A molecular probe, its preparation method and application

Abstract

The application discloses a molecular probe and a preparation method and application thereof, and relates to the technical field of fluorescent probes.The structural formula of the molecular probe is as follows: the application takes two identical corrole units covalently connected as a parent body, and simultaneously introduces a reduced glutathione response group into the two corrole units, so that a molecular probe, namely a double-corrole molecular probe, which can remove reduced glutathione and realize NIR-II fluorescent signal opening, is constructed.The molecular probe can induce cell ferroptosis by consuming reduced glutathione in tumor cells.In addition, the molecular probe itself has a high photothermal conversion efficiency, and can perform photothermal treatment on tumor cells.Therefore, the molecular probe provided by the application is a companion diagnostic reagent integrating NIR-II fluorescent response, ferroptosis induction and photothermal treatment, has excellent antitumor effect, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of fluorescent probe technology, and in particular to a molecular probe, its preparation method, and its application. Background Technology

[0002] Tumor heterogeneity and individual variability in the tumor microenvironment limit cancer treatment. Companion diagnostics (CDx) are diagnostic technologies, often developed in conjunction with specific therapeutic drugs, used to determine a patient's suitability for a particular treatment. CDx help physicians select the most effective treatment options by detecting a patient's biomarkers, improving treatment safety and efficacy, and reducing unnecessary medical expenses. However, most FDA-approved CDx reagents only allow for in vitro detection of biomarkers, making it difficult to dynamically monitor changes in in vivo targets.

[0003] Fluorescence imaging is a non-invasive bioimaging technique characterized by rapid response, high sensitivity, and high spatiotemporal resolution. Compared to the visible or near-infrared I region, near-infrared II (NIR-II) light has deeper tissue penetration, lower tissue absorption and scattering, and negligible tissue autofluorescence in the NIR-II band, resulting in superior imaging performance. Therefore, the development of CDx reagents based on NIR-II fluorescence imaging holds immense promise for applications. Furthermore, the separation of imaging and therapeutic agents reduces the accuracy of treatment assessment. Therefore, it is essential to develop CDx reagents that integrate in vivo imaging and treatment. Summary of the Invention

[0004] Based on the shortcomings of the existing technology, the purpose of this invention is to provide a molecular probe, its preparation method and application, aiming to provide an integrated CDx reagent that combines NIR-II fluorescence response, ferroptosis induction and photothermal therapy (PTT), and then monitor and regulate the cancer treatment process through companion diagnostics, 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, the present invention provides a molecular probe, wherein the molecular probe has the following structural formula:

[0007]

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

[0009] In an inert atmosphere, 2-Dicyanomethylene-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, the intermediate compound and 3,5-bis(trifluoromethyl)benzylthiophenol were added to a second solvent, and the mixture was stirred at a second preset temperature for a second preset time to obtain the molecular probe.

[0011] The structural formula of the intermediate compound is as follows:

[0012]

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

[0014] The first preset temperature is 60-100℃, and the first preset time is 12-24h.

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

[0016] The second preset temperature is 10-50℃, and the second preset time is 5-15h.

[0017] A third aspect of the present invention provides the application of the molecular probe of the present invention as described above in the detection of reduced glutathione for non-disease diagnostic purposes.

[0018] A fourth aspect of the present invention provides the application of the molecular probe of the present invention as described above in the detection of reduced glutathione concentration for non-disease diagnostic purposes.

[0019] A fifth aspect of the present invention provides the application of the molecular probe described above in the preparation of companion diagnostic reagents.

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

[0021] Prepare solutions of reduced glutathione at different concentrations;

[0022] The molecular probes described above were added to solutions of reduced glutathione at different concentrations, and fluorescence intensity was detected to obtain fluorescence intensity values ​​corresponding to solutions of reduced glutathione at different concentrations. A standard curve was obtained by plotting the reduced glutathione concentration and fluorescence intensity values.

[0023] Provide the test solution;

[0024] The molecular probe is added to the test solution, and the fluorescence intensity is detected to obtain the first fluorescence intensity value;

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

[0026] A seventh aspect of the present invention provides a nanoparticle comprising 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: This invention uses two covalently linked identical cyanine units as the parent compound and simultaneously introduces reduced glutathione (GSH) responsive groups into both cyanine units to construct a molecular probe, namely a dicyanine molecular probe, that can scavenge GSH while simultaneously activating NIR-II fluorescence signaling. This molecular probe can induce ferroptosis in tumor cells by consuming GSH within them. Furthermore, the molecular probe itself has high photothermal conversion efficiency, enabling photothermal therapy of tumor cells. Therefore, the molecular probe provided by this invention is a CDx reagent integrating NIR-II fluorescence response, ferroptosis induction, and photothermal therapy, exhibiting excellent anti-tumor effects and broad application prospects. Attached Figure Description

[0029] Figure 1a The image shows the ESI-MS mass spectrum of compound 2 from Example 1. Figure 1b The image shows the 1H NMR spectrum of compound 2 from Example 1. Figure 1c The image shows the carbon NMR spectrum of compound 2 in Example 1.

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

[0031] Figure 3 The figure shows the feasibility verification results of using LET-13 for fluorescence imaging detection of GSH in Example 2. In the figure, a is the fluorescence spectrum of the mixture over time, and b is the result of the fluorescence intensity of the mixture at 928 nm over time.

[0032] Figure 4The figure shows the feasibility verification results of using LET-13 for fluorescence imaging to detect GSH concentration in Example 3. In the figure, a is the fluorescence spectrum of the mixture containing different concentrations of GSH, and b is the result of the fluorescence intensity of the mixture at 928 nm changing with the GSH concentration.

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

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

[0035] Figure 7 The figures shown are the photothermal performance test results of LET-13 nanoparticles in Example 5. In the figure, a is the temperature change of the solution containing different concentrations of LET-13 nanoparticles with light irradiation time; b is the temperature change of the solution containing 20 μM LET-13 nanoparticles with light irradiation time under different laser powers; c is the photothermal cycle diagram of the solution containing 20 μM LET-13 nanoparticles; and d is the temperature change curve of the solution containing 20 μM LET-13 nanoparticles after laser irradiation with time and the linear fitting diagram of the negative logarithmic function of time and temperature change.

[0036] Figure 8 The graph shows the results of the tumor cell killing test. In the graph, a represents the cell survival rate of 4T1 cells treated with LET-13 nanoparticles under different concentrations of LET-13 nanoparticles, and b represents the cell survival rate of 4T1 cells treated with LET-13 nanoparticles under and without laser irradiation.

[0037] Figure 9 Figure 1 shows the results of GSH scavenging capacity and lipid peroxidation tests. In figure 1, a represents the cell viability of 4T1 cells treated with LET-13 nanoparticles under laser irradiation and without laser irradiation; b represents the intracellular lipid peroxidation staining of 4T1 cells treated with LET-13 nanoparticles under laser irradiation and without laser irradiation; and c represents the quantitative graph of intracellular lipid peroxidation concentration of 4T1 cells treated with LET-13 nanoparticles under laser irradiation and without laser irradiation.

[0038] Figure 10 The images show the visualization results of the GSH test. In the image, a is the NIR-II fluorescence imaging of tumor-bearing mice over time after LET-13 nanoparticles were administered via the tail vein, and b is the quantification of the NIR-II fluorescence signal at the tumor site.

[0039] Figure 11 The figures show the results of tumor treatment tests. In figure a, the tumor volume changes after treatment with LET-13 nanoparticles are shown; in figure b, the tumor weight changes after 15 days of treatment are shown; and in figure c, the mouse body weight changes after treatment are shown.

[0040] Figure 12 H&E staining images of major organs in mice from each group after tumor treatment testing. Detailed Implementation

[0041] This invention provides a molecular probe and its preparation method. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0042] If the embodiments of the present invention involve descriptions such as "first" or "second", such descriptions are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated.

[0043] The molecular probe provided in this invention is the first diflorin CDx reagent capable of integrating NIR-II fluorescence response, ferroptosis induction, and photothermal therapy. Specifically, the structural formula of the molecular probe is:

[0044]

[0045] This invention uses two covalently linked identical cyanine units as the parent compound and simultaneously introduces GSH-responsive groups into both cyanine units to construct a molecular probe, namely a dicyanine molecular probe, that can scavenge GSH while simultaneously activating NIR-II fluorescence signal. This molecular probe can achieve specific detection of GSH and induce ferroptosis in tumor cells by consuming GSH within them. As shown in the reaction formula below, the molecular probe (LET-13) provided by this invention can specifically bind to GSH, consuming two molecules of GSH while generating the product LET-13G, which has a strong fluorescence signal. Therefore, it can achieve specific detection of GSH and induce ferroptosis in tumor cells by consuming GSH within them.

[0046]

[0047] Furthermore, this molecular probe itself possesses high photothermal conversion efficiency, enabling photothermal therapy of tumor cells. In summary, the molecular probe provided by this invention is a CDx reagent integrating NIR-II fluorescence response, ferroptosis induction, and photothermal therapy, exhibiting excellent anti-tumor effects and broad application prospects.

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

[0049] In this embodiment of the invention, the molecular probe is specific to GSH and is not affected 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), and L-dithiothreitol (DTT).

[0050] This invention also provides a method for preparing the molecular probe described above, comprising the following steps:

[0051] S11. Under an inert atmosphere, 2-Dicyanomethylene-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, the intermediate compound and 3,5-bis(trifluoromethyl)benzylthiophenol (BTBT) are added to a second solvent, and stirred at a second preset temperature for a second preset time to obtain the molecular probe.

[0053] The structural formula of the intermediate compound is as follows:

[0054]

[0055] The preparation method provided by this invention is simple, safe, has high yield, uses inexpensive raw materials, and is easy to industrialize. In this embodiment, an intermediate compound is first prepared, and then the intermediate compound undergoes a nucleophilic reaction with BTBT to obtain a molecular probe. The intermediate compound has a maximum absorption peak at 900 nm, a maximum emission wavelength at 925 nm with a tailing to over 1000 nm, and good photostability, making it an ideal fluorescent parent structure. The molecular probe has a very weak fluorescence signal, but in the presence of GSH, it can specifically bind to two molecules of GSH, generating a product, LET-13G, with a strong fluorescence signal. This allows the molecular probe to be used as a GSH probe for fluorescence imaging, simultaneously consuming two molecules of GSH to induce ferroptosis. Furthermore, the molecular probe itself has high photothermal conversion efficiency, enabling photothermal therapy of tumor cells. In other words, the molecular probe prepared in this embodiment can consume intracellular GSH to induce ferroptosis, simultaneously achieving a GSH fluorescence response, and can be used for photothermal therapy of tumors.

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

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

[0058] In some embodiments, the first preset temperature is 60-100℃, for example, it can be 60℃, 70℃, 80℃, 90℃ or 100℃; 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.

[0059] In step S12, the inert atmosphere is one of nitrogen atmosphere, helium atmosphere, argon atmosphere and 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℃, for example, it can be 10℃, 20℃, 30℃, 40℃ or 50℃; the second preset time is 5-15h, for example, it can be 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h or 15h.

[0062] This invention also provides an application of the molecular probe described above in the detection of GSH for non-disease diagnostic purposes. This molecular probe can specifically bind to GSH and generate LET-13G with a strong fluorescent signal, thus enabling the detection of GSH.

[0063] Currently, the main method for determining GSH concentration or content is high-performance liquid chromatography (HPLC). However, this method usually requires complex sample pretreatment, which is time-consuming and results in a long sample detection time. Therefore, this invention also provides an application of the molecular probe described above in the detection of GSH concentration for non-disease diagnostic purposes. In a system containing the molecular probe and GSH, the fluorescence intensity increases with increasing GSH concentration, and the relationship between GSH concentration and fluorescence intensity is linear. Therefore, the molecular probe can be used to detect GSH concentration conveniently and quickly, without requiring sample pretreatment.

[0064] This invention also provides a method for detecting GSH concentration, comprising the following steps:

[0065] S21. Prepare GSH solutions of different concentrations;

[0066] S22. The molecular probes described above in the embodiments of the present invention are added to GSH solutions of different concentrations, and the fluorescence intensity is detected to obtain the fluorescence intensity values ​​corresponding to different concentrations of GSH solutions. The glutathione concentration and fluorescence intensity values ​​are plotted to obtain a standard curve.

[0067] S23. Provide the solution to be tested;

[0068] S24. Add the molecular probe to the test solution and perform fluorescence intensity detection to obtain the first fluorescence intensity value;

[0069] S25. Based on the first fluorescence intensity value and the standard curve, the concentration of GSH in the test solution is obtained.

[0070] The detection method provided in this invention is convenient, efficient, requires no sample pretreatment, and has a short detection time.

[0071] This invention also provides an application of the molecular probe described above in the preparation of CDx reagents. The molecular probe provided by this invention simultaneously functions as an imaging agent and a therapeutic agent, enabling its use in the preparation of CDx reagents that integrate in vivo imaging and therapy.

[0072] This invention also provides a nanoparticle, which includes the molecular probe described above in this invention, and liposomes encapsulating the molecular probe.

[0073] In some embodiments, the liposomes comprise 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 includes the following steps:

[0076] Molecular probe, distearate phosphatidylethanolamine-polyethylene glycol 2000, and folic acid-functionalized distearate phosphatidylethanolamine-polyethylene glycol 2000 were dissolved in tetrahydrofuran (THF), sonicated, and then deionized water was added to obtain a mixture.

[0077] Argon gas was introduced into the mixture 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 through specific embodiments.

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

[0080] Example 1

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

[0082]

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

[0084] Compound 1 (717 mg, 1.0 mmol) and TCF (1 g, 5.0 mmol) were dispersed in anhydrous ethanol (20 mL) under N2 atmosphere, 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] Ethanol was evaporated under vacuum and then washed three times with dichloromethane (DCM). The crude product was further purified by column chromatography using DCM / methanol (10 / 1, v / v) as the eluent to give the intermediate compound, compound 2 (708 mg, yield 64%).

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

[0087] test:

[0088] Compound 2 from Example 1 was dissolved in methanol and subjected to ESI-MS (electrospray ionization mass spectrometry) analysis. The resulting ESI-MS mass spectrum is shown below. Figure 1a As shown, its mass-to-charge ratio was measured to be 532.14240.

[0089] 5 mg of compound 2 from Example 1 was dissolved in 0.6 mL of deuterated dimethyl sulfoxide, and a 1H NMR spectrum was obtained. The 1H NMR spectrum is shown below. Figure 1b As shown.

[0090] 10 mg of compound 2 from Example 1 was dissolved in 0.6 mL of deuterated dimethyl sulfoxide, and carbon NMR spectroscopy was performed. The resulting carbon NMR spectrum is shown below. Figure 1c As shown.

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

[0092] 5 mg of LET-13 from Example 1 was dissolved in 0.6 mL of deuterated dimethyl sulfoxide, and a 1H NMR spectrum was obtained. The 1H NMR spectrum is shown below. Figure 2b As shown.

[0093] 10 mg of LET-13 from Example 1 was dissolved in 0.6 mL of deuterated dimethyl sulfoxide, and carbon NMR spectroscopy was performed. The resulting carbon NMR spectrum is shown below. Figure 2c As shown.

[0094] 10 mg of LET-13 from Example 1 was dissolved in 0.6 mL of deuterated dimethyl sulfoxide, and nuclear magnetic resonance (NMR) fluorine spectroscopy was performed. The resulting NMR fluorine spectrum is shown below. Figure 2d As shown.

[0095] Example 2: Feasibility Verification of LET-13 for Fluorescence Imaging Detection of GSH

[0096] The LET-13 from 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 buffer (PBS) and dimethyl sulfoxide (DMSO), and the volume ratio of PBS to DMSO was 1:1.

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

[0098] The results are as follows Figure 3 As shown, it can be seen that the fluorescence emission signal (928nm) of LET-13 increases significantly over time in the presence of GSH, 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 from Example 1 was dispersed in a mixed solvent (composed of PBS and DMSO in a volume ratio of 1:1) to obtain a probe solution with a LET-13 concentration of 10 μM.

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

[0102] After incubating the mixture at 37°C for 20 minutes, the fluorescence spectrum of the mixture was measured. Figure 4 As shown in a; a graph is plotted between fluorescence intensity at 928 nm and GSH concentration, as shown in Figure a. Figure 4 As shown in b, the two have a linear relationship, yielding the standard curve y = 231.81x + 1155 (where R0...). 2 =0.997, detection limit LOD =0.21μM), x is the GSH concentration, and y is the fluorescence intensity at 928nm.

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

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

[0105] The LET-13 from Example 1 was dispersed in a mixed solvent (composed of PBS and DMSO in 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, with a final concentration of 50 μM for each amino acid in the probe solution) were added to the above probe solutions to obtain various mixtures. After incubation at 37°C for 20 min, the fluorescence intensity of each mixture at 928 nm was measured under 808 nm laser irradiation, and NIR-II fluorescence imaging was performed on each solution. The results are as follows: Figure 5 As shown, LET-13 is not easily interfered with by other amino acids, proving that LET-13 has specificity for the detection of GSH.

[0107] Example 5

[0108] LET-13 from Example 1 was used with distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-mPEG). 2000 ) and 5% folic acid-functionalized DSPE-mPEG2000 (DSPE-mPEG) 2000 -FA (obtained commercially) is encapsulated into nanoparticles (denoted as LET-13 nanoparticles) specifically including the following steps:

[0109] LET-13 (1 mg) and DSPE-mPEG 2000 (3.8 mg) and DSPE-mPEG 2000 -FA (0.2 mg) was dissolved in THF (10 mL), sonicated (180 W), and then deionized water (30 mL) was added to obtain a mixture.

[0110] Argon gas was introduced into the resulting mixture to remove THF, and free molecules were washed away 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 transmission electron microscopy, the hydrated particle size of LET-13 nanoparticles was measured using a dynamic light scattering particle size analyzer, and the absorption spectrum of LET-13 nanoparticles was detected using a UV-Vis-NIR spectrophotometer. The results are as follows: Figure 6 As shown, the particle size of the formed LET-13 nanoparticles is mainly distributed around 137.6 nm, which is conducive to tumor accumulation. Furthermore, they exhibit a strong absorption peak at 808 nm, which is beneficial for subsequent photothermal therapy research.

[0114] (2) Photothermal performance test

[0115] LET-13 nanoparticles were dispersed in an aqueous solution to prepare LET-13 solutions with concentrations of 5 μM, 10 μM, 20 μM, and 30 μM. The solutions were then analyzed using an 808 nm laser at 0.6 W / cm². 2 After irradiating with light density for 5 minutes, the photothermal heating curves of LET-13 solutions with different concentrations were obtained, as shown below. Figure 7 As shown in 'a'.

[0116] LET-13 nanoparticles were dispersed in an aqueous solution to prepare a 20 μM LET-13 solution. The solution was then analyzed using an 808 nm laser at concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0 W / cm². 2 After irradiating with light density for 5 minutes, the photothermal heating curves of the LET-13 solution under different laser powers were obtained, as shown below. Figure 7 As shown in b in the figure.

[0117] LET-13 nanoparticles were dispersed in an aqueous solution to prepare a 20 μM LET-13 solution, and then analyzed using an 808 nm laser at 0.6 W / cm². 2 Irradiation with light density for 5 minutes, followed by cooling for 10 minutes, was repeated 5 times to obtain the photothermal cycling curve of the LET-13 solution, as shown below. Figure 7 As shown in c in the figure.

[0118] LET-13 nanoparticles were dispersed in an aqueous solution to prepare a 20 μM LET-13 solution, and then analyzed using an 808 nm laser at 0.6 W / cm². 2 Irradiate the solution with light density for 10 minutes, then allow it to cool naturally. Obtain the photothermal cooling curve of the LET-13 solution and calculate the photothermal conversion efficiency. 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] As can be seen, the LET-13 nanoparticles in Example 5 have good photothermal properties under 808nm laser irradiation.

[0120] (3) Tumor cell killing ability test

[0121] LET-13 nanoparticles were dispersed in 4T1 cells at concentrations (0 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, and 160 μg / mL) and incubated in the dark for 24 h. Cell viability was assessed using the standard MTT assay. The results are as follows: 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. The cells were then subjected to both laser irradiation (0.4 W / cm²) and laser irradiation (without laser light). 2 Or 0.6W / cm 2 After 5 minutes, the cells were incubated for 0 hours, 4 hours, 12 hours, and 24 hours, respectively. Cell viability was then tested using the standard MTT assay. The results are as follows: Figure 8 As shown in b, it can be seen that the killing ability of LET-13 nanoparticles against tumor cells is further enhanced under laser irradiation.

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

[0124] a. LET-13 nanoparticles were dispersed in 4T1 cells at a concentration of 20 μg / mL and incubated in the dark for 4 h. The cells were then subjected to both laser irradiation (0.4 W / cm²) and laser irradiation (without laser light). 2 Or 0.6W / cm 2 After 5 minutes, incubation continued for 1 hour, and intracellular GSH concentration was measured. Simultaneously, PBS was dispersed in 4T1 cells, and incubation was performed under both laser-free and laser-irradiated conditions (wavelength 808 nm, 0.6 W / cm²). 2 After 5 minutes, continue incubation for 1 hour and test the intracellular GSH concentration.

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

[0126] b. Disperse LET-13 nanoparticles at a concentration (20 μg / mL) in 4T1 cells and incubate in the dark for 4 h, with and without laser irradiation (0.4 W / cm²). 2 Or 0.6W / cm 2 After 5 minutes, continue incubation for 2 hours; simultaneously, PBS is dispersed in 4T1 cells, and the cells are incubated under conditions of no laser irradiation and irradiation with 808nm laser (0.6W / cm²). 2 After 5 minutes, continue incubation for 1 hour. Use C11-BODIPY. 581 / 591 The dye stained intracellular lipid peroxidation (LPO). Microscopic fluorescence imaging was performed using a high-content analysis system, and intracellular LPO levels were calculated. The results are as follows: Figure 9 As shown in b and c (where ** indicates p < 0.01 and *** indicates p < 0.001), it can be seen that LET-13 nanoparticles can increase intracellular LPO, and LPO increases further under 808nm laser irradiation.

[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. Real-time fluorescence imaging of the mouse dorsal region was performed under 808 nm laser irradiation, and the signal intensity in the tumor area was statistically analyzed. Results... Figure 10 As shown, the results indicate that LET-13 nanoparticles can visualize GSH within tumors in real time at the in vivo level.

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

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

[0131] Tumor-bearing mice were randomly divided into 5 groups. Group 1 mice received a tail vein injection of PBS; Group 2 mice received a tail vein injection of PBS, and 12 hours later were treated with an 808nm laser at 0.6W / cm². 2 The mice in the first group were irradiated with light at a specific intensity for 10 minutes; the mice in the second group were injected intravenously with LET-13 solution at a dose of 10 mg / kg via the tail vein; the mice in the third group were first injected intravenously with LET-13 solution at a dose of 10 mg / kg via the tail vein, and then irradiated with an 808 nm laser at a intensity of 0.4 W / cm² 12 hours later. 2 The mice were irradiated with light at a specific density for 10 minutes; the fifth group of mice were first injected intravenously with LET-13 solution at a dose of 10 mg / kg, and 12 hours later were treated with an 808 nm laser at 0.6 W / cm². 2 Irradiate with light density for 10 minutes.

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

[0133] Mice in each group were dissected after tumor treatment, and hematoxylin and eosin (H&E) staining images of major organs (heart, liver, spleen, lungs, and kidneys) were obtained, as shown below. Figure 12 As shown, no lesions were found in the major organs, demonstrating the excellent biocompatibility of LET-13.

[0134] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

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, Includes the following steps: In an inert atmosphere, 2-Dicyanomethylene-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, the intermediate compound and 3,5-bis(trifluoromethyl)benzylthiophenol were added to a second solvent, and the mixture was stirred at a second preset temperature for a second preset time to obtain the molecular probe. The structural formula of the intermediate compound is as follows:

3. The preparation method according to claim 2, characterized in that, The first solvent includes at least one of acetic anhydride and alcohol solvents; and / or, The first preset temperature is 60-100℃, and the first preset time is 12-24h.

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℃, and the second preset time is 5-15h.

5. The use of the molecular probe of claim 1 in the detection of reduced glutathione for non-disease diagnostic purposes.

6. The use of the molecular probe of claim 1 in detecting the concentration of reduced glutathione for non-disease diagnostic purposes.

7. The use of the molecular probe of claim 1 in the preparation of companion diagnostic reagents.

8. A method for detecting the concentration of reduced glutathione for non-disease diagnostic purposes, characterized in that, Includes the following steps: Prepare solutions of reduced glutathione at different concentrations; The molecular probe described in claim 1 was added to solutions of reduced glutathione at different concentrations, and the fluorescence intensity was detected to obtain the fluorescence intensity values ​​corresponding to solutions of reduced glutathione at different concentrations. The concentration of reduced glutathione and the fluorescence intensity values ​​were plotted to obtain a standard curve. Provide the test solution; The molecular probe is added to the test solution, and the fluorescence intensity is detected to obtain the first fluorescence intensity value; The concentration of reduced glutathione in the test solution is obtained based on the first fluorescence intensity value and the standard curve.

9. A nanoparticle, characterized in that, It includes the molecular probe of claim 1, and liposomes encapsulating the molecular probe.

10. The nanoparticles 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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