent to obtain LET-13 as a dark green solid (210 mg, 82% yield).

[0087] test:

[0088] The compound 2 in Example 1 was dissolved in methanol and subjected to ESI-MS (electrospray ionization mass spectrometry) test to obtain an ESI-MS mass spectrum as shown below: Figure 1a As shown, its mass-to-charge ratio was measured to be 532.14240.

[0089] 5 mg of compound 2 in Example 1 was dissolved in 0.6 mL of deuterated dimethyl sulfoxide and subjected to H NMR spectrum test to obtain the following H NMR spectrum: Figure 1b shown.

[0090] 10 mg of compound 2 in Example 1 was dissolved in 0.6 mL of deuterated dimethyl sulfoxide and subjected to a carbon NMR spectrum test to obtain a carbon NMR spectrum as shown below: Figure 1c shown.

[0091] The LET-13 in Example 1 was dissolved in methanol and subjected to ESI-MS test to obtain an ESI-MS mass spectrum as shown below: Figure 2a As shown, its mass-to-charge ratio was measured to be 742.16181.

[0092] 5 mg of LET-13 in Example 1 was dissolved in 0.6 mL of deuterated dimethyl sulfoxide and subjected to H NMR spectrum test to obtain the following H NMR spectrum: Figure 2b shown.

[0093] 10 mg of LET-13 in Example 1 was dissolved in 0.6 mL of deuterated dimethyl sulfoxide and subjected to a carbon NMR spectrum test to obtain a carbon NMR spectrum as shown below: Figure 2c shown.

[0094] 10 mg of LET-13 in Example 1 was dissolved in 0.6 mL of deuterated dimethyl sulfoxide and subjected to a nuclear magnetic resonance fluorine spectrum test to obtain a nuclear magnetic resonance fluorine spectrum as shown below: Figure 2d shown.

[0095] Example 2 Feasibility verification of LET-13 for fluorescence imaging detection of GSH

[0096] The LET-13 in Example 1 was dispersed in a mixed solvent to prepare a probe solution with a LET-13 concentration of 10 μM; wherein the mixed solvent consisted of phosphate buffered saline (PBS) and dimethyl sulfoxide (DMSO), and the volume ratio of PBS to DMSO was 1:1.

[0097] GSH (40 μM) was added to the above probe solution to obtain a mixed solution, which was incubated at 37° C. and the fluorescence spectrum of the mixed solution was measured over time.

[0098] The results are as follows Figure 3 As shown, it can be seen that in the presence of GSH, the fluorescence emission (928 nm) signal of LET-13 significantly increases over time, proving the feasibility of using LET-13 for fluorescence imaging detection of GSH.

[0099] Example 3 Feasibility verification of LET-13 for fluorescence imaging detection of GSH concentration

[0100] The LET-13 in Example 1 was dispersed in a mixed solvent (composed of PBS and DMSO, with a volume ratio of 1:1) to obtain a probe solution with a LET-13 concentration of 10 μM;

[0101] Adding different concentrations of GSH to the above probe solution (i.e., the final concentrations of GSH in the probe solution are 0 μM, 4 μM, 8 μM, 12 μM, 16 μM and 20 μM, respectively) to obtain a mixed solution;

[0102] After the mixture was incubated at 37°C for 20 min, the fluorescence spectrum of the mixture was measured. Figure 4 As shown in a; the fluorescence intensity at 928nm is plotted against the GSH concentration, as shown in Figure 4 As shown in b, there is a linear relationship between the two, and the standard curve y = 231.81x + 1155 (where R 2 =0.997, detection limit LOD=0.21 μM), x is the GSH concentration, y is the fluorescence intensity value at 928 nm.

[0103] Based on the linear relationship between fluorescence intensity and GSH concentration, it was demonstrated that LET-13 can be used to detect the concentration of GSH.

[0104] Example 4 Verification of the detection specificity of LET-13 for GSH

[0105] The LET-13 in Example 1 was dispersed in a mixed solvent (composed of PBS and DMSO, with a volume ratio of 1:1) to obtain a probe solution with a LET-13 concentration of 10 μM;

[0106] Various amino acids (PBS, GSH, Cys, Hcy, Met, Phe, Gly, Gln, Lys, Glu, His, Ser, Ala, Arg and DTT, the final concentration of each amino acid in the probe solution is 50 μM) were added to the above probe solution to obtain each mixed solution, which was incubated at 37°C for 20 min, and then the fluorescence intensity of each mixed solution at 928 nm was measured under 808 nm laser irradiation, and NIR-II fluorescence imaging was performed on each solution. The results are shown in Figure 2. Figure 5 As shown, it can be seen that LET-13 is not easily interfered by other amino acids, proving that LET-13 has specificity for the detection of GSH.

[0107] Example 5

[0108] The LET-13 in Example 1 was treated with distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-mPEG 2000 ) and 5% folic acid functionalized DSPE-mPEG2000 (DSPE-mPEG 2000 -FA, obtained through commercial channels) encapsulated into nanoparticles (referred to as LET-13 nanoparticles) specifically comprises the following steps:

[0109] LET-13 (1 mg), DSPE-mPEG 2000 (3.8 mg) and DSPE-mPEG 2000 -FA (0.2 mg) was dissolved in THF (10 mL), and after ultrasonication (180 W), deionized water (30 mL) was added to obtain a mixed solution;

[0110] Argon gas was passed into the obtained mixed solution to remove THF, and free molecules were washed out using an ultrafiltration tube (3.5 kDa) to obtain LET-13 nanoparticles.

[0111] The LET-13 nanoparticles in Example 5 were tested as follows:

[0112] (1) Morphology, particle size and absorption spectrum testing

[0113] The morphology of LET-13 nanoparticles was tested using a transmission electron microscope, the hydrated particle size of LET-13 nanoparticles was tested using a dynamic light scattering particle size analyzer, and the absorption spectrum of LET-13 nanoparticles was detected using a UV-visible-near infrared photometer. The results are as follows: Figure 6 As shown. It can be seen that the particle size of the formed LET-13 nanoparticles is mainly distributed around 137.6nm, which is conducive to tumor accumulation, and has a strong absorption peak at 808nm, which is conducive to subsequent photothermal therapy research.

[0114] (2) Photothermal performance test

[0115] The LET-13 nanoparticles were dispersed in aqueous solution to prepare LET-13 solutions with concentrations of 5 μM, 10 μM, 20 μM, and 30 μM, respectively. The LET-13 solutions were irradiated with an 808 nm laser at 0.6 W / cm 2 The photothermal temperature rise curves of LET-13 solutions with different concentrations were obtained by irradiating with an optical density of 5 min. Figure 7 As shown in a in .

[0116] The LET-13 nanoparticles were dispersed in an aqueous solution to prepare a 20 μM LET-13 solution. The solution was irradiated with an 808 nm laser at 0.2, 0.4, 0.6, 0.8, and 1.0 W / cm 2 The photothermal temperature rise curves of LET-13 solution under different laser powers were obtained. Figure 7 As shown in b.

[0117] The LET-13 nanoparticles were dispersed in an aqueous solution to prepare a 20 μM LET-13 solution. The solution was then irradiated with an 808 nm laser at 0.6 W / cm 2 The photothermal cycle curve of the LET-13 solution was obtained by irradiating with an optical density of 5 min and cooling for 10 min, and this cycle was repeated 5 times. Figure 7 As shown in c.

[0118] The LET-13 nanoparticles were dispersed in an aqueous solution to prepare a 20 μM LET-13 solution. The solution was then irradiated with an 808 nm laser at 0.6 W / cm 2 The photothermal cooling curve of LET-13 solution was obtained and the photothermal conversion efficiency was calculated. Figure 7 As shown in d (lnθ is the logarithm of temperature), the photothermal efficiency η = 97%, and the system time constant τ = 212s.

[0119] It can be seen that the LET-13 nanoparticles in Example 5 have good photothermal properties under 808nm laser irradiation.

[0120] (3) Tumor cell killing test

[0121] LET-13 nanoparticles were dispersed in 4T1 cells at different concentrations (0 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, and 160 μg / mL), incubated in the dark for 24 h, and the cell viability was tested using the standard MTT assay. The results are shown in Figure 8 As shown in a, it can be seen that LET-13 nanoparticles have the ability to kill tumor cells.

[0122] LET-13 nanoparticles were dispersed in 4T1 cells at a concentration of 20 μg / mL and incubated in the dark for 12 h. 2 or 0.6W / cm 2 )5min later, the cells were incubated for 0h, 4h, 12h, and 24h, and the cell survival rate was tested using the standard MTT assay. The results were as follows Figure 8 As shown in b, it can be seen that under laser irradiation, the killing ability of LET-13 nanoparticles on tumor cells is further enhanced.

[0123] (4) GSH scavenging ability and lipid peroxidation test

[0124] a. Disperse LET-13 nanoparticles in 4T1 cells at a concentration of 20 μg / mL and incubate in the dark for 4 h. 2 or 0.6W / cm 2 ) for 5 min, and then continue to incubate for 1 h to test the intracellular GSH concentration; at the same time, PBS was dispersed in 4T1 cells and the cells were exposed to laser irradiation (wavelength 808 nm, 0.6 W / cm 2 ) After 5 min, the cells were incubated for 1 h and the intracellular GSH concentration was tested.

[0125] The results are as follows Fig. 9 As shown in a (where *** indicates p<0.001), it can be seen that LET-13 nanoparticles can clear GSH in cells, and their GSH clearing ability is further enhanced under 808nm laser irradiation.

[0126] b. LET-13 nanoparticles were dispersed in 4T1 cells at a concentration of 20 μg / mL and incubated in the dark for 4 h. 2 or 0.6W / cm 2 ) for 5 min, and then continue to incubate for 2 h. Meanwhile, PBS was dispersed in 4T1 cells and incubated under conditions of no laser irradiation, 808 nm laser irradiation (0.6 W / cm 2 )5min later, continue incubation for 1h. 581 / 591 The dye stains intracellular lipid peroxidation (LPO). The high-content analysis system is used for microscopic fluorescence imaging and the intracellular LPO level is calculated. Fig. 9 As shown in b and c (where ** indicates p<0.01, *** indicates p<0.001), it can be seen that LET-13 nanoparticles can increase LPO in cells, and under 808nm laser irradiation, LPO further increases.

[0127] (5) GSH visualization test

[0128] LET-13 nanoparticles were dispersed in PBS to prepare a 200 μg / mL solution, which was then injected into 4T1 tumor-bearing mice via the tail vein at a dose of 10 mg / kg. Under 808 nm laser irradiation, real-time fluorescence imaging of the mouse back was taken, and the signal intensity of the tumor area was counted. Fig.10 As shown, the results show that LET-13 nanoparticles can visualize GSH in tumors in real time at the in vivo level.

[0129] (6) Tumor treatment testing and safety verification

[0130] The LET-13 nanoparticles were dispersed in PBS to prepare a 200 μg / mL solution (referred to as LET-13 solution).

[0131] The tumor-bearing mice were randomly divided into 5 groups. The mice in the first group were injected with PBS through the tail vein. The mice in the second group were injected with PBS through the tail vein. Twelve hours later, the mice were laser-exposed at 808 nm at 0.6 W / cm 2 The mice in the third group were injected with LET-13 solution at a dose of 10 mg / kg into the tail vein. The mice in the fourth group were first injected with LET-13 solution at a dose of 10 mg / kg into the tail vein, and then irradiated with 808 nm laser at 0.4 W / cm 2 The fifth group of mice were first injected with LET-13 solution at a dose of 10 mg / kg into the tail vein, and then irradiated with 808 nm laser at 0.6 W / cm 2 The optical density was irradiated for 10 min.

[0132] The weight of the mice and the volume of the tumor were recorded, and the tumor weight was measured after 15 days. Fig.11 As shown (where *** indicates p<0.001), it can be seen that at the in vivo level, LET-13 nanoparticles have excellent therapeutic effects on tumors.

[0133] The mice in each group after tumor treatment were dissected to obtain hematoxylin-eosin (H&E) staining images of major organs (heart, liver, spleen, lung and kidney). Fig.12 As shown, it can be seen that no lesions occurred in major organs, proving the excellent biological safety of LET-13.

[0134] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A molecular probe, characterized in that The structural formula of the molecular probe is:

2. A method for preparing the molecular probe according to claim 1, characterized in that: The steps include: Under an inert atmosphere, 2-dicyanomethylidene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran, sodium acetate and a first solvent are mixed and reacted at a first preset temperature for a first preset time to obtain an intermediate compound; Under an inert atmosphere, adding the intermediate compound and 3,5-bis(trifluoromethyl)benzenethiol to a second solvent, stirring at a second preset temperature for a second preset time, to obtain the molecular probe; Among them, the structural formula of the intermediate compound is:

3. The preparation method according to claim 2, characterized in that: The first solvent comprises at least one of acetic anhydride and an alcohol solvent; and / or, The first preset temperature is 60-100° C., and the first preset time is 12-24 hours.

4. The preparation method according to claim 2, characterized in that: The second solvent is N,N-dimethylformamide; and / or, The second preset temperature is 10-50° C., and the second preset time is 5-15 hours.

5. Use of the molecular probe according to claim 1 in detecting reduced glutathione for purposes other than disease diagnosis.

6. Use of the molecular probe according to claim 1 in detecting the concentration of reduced glutathione for purposes other than disease diagnosis.

7. Use of the molecular probe according to claim 1 in the preparation of companion diagnostic reagents.

8. A method for detecting reduced glutathione concentration, characterized in that: The steps include: Prepare reduced glutathione solutions of different concentrations; The molecular probe according to claim 1 is added to reduced glutathione solutions of different concentrations, and fluorescence intensity is detected to obtain fluorescence intensity values ​​corresponding to reduced glutathione solutions of different concentrations, and the reduced glutathione concentration and the fluorescence intensity value are plotted to obtain a standard curve; Provide the test fluid; Adding the molecular probe into the test solution, performing fluorescence intensity detection, and obtaining a first fluorescence intensity value; The concentration of reduced glutathione in the test solution is obtained according to the first fluorescence intensity value and the standard curve.

9. A nanoparticle, characterized in that: The invention comprises the molecular probe according to claim 1, and a liposome encapsulating the molecular probe.

10. The nanoparticle according to claim 9, characterized in that The liposomes include distearate phosphatidylethanolamine-polyethylene glycol 2000 and folic acid functionalized distearate phosphatidylethanolamine-polyethylene glycol 2000.

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