A slightly acidic environment-responsive AIE fluorescent probe and its preparation method and application

Abstract

The present invention discloses a kind of slightly acidic environment response AIE fluorescent probe and its preparation method and application, comprise the following steps: step one: benzenesulfonamide, sulfacetamide or sulfadoxine are dissolved in a certain amount of solvent, and triethylamine is added in proportion, is placed in ice-water bath and stirred, and obtains the first solution;Step 2: a certain amount of acyl chloride solid is dissolved in a certain amount of solvent to obtain the second solution, and is added dropwise to the first solution of step one to obtain a mixed solution;Step 3: after the second solution is added dropwise, the mixed solution obtained by step 2 is stirred at a specific temperature for 48 72 h, and the residue after evaporating the solvent is dried by purification to obtain a fluorescent probe compound. The present invention combines TPE with sulfonamide compounds by amidation reaction, and successfully synthesizes an AIE fluorescent probe, which shows sensitive pH response and superior AIE effect, and can achieve safe and effective selective fluorescence imaging of tumor cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent probes, and in particular relates to a slightly acidic environment responsive AIE fluorescent probe and a preparation method and application thereof. Background Art

[0002] Bioimaging is a powerful tool in biomedical research and is of great significance in the early diagnosis and treatment of diseases and the development of basic drugs. Bioimaging techniques include magnetic resonance imaging, X-ray computed tomography, fluorescence imaging, radionuclide imaging, and photoacoustic tomography. Fluorescence imaging has the advantages of simple operation, fast response, and high selectivity, and has become an indispensable bioimaging tool. In the past few decades, various optical reagents such as fluorescent proteins, quantum dots (QDs), and conventional organic dyes have been widely used in the field of fluorescence imaging. However, there are some problems, such as the presence of heavy metal ions in quantum dots that may cause certain cytotoxicity, and the complex and time-consuming fluorescent protein transfection procedure.

[0003] Organic fluorescent materials have great potential for application in optoelectronic devices, detection, bioimaging, and biotherapy due to their low cost, low toxicity, simple operation, easily tunable structure, high signal-to-noise ratio, good photostability, excellent fluorescence quantum yield, deeper tissue penetration, and higher spatial resolution. They have become powerful tools for microbiological and pathological research. However, most traditional organic fluorescent molecules have a planar conjugated structure, which is prone to π-π stacking in the aggregated state. This leads to severe energy loss in the excited state, resulting in poor luminescence performance and aggregation-induced quenching (ACQ). Although many types of fluorophores have been commercially applied in bioimaging, their further development is hindered by their poor photostability, aggregation-induced quenching (ACQ), and complex fluorophore synthesis.

[0004] In recent years, acid-responsive AIE fluorescent probes have provided an effective strategy to avoid the ACQ problem. A new type of small molecule fluorescent probe with aggregation-induced emission (AIE) properties has effectively overcome the defects of traditional fluorescent dyes. AIE fluorescent probes have many advantages, such as excellent brightness in the aggregated state, large Stokes shift deviation, good photostability and high signal-to-noise ratio. They can sensitively respond to the characteristics of the tumor microenvironment for tumor tissue imaging. The acidic microenvironment is combined with ionizable chemical groups to respond to changes in environmental pH by protonation or deprotonation. However, commonly used ionizable groups, such as carboxylic acid and tertiary amino groups, are protonated or deprotonated in the pH range of 5.0-6.5, which is lower than the pH of the acidic TME (pH≈6.2-6.9) and cannot be applied to the tumor microenvironment. Although fluorescent probes have certain advantages, they have problems such as a wide response range, a lack of a clear pH transition point, and an inability to accurately distinguish normal tissue from tumor tissue by utilizing the differences in the slightly acidic environment of the tumor.

[0005] Prior art 1 (CN117586205A) discloses a lysosomal pH fluorescent probe, its preparation method, and application. It uses 1,4-cyclohexanedione-2,5-dicarboxylic acid dimethyl ester as a skeleton and forms an acid-base sensitive pH fluorescent probe through protonation with a morpholine group. The probe can achieve high-efficiency luminescence in both solid and liquid phases, has a high fluorescence quantum yield, and gradually weakens with increasing pH. It has good anti-interference ability and resistance to photobleaching, and has low cytotoxicity. Cell imaging experiments have shown that the probe can effectively locate in lysosomes and can detect changes in different pH values ​​in lysosomes and zebrafish. However, this pH fluorescent probe does not have a clear pH transition point. Although it can target lysosomes, it cannot effectively distinguish between normal cells and tumor cells, and its application prospects are limited.

[0006] Prior art 2 (CN117777018A) discloses a dual-channel fluorescent probe for detecting viscosity and pH, its preparation method, and its application in selective dynamic visualization of viscosity and pH in biological cell systems and in the preparation of tumor cell imaging detection preparations. The fluorescent probe prepared in this invention exhibits strong specificity, good selectivity, excellent photostability, and excellent dual-targeting capabilities for mitochondria and lysosomes. However, the synthesis steps of this dual-channel fluorescent probe for detecting viscosity and pH are relatively complex and the conditions are relatively harsh.

[0007] Therefore, combined with the above analysis, the AIE fluorescent probes in the relevant existing technologies still have the following defects that need to be improved: First, the fluorescent probes after group modification of the AIE molecules are difficult to achieve response aggregation in the acidic part of the tumor, but instead show the characteristics of alkaline aggregation and acidic dispersion, and their fluorescence intensity is weak at pH 7.4 in the normal physiological environment and is far from meeting the requirements of cell imaging. The reason is that a single simple group modification cannot effectively regulate the intramolecular rotation process of tetraphenylethylene (TPE), resulting in the inability to obtain the ideal pH transition point and transition range. Second, although the fluorescent probes developed using near-infrared excitation, ultrasound, protein labeling and other conditions can achieve the process of tumor cell imaging, the response range is wide and there is a lack of a clear pH transition point. The preparation process is relatively cumbersome and causes irreversible damage to the human body. The reason is that the introduction of multiple properties at the same time makes the preparation process of the fluorescent probe complicated, and it is necessary to rely on relatively strong external stimulation to achieve the purpose of in vivo imaging.

[0008] Therefore, in view of the shortcomings of existing fluorescence imaging technologies such as low signal-to-noise ratio, high cytotoxicity and aggregation-induced quenching, it is necessary to develop an AIE fluorescent probe that responds to the acidic microenvironment of tumors, so as to solve the problem of low imaging sensitivity in tumor sites, so as to achieve pH-responsive fluorescence imaging effects and provide an effective method for targeting the acidic microenvironment of tumors. Summary of the Invention

[0009] To address at least one of the above-mentioned issues, the present invention provides a slightly acidic environment-responsive AIE fluorescent probe, its preparation method, and its application. The small-molecule fluorescent probe prepared in this solution utilizes a slightly acidic pH-responsive group design to enhance its detection sensitivity in tumor imaging. When the fluorescent probe reaches the tumor site, the slightly acidic environment of the tumor causes the amide bond to protonate, resulting in overall charge neutrality. In the aggregated state, it emits bright fluorescence within the cell, enabling safe and effective selective fluorescence imaging of tumor cells.

[0010] One of the purposes of the present invention is to provide a slightly acidic environment-responsive AIE fluorescent probe, the general structural formula of which is:

[0011]

[0012] Wherein, R is selected from One of them.

[0013] As a preferred embodiment, the structural formula of the fluorescent probe is:

[0014]

[0015] A second object of the present invention is to provide a method for preparing a slightly acidic environment-responsive AIE fluorescent probe, comprising the following steps:

[0016] Step 1: dissolving the sulfonamide compound in a certain amount of solvent, adding triethylamine in proportion, placing in an ice-water bath and stirring for 10-30 minutes to obtain a first solution;

[0017] Step 2: dissolving a certain amount of acyl chloride solid in a certain amount of solvent to obtain a second solution, and adding the second solution dropwise to the first solution prepared in step 1 to prepare a mixed solution;

[0018] Step 3: After the second solution is added dropwise, the mixed solution prepared in step 2 is stirred at a certain reaction temperature for 48-72 hours, and the residue after evaporating the solvent of the mixed solution after the reaction is completed is purified and dried to obtain a fluorescent probe compound.

[0019] As a preferred embodiment, in step 2, the preparation method of the acyl chloride solid is as follows: dissolving 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene in dichloride according to a proportion; and reacting the resulting solution at reflux temperature for 24-48 hours; after the reaction is completed, cooling the reaction solution to room temperature, and then removing the solvent to obtain a solid acyl chloride.

[0020] As a preferred embodiment, 1 g of 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene is added to 70-100 mL of thionyl chloride.

[0021] As a preferred embodiment, the molar ratio of 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene:sulfonamide compound:triethylamine is in the range of 1:4-8:8-12, and the sulfonamide compound is selected from one of benzenesulfonamide, sulfacetamide or sulfadoxine; preferably, the sulfonamide compound is sulfadoxine, and the molar ratio of 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene:sulfadoxine:triethylamine is in the range of 1:4-8:8-12.

[0022] As a preferred embodiment, in the step 3, the reaction temperature is 25-50°C.

[0023] As a preferred solution, the following steps are included:

[0024] Step 1: dissolving sulfadoxine in a certain amount of tetrahydrofuran, adding triethylamine in proportion, placing in an ice-water bath and stirring for 10-30 minutes to obtain a first solution;

[0025] Step 2: dissolving a certain amount of acyl chloride solid in a certain amount of tetrahydrofuran to obtain a second solution, and adding the second solution dropwise to the first solution prepared in step 1 to prepare a mixed solution;

[0026] Step 3: After the second solution is added dropwise, the mixed solution prepared in step 2 is stirred at 50° C. for 48-72 hours, and the residue after evaporating the solvent from the mixed solution is purified and dried by silica gel column chromatography to obtain the fluorescent probe compound TPE-SDOX.

[0027] A third object of the present invention is to provide an application of a slightly acidic environment-responsive AIE fluorescent probe in tumor cell imaging.

[0028] As a preferred solution, the small molecule fluorescent probe and tumor cells are co-cultured. Preferably, the small molecule fluorescent probe is TPE-SDOX.

[0029] Compared with the prior art, the present invention has one of the following beneficial effects:

[0030] First, the fluorescent probes in this scheme exhibit good biosafety and low toxic side effects, can target the acidic microenvironment of tumors for cell fluorescence imaging, and have excellent cell penetration and uptake capabilities, providing an effective fluorescent probe compound for targeting the acidic microenvironment of tumors. The fluorescent probe compounds in this scheme utilize the different pKas of sulfonamide derivatives within the physiological range. Through the protonation and deprotonation process of the amide group of the small molecule fluorescent probe compound in different pH environments, the fluorescent probe responds to the acidic microenvironment of tumors for live cell fluorescence imaging, and successfully adjusts the pH transition point to the acidic microenvironment of tumors.

[0031] Secondly, this scheme optimizes the preparation process of fluorescent probe compounds, selects tetraphenylethylene as the AIE rotor, and prepares an AIE fluorescent probe that responds to the acidic microenvironment of the tumor. The AIE molecules represented by 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene (H4TCPE) undergo amidation reaction with sulfonamide molecules to successfully prepare acid-responsive AIE small molecule fluorescent probes, tetraphenylethylene-benzenesulfonamide (TPE-SA), tetraphenylethylene-sulfacetamide (TPE-SAC) and tetraphenylethylene-sulfadoxine (TPE-SDOX). By strictly controlling the temperature index and reaction time, the solvent is evaporated under reduced pressure and the residue is purified and dried to obtain the small molecule fluorescent probe compound of the present application. The small molecule fluorescent probe prepared by this scheme is designed with a slightly acidic pH response group to improve its detection sensitivity in tumor imaging.

[0032] Third, the fluorescent probe prepared by the present invention exhibits good biosafety and low toxic side effects. It can target the acidic microenvironment of tumors for cell fluorescence imaging and has excellent cell penetration and uptake ability. This provides an effective method for targeting the acidic microenvironment of tumors. By co-culturing the fluorescent probe with tumor cells and adjusting the pH value of the culture medium, the effect of aggregation-induced luminescence imaging is achieved. Specifically, in the slightly acidic environment of the tumor, the amide bond is protonated, resulting in overall neutrality. In the aggregated state, it emits bright fluorescence within the cell, achieving safe and effective selective fluorescence imaging of tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is the H NMR spectrum of H4TCPE in the present invention;

[0034] Figure 2 This is the H NMR spectrum of TPE-SA prepared in the embodiment of the present invention;

[0035] Figure 3 This is the H NMR spectrum of TPE-SAC prepared in an embodiment of the present invention;

[0036] Figure 4 This is the H NMR spectrum of TPE-SDOX prepared in the embodiment of the present invention;

[0037] Figure 5 FL spectra of H4TCPE in DMSO / H2O mixtures containing different volume fractions of water;

[0038] Figure 6 The relative FL intensity diagram of H4TCPE in DMSO / H2O mixtures containing different volume fractions of water;

[0039] Figure 7 FL spectra of TPE-SA in DMSO / H2O mixtures containing different volume fractions of water;

[0040] Figure 8 is the relative FL intensity diagram of TPE-SA in DMSO / H2O mixtures containing different volume fractions of water;

[0041] Figure 9 FL spectra of TPE-SAC in DMSO / H2O mixtures containing different volume fractions of water;

[0042] Figure 10 The relative FL intensity diagram of TPE-SAC in DMSO / H2O mixtures containing different volume fractions of water;

[0043] Figure 11 FL spectra of TPE-SDOX in DMSO / H2O mixtures containing different volume fractions of water;

[0044] Figure 12 is the relative FL intensity diagram of TPE-SDOX in DMSO / H2O mixtures containing different volume fractions of water;

[0045] Figure 13 is the fluorescence spectra of H4TCPE in buffers with different pH values;

[0046] Figure 14 is the graph showing the change of H4TCPE fluorescence intensity with pH value;

[0047] Figure 15 The fluorescence characteristics of H4TCPE in buffer solutions with different pH values.

[0048] Figure 16 is the fluorescence spectra of TPE-SA in buffers with different pH values;

[0049] Figure 17 is the graph showing the change of TPE-SA fluorescence intensity with pH value;

[0050] Figure 18 is the fluorescence characteristics of TPE-SA in different pH buffers;

[0051] Figure 19 Fluorescence spectra of TPE-SAC in buffers with different pH values;

[0052] Figure 20 is the graph showing the change of TPE-SAC fluorescence intensity with pH value;

[0053] Figure 21 is the fluorescence characteristics of TPE-SAC in different pH buffers;

[0054] Figure 22 Fluorescence spectra of TPE-SDOX in buffers with different pH values;

[0055] Figure 23 is the graph showing the change of TPE-SDOX fluorescence intensity with pH value;

[0056] Figure 24 is the fluorescence characteristics of TPE-SDOX in different pH buffers;

[0057] Figure 25 The H4TCPE stock solution was mixed with PBS buffer solution at pH 4.96 and DMEM solution to prepare a solution with a water content of 98%; the relationship between the fluorescence intensity and time was plotted based on the maximum fluorescence emission intensity;

[0058] Figure 26The TPE-SA mother solution was mixed with a PBS buffer solution with a pH of 5.75 and a DMEM solution to prepare a solution with a water content of 98%; a graph showing the relationship between fluorescence intensity and time was drawn based on the maximum fluorescence emission intensity;

[0059] Figure 27 TPE-SAC was mixed with PBS buffer solution at pH 6.27 and DMEM solution to prepare a solution with a water content of 98%; the relationship between fluorescence intensity and time was plotted based on the maximum fluorescence emission intensity;

[0060] Figure 28 TPE-SDOX was mixed with PBS buffer solution at pH 6.7 and DMEM solution to prepare a solution with a water content of 98%; the relationship between fluorescence intensity and time was plotted based on the maximum fluorescence emission intensity;

[0061] Figure 29 The relationship between fluorescence intensity and cycle number was plotted based on the maximum fluorescence emission intensity for H4TCPE stock solution between pH 4.0 and 5.5;

[0062] Figure 30 The relationship between fluorescence intensity and cycle number was plotted based on the maximum fluorescence emission intensity for TPE-SA stock solution between pH 5.0 and 6.0;

[0063] Figure 31 The relationship between fluorescence intensity and cycle number was plotted based on the maximum fluorescence emission intensity for TPE-SAC stock solution between pH 5.5 and 6.5;

[0064] Figure 32 The relationship between fluorescence intensity and cycle number was plotted based on the maximum fluorescence emission intensity for TPE-SDOX stock solution between pH 6.5 and 7.4;

[0065] Figure 33 This is a graph showing the effects of different concentrations of small molecule fluorescent probes on the cytotoxicity of L02 cells;

[0066] Figure 34 This is a graph showing the effect of different concentrations of small molecule fluorescent probes on the cytotoxicity of HepG2 cells;

[0067] Figure 35 Fluorescence images of HepG2 and L02 cells after H4TCPE treatment; scale bar: 20 μm;

[0068] Figure 36 Fluorescence images of HepG2 and L02 cells after TPE-SDOX treatment; scale bar: 20 μm. DETAILED DESCRIPTION

[0069] In order to make the technical means, creative features, objectives and beneficial effects achieved by the present invention easier to understand, the present invention is further described below in conjunction with specific implementation methods.

[0070] In addition, in order to better illustrate the present invention, numerous specific details are given in the specific embodiments below. Those skilled in the art should understand that the present invention can also be implemented without certain specific details. In other embodiments, methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present invention.

[0071] The present invention provides an AIE fluorescent probe and a preparation method of the slightly acidic environment-responsive AIE fluorescent probe, comprising the following steps: step 1, dissolving a sulfonamide compound in a certain amount of anhydrous tetrahydrofuran, adding triethylamine according to a proportion, placing the mixture in an ice-water bath and stirring for 10-30 minutes to obtain a first solution; step 2, dissolving a certain amount of acyl chloride solid in a certain amount of anhydrous tetrahydrofuran to obtain a second solution, and dropwise adding the second solution to the first solution obtained in step 1, thereby preparing a mixed solution. The preparation method of the acyl chloride solid used in this step 2 is as follows: 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene is dissolved in SOCl2, wherein each 1g of 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene is added to 70-100mL of dichlorothionyl (SOCl2), and the mixture is reacted at reflux temperature for 24-48h for activation; after the activation reaction is completed, the mixture is cooled to room temperature, and the solvent is evaporated under reduced pressure to obtain a solid acyl chloride; in step 3, after the second solution is added dropwise, the mixed solution prepared in step 2 is stirred at 25-50°C for 48-72h, and the residue after evaporation of the solvent is purified and dried by silica gel column chromatography to obtain the corresponding fluorescent probe compound.

[0072] The molar ratio of 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene:sulfonamide compound:triethylamine in the above steps is in the range of 1:4-8:8-12, and the sulfonamide compound is selected from one of benzenesulfonamide, sulfacetamide or sulfadoxine; preferably, the sulfonamide compound is sulfadoxine.

[0073] In the present invention, the sulfonamide compound is benzenesulfonamide. After the solvent of the product obtained in step 3 is evaporated under reduced pressure, the residue is purified and dried by silica gel column chromatography (DCM: MeOH = 5: 1) to obtain TPE-SA.

[0074] In the present invention, the sulfonamide compound is sulfacetamide. After the solvent of the product obtained in step 3 is evaporated under reduced pressure, the residue is purified and dried by silica gel column chromatography (DCM: MeOH = 5: 1) to obtain TPE-SAC.

[0075] Preferably, the sulfonamide compound is sulfadoxine. After evaporating the solvent under reduced pressure, the residue of the product obtained in step 3 is purified and dried by silica gel column chromatography (EtOAc:DCM=10:1) to obtain TPE-SDOX.

[0076] Preferably, the working concentration of the fluorescent probe solution is: the fluorescent probe synthesized by TPE molecules and sulfonamide compounds is dissolved in DMSO at a concentration of 20 μM.

[0077] Example 1

[0078] 1,1,2,2-Tetrakis(4-carboxyphenyl)ethylene (0.1 g, 0.196 mmol) was dissolved in SOCl2 (8 mL) and reacted at reflux temperature for 48 h. The mixture was cooled to room temperature and the solvent was evaporated under reduced pressure to obtain a solid acid chloride.

[0079] Example 2

[0080] 1,1,2,2-Tetrakis(4-carboxyphenyl)ethylene (0.1 g, 0.196 mmol) was dissolved in SOCl2 (10 mL) and reacted at reflux temperature for 24 h. The mixture was cooled to room temperature and the solvent was evaporated under reduced pressure to obtain solid acid chloride.

[0081] Example 3

[0082] 1,1,2,2-Tetrakis(4-carboxyphenyl)ethylene (0.1 g, 0.196 mmol) was dissolved in SOCl2 (7 mL) and reacted at reflux temperature for 48 h. The mixture was cooled to room temperature and the solvent was evaporated under reduced pressure to obtain a solid acid chloride.

[0083] Wherein, the reaction equation of embodiment 1-3 is as follows:

[0084]

[0085] Example 4

[0086] Dissolve 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene (0.1 g, 0.196 mmol) in SOCl2 (8 mL) and react at reflux (80°C) for 48 h. Cool to room temperature and evaporate the solvent under reduced pressure to obtain a solid acid chloride. Dissolve sulfadoxine (0.366 g, 1.176 mmol) in anhydrous tetrahydrofuran (3 mL) and add triethylamine (0.218 mL, 1.568 mmol) to obtain a first solution. Place the first solution in an ice-water bath and stir for 30 min. Dissolve the solid acid chloride prepared above in 3 mL of anhydrous tetrahydrofuran to obtain a second solution. Add the second solution dropwise to the first solution to obtain a mixed solution. Stir the mixed solution at 50°C for 72 h, then evaporate the solvent under reduced pressure. Subsequently, the residue after evaporation of the solvent was purified and dried by silica gel column chromatography (EtOAc:DCM=10:1) to obtain TPE-SDOX with a mass of 0.257 g, a yield of 77.85%, and a purity of 85.26%.

[0087] Example 5

[0088] Dissolve 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene (0.1 g, 0.196 mmol) in SOCl2 (8 mL) and react at reflux temperature (80°C) for 48 h. Cool to room temperature and evaporate the solvent under reduced pressure to obtain a solid acid chloride. Dissolve sulfadoxine (0.244 g, 0.784 mmol) in anhydrous tetrahydrofuran (3 mL) and add triethylamine (0.272 mL, 1.96 mmol) to obtain a first solution. Place the first solution in an ice-water bath and stir for 30 min. Dissolve the solid acid chloride prepared above in 3 mL of anhydrous tetrahydrofuran to obtain a second solution. Add the second solution dropwise to the first solution to obtain a mixed solution. Stir the mixed solution at 50°C for 60 h, then evaporate the solvent under reduced pressure. Subsequently, the residue after evaporation of the solvent was purified and dried by silica gel column chromatography (EtOAc:DCM=10:1) to obtain TPE-SDOX with a mass of 0.2214 g, a yield of 67.07%, and a purity of 80.26%.

[0089] Example 6

[0090] Dissolve 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene (0.1 g, 0.196 mmol) in SOCl2 (8 mL) and react at reflux for 48 h. Cool to room temperature and evaporate the solvent under reduced pressure to obtain a solid acid chloride. Dissolve sulfadoxine (0.366 g, 1.176 mmol) in anhydrous tetrahydrofuran (3 mL) and add triethylamine (0.218 mL, 1.568 mmol) to obtain a first solution. Place the first solution in an ice-water bath and stir for 30 min. Dissolve the solid acid chloride prepared above in 3 mL of anhydrous tetrahydrofuran to obtain a second solution. Add the second solution dropwise to the first solution to obtain a mixed solution. Stir the mixed solution at 50°C for 48 h, then evaporate the solvent under reduced pressure. Subsequently, the residue after evaporation of the solvent was purified and dried by silica gel column chromatography (EtOAc:DCM=10:1) to obtain TPE-SDOX with a mass of 0.269 g, a yield of 81.49%, and a purity of 89.6%.

[0091] Example 7

[0092] Dissolve 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene (0.1 g, 0.196 mmol) in SOCl2 (8 mL) and react at reflux for 48 h. Cool to room temperature and evaporate the solvent under reduced pressure to obtain a solid acid chloride. Dissolve sulfadoxine (0.488 g, 1.568 mmol) in anhydrous tetrahydrofuran (3 mL) and add triethylamine (0.327 mL, 2.352 mmol) to obtain a first solution, which is stirred in an ice-water bath for 30 min. Dissolve the resulting solid acid chloride in 3 mL of anhydrous tetrahydrofuran to obtain a second solution, which is added dropwise to the first solution to obtain a mixed solution. Stir the mixed solution at 50°C for 72 h, and then evaporate the solvent under reduced pressure. Subsequently, the residue after evaporation of the solvent was purified and dried by silica gel column chromatography (EtOAc:DCM=10:1) to obtain TPE-SDOX with a mass of 0.249 g, a yield of 75.4%, and a purity of 87.9%.

[0093] Wherein, the reaction formula of Example 4-7 is as follows:

[0094]

[0095] Figure 4 This is the H NMR spectrum of TPE-SDOX prepared in Example 7 (using deuterated dimethyl sulfoxide as solvent).

[0096] Example 8

[0097] 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene (0.1 g, 0.196 mmol) was dissolved in SOCl2 (8 mL) and reacted at reflux temperature for 48 h. After cooling to room temperature, the solvent was evaporated under reduced pressure to obtain a solid acid chloride. Benzenesulfonamide (0.185 g, 1.176 mmol) was dissolved in anhydrous tetrahydrofuran (3 mL), and triethylamine (0.218 mL, 1.568 mmol) was added to obtain a first solution. The obtained first solution was placed in an ice-water bath and stirred for 30 min. The acid chloride solid prepared above was completely dissolved in 3 mL of anhydrous tetrahydrofuran to obtain a second solution. The obtained second solution was added dropwise to the above-mentioned first solution to obtain a mixed solution. The mixed solution was stirred at room temperature for 72 h, and the solvent was evaporated under reduced pressure. Subsequently, the residue after evaporation of the solvent was purified and dried by silica gel column chromatography (DCM:MeOH=5:1) to obtain TPE-SA. The product mass was 0.165 g, the product yield was 78.7%, and the product purity was 91.6%. The reaction equation is as follows:

[0098]

[0099] Figure 2 This is the H NMR spectrum of TPE-SA prepared in Example 8 (using deuterated dimethyl sulfoxide as solvent).

[0100] Example 9

[0101] Dissolve 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene (0.1 g, 0.196 mmol) in SOCl2 (8 mL) and react at reflux for 48 h. Cool to room temperature and evaporate the solvent under reduced pressure to obtain a solid acid chloride. Dissolve sulfacetamide (0.253 g, 1.176 mmol) in anhydrous tetrahydrofuran (3 mL) and add triethylamine (0.218 mL, 1.568 mmol) to obtain a first solution. Place the first solution in an ice-water bath and stir for 30 min. Dissolve the solid acid chloride prepared above in 3 mL of anhydrous tetrahydrofuran to obtain a second solution. Add the second solution dropwise to the first solution to obtain a mixed solution. Stir the mixed solution at 50°C for 72 h, then evaporate the solvent under reduced pressure. Subsequently, the solvent was evaporated and the residue was purified and dried via silica gel column chromatography (DCM:MeOH = 5:1) to obtain TPE-SAC. The product mass was 0.179 g, the yield was 70.3%, and the purity was 86.4%. The reaction equation is as follows:

[0102]

[0103] Figure 3 This is the H NMR spectrum of TPE-SAC prepared in Example 9 (using deuterated dimethyl sulfoxide as solvent).

[0104] Comparative Example 1

[0105] 1,1,2,2-Tetrakis(4-carboxyphenyl)ethylene (0.1 g, 0.196 mmol) was dissolved in SOCl2 (8 mL) and reacted at reflux temperature for 48 h. After cooling to room temperature, the solvent was evaporated under reduced pressure to obtain a solid acid chloride. Sulfathiazole (0.301 g, 1.176 mmol) was dissolved in anhydrous tetrahydrofuran (3 mL), and triethylamine (0.218 mL, 1.568 mmol) was added to obtain a first solution. The obtained first solution was placed in an ice-water bath and stirred for 30 min. The obtained solid acid chloride was completely dissolved in 3 mL of anhydrous tetrahydrofuran to obtain a second solution. The obtained second solution was added dropwise to the above-mentioned first solution to obtain a mixed solution. The mixed solution was stirred at 50°C for 72 h, and the solvent was evaporated under reduced pressure. Subsequently, the residue after evaporation of the solvent was purified and dried by silica gel column chromatography to obtain TPE-STZ with a product mass of 0.207 g, a product yield of 72%, and a product purity of 79.94%. The reaction formula is as follows:

[0106] Comparative Example 2

[0107] 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene (0.1 g, 0.196 mmol) was dissolved in SOCl2 (8 mL) and reacted at reflux temperature for 48 h. After cooling to room temperature, the solvent was evaporated under reduced pressure to obtain a solid acid chloride. Sulfadimethazine (0.328 g, 1.176 mmol) was dissolved in anhydrous tetrahydrofuran (3 mL), and triethylamine (0.218 mL, 1.568 mmol) was added to obtain a first solution, which was placed in an ice-water bath and stirred for 30 min. The above-obtained solid acid chloride was completely dissolved in 3 mL of anhydrous tetrahydrofuran to obtain a second solution, which was added dropwise to the above-obtained first solution to obtain a mixed solution. The mixed solution was stirred at 50 ° C for 72 h, and the solvent was evaporated under reduced pressure. Subsequently, the residue was purified and dried by silica gel column chromatography to obtain TPE-SMZ, with a product mass of 0.235 g, a product yield of 77.02%, and a product purity of 67.63%. The reaction equation is as follows:

[0108]

[0109] This program, through 1H NMR and FTIR characterizations confirmed the successful preparation of the fluorescent probes. The fluorescence intensities of solutions with different water contents were measured by a fluorescence spectrophotometer, confirming that TPE-SA, TPE-SAC, and TPE-SDOX had excellent AIE effects. Further measurements of fluorescence intensity in buffer solutions with different pH values ​​revealed that the pH transition points of TPE-SA, TPE-SAC, and TPE-SDOX were 5.76±0.02, 6.09±0.03, and 6.83±0.02, respectively, demonstrating that the pH transition point of the H4TCPE aggregation luminescence response could be adjusted to the tumor microenvironment by modifying the TPE molecules with sulfonamide groups.

[0110] In this scheme, the above-mentioned Examples 4-9 combine TPE with sulfonamide compounds through an amidation reaction to successfully synthesize TPE-SA, TPE-SAC and TPE-SDOX. Among them, the TPE-SDOX in Example 4-7 adjusts the pH transition point to the slightly acidic environment of the tumor, so that the AIE phenomenon occurs at the tumor site for tumor cell imaging. The obtained fluorescent probe compound is dissolved in a solvent and injected into the tumor site to prepare a slightly acid-responsive intelligent fluorescent imaging probe. This scheme uses a small molecule fluorescent probe to co-incubate with tumor cells, which can achieve rapid charge conversion to enhance cell uptake and accurate fluorescence imaging when incubated with cancer cells. The small molecule fluorescent probe reaches the tumor site and, in the slightly acidic environment of the tumor, causes the amide bond to be protonated to form CONH 2+ , introducing a positive charge, while C=O and S=O=S are electron-withdrawing groups, resulting in overall neutrality. In the aggregated state, intramolecular rotation is restricted, resulting in the release of excited-state energy as luminescence, leading to bright fluorescence within cells. The TPE-SDOX probe exhibits sensitive pH response and a superior AIE effect, enabling safe and effective selective fluorescence imaging of tumor cells.

[0111] Biocompatibility evaluation

[0112] The present invention co-cultures small molecule fluorescent probes with cells. HepG2 and L02 cells were cultured separately at 37°C in a 5% CO2 incubator in DMEM medium containing 10% FBS and 1% antibiotics (including 100 U / mL penicillin and 100 μg / mL streptomycin). By co-culturing the small molecule fluorescent probes with tumor cells and adjusting the pH of the culture medium, the present invention achieves aggregation-induced luminescence imaging (AIE) effects.

[0113] MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) is a commonly used cytotoxicity assay method for evaluating the effects of small molecule fluorescent probes on cell viability. When L02 and HepG2 cells in a T75 culture box grew to 80%, trypsin was added to digest and wash the adherent cells. After adding fresh culture medium to fully disperse the cells, they were seeded into a 96-well plate at a concentration of 8000 cells / well, maintaining a final culture medium volume of 200μL per well. After culturing for 24 hours, the cell density in the well was observed to reach 70%. Subsequently, different concentrations of H4TCPE, TPE-SA, TPE-SAC, and TPE-SDOX were mixed with the cells and incubated for 24 hours, maintaining the final culture medium concentration at 10, 20, 40, 60, 80, and 100μM. Subsequently, 10μL MTT (5mg / mL) was added to each well. After incubation at 37°C in a CO2 incubator for 4 hours, the MTT reagent and the metabolites produced by the cells will form blue-purple formazan crystals scattered on the bottom of the culture dish. Then, the culture medium is removed and the blue-purple formazan crystals at the bottom are dissolved with 180 μL of isopropanol. The absorbance (OD) value at 570 nm is measured using an ELISA plate reader. The cell viability (%) is calculated as follows:

[0114]

[0115] OD sample is the absorbance after treatment with fluorescent probe, OD control The absorbance is the value before treatment with fluorescent probe. The cytotoxicity experiment was repeated three times.

[0116] like Figure 33 、 34 As shown in Figure 2, this example demonstrates in vitro biocompatibility testing, using the MTT assay to investigate the biocompatibility of H4TCPE and the small molecule fluorescent probes from Examples 7, 8, and 9 with L02 and HepG2 cell lines. As shown in the figure, after 24 hours of co-culture with HepG2 and L02 cells, cell viability remained above 90%, even at concentrations as high as 100 μM. These results demonstrate that the fluorescent probes exhibit minimal cellular toxicity. Even at high concentrations, the AIE fluorescent probes maintain cell viability, providing excellent biosafety for cell imaging.

[0117] Evaluation of cellular uptake

[0118] In order to evaluate the cellular uptake of H4TCPE and TPE-SDOX fluorescent probes, the present invention used a fluorescent inverted microscope to observe L02 and HepG2 cells. First, the coverslip was soaked in aqua regia and sterilized with high-pressure steam. The coverslip was then placed on a well plate and rinsed with PBS solution to prevent bilateral cell growth from affecting the sealing and imaging effects. Subsequently, 0.5 mL of culture medium was added to each well to ensure that the cells were evenly dispersed in the 24-well plate. Each well was inoculated with 2×10 5 When the cell density in the well plate reaches approximately 70%, the original culture medium can be replaced with DMEM adjusted to pH 6.5 and 20 μL of H4TCPE or TPE-SDOX in DMSO can be added. (The entire process is: aspirate the original culture medium, add 980 μL of pH 6.5 culture medium, and add 20 μL of the probe. The total volume is 1 mL, and the final concentration of the fluorescent probe is 20 μM.) During the culture medium replacement process, the well plate is washed with sterile PBS to reduce serum residue. After incubating the cells in a CO2 incubator for various periods of time, the culture medium is aspirated and the well plate is washed three times with PBS. Subsequently, paraformaldehyde solution (4% PFA) suitable for covering a coverslip is added and incubated for 10 minutes to fix the cells. After aspirating the PFA, the cells are washed three times with cell-grade PBS buffer (buffer model P1020). 20 μL of anti-quenching mounting solution is applied to the surface of the slide. The coverslip is removed using a cell clamp and placed on an inverted slide. Subsequently, non-fluorescent microscope oil was dropped on the surface of the coverslip, and the cell uptake was observed using a fluorescence inverted microscope.

[0119] like Figure 35 、 36 As shown, this embodiment is a cell uptake performance experimental embodiment, which uses a cell uptake experiment to evaluate the fluorescence imaging effect of the small molecule fluorescent probe TPE-SDOX prepared in Example 7 on L02 normal cells and HepG2 cancer cells. Compared with the treated L02 cells, the HepG2 cells treated with TPE-SDOX showed a significantly increased fluorescence brightness. However, neither the L02 nor the HepG2 cells treated with H4TCPE showed obvious fluorescence signals. The tumor microenvironment is often acidic (about 6.2-6.9). When TPE-SDOX reaches the tumor site, it is closer to the pH conversion point of TPE-SDOX, and therefore can produce a stronger and more obvious fluorescence signal.

[0120] AIE characteristics evaluation

[0121] like Figure 5-12As shown, the FL spectra and relative FL intensity diagram (fw) of H4TCPE (a, b), TPE-SA prepared in Example 8 (c, d), TPE-SAC prepared in Example 9 (e, f) and TPE-SDOX prepared in Example 7 (g, h) in DMSO / H2O mixtures containing different volume fractions of water, respectively, where Figure 6 、 8 The inset images in Figures 10 and 12 correspond to the fluorescence images of H4TCPE, TPE-SA, TPE-SAC and TPE-SDOX in pure DMSO (the volume fraction of water is fw = 0%) and a certain volume of DMSO / H2O mixture (the volume fraction of water is fw = 98%), respectively. Figure 5-12 The detection conditions are as follows: excitation wavelength: 365 nm, probe concentration in solution: 20 μM. Detection equipment: F96 Pro fluorescence spectrophotometer (Shanghai Lingguang Technology Co., Ltd.).

[0122] As can be seen from the figure, in the good solvent pure DMSO, the small molecule fluorescent probe will produce weak fluorescence emission. However, when the poor solvent water is introduced, the fluorescence intensity of the small molecule fluorescent probe is significantly enhanced. When the water content continues to increase, the fluorescence intensity of H4TCPE, TPE-SA, TPE-SAC and TPE-SDOX shows a clear upward trend. The volume fraction of water is fw = 98%. The fluorescence intensity of each fluorescent probe solution is 131, 98, 139 and 265 times the corresponding fluorescence intensity value in the fluorescent probe solution containing pure DMSO. Compared with the fluorescent probe solution of pure DMSO solvent (that is, the volume fraction of water is fw = 0%), the small molecule fluorescent probe with DMSO / H2O mixture (the volume fraction of water is fw = 98%) as the solvent exhibits stronger fluorescence intensity and has unique AIE characteristics. The AIE characteristics of H4TCPE, TPE-SA, TPE-SAC and TPE-SDOX solutions were observed by irradiation with 365nm ultraviolet light. As shown Figure 6 、 8 As shown in the insets in Figures 10, 12, H4TCPE, TPE-SA, TPE-SAC, and TPE-SDOX exhibit almost no visible fluorescence in pure DMSO, but exhibit significant fluorescence emission in a DMSO / H2O mixture (water volume fraction fw = 98%). These results demonstrate that the small-molecule fluorescent probes synthesized by amidation of various sulfonamide groups with TPE retain the excellent AIE properties inherent in the TPE molecule.

[0123] Evaluation of fluorescence intensity of fluorescent probes under different pH solution conditions

[0124] like Figure 13 、 16, 19, 22 show the fluorescence spectra of H4TCPE, TPE-SA, TPE-SAC and TPE-SDOX in buffers with different pH values. In the pH range of 8.98-1.06, the fluorescence intensities of H4TCPE, TPE-SA, TPE-SAC and TPE-SDOX conform to the Sigmoid Boltzmann function (e.g. Figure 14 、 17 , 20, 23). Figure 13-24 In the case of , the excitation wavelength is 365 nm, Figure 14 As shown in Figure 3, the pH transition point of H4TCPE is around pH 4.91, which is beyond the normal physiological range (tumor microenvironment pH 6.2-6.9).

[0125] The assay stock solutions for this protocol were prepared as follows: 10.5 mg of TPE-SDOX was dissolved in 1 mL of DMSO to obtain a 6.25 mM stock solution C1; 5 mg of TPE-SA was dissolved in 0.6183 mL of DMSO to obtain a 6.25 mM stock solution C1; and 3 mg of H4TCPE was dissolved in 0.944 mL of DMSO to obtain a 6.25 mM stock solution C1. Fluorescence spectrometry detection was performed as follows: First, 320 μL of the corresponding 6.25 mM stock solution C1 was dissolved in 1.68 mL of DMSO to dilute the stock solution C2 to a 1 mM concentration. Then, 60 μL of the 1 mM stock solution C2 was added to 2.94 mL of PBS solutions (DMSO / PBS = 2 / 98) at different pH values ​​(4.96, 5.75, 6.27, and 6.7). The final concentration was 20 μM, and the excitation wavelength was 365 nm. In this protocol, PBS buffer solutions of varying pH values ​​were prepared by preparing 0.01M standard PBS solutions of pH 7.2-7.4, adding 1M HCl, adjusting the pH, and measuring the pH using a pH meter. In this protocol, PBS buffer solutions or DMEM solutions with pH values ​​of 4.96, 5.75, 6.27, and 6.7 were prepared as follows: varying amounts of 1M HCl were added to purchased standard PBS solutions / DMEM to adjust the solutions to the corresponding pH values, and the pH values ​​were measured using a pH meter.

[0126] The present invention, Figure 14 、 17, 20, 23 are plotted based on the following test data. The pH values ​​of the fluorescence intensity measured by H4TCPE at different pH values ​​are 1.06, 1.56, 2.06, 2.55, 3.07, 3.5, 4.04, 4.57, 4.96, 5.49, 6.08, 6.52, 7.02, 7.4, 8.0, 8.49, and 8.98, respectively. The pH values ​​of the fluorescence intensity measured by TPE-SA at different pH values ​​are 1.06, 2.06, 3.07, 3.5, 4.00, 4.57, 4.96, 5.49, 5.78, 6.27, 6.52, 7.02, 7.4, 8.0, 8.49, and 8.98, respectively. The pH values ​​of the fluorescence intensity at different pH values ​​measured by TPE-SAC were 1.06, 2.06, 3.07, 3.88, 4.34, 4.96, 5.49, 5.78, 6.08, 6.27, 6.52, 7.4, 8.0, and 8.98, respectively. The pH values ​​of the fluorescence intensity at different pH values ​​measured by TPE-SDOX were 1.06, 2.06, 3.07, 3.43, 3.88, 4.34, 4.77, 5.26, 5.8, 5.95, 6.27, 6.58, 6.77, 6.95, 7.4, 7.6, 8.0, 8.49, and 8.98, respectively. The detection conditions were: excitation wavelength: 365 nm, and the concentration of the probe in the solution was 20 μM. The absorbance detection equipment used in this application was a F96Pro fluorescence spectrophotometer (Shanghai Lingguang Technology Co., Ltd.).

[0127] pH transition point of each fluorescent probe

[0128] This scheme successfully synthesized TPE-SA, TPE-SAC and TPE-SDOX compounds at the molecular level through precise modification of the sulfonamide group. By detecting the FL of TPE-SA and TPE-SAC in solutions with different pH values ​​and analyzing the relationship between FLmax and the different pH values ​​of the small molecule fluorescent probe, the TPE-SA prepared in Example 8 and the TPE-SAC prepared in Example 9 had obvious pH turning points of 5.76±0.02 and 6.09±0.03, respectively. However, these two compounds still failed to reach the expected pH variation range (tumor microenvironment pH 6.2-6.9) and therefore could not be used for tumor microenvironment detection. In contrast, Figure 22 、 23As shown in Figures 24 and 25, the TPE-SDOX prepared in Examples 4-7 showed a highly sensitive response to pH, with a pH transition point of 6.83±0.02. TPE-SDOX exhibited obvious AIE properties and sensitive pH response, and can be applied to target the tumor microenvironment, achieving the expected pH change range (pH 6.2-6.9). The pH response areas of H4TCPE, TPE-SA, TPE-SAC and TPE-SDOX were 4.59-5.24 (ΔpHt=0.65), 5.55-5.98 (ΔpHt=0.43), 5.78-6.43 (ΔpHt=0.65) and 6.59-7.08 (ΔpHt=0.49), respectively. These results indicate that H4TCPE, TPE-SA, TPE-SAC and TPE-SDOX can achieve sensitive pH detection over a wide pH range.

[0129] The pH transition point of the TPE-STZ obtained in the aforementioned Comparative Example 1 was measured to be 6.99±0.03 according to the above-mentioned detection method, and the pH transition point of the TPE-SMZ obtained in the aforementioned Comparative Example 2 was measured to be 7.24±0.03 according to the above-mentioned detection method. Therefore, both the TPE-STZ obtained in Comparative Example 1 and the TPE-SMZ obtained in Comparative Example 2 did not reach the expected pH variation range (pH 6.2-6.9).

[0130] Photostability evaluation

[0131] like Figure 25 、 26 As shown in Figures 27 and 28, H4TCPE, TPE-SA, TPE-SAC, and TPE-SDOX stock solutions C2 were mixed with PBS buffer or DMEM solutions at pH values ​​of 4.96, 5.75, 6.27, and 6.7 to prepare solutions with a water content of 98%. Fluorescence intensity of these solutions was measured at different time points, and the relationship between fluorescence intensity and time was plotted based on the maximum fluorescence emission intensity.

[0132] Since photostability is an important parameter for evaluating small molecule fluorescent probes, the FL changes of H4TCPE, TPE-SA, TPE-SAC and TPE-SDOX in DMEM and PBS solutions within 72 hours were detected by fluorescence spectrophotometer to investigate the photostability of small molecule fluorescent probes. Figures 25-28The resulting characterization graph shows that the small molecule fluorescent probe exhibits excellent fluorescence stability, as evidenced by the stable fluorescence intensity observed over 72 hours. These results demonstrate that the TPE fluorescent probe exhibits excellent stability. Unlike traditional fluorescent molecules, the small molecule fluorescent probe exhibits flexibility in solution and transforms into a rigid state upon aggregation. This carefully designed configuration enhances the stability of the molecule during aggregation and enables it to maintain a high level of fluorescence intensity for a long period of time.

[0133] Photoreversibility evaluation

[0134] like Figure 29 、 30 As shown in Figures 31 and 32, the H4TCPE, TPE-SA, TPE-SAC and TPE-SDOX stock solutions were mixed with PBS buffer solutions of different pH values ​​to prepare solutions with a water content of 98%, and 0.1 M HCl or NaOH was added sequentially to adjust the pH value of the small molecule fluorescent probe solution. The cycle was repeated for 5 cycles, and the fluorescence intensity of the small molecule fluorescent probe after each pH adjustment was measured. The relationship between the fluorescence intensity and the number of cycles was plotted based on the maximum fluorescence emission intensity.

[0135] In this scheme, the preparation method of the solution with a water content of 98% is as follows: 10.5 mg of TPE-SDOX was dissolved in 1 mL of DMSO to prepare a 6.25 mM mother solution C1; 5 mg of TPE-SA was dissolved in 0.751 mL of DMSO, and 5 mg of TPE-SAC was dissolved in 0.6185 mL of DMSO to prepare a 6.25 mM mother solution C1; 3 mg of H4TCPE was dissolved in 0.944 mL of DMSO to prepare a 6.25 mM mother solution C1; 320 uL of 6.25 mM H4TCPE, TPE-SA, TPE-SAC and TPE-SDOX mother solution C1 were respectively dissolved in 1.68 mL DMSO was diluted to obtain a 1 mM stock solution C2; 60 uL of the above 1 mM stock solution C2 was added to 2.94 mL of PBS solution of different pH values ​​(DMSO / PBS = 2 / 98) [water content 98%] to a final concentration of 20 uM, with an excitation wavelength of 365 nm.

[0136] According to Figure 29 、 30, 31, 32 analysis, the following conclusions were drawn: Based on the fact that photoreversibility is another important parameter for evaluating small molecule fluorescent probes, we adjusted the pH value of the solution by adding 0.1M HCl or NaOH, and studied the responses of H4TCPE, TPE-SA, TPE-SAC and TPE-SDOX to different pH values. The results showed that at different pH values, the above fluorescent probes all exhibited obvious reversible conversion processes and maintained effective fluorescence response conversion capabilities. Further observations found that even after multiple cycle experiments, the above small molecule fluorescent probes were still able to maintain their special reversibility and repeatable responsiveness. Whether under acidic or alkaline conditions, they exhibited the ability to stably and efficiently convert into corresponding fluorescent signals. This special reversibility and repeatable responsiveness make the above small molecule fluorescent probes ideal candidate substances that can be used in the biomedical field to monitor and diagnose cancer and other related diseases.

[0137] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as a preferred embodiment, it is not intended to limit the present invention. Any technician familiar with the present profession can make some changes or modifications to equivalent embodiments of equivalent changes using the technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A slightly acidic environment-responsive AIE fluorescent probe, characterized by: Its general structural formula is: Wherein, R is selected from ; or One of them.

2. The slightly acidic environment-responsive AIE fluorescent probe according to claim 1, characterized in that: The structural formula of the fluorescent probe is: The code name of the fluorescent probe in the above formula is TPE-SDOX.

3. The method for preparing a slightly acidic environment-responsive AIE fluorescent probe according to claim 1 or 2, wherein: The steps include: Step 1: dissolving the sulfonamide compound in a certain amount of solvent, adding triethylamine in proportion, placing in an ice-water bath and stirring for 10-30 minutes to obtain a first solution; Step 2: dissolving a certain amount of solid acyl chloride in a certain amount of solvent to obtain a second solution, and adding the second solution dropwise to the first solution prepared in step 1 to prepare a mixed solution; the preparation method of the solid acyl chloride is as follows: dissolving 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene in dichloride according to a certain proportion; and reacting the resulting solution at reflux temperature for 24-48 hours; after the reaction is completed, cooling the reaction solution to room temperature, and then removing the solvent to obtain solid acyl chloride; the molar ratio of 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene: sulfonamide compound: triethylamine is in a range of 1:4-8:8-12, and the sulfonamide compound is selected from one of benzenesulfonamide, sulfacetamide, or sulfadoxine; Step 3: After the second solution is added dropwise, the mixed solution prepared in step 2 is stirred at a certain reaction temperature for 48-72 hours, and the residue after evaporating the solvent of the mixed solution after the reaction is completed is purified and dried to obtain a fluorescent probe compound.

4. The method for preparing a slightly acidic environment-responsive AIE fluorescent probe according to claim 3, wherein: Add 1g of 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene into 70-100 mL of thionyl chloride.

5. The method for preparing a slightly acidic environment-responsive AIE fluorescent probe according to claim 3, wherein: The sulfonamide compound is sulfadoxine.

6. The method for preparing a slightly acidic environment-responsive AIE fluorescent probe according to claim 3, wherein: In the step 3, the reaction temperature is 25-50°C.

7. The method for preparing a slightly acidic environment-responsive AIE fluorescent probe according to claim 3, wherein: The steps include: Step 1: dissolving sulfadoxine in a certain amount of tetrahydrofuran, adding triethylamine in proportion, placing in an ice-water bath and stirring for 10-30 minutes to obtain a first solution; Step 2: dissolving a certain amount of acyl chloride solid in a certain amount of tetrahydrofuran to obtain a second solution, and adding the second solution dropwise to the first solution prepared in step 1 to prepare a mixed solution; Step 3: After the second solution is added dropwise, the mixed solution prepared in step 2 is stirred at a certain reaction temperature for 48-72 h, and the residue after evaporating the solvent of the mixed solution after the reaction is completed is purified and dried by silica gel column chromatography to obtain the fluorescent probe compound TPE-SDOX.

8. Use of a slightly acidic environment-responsive AIE fluorescent probe according to claim 1 or 2 in tumor cell imaging, wherein the use is for purposes other than diagnosis or treatment of a disease.

9. Use of a slightly acidic environment-responsive AIE fluorescent probe in tumor cell imaging according to claim 8, characterized in that: The small molecule fluorescent probe and tumor cells are co-cultured.

10. Use of a slightly acidic environment responsive AIE fluorescent probe in tumor cell imaging according to claim 9, characterized in that: The small molecule fluorescent probe is TPE-SDOX.

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