CN118772066A - A slightly acidic environment responsive AIE fluorescent probe and its preparation method and application - Google Patents

Abstract

The invention discloses a weak acid environment response AIE fluorescent probe and a preparation method and application thereof, comprising the following steps: step one: dissolving benzenesulfonamide, sulfacetamide or sulfadoxine in a certain amount of solvent, adding triethylamine according to a proportion, and placing the mixture in an ice water bath for stirring to obtain a first solution; step two: dissolving a certain amount of acyl chloride solid in a certain amount of solvent to obtain a second solution, and dripping the second solution into the first solution in the first step to obtain a mixed solution; step three: after the second solution is added dropwise, stirring the mixed solution obtained in the second step at a specific temperature for 48-72 h, and purifying and drying residues obtained after solvent evaporation to obtain the fluorescent probe compound. According to the invention, TPE and sulfonamide compounds are combined through amidation reaction, so that an AIE fluorescent probe is successfully synthesized, the probe shows sensitive pH response and excellent AIE effect, and safe and effective selective fluorescent imaging on tumor cells can be realized.

Description

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

Technical Field

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

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 speed, 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 complicated and time-consuming fluorescent protein transfection procedure.

Organic fluorescent materials have good application prospects in optoelectronic devices, detection, bioimaging and biotherapy due to their advantages such as low price, low toxicity, simple operation, easy structure adjustment, 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 planar conjugated structures, which are prone to π-π stacking in the aggregated state. In the excited state, the energy loss is serious, resulting in poor luminescence performance and aggregation-induced quenching (ACQ) problems. Although many types of fluorophores have been commercially used in bioimaging, their poor photostability, aggregation-induced quenching (ACQ) and complex fluorophore synthesis have hindered their further development.

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) characteristics 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 offset, good photostability and high signal-to-noise ratio, and can sensitively respond to tumor microenvironment characteristics for tumor tissue imaging. The acidic microenvironment is combined with ionizable chemical groups to respond to environmental pH changes through protonation or deprotonation. However, commonly used ionizable groups, such as carboxylic acids 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, lack of a clear pH transition point, and inability to accurately distinguish normal tissue from tumor tissue using differences in the slightly acidic environment of the tumor.

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

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

Therefore, combined with the above analysis, the AIE fluorescent probes in the related prior art still have the following defects to be improved: First, the fluorescent probes after the AIE molecules are modified with groups are difficult to achieve response aggregation in the acidic part of the tumor, but show the characteristics of alkaline aggregation and acidic dispersion, and the fluorescence intensity is weak at pH 7.4 in the normal physiological environment and far from meeting the needs of cell imaging. The reason is: 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 interval. Second, the fluorescent probes developed using near-infrared excitation, ultrasound, protein labeling and other conditions can achieve the process of tumor cell imaging, but 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: the introduction of multiple properties at the same time makes the preparation process of the fluorescent probe complicated, and it is necessary to use a relatively strong external stimulus to achieve the purpose of in vivo imaging.

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 the tumor, so as to solve the problem of low imaging sensitivity in the tumor site, so as to achieve pH-responsive fluorescence imaging effect and provide an effective method for targeting the acidic microenvironment of the tumor.

Summary of the invention

In order to solve at least one of the above problems, the present invention provides a slightly acidic environment responsive AIE fluorescent probe and its preparation method and application. The small molecule fluorescent probe prepared in this scheme is designed with a slightly acidic pH responsive group to improve its detection sensitivity in tumor imaging. When the fluorescent probe reaches the tumor site, in the slightly acidic environment of the tumor, the amide bond is protonated and the whole is electrically neutral. In the aggregated state, bright fluorescence is emitted in the cell, realizing safe and effective selective fluorescence imaging of tumor cells.

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:

Among them, R is selected from One of them.

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

The 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:

Step 1, dissolving the sulfonamide compound in a certain amount of solvent, adding triethylamine according to a 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 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;

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.

As a preferred embodiment, in step 2, the preparation method of the solid acyl chloride is as follows: dissolving 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene in dichlorothionyl according to a proportion; and reacting the obtained 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.

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

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.

As a preferred embodiment, in the step three, the reaction temperature is 25-50°C.

As a preferred solution, the following steps are included:

Step 1, dissolving sulfadoxine in a certain amount of tetrahydrofuran, adding triethylamine according to a 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 50° C. for 48-72 hours, 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.

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

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

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

First, the fluorescent probe of this scheme exhibits good biosafety and low toxic side effects, can target the acidic microenvironment of tumors for cell fluorescence imaging, and has excellent cell penetration and uptake ability, providing an effective fluorescent probe compound for targeting the acidic microenvironment of tumors. The fluorescent probe compound of this scheme utilizes the different pKa of sulfonamide derivatives within the physiological range, and through the process of protonation and deprotonation of the amide group of the small molecule fluorescent probe compound in different pH environments, the process of fluorescent probe responding to the acidic microenvironment of tumors for live cell fluorescence imaging is realized, and the pH transition point is successfully adjusted to the acidic microenvironment of tumors.

Second, 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 tumors. The AIE molecules represented by 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene (H4TCPE) can successfully prepare acid-responsive AIE small molecule fluorescent probes, tetraphenylethylene-benzenesulfonamide (TPE-SA), tetraphenylethylene-sulfonic acetamide (TPE-SAC) and tetraphenylethylene-sulfadoxine (TPE-SDOX), by undergoing amidation reaction with sulfonamide molecules. The small molecule fluorescent probe compounds of this application are obtained by strictly controlling the temperature index and reaction time, removing the solvent under reduced pressure, and purifying and drying the residue. 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.

Third, the fluorescent probe prepared by the present invention exhibits good biosafety and low toxic side effects, can target the acidic microenvironment of tumors for cell fluorescence imaging, and has excellent cell penetration and uptake ability, providing an effective method for targeting the acidic microenvironment of tumors. By co-culturing fluorescent probes with tumor cells, the pH value of the culture medium is adjusted to achieve the effect of aggregation-induced luminescence imaging, specifically in the slightly acidic environment of tumors, resulting in the protonation of amide bonds and overall neutrality. Bright fluorescence is emitted in the cell in the aggregated state, achieving safe and effective selective fluorescence imaging of tumor cells.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is the nuclear magnetic hydrogen spectrum of H4TCPE in the present invention;

FIG2 is a hydrogen NMR spectrum of TPE-SA prepared in an embodiment of the present invention;

FIG3 is a hydrogen NMR spectrum of TPE-SAC prepared in an embodiment of the present invention;

FIG4 is a hydrogen NMR spectrum of TPE-SDOX prepared in an embodiment of the present invention;

FIG5 is a FL spectrum of H4TCPE in DMSO/H2O mixtures containing different volume fractions of water;

FIG6 is a graph showing the relative FL intensity of H4TCPE in DMSO/H2O mixtures containing different volume fractions of water;

FIG7 is a FL spectrum of TPE-SA in DMSO/H2O mixtures containing different volume fractions of water;

FIG8 is a graph showing the relative FL intensity of TPE-SA in DMSO/H2O mixtures containing different volume fractions of water;

FIG9 is a FL spectrum of TPE-SAC in DMSO/H2O mixtures containing different volume fractions of water;

FIG10 is a graph showing the relative FL intensity of TPE-SAC in DMSO/H2O mixtures containing different volume fractions of water;

FIG11 is a FL spectrum of TPE-SDOX in DMSO/H2O mixtures containing different volume fractions of water;

FIG12 is a graph showing the relative FL intensity of TPE-SDOX in DMSO/H2O mixtures containing different volume fractions of water;

FIG13 is the fluorescence spectra of H4TCPE in buffers with different pH values;

FIG14 is a graph showing the change in fluorescence intensity of H4TCPE with pH value;

FIG15 shows the fluorescence characteristics of H4TCPE in buffer solutions with different pH values.

FIG16 is the fluorescence spectra of TPE-SA in buffers with different pH values;

FIG17 is a graph showing the change in fluorescence intensity of TPE-SA with pH value;

FIG18 shows the fluorescence characteristics of TPE-SA in different pH buffers;

FIG19 is the fluorescence spectra of TPE-SAC in buffers with different pH values;

FIG20 is a graph showing the change in fluorescence intensity of TPE-SAC with pH value;

FIG21 shows the fluorescence characteristics of TPE-SAC in different pH buffers;

FIG22 is the fluorescence spectra of TPE-SDOX in buffers with different pH values;

FIG23 is a graph showing the change in fluorescence intensity of TPE-SDOX with pH value;

FIG24 shows the fluorescence characteristics of TPE-SDOX in different pH buffers;

FIG25 is a graph showing the relationship between fluorescence intensity and time obtained by mixing H4TCPE mother solution with PBS buffer solution having a pH of 4.96 and DMEM solution to prepare a solution having a water content of 98%; FIG25 is a graph showing the relationship between fluorescence intensity and time obtained by plotting the maximum fluorescence emission intensity;

FIG26 is a graph showing the relationship between fluorescence intensity and time, in which the TPE-SA mother solution was mixed with a PBS buffer solution having a pH of 5.75 and a DMEM solution to prepare a solution having a water content of 98%; and the maximum fluorescence emission intensity was plotted;

FIG27 is a graph showing the relationship between fluorescence intensity and time, in which TPE-SAC is mixed with a PBS buffer solution having a pH of 6.27 and a DMEM solution to prepare a solution having a water content of 98%; and the maximum fluorescence emission intensity is plotted;

FIG28 is a graph showing the relationship between fluorescence intensity and time, in which TPE-SDOX is mixed with a PBS buffer solution having a pH of 6.7 and a DMEM solution to prepare a solution having a water content of 98%; and the maximum fluorescence emission intensity is plotted;

FIG29 is a graph showing the relationship between fluorescence intensity and cycle number when the H4TCPE mother solution is between pH 4.0 and 5.5 and plotted based on the maximum fluorescence emission intensity;

FIG30 is a graph showing the relationship between fluorescence intensity and cycle number when the maximum fluorescence emission intensity is plotted for TPE-SA mother solution between pH 5.0 and 6.0;

FIG31 is a graph showing the relationship between fluorescence intensity and cycle number when the TPE-SAC mother solution is between pH 5.5 and 6.5 and plotted based on the maximum fluorescence emission intensity;

FIG32 is a graph showing the relationship between fluorescence intensity and cycle number when the TPE-SDOX mother solution is between pH 6.5 and 7.4 and plotted based on the maximum fluorescence emission intensity;

FIG33 is a graph showing the effect of different concentrations of small molecule fluorescent probes on the cytotoxicity of L02 cells;

FIG34 is a graph showing the effect of different concentrations of small molecule fluorescent probes on the cytotoxicity of HepG2 cells;

FIG35 is a fluorescence image of HepG2 and L02 cells after H4TCPE treatment; scale bar: 20 μm;

FIG. 36 is a fluorescence image of HepG2 and L02 cells after TPE-SDOX treatment; scale bar: 20 μm.

DETAILED DESCRIPTION

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

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.

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 dropping the second solution into the first solution obtained in step 1 to prepare a mixed solution. The preparation method of the acyl chloride solid used in step 2 is as follows: dissolving 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene in SOCl ₂ , wherein 1g of 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene is added into 70-100mL of dichlorothionyl (SOCl ₂ ), and reacting at reflux temperature for 24-48h for activation; after the activation reaction is completed, cooling to room temperature, and evaporating under reduced pressure to remove the solvent to obtain solid acyl chloride; step 3, after the second solution is added dropwise, stirring the mixed solution prepared in step 2 at 25-50° C. for 48-72h, and purifying the residue after evaporating the solvent by silica gel column chromatography and drying to obtain the corresponding fluorescent probe compound.

Wherein, 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.

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

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

Preferably, the sulfonamide compound is sulfadoxine. After the solvent is evaporated 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.

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.

Example 1

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

Example 2

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

Example 3

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

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

Example 4

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

Example 5

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

Example 6

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

Example 7

Dissolve 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene (0.1 g, 0.196 mmol) in SOCl ₂ (8 mL) and react at reflux temperature 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 placed in an ice-water bath and stirred for 30 min. Dissolve the obtained solid acid chloride in 3 mL of anhydrous tetrahydrofuran to obtain a second solution, and add the obtained second solution dropwise to the above-mentioned first solution to obtain a mixed solution. Stir the mixed solution at 50°C for 72 h and evaporate the solvent under reduced pressure. Subsequently, the residue after evaporating the solvent was purified and dried by silica gel column chromatography (EtOAc:DCM=10:1) to obtain TPE-SDOX. The product mass was 0.249 g, the product yield was 75.4%, and the product purity was 87.9%.

Wherein, the reaction formula of embodiment 4-7 is as follows:

FIG4 is a hydrogen NMR spectrum of TPE-SDOX prepared in Example 7 (using deuterated dimethyl sulfoxide as solvent).

Example 8

Dissolve 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene (0.1 g, 0.196 mmol) in SOCl ₂ (8 mL) and react at reflux temperature for 48 h. Cool to room temperature, evaporate the solvent under reduced pressure to obtain solid acid chloride. Dissolve benzenesulfonamide (0.185 g, 1.176 mmol) in anhydrous tetrahydrofuran (3 mL), add triethylamine (0.218 mL, 1.568 mmol) to obtain a first solution, and place the obtained first solution in an ice-water bath and stir for 30 min. Dissolve all the acid chloride solids prepared above in 3 mL of anhydrous tetrahydrofuran to obtain a second solution, and add the obtained second solution dropwise to the above first solution to obtain a mixed solution. Stir the mixed solution at room temperature for 72 h and evaporate the solvent 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:

FIG2 is a hydrogen NMR spectrum of TPE-SA prepared in Example 8 (using deuterated dimethyl sulfoxide as solvent).

Example 9

Dissolve 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene (0.1 g, 0.196 mmol) in SOCl ₂ (8 mL) and react at reflux temperature 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, and place the obtained first solution in an ice-water bath and stir for 30 min. Dissolve all the solid acid chloride prepared above in 3 mL of anhydrous tetrahydrofuran to obtain a second solution, and add the obtained second solution dropwise to the above first solution to obtain a mixed solution. Stir the mixed solution at 50°C for 72 h and evaporate the solvent 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-SAC, with a product mass of 0.179 g, a product yield of 70.3%, and a product purity of 86.4%. The reaction formula is as follows:

FIG3 is a hydrogen NMR spectrum of TPE-SAC prepared in Example 9 (using deuterated dimethyl sulfoxide as solvent).

Comparative Example 1

1,1,2,2-tetrakis(4-carboxyphenyl)ethylene (0.1g, 0.196mmol) was dissolved in SOCl ₂ (8mL) and reacted at reflux temperature for 48h. After cooling to room temperature, the solvent was evaporated under reduced pressure to obtain a solid acid chloride. Sulfathiazole (0.301g, 1.176mmol) was dissolved in anhydrous tetrahydrofuran (3mL), and triethylamine (0.218mL, 1.568mmol) was added to obtain a first solution, and the obtained first solution was placed in an ice-water bath and stirred for 30min. The above-obtained solid acid chloride was completely dissolved in 3mL of anhydrous tetrahydrofuran to obtain a second solution, and the obtained second solution was added dropwise to the above-obtained first solution to obtain a mixed solution. The mixed solution was stirred at 50°C for 72h 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 0.207g, a product yield of 72%, and a product purity of 79.94%. The reaction formula is as follows:

Comparative Example 2

1,1,2,2-tetrakis(4-carboxyphenyl)ethylene (0.1 g, 0.196 mmol) was dissolved in SOCl ₂ (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. Sulfadimethoxine (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, and the obtained first solution 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, and the obtained second solution 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 then 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:

In this scheme, the successful preparation of the fluorescent probe was confirmed by ¹ H NMR and FTIR characterization; the fluorescence intensity of solutions with different water contents was measured by fluorescence spectrophotometer, confirming that TPE-SA, TPE-SAC and TPE-SDOX had excellent AIE effect; the fluorescence intensity was further measured in different pH buffer solutions, and it was found 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, proving that the pH transition point of the H4TCPE aggregation luminescence response was adjusted to the tumor microenvironment by modifying the TPE molecules with sulfonamide groups.

In this scheme, the above-mentioned Example 4-9 combines TPE with sulfonamide compounds through an amidation reaction, and successfully synthesizes TPE-SA, TPE-SAC and TPE-SDOX, wherein 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, which is used 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 adopts the method of co-incubating small molecule fluorescent probes with tumor cells to achieve rapid charge conversion to enhance cell uptake and accurate fluorescence imaging during incubation with cancer cells. When the small molecule fluorescent probe reaches the tumor site, it causes the amide bond to be protonated in the slightly acidic environment of the tumor, forming CONH ²⁺ , introducing a positive charge, while C=O and S=O=S are electron-withdrawing groups, and the whole is electrically neutral. In the aggregated state, the intramolecular rotation is restricted, resulting in the release of the energy of the excited state in the form of luminescence, resulting in bright fluorescence in the cell. The TPE-SDOX probe exhibits sensitive pH response and superior AIE effect, achieving safe and effective selective fluorescence imaging of tumor cells.

Biocompatibility evaluation

The present invention co-cultures small molecule fluorescent probes with cells, and the culture method is as follows: HepG2 cells and L02 cells are cultured in an incubator at 37°C and 5% _CO2 , respectively, and the DMEM culture medium contains 10% FBS and 1% antibiotics (containing 100U/mL penicillin and 100μg/mL streptomycin). The present invention co-cultures small molecule fluorescent probes with tumor cells and adjusts the pH value of the culture medium to achieve the effect of aggregation-induced luminescence imaging.

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) is a commonly used cytotoxicity assay to evaluate the effects of small molecule fluorescent probes on cell viability. When L02 and HepG2 cells in the 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 inoculated into 96-well plates at a concentration of 8000 cells/well, keeping the final culture medium volume of each well at 200μL. After 24h of culture, it was observed that the cell density in the well reached 70%. Subsequently, different concentrations of H4TCPE, TPE-SA, TPE-SAC and TPE-SDOX were mixed with the cells and incubated for 24h, keeping the final concentration of the culture medium at 10, 20, 40, 60, 80 and 100μM. Subsequently, 10μMTT (5mg/mL) was added to each well. After incubation at 37°C for 4 hours in a _CO2 incubator, the MTT reagent and the metabolites produced by the cells will form blue-purple crystalline formazan scattered on the bottom of the culture dish. Then, remove the culture medium and use 180 μL of isopropanol to dissolve the blue-purple formazan crystals deposited at the bottom. Use an ELISA plate reader to measure the absorbance (OD) value at 570 nm. The cell activity (%) calculation formula is as follows:

OD _sample is the absorbance after treatment with the fluorescent probe, and OD _control is the absorbance without treatment with the fluorescent probe. The cytotoxicity experiment was repeated three times.

As shown in Figures 33 and 34, this embodiment is an in vitro biocompatibility experiment embodiment, and the MTT method is used to study the biocompatibility of H4TCPE, small molecule fluorescent probes of Examples 7, 8, and 9 on L02 and HepG2 cell lines. As can be seen from the figure analysis, after the small molecule fluorescent probe was co-cultured with HepG2 and L02 cells for 24 hours, the cell survival rate remained above 90% even at a concentration of up to 100 μM. The results show that the fluorescent probe has less toxicity to cells. Even at high concentrations, the AIE fluorescent probe can maintain cell viability, providing good biosafety for cell imaging.

Evaluation of cellular uptake

In order to evaluate the cellular uptake of H4TCPE and TPE-SDOX fluorescent probes, the present invention uses a fluorescent inverted microscope to observe L02 and HepG2 cells. First, the coverslip is soaked in aqua regia and sterilized with high-pressure steam. Then the coverslip is placed on the well plate and rinsed with PBS solution to avoid bilateral cell growth affecting the sealing and imaging effects. Subsequently, 0.5mL of culture medium is added to each well to ensure that the cells can be evenly dispersed in the 24-well plate. Each well is inoculated with 2×10 ⁵ cells, and the cell density in the well plate reaches about 70%. The original culture medium can be replaced with DMEM culture medium adjusted to pH 6.5, and 20μL of H4TCPE or TPE-SDOX DMSO solution is added (the whole process is: aspirate the original culture medium, add 980uL of pH6.5 culture medium, and add 20uL probe. The total volume of the whole is 1mL, and the final concentration of the fluorescent probe is 20uM). In the process of replacing the culture medium, the well plate is washed with sterile PBS to reduce serum residues. After the cells were incubated in a _CO2 incubator for different periods of time, the culture medium in the well plate was aspirated and washed three times with PBS. Subsequently, a polyformaldehyde solution (4%, PFA) that can cover the coverslip was added and incubated for 10 minutes to fix the cells. After the PFA was aspirated, the cells were washed three times with a cell-grade PBS buffer solution (buffer model P1020). 20 μL of anti-fluorescence quenching sealing solution was dripped on the surface of the slide, and the coverslip was removed and inverted on the slide using a cell clamp. Subsequently, non-fluorescence microscope oil was dripped on the surface of the coverslip, and the cell uptake was observed using a fluorescent inverted microscope.

As shown in Figures 35 and 36, 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, so it can produce a stronger and more obvious fluorescence signal.

AIE characteristics evaluation

As shown in Figures 5-12, the FL spectra and relative FL intensity diagrams (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/H ₂ O mixtures containing different volume fractions of water are shown, respectively, where the inset pictures of Figures 6, 8, 10 and 12 correspond to the fluorescence diagrams of H4TCPE, TPE-SA, TPE-SAC and TPE-SDOX in pure DMSO (volume fraction of water is fw = 0%) and a certain volume of DMSO/H ₂ O mixture (volume fraction of water is fw = 98%). The detection conditions of Figures 5-12 are: excitation wavelength: 365nm, and the concentration of the probe in the solution is: 20μM. Detection equipment: F96Pro fluorescence spectrophotometer (Shanghai Lingguang Technology Co., Ltd.).

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 fluorescence intensity of each fluorescent probe solution with a water volume fraction of fw = 98% 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 (i.e., the volume fraction of water is fw = 0%), the small molecule fluorescent probe with DMSO/H ₂ O mixture (the volume fraction of water is fw = 98%) as solvent shows 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 in the insets of Figures 6, 8, 10, and 12, in pure DMSO solvent, H4TCPE, TPE-SA, TPE-SAC, and TPE-SDOX have almost no fluorescence visible to the naked eye, but have obvious fluorescence emission when DMSO/H ₂ O mixture (the volume fraction of water is fw=98%) is used as solvent. The results show that the small molecule fluorescent probe synthesized by amidation reaction of different sulfonamide groups with TPE maintains the excellent AIE properties of the TPE molecule itself.

Evaluation of fluorescence intensity of fluorescent probes under different pH solution conditions

As shown in Figures 13, 16, 19, and 22, the fluorescence spectra of H4TCPE, TPE-SA, TPE-SAC, and TPE-SDOX in buffers with different pH values, in the range of pH 8.98-1.06, the fluorescence intensity of H4TCPE, TPE-SA, TPE-SAC, and TPE-SDOX conforms to the Sigmoid Boltzmann function (as shown in Figures 14, 17, 20, and 23). In Figures 13-24, the excitation wavelength is 365nm. As shown in Figure 14, the pH transition point of H4TCPE is around pH 4.91, which is beyond the physiological normal range (tumor microenvironment pH 6.2-6.9).

The preparation method of the detection mother solution of this scheme is as follows: weigh 10.5mg TPE-SDOX and dissolve it in 1mL DMSO to prepare 6.25mM mother solution C ₁ ; weigh 5mg TPE-SA and dissolve it in 0.6183mL DMSO to prepare 6.25mM mother solution C ₁ ; weigh 3mg H4TCPE and dissolve it in 0.944mL DMSO to prepare 6.25mM mother solution C _1. The fluorescence spectrum detection method is as follows: first take 320uL of 6.25mM corresponding mother solution C ₁ and dissolve it in 1.68mL DMSO, dilute to the concentration of mother solution C ₂ is 1mM; then take 60uL of 1mM mother solution C ₂ and add it to 2.94mL PBS solution (DMSO/PBS=2/98) with different pH (4.96, 5.75, 6.27, 6.7). The final concentration is 20uM, and the excitation wavelength is 365nm. In this scheme, PBS buffer solutions of different pH are prepared by preparing 0.01M standard PBS solution of pH7.2-7.4, adding 1M HCl, adjusting the pH value and measuring its pH value by a pH meter. In this scheme, the PBS buffer or DMEM solution with a pH of 4.96, 5.75, 6.27, and 6.7 is prepared as follows: add different amounts of 1M HCl to the purchased standard PBS solution/DMEM so that the solution is adjusted to the corresponding pH value, and measure its pH value by a pH meter.

In the present invention, Figures 14, 17, 20, and 23 are drawn based on the following test data. The pH values of the fluorescence intensity at different pH values measured by H4TCPE 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 at different pH values measured by TPE-SA 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 of the present application was a F96Pro fluorescence spectrophotometer (Shanghai Lingguang Technology Co., Ltd.).

pH transition point of each fluorescent probe

This scheme successfully synthesized TPE-SA, TPE-SAC and TPE-SDOX compounds at the molecular level by precise modification of the sulfonamide group. By detecting the FL of TPE-SA and TPE-SAC in different pH solutions and analyzing the relationship between FLmax and different pH of small molecule fluorescent probes, the TPE-SA prepared in Example 8 and the TPE-SAC prepared in Example 9 had obvious pH turning points, which were 5.76±0.02 and 6.09±0.03, respectively. However, these two compounds still failed to reach the expected pH range (tumor microenvironment pH 6.2-6.9), and therefore could not be used for tumor microenvironment detection. In contrast, as shown in Figures 22, 23 and 24, 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 exhibits obvious AIE characteristics and sensitive pH response, and can be used to target tumor microenvironments to achieve the expected pH range (pH 6.2-6.9). The pH response ranges 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 in a wide pH range.

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

Photostability evaluation

As shown in Figures 25, 26, 27, and 28, H4TCPE, TPE-SA, TPE-SAC, and TPE-SDOX mother solutions _C2 were mixed with PBS buffer or DMEM solution at pH 4.96, 5.75, 6.27, and 6.7 to prepare solutions with a water content of 98%. The fluorescence intensity of the above solutions was measured at different time points, and the relationship between the fluorescence intensity and time was plotted according to the maximum fluorescence emission intensity.

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. As shown in the figure, according to the characterization diagrams obtained in Figures 25-28, it can be seen that the fluorescence intensity of the small molecule fluorescent probe remained stable within 72 hours, indicating that it has good fluorescence stability. The above results show that the TPE fluorescent probe exhibits good stability. Unlike traditional fluorescent molecules, small molecule fluorescent probes show flexibility in solution and transform into a rigid state after aggregation. This carefully designed configuration enhances the stability of the molecule during the aggregation process and enables it to maintain a high level of fluorescence intensity for a long time.

Photoreversibility evaluation

As shown in Figures 29, 30, 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 in sequence 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.

In this scheme, the preparation method of the solution with a water content of 98% is as follows: 10.5 mg TPE-SDOX is weighed and dissolved in 1 mL DMSO to prepare a 6.25 mM mother solution C ₁ ; 5 mg TPE-SA is weighed and dissolved in 0.751 mL DMSO, 5 mg TPE-SAC is weighed and dissolved in 0.6185 mL DMSO to prepare a 6.25 mM mother solution C ₁ ; 3 mg H4TCPE is weighed and dissolved in 0.944 mL DMSO to prepare a 6.25 mM mother solution _{C 1 ;} 320 uL of 6.25 mM H4TCPE, TPE-SA, TPE-SAC and TPE-SDOX mother solutions C ₁ are respectively taken and dissolved in 1.68 mL DMSO, and diluted to obtain a 1 mM mother solution C ₂ ; 60 uL of the above mother solutions C 1 with a concentration of 1 mM is taken ₂ Add 2.94 mL of PBS solution with different pH values (DMSO/PBS=2/98) [water content 98%], the final concentration reaches 20 uM, and the excitation wavelength is 365 nm.

According to the analysis shown in Figures 29, 30, 31, and 32, the following conclusions were obtained: 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 response 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 showed 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 exhibit 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.

The above description is only 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 as above, it is not intended to limit the present invention. Any technician familiar with the 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 modification, equivalent change and modification 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 still falls within the scope of the technical solution of the present invention.

Claims (10)

1. A slightly acidic environment responsive AIE fluorescent probe, characterized in that: its general structural formula is: Among them, R is selected from One of them. 2. A slightly acidic environment responsive AIE fluorescent probe according to claim 1, characterized in that: the structural formula of the fluorescent probe is: 3. A method for preparing a slightly acidic environment responsive AIE fluorescent probe, characterized in that it comprises the following steps: Step 1, dissolving the sulfonamide compound in a certain amount of solvent, adding triethylamine according to a 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 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; 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. According to the method for preparing a slightly acidic environment responsive AIE fluorescent probe according to claim 3, it is characterized in that: in step 2, the preparation method of the acyl chloride solid is as follows: 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene is dissolved in dichloride according to a proportion; and the resulting solution is reacted at reflux temperature for 24-48h; after the reaction is completed, the reaction solution is cooled to room temperature, and then the solvent is removed to obtain a solid acyl chloride. 5. A method for preparing a slightly acidic environment responsive AIE fluorescent probe according to claim 4, characterized in that: 1 g of 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene is added to 70-100 mL of dichlorothionyl. 6. A method for preparing a slightly acidic environment responsive AIE fluorescent probe according to claim 3, characterized in that 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. 7. The method for preparing a slightly acidic environment responsive AIE fluorescent probe according to claim 3, characterized in that: in the step 3, the reaction temperature is 25-50°C. 8. The method for preparing a slightly acidic environment responsive AIE fluorescent probe according to claim 6, characterized in that it comprises the following steps: Step 1, dissolving sulfadoxine in a certain amount of tetrahydrofuran, adding triethylamine according to a 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 for 48-72 hours under a certain reaction stability, 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. 9. Use of the slightly acidic environment responsive AIE fluorescent probe according to claim 1 or 2 in tumor cell imaging. 10. The 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 and tumor cells are co-cultured, and preferably, the small molecule fluorescent probe is TPE-SDOX.