A tumor-targeted fluorescent probe and its preparation method and application

Abstract

The present invention discloses a tumor-targeted fluorescent probe having a structure shown in formula (I). The fluorescent probe uses a maleimide-modified alkyl chain as a bridging segment, and modifies a group targeting SLC7A5 on an ICG fluorescent dye by coupling cysteine ​​with a thiol group, thereby achieving targeted fluorescent imaging of tumor tissues by targeting SLC7A5 overexpressed in tumor cells. The present invention also discloses a preparation method and application of the fluorescent probe.

Description

Technical Field

[0001] The invention belongs to the technical field of biological and medical nanomaterials, and relates to a tumor-targeted fluorescent probe, and a preparation method and application thereof. Background Art

[0002] With the widespread popularization and rapid development of surgical operations, the requirements for accurate positioning of lesions are becoming increasingly higher. Among them, intraoperative fluorescence visualization technology has achieved vigorous development and has been widely used in fields such as tumor resection surgery. Near-infrared fluorescence imaging technology has been widely used in the field of medical biology in recent years. Among them, indocyanine green (ICG), a representative of fluorescent contrast agents, has become an ideal real-time detection tool for intraoperative tumor lesion positioning and edge judgment. Most importantly, achieving specific binding of ICG to tumors is the key to its clinical application.

[0003] L-type amino acid transporter 1 (LAT1), also known as SLC7A5, is an important member of the L-type amino acid transporter, which is mainly responsible for the transport of amino acids such as methionine, leucine, isoleucine, and tryptophan. It has been reported that SLC7A5 is abnormally highly expressed in some tumor tissues. In models of early and late metastatic disease, the glutamine antiporter SLC7A5 is essential for the occurrence of colorectal tumors (reference: The aminoacid transporter SLC7A5 is required for efficient growth of KRAS-mutant colorectal cancer, Nature 2021). Tamoxifen-resistant breast cancer has a large leucine uptake, and the reduction of leucine levels significantly inhibits the division of estrogen receptor (ER)-positive breast cancer cells. SLC7A5 is necessary for the transport of leucine into cells, so the SLC7A5 expression level of tamoxifen-resistant ER+ breast cancer cells is significantly higher than that of non-resistant cells (reference: LLGL2 rescues nutrient stress by promoting leucine uptake in ER breast cancer, Nature 2019). The above characteristics revealed by the study provide a basis for the specific fluorescent imaging of tumor cells. Summary of the invention

[0004] The object of the present invention is to provide a tumor-targeted fluorescent probe and a preparation method thereof. The fluorescent probe is based on ICG fluorescent dyes, can target tumor cells with high expression of SLC7A5, and has strong fluorescence intensity and good stability.

[0005] Another object of the present invention is to provide an application of the fluorescent probe.

[0006] In order to achieve the above-mentioned invention object, the present invention adopts the following technical scheme:

[0007] A tumor-targeting fluorescent probe, characterized in that it has a structure as shown in the following formula (I):

[0008]

[0009] Wherein, n is a positive integer of 1-14, and X is Na + , K + .

[0010] Preferably, the n is 3-11, and more preferably, the n is 9.

[0011] The preparation method of the fluorescent probe, the synthesis path is as follows Figure 1 As shown, the following steps are included:

[0012] 1) Preparation of maleimide-modified N-acylethylenediamine derivatives

[0013] C5-18 straight chain alkyl dibasic acid (1), N-(2-aminoethyl) maleimide (2) and N-fluorenylmethoxycarbonylethylenediamine (3) are dissolved in a solvent and reacted at room temperature under the action of a coupling agent to obtain a maleimide-modified N-acylethylenediamine derivative of formula (II) (denoted as NH2-(CH2)m-MAL, where m=n+6 represents the total number of methylene groups)

[0014]

[0015] 2) Preparation of tumor-targeted fluorescent probes

[0016] The NH2-(CH2)m-MAL (II) prepared in step 1) and the indocyanine green derivative (denoted as Cl-IR-781) of the structure of formula (4) are dissolved in a solvent, heated under reflux reaction under the action of a coupling agent, and then Fmoc-protected cysteine ​​(5) is added to the reaction solution, and the heating reflux reaction is continued to obtain a coupled fluorescent dye (denoted as Cys-(CH2)m-IR-781) of the structure of formula (I), i.e., the tumor-targeted fluorescent probe

[0017]

[0018] Wherein, n is a positive integer of 1-14, and X is Na + , K + .

[0019] In step 1), the molar ratio of the C5-C18 straight-chain alkyl dibasic acid, N-(2-aminoethyl)maleimide and N-fluorenylmethoxycarbonylethylenediamine is preferably 1:1:1.

[0020] In step 1), the coupling agent is preferably N,N'-diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBT).

[0021] In step 1), the solvent is preferably N,N-dimethylformamide (DMF).

[0022] In step 2), the molar ratio of NH2-(CH2)m-MAL to Cl-IR-781 is preferably 1:1-5.

[0023] In step 2), the solvent is preferably methanol.

[0024] In step 2), the coupling agent is preferably N,N-diisopropylethylamine (DIPEA).

[0025] In step 2), the temperature of the heating reflux reaction is preferably 40-70°C.

[0026] The tumor-targeted fluorescent probe has an emission wavelength of 760-810 nm, has strong fluorescence intensity, high stability, is not easily quenched, and is suitable as a near-infrared fluorescent probe.

[0027] The tumor-targeted fluorescent probe is a cysteine-modified ICG fluorescent dye. The cysteine ​​with an alkyl chain structure is a substrate of SLC7A5, which can target SLC7A5-highly expressed tumor cells, and is transported into tumor cells through the overexpressed SLC7A5 on the cell membrane, thereby realizing fluorescent imaging of tumor cells. The alkyl chain modified with maleimide in the molecular structure of the fluorescent probe is a bridging segment, and cysteine ​​is coupled to the thiol group. After contacting with the SLC7A5 protein ligand, when the alkane chain contains 3-16 carbon atoms, it forms a good combination with the SLC7A5 protein ligand structure, especially when the alkane chain length is 11 carbon atoms, the targeting performance is optimal (the calculated value of the hydrogen bond intermolecular force is also optimal).

[0028] The invention also relates to the application of the tumor-targeted fluorescent probe in the preparation of contrast agents.

[0029] Furthermore, the contrast agent is used for fluorescent imaging of tumor tissues, and the tumors include breast cancer, rectal tumors, cervical cancer, lung cancer, glioma, nasopharyngeal carcinoma, etc.

[0030] The fluorescent probe modifies the ICG fluorescent dye with a group targeting SLC7A5, thereby achieving targeted imaging of tumor cells. Existing studies have confirmed that SLC7A5 is associated with many cancers, and is abnormally highly expressed in tumor tissues such as breast cancer, cervical cancer, nasopharyngeal carcinoma, lung cancer, and glioma, while being lowly expressed in corresponding normal tissues. The contrast agent based on the fluorescent probe can be used for the diagnosis and treatment of many tumors.

[0031] Beneficial effects: The tumor-targeted fluorescent probe according to the present invention has a strong fluorescence intensity, good stability and is not easy to quench. The fluorescent probe is modified with a group targeting SLC7A5 on the ICG fluorescent dye, and targeted fluorescence imaging of tumor tissue is achieved by targeting SLC7A5 overexpressed in tumor cells. By constructing a contrast agent based on the fluorescent probe, real-time tumor imaging in vivo can be achieved, thereby providing more comprehensive and accurate information for tumor diagnosis and treatment, realizing intraoperative navigation during tumor surgery, and facilitating complete resection of the tumor. The tumor-targeted fluorescent probe of the present invention is of great significance to the application of ICG fluorescence visualization technology in tumor diagnosis and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 .Cys-(CH2) 15 -Synthesis route of IR-781 near-infrared fluorescent dye;

[0033] Figure 2 .Cys-(CH2) 15 -IR-781 fluorescent dye 1 H NMR spectrum;

[0034] Figure 3 .Cys-(CH2) 15 - Ultraviolet-visible (UV-Vis) absorption spectrum of IR-781;

[0035] Figure 4 .Cys-(CH2) 15 - Fluorescence spectrum of IR-781;

[0036] Figure 5 .Western blot protein bands of SLC7A5 on the surface of MCF7 and MCF7 / Tam cells;

[0037] Figure 6 .SLC7A5 expression level on the surface of MCF7 and MCF7 / Tam cells;

[0038] Figure 7 .RT-qPCR detection of SLC7A5 knockout efficiency in MCF7 / Tam cells;

[0039] Figure 8.MCF7 / Tam cells to Cys-(CH2) 15 - Uptake of IR-781 fluorescent probe;

[0040] Fig. 9 .Schematic diagram of three different coupling methods;

[0041] Fig.10 .Cys-(CH2) 15 -Diagram of the binding pattern of IR-781 and SLC7A5 protein. DETAILED DESCRIPTION

[0042] The technical solution of the present invention is further described below through specific embodiments. However, it is necessary to point out that the following embodiments are only used to describe the content of the present invention and do not constitute a limitation on the protection scope of the present invention.

[0043] Example 1. Cys-(CH2) 15 -IR-781 long-chain fluorescent dye and its synthesis

[0044] A tumor-targeted fluorescent probe comprising a long alkyl chain and a synthesis method thereof are as follows:

[0045] 1) Preparation of ICG derivative Cl-IR-781(4)

[0046] Cl-IR-781 was prepared in-house. The preparation method thereof can be found in SA Hilderbrand, et al. Bioconjugate Chem. 2005, 16: 1275-1281. The specific synthesis steps are as follows: Under argon protection, potassium salt of 2,3,3-trimethylindole-5-sulfonic acid and 1,4-butanesulfonic acid lactone in a molar ratio of 1:3 were taken, condensed and refluxed at 120°C in o-dichlorobenzene solvent, and reacted for 16 hours. After the solution changed from yellow to rose red, the o-dichlorobenzene solvent was removed by vacuum rotary evaporation, and purified and collected by C18 gel chromatography column to obtain a red solid. At the same time, phosphorus oxychloride, N,N-dimethylformamide, and dichloromethane in a molar ratio of 1:1:1 were stirred and reacted under ice bath for 30 minutes, and then cyclohexanone in a molar ratio of 1:1 was added, heated to 80°C, refluxed for 3 hours, and then the reaction was cooled to 0°C overnight, and then filtered to obtain a yellow solid. The red and yellow solids with a molar ratio of 2:1 were dissolved in a mixed solution of ethyl acetate and methanol with a volume ratio of 1:1, condensed and refluxed at 80°C for 3 hours, and the product was purified and collected by C18 column gel chromatography to obtain a dark green solid, namely Cl-IR-781.

[0047] 2) Maleimide-modified N-acylethylenediamine derivatives NH2-(CH2) 15 Preparation of -MAL (II, n = 9)

[0048] Take tridecanedioic acid, N-(2-aminoethyl)maleimide hydrochloride and N-fluorenylmethoxycarbonylethylenediamine hydrochloride (N-Fmoc-ethylenediamine hydrochloride) in a molar ratio of 1:1:1, dissolve in N,N-dimethylformamide (DMF) solution containing 6 times the molar amount of N,N'-diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBT), react at room temperature for 16 hours, and then purify by C18 column gel chromatography, the mobile phase is methanol and ethyl acetate in a volume ratio of 1:1-5, and collect the maleimide-modified N-acylethylenediamine derivative NH2-(CH2) 15 -MAL.

[0049] 3) Cysteine-modified long-chain coupled fluorescent dye Cys-(CH2) 15 - Preparation of IR-781 (Ⅰ, n=9)

[0050] Take NH2-(CH2) 15 -MAL and Cl-IR-781 were added to a mixed solution of methanol and N,N-diisopropylethylamine in a volume ratio of 1:1 in a molar ratio of 1:2, and heated under reflux at 55°C for 3 hours to obtain an intermediate product - a C-long-chain coupled ICG derivative (CH2) 15 -IR-781; then, Fmoc-protected cysteine ​​dissolved in methanol was added to the reaction solution, and the reaction was continued by heating and reflux for 3 hours. The reaction product was purified by C18 column gel chromatography, and the mobile phase was methanol, ethyl acetate and water in a volume ratio of 1:1-5:0.05, and the cysteine-modified long-chain coupled fluorescent dye Cys-(CH2) was collected. 15 -IR-781.

[0051] That 1 H NMR spectrum is attached Figure 2 , specifically 9.81-9.93CH2CH2NHCO,CHCOOH,8.37-8.62OCH2CH2NHCO,SCH2CHNH2,7.15-7.47C CHCHC,C CHCC,5.72-5.85(CH2)NHCH2,2.91-3.85NHCH2CH2NH,CCHCHC,NCH2CH2CH2CH2S,COCHCH2CO,2.39-2.61SCH2(CH2)NH2,NCH2CH2CO,CCHCHC,CCCH2CH2 1.24-1.93COCH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2,CCCH2CH2 CH3C(CH3)C,NCH2CH2CH2CH2S.

[0052] Example 2. Cys-(CH2) 15-UV-visible absorption spectrum of IR-781 fluorescent probe

[0053] Take 10 μg of fluorescent probe Cys-(CH2) 15 -IR-781 was dissolved in 1 mL of PBS (0.1 M, pH 7.2) buffer solution, mixed evenly, and added to a quartz cuvette and allowed to stand for 5 min. Subsequently, the cuvette was placed in a UV-Vis full-wavelength spectrophotometer (UV-3600, Shimadzu, Japan) to detect Cys-(CH2) 15 - Absorption spectrum of IR-781 probe, the results are shown in the attached Figure 3 As shown. Cys-(CH2) 15 -The absorption peak of IR-781 appears at 741 nm.

[0054] Example 3. Cys-(CH2) 15 -Fluorescence spectrum of IR-781 fluorescent probe

[0055] Take the fluorescent probe Cys-(CH2) 15 10 μg of IR-781 probe was dissolved in 1 mL of PBS (0.1 M, pH 7.2) buffer solution, mixed evenly and added to a quartz cuvette for 5 min. Subsequently, the emission spectrum of the probe at an excitation wavelength of 760 nm was detected using a fluorescence full-wavelength detector (Hitachi F-4600, Japan). The results are shown in the attached figure. Figure 4 , Cys-(CH2) 15 -The emission peak of IR-781 appears at 788nm.

[0056] Example 4. SLC7A5 expression level in tamoxifen-resistant breast cancer cells

[0057] Western blot was used to detect the expression of human breast cancer cells MCF7 and tamoxifen-resistant breast cancer cells MCF7 / Tam. First, the total protein of MCF7 and MCF7 / Tam cells was extracted by RIPA lysis buffer (Shanghai Biyuntian Biotechnology Co., Ltd.) and the protein content was detected using a BCA protein kit (Shanghai Biyuntian Biotechnology Co., Ltd.). Subsequently, protein loading buffer (Suzhou Xinsaimei Biotechnology Co., Ltd.) was added to these protein samples, mixed and placed in a 99℃ metal bath for 10 minutes to fully denature the protein, and then immediately placed on ice for cooling and stored at -20℃ for use. The protein samples were separated by 10% SDS-PAGE gel electrophoresis and transferred to a PVDF membrane (BIO-RAD, USA). After adding a rapid blocking solution (Shanghai Biyuntian Biotechnology Co., Ltd.) and blocking at room temperature for 15 minutes. The primary antibody was diluted to an appropriate working concentration using the primary antibody diluent and incubated overnight at 4℃ in an incubation box with the PVDF membrane. Subsequently, the secondary antibody was incubated on a shaker at room temperature for 1 hour, and then washed three times with 1 shaker, each time for 10 minutes, and developed with ECL supersensitive luminescent solution for 3 minutes. Finally, the Bio-Rad chemiluminometer was adjusted to the filter-free mode for color development. The obtained band graph was detected by Image J software for grayscale value, and the data was statistically analyzed and then a bar graph was drawn. The primary antibodies are as follows: Mouse anti-β-actin (1:5000, Abcam, USA), rabbit anti-SLC7A5 (1:1000, Abcam, USA); the secondary antibodies are as follows: goat anti-mouse IgG HRP-conjugated antibody (CST, USA), goat anti-rabbit IgG HRP-conjugated antibody (CST, USA).

[0058] As attached Figure 5 And attached Figure 6 As shown, quantitative analysis showed that the gray value ratios of SLC7A5 expression in MCF7 and MCF7 / Tam compared with the internal reference were 0.707 and 1, respectively. Therefore, the expression of SLC7A5 was significantly increased in tamoxifen-resistant breast cancer cell lines.

[0059] Example 5. Cys-(CH2) 15 -IR-781 fluorescent probe targets and tracks drug-resistant breast cancer cells

[0060] To investigate the effect of SLC7A5 expression level on the expression of Cys-(CH2) in MCF7 / Tam 15 - Effect of IR-781 uptake, using siRNA to silence SLC7A5. Take 2 × 10 5MCF7 / Tam cells at a concentration of 100 μg / mL were inoculated in a 24-well plate, and transfection was performed after the degree of confluence reached about 40%. Take tube a and configure 125 μL Opti-MEM buffer + 10 μL siRNA or control NC, and add 125 μL Opti-MEM + 10 μL lipo8000 to tube b. After tube a and tube b were allowed to stand at room temperature for 5 minutes, tube a was added to tube b and mixed, and allowed to stand at room temperature for 20 minutes. Subsequently, 25 μL of the mixture was added to each well of the 24-well plate, and the transfection reagent mixture was shaken. After the cell plate was placed in a 37°C, 5% CO2 incubator for 72 hours, the silencing effect was detected using RT-qPCR experiments. Specifically, the total cell RNA was extracted using the TRIzol method, and after the spectrophotometer was zeroed with DEPC water, 1 μL of RNA sample was aspirated to detect the concentration of the RNA sample at OD260. The ratio of OD260 / OD280 was recorded at wavelengths of 260 nm and 280 nm to measure the purity of the RNA sample. Subsequently, the cDNA obtained by reverse transcription was stored at -20°C.

[0061] The sequence of human SLC7A5 was searched in the database, and the primer sequences of SLC7A5 were selected: siRNA-1 (upstream primer: CCUACAUGCUGGAGGUCUATT; downstream primer: UAGACCUCCAGCAUGUAGGTT), siRNA-2 (upstream primer: UCACAGAGGAAAUGAUCAATT; downstream primer: UUGAUCAUUUCCUCUGUG ATT), siRNA-3 (upstream primer: GUGAACUGCUACAGCGUGATT; downstream primer: UCACGCUG UAGCAGUUCACTT). Subsequently, the RT-qPCR reaction system was prepared, added to 8-tube strips, mixed and centrifuged, and amplified using an RT-qPCR instrument. The expression level of mRNA was relatively quantitatively analyzed using the 2-△△Ct method. The Ct value is the number of cycles used to reach the threshold set by the RT-qPCR instrument. △△Ct=△Ct(experimental group)-△Ct(control group)=(average Ct value of target gene in experimental group-average Ct value of reference gene in experimental group)-(average Ct value of target gene in control group-average Ct value of reference gene in control group). Transfection efficiency is shown in the attached figure. Figure 7 As shown, the results showed that the SLC7A5 gene was knocked down by siRNA-1, siRNA-2 and siRNA-3, among which siRNA-1 had the highest knockdown efficiency.

[0062] MCF7 / Tam cells were seeded in 96-well plates, and siRNA-1 was selected to knock down SLC7A5 in MCF7 / Tam cells. 15-IR-781 was used to incubate MCF7 / Tam cells and MCF7 / Tam cells treated with siRNA-1 for 6 h, and the cell supernatant was removed and washed three times with PBS. The 96-well plate was placed in a microplate reader (SpectraMax XPS, Molecular Devices, USA), after setting the excitation wavelength and emission wavelength to 750nm and 800nm, the fluorescence intensity of the cell samples was detected and recorded. The results are shown in the attached Figure 8 As shown in Figure 2, the fluorescence signal intensity of MCF7 / Tam cells after SLC7A5 knockdown was significantly reduced (p<0.05, Student's T-test), indicating that SLC7A5 mediates the cellular uptake of Cys-(CH2) 15 -IR-781.

[0063] Example 6. Cys-(CH2)9-IR-781 fluorescent probe and its synthesis

[0064] A tumor-targeted fluorescent probe and its synthesis method are as follows:

[0065] 1) Prepare the ICG derivative Cl-IR-781 according to the method of Example 1

[0066] 2) Preparation of maleimide-modified N-acylethylenediamine derivative NH2-(CH2)9-MAL

[0067] Take pimelic acid, N-(2-aminoethyl)maleimide hydrochloride and N-fluorenylmethoxycarbonylethylenediamine hydrochloride in a molar ratio of 1:1:1, dissolve them in a DMF solution containing 6 times the molar amount of DIC and HOBT, react at room temperature for 8-24 hours, and then purify it through a C18 column gel chromatography column to collect the maleimide-modified N-acylethylenediamine derivative NH2-(CH2)9-MAL.

[0068] 3) Preparation of Cysteine-modified Coupled Fluorescent Dye Cys-(CH2)9-IR-781 (Ⅰ, n=3)

[0069] Take NH2-(CH2)9-MAL and Cl-IR-781 in a molar ratio of 1:2, add them to a mixed solution of methanol and N,N-diisopropylethylamine in a volume ratio of 1:1, heat under reflux at 45°C for 2-4 hours to obtain the intermediate product (CH2)9-IR-781; then add Fmoc-protected cysteine ​​dissolved in methanol to the reaction solution, continue to heat under reflux for 2-4 hours, and the reaction product is purified by C18 column gel chromatography to collect the cysteine-modified coupled fluorescent dye Cys-(CH2)9-IR-781.

[0070] Example 7. Cys-(CH2) 17-IR-781 long-chain fluorescent probe and its synthesis

[0071] A tumor-targeted fluorescent probe and its synthesis method are as follows:

[0072] 1) Prepare the ICG derivative Cl-IR-781 according to the method of Example 1

[0073] 2) Maleimide-modified N-acylethylenediamine derivatives NH2-(CH2) 17 Preparation of -MAL

[0074] Take 15-decanedioic acid, N-(2-aminoethyl)maleimide hydrochloride and N-fluorenylmethoxycarbonylethylenediamine hydrochloride in a molar ratio of 1:1:1, dissolve in a DMF solution containing 6 times the molar amount of DIC and HOBT, react at room temperature for 8-24 hours, and then purify by C18 column gel chromatography to collect the maleimide-modified N-acylethylenediamine derivative NH2-(CH2) 17 -MAL.

[0075] 3) Cysteine-modified long-chain fluorescent dye Cys-(CH2) 17 - Preparation of IR-781 (Ⅰ, n=11)

[0076] Take NH2-(CH2) 17 -MAL and Cl-IR-781 were added to a mixed solution of methanol and N,N-diisopropylethylamine in a volume ratio of 1:1 in a molar ratio, and heated under reflux at 65°C for 3 hours to obtain the intermediate product (CH2) 17 -IR-781; then, Fmoc-protected cysteine ​​dissolved in methanol was added to the reaction solution, and the reaction was continued by heating and reflux for 3 hours. The reaction product was purified by C18 column gel chromatography to collect the cysteine-modified long-chain fluorescent dye Cys-(CH2) 17 -IR-781.

[0077] Example 8. Simulation experiment of docking of Cys-(CH2)m-IR-781 fluorescent probe with SLC7A5 molecule

[0078] There are three coupling sites on sulfur-containing amino acids such as methionine and cysteine: sulfhydryl (-SH), amino (-NH2), and carboxyl (-COOH). First, three molecules are randomly selected after one of the three sites is coupled, and the binding of these three structures with the SLC7A5 protein ligand is simulated, so as to test the binding force and binding mode of the two by calculating the physicochemical parameters. The specific method is to use the SLC7A5 protein ligand structure on the RCSB PDB protein structure database, and prepare molecules with three different coupling sites ( Fig. 9 ) and the docking structure of SLC7A5 protein ligand, and molecular docking experiments were performed using Watvina software. Fig. 9 After the sulfhydryl group of the (homo)cysteine ​​molecule is coupled, the atomic distance between it and the SLC7A5 ligand is the smallest, that is, the root mean square deviation (RMSD) between the two is the smallest, indicating that the sulfhydryl group of the cysteine ​​molecule is the optimal coupling site, and the coupled molecule has the best binding ability with the SLC7A5 ligand.

[0079] Considering the steric hindrance of the binding of the fluorescent probe to the SLC7A5 protein ligand, further, the length of the bridging chain segment of the thiol group of the coupled cysteine ​​molecule and the fluorescent group of the probe, and its overall binding force and binding mode with the SLC7A5 protein ligand under this length were simulated. The results showed that for a series of alkane chains of different lengths (CH2 repeating units 3-16), the fluorescent probe of formula I showed good binding ability with the SLC7A5 ligand, among which the alkane chain with 11 repeating units (n=9), as the bridging molecular chain segment between the cysteine ​​molecule and the fluorescent group, made the final structure after the cysteine ​​molecule and the fluorescent group coupled have the best binding force with the SLC7A5 protein ligand, and the experimental score was -8.078 (Appendix Fig.10 ). Regarding the targeting performance of the Cys-(CH2)m-IR-781 fluorescent probe, the fluorescent probe targeted tracing of drug-resistant breast cancer cells experiment was consistent with the simulation results, and the fluorescent probe of Example 1 was better than that of Examples 6 and 7.

Claims

1. A tumor-targeting fluorescent probe, characterized in that: It has the structure shown in the following formula (I), (Ⅰ) Wherein, n is a positive integer of 1-14, and X is Na + , K + .

2. The tumor-targeting fluorescent probe according to claim 1, characterized in that: The n is a positive integer of 3-11.

3. A method for preparing the fluorescent probe according to claim 1, characterized in that: The following steps are involved: 1) Preparation of maleimide-modified N-acylethylenediamine derivatives (II) C5-18 straight chain alkyl dicarboxylic acid (1), N-(2-aminoethyl) maleimide (2) and N-fluorenylmethoxycarbonylethylenediamine (3) are dissolved in a solvent N, N-dimethylformamide, and reacted at room temperature under the action of coupling agents N, N'-diisopropylcarbodiimide and 1-hydroxybenzotriazole to obtain a maleimide-modified N-acylethylenediamine derivative of formula (II), which is denoted as NH2-(CH2)m-MAL. （Ⅱ） 2) Preparation of tumor-targeted fluorescent probes The NH2-(CH2)m-MAL (II) prepared in step 1) and the indocyanine green derivative of the structure of formula (4), denoted as Cl-IR-781, are dissolved in a solvent of methanol, and heated under reflux reaction under the action of a coupling agent N,N-diisopropylethylamine, and then Fmoc-protected cysteine ​​(5) is added to the reaction solution, and the heating reflux reaction is continued to obtain the tumor-targeted fluorescent probe (I); （4） Wherein, n is a positive integer of 1-14, and X is Na + , K + .

4. The method for preparing a fluorescent probe according to claim 3, characterized in that: In step 1), the molar ratio of the C5-18 straight-chain alkyl dibasic acid (1), N-(2-aminoethyl)maleimide (2) and N-fluorenylmethoxycarbonylethylenediamine (3) is 1:1:

1.

5. The method for preparing a fluorescent probe according to claim 3, characterized in that: In step 2), the molar ratio of NH2-(CH2)m-MAL (II) to Cl-IR-781 (4) is 1:1-5.

6. The method for preparing a fluorescent probe according to claim 3, characterized in that: In step 2), the temperature of the heating reflux reaction is 40-70°C.

7. Use of the tumor-targeted fluorescent probe according to claim 1 in the preparation of contrast agents.

8. The use according to claim 7, characterized in that: The contrast agent is used for fluorescent imaging of tumor tissue, and the tumor is breast cancer, rectal tumor, cervical cancer, lung cancer, glioma or nasopharyngeal carcinoma.

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