A specific targeting lipid raft fluorescent probe and its preparation method and application

Abstract

The present invention belongs to the field of fluorescent probes, and specifically relates to a specific targeting lipid raft fluorescent probe and its preparation method and application. In view of the characteristics of visualization, non-invasiveness, high sensitivity, etc., fluorescence technology has become an indispensable means of modern biological and medical research, and has played an increasingly important role in the early diagnosis of tumors and surgical navigation. Lipid rafts are microdomains rich in cholesterol and sphingomyelin on the plasma membrane, which play a key role in cancer cell adhesion and migration, and are overexpressed on the membranes of many cancer cells. Therefore, the present invention has developed a probe that can specifically target lipid rafts. The probe can use the characteristic that cancer cells overexpress lipid rafts compared to normal cells to achieve the distinction between the two, and further serve as a fluorescent surgical navigation reagent to guide the removal of tumors in SKOV3 tumor-bearing mice. In addition, the reagent also has the characteristics of short action time and no need for washing.

Description

Technical Field

[0001] The invention belongs to the field of fluorescent probes, and in particular relates to a specific targeted lipid raft fluorescent probe and a preparation method and application thereof. Background Art

[0002] Cancer refers to malignant tumors. Early detection, diagnosis and treatment of cancer are important means to improve its cure rate and survival rate and reduce mortality. How to distinguish cancer cells / cancer tissues from normal cells / normal tissues is the key to early diagnosis and treatment of cancer. In view of its visualization, non-invasiveness and high sensitivity, fluorescence technology has become an indispensable means in modern biological and medical research, and has played an increasingly important role in the early diagnosis and surgical navigation of tumors. In addition, in view of its advantages such as high spatiotemporal resolution, activatability, no radiation, no drug resistance, and repeated diagnosis and treatment, this technology has also shown great potential in photodynamic therapy. Compared with normal cells and their surrounding tissues, cancer cells have unique microstructures and microenvironments, such as overexpression of certain proteins on the cell membrane surface, tissue hypoxia, decreased intracellular and extracellular pH, and increased levels of reactive sulfur (RSS) and reactive oxygen (ROS). These characteristics have an important impact on tumor proliferation, invasion, migration, adhesion ability and the formation of new blood vessels. Among them, lipid rafts are microdomains rich in cholesterol and sphingomyelin on the plasma membrane. They play a key role in cancer cell adhesion and migration, and are overexpressed on the membranes of many cancer cells. Therefore, the use of fluorescent probes that specifically target lipid rafts is expected to achieve the distinction between cancer cells / cancer tissues and normal cells / normal tissues. Summary of the invention

[0003] In view of the above problems, the present invention provides a specific targeting lipid raft fluorescent probe and its preparation method and application. The probe can specifically target the ester rafts overexpressed by cancer cells, and thus can be used to distinguish cancer cells / tissues from normal cells / tissues. In addition, the reagent can also guide the intraoperative removal of tumors in SKOV3 tumor-bearing mice, providing a potential fluorescent surgical navigation reagent for clinicians when surgically removing tumors.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions:

[0005] A specific targeting lipid raft fluorescent probe, the structural formula of which is:

[0006]

[0007] A method for preparing a specific lipid raft-targeting fluorescent probe comprises the following steps:

[0008]

[0009] (1) Under nitrogen protection, methyl 2,3,3-trimethyl-3H-indole-5-carboxylate (Compound 1), 2-(2-bromoethyl)-1,3-dioxane (Compound 2), NaHCO3 and NaI were dissolved in anhydrous acetonitrile, stirred for reaction, and after the reaction, the reaction solution was cooled to room temperature, the solvent was dried, and the crude product was separated and purified by column chromatography to obtain Compound 3;

[0010] (2) Compound 3, 3-formyl-4-chlorocoumarin (Compound 4), triethylamine hydrochloride and piperidine are dissolved in acetonitrile in sequence, and refluxed under vigorous stirring. After the reaction, the reaction solution is cooled to room temperature, and the solvent is dried by rotary evaporation to obtain an intermediate; the intermediate is redissolved in a mixed solvent of dichloromethane and concentrated hydrochloric acid and the reaction is continued until the reaction of the raw materials is complete as monitored by TLC. After the reaction, the dichloromethane is dried by rotary evaporation, and the remaining acid solution is adjusted to pH 6-7 with a Na2CO3 aqueous solution, extracted, and dried to obtain a crude product. The crude product is separated and purified by column chromatography to obtain the target compound CRCHC;

[0011] Furthermore, in the step (1), the molar ratio of 2,3,3-trimethyl-3H-indole-5-carboxylic acid methyl ester, 2-(2-bromoethyl)-1,3-dioxane, NaHCO3 and NaI is 2:3:4:3.

[0012] Furthermore, the stirring reaction temperature in step (1) is 95° C. and the time is 18 hours.

[0013] Furthermore, the developing solvent for column chromatography separation and purification in step (1) is petroleum ether:ethyl acetate=5:1 (v / v).

[0014] Furthermore, in the step (2), the molar ratio of compound 3, 3-formyl-4-chlorocoumarin and triethylamine hydrochloride is 1:1:1, and the volume ratio of dichloromethane and concentrated hydrochloric acid is 50 mL:15 mL.

[0015] Furthermore, in the step (2), the temperature of the reflux reaction is 82° C. (boiling point of acetonitrile), the time is 12 h, and the temperature of the continued reaction is 50° C.

[0016] Furthermore, the developing solvent for column chromatography separation and purification in step (2) is CH2Cl2:MeOH:TFA=400:10:1 (v / v / v).

[0017] A fluorescent probe is used to distinguish cancer cells / tissues from normal cells / tissues, and is used in guided surgery to remove tumors in SKOV3 tumor-bearing mice.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] Compared with normal cells, cancer cells consume a large amount of glucose and produce a large amount of lactic acid through aerobic glycolysis. This metabolic mode will lead to a special microenvironment of cancer cells, such as hypoxia, reduced pH, increased levels of reactive sulfur (RSS) and reactive oxygen (ROS), and reduced polarity. However, the fluorescent probes designed based on these characteristics need to connect the cancer cell-specific reaction sites with the fluorophores, and the synthesis steps are cumbersome and the sensitivity is not high. The present invention has developed a probe that can specifically target lipid rafts. The probe can use the characteristic that cancer cells overexpress lipid rafts compared to normal cells to achieve the distinction between the two, and further serve as a fluorescent surgical navigation reagent to guide the removal of tumors in SKOV3 tumor-bearing mice. In addition, the reagent also has the characteristics of short action time and no need for washing. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 NMR and HRMS diagrams of compound 3 of the present invention;

[0021] Figure 2 NMR and HRMS diagrams of the probe CRCHC of the present invention;

[0022] Figure 3 Cytotoxicity experiments were performed on A549 cells after treatment with different concentrations (0 μM, 1.0 μM, 2.0 μM, 3.0 μM, 4.0 μM and 5.0 μM) of CRCHC for 24 hours;

[0023] Figure 4 Cell images of A549 cells treated with CRCHC (0.5 μM, 5 min), with an excitation wavelength of 561 nm and a collection wavelength of 570-740 nm. Scale bar: 20 μm.

[0024] Figure 5 A: A549 cells were incubated with CRCHC (0.2 μM, 5 min) and commercial cell membrane probe DiO (5 μM, 10 min). Figure 5 B is the cell image of A549 cells after incubation with CRCHC (0.2μM, 5min) and commercial lipid raft probe CLS-PEG-FITC (0.5μM, 10min). For CRCHC, the collection wavelength was 570-740nm (λ ex =561 nm), for DiO and CLS-PEG-FITC, the collection wavelength was 490-560 nm (λ ex =488nm), scale bar: 20μm;

[0025] Figure 6Fluorescence imaging of A549 cells, HepG2 cells, LO2 cells, and BEAS-2B cells treated with CRCHC (0.5 μM) for 5 minutes, with a collection wavelength of 570–750 nm (λex = 561 nm), and a scale bar of 20 μm;

[0026] Figure 7 Fluorescence imaging of tumor tissue sections and normal organ tissue sections of tumor-bearing mice treated with CRCHC (1.0 μM) for 5 minutes, with a collection wavelength of 570–740 nm (λex = 561 nm), and a scale bar of 20 μm;

[0027] Figure 8 In vivo imaging of tumors and surrounding normal tissues in SKOV3 tumor-bearing mice exposed 5 minutes after spraying CRCHC (20 μM). DETAILED DESCRIPTION

[0028] Example 1

[0029] A specific targeting lipid raft fluorescent probe, the structural formula of which is:

[0030]

[0031] A method for preparing a specific lipid raft-targeting fluorescent probe comprises the following steps:

[0032] (1) Under nitrogen protection, 2,3,3-trimethyl-3H-indole (compound 1, 2.0 mmol), 2-(2-bromoethyl)-1,3-dioxane (compound 2, 3.0 mmol), NaHCO3 (4.0 mmol) and NaI (3.0 mmol) were dissolved in anhydrous acetonitrile, and the mixture was stirred at 95° C. for 18 hours. After the reaction, the reaction solution was cooled to room temperature, the solvent was dried, and the crude product was separated and purified by column chromatography (developing solvent: petroleum ether / ethyl acetate = 5:1, v / v) to obtain compound 3 (0.331 g, yield 52.2%). Figure 1 NMR and HRMS spectra of compound 3.

[0033] 1 H NMR (600MHz, CDCl3) δ7.91 (d, J = 6.6Hz, 1H), 7.75 (s, 1H), 6.62 (s, 1H), 4.92 (s ,1H),4.08(s,1H),4.00-3.88(m,8H),3.72(s,2H),2.03(s,2H),1.35(s,6H); 13C NMR (150MHz, CDCl3) δ167.5,160.6,149.7,137.5,131.0,123.2,120.0,104.5,102.4,76.3,65.0,51.7,43.7,37.2,30.1,29.9; ESI-MS[M+H] + :calculated for318.1700,found318.1700.

[0034] (2) Compound 3 (1 mmol), 3-formyl-4-chlorocoumarin (compound 4, 1 mmol), triethylamine hydrochloride (1 mmol) and a few drops of piperidine were dissolved in acetonitrile (50 mL) in sequence, and the mixture was refluxed at 82° C. for 12 hours under vigorous stirring. After the reaction, the reaction solution was cooled to room temperature and the solvent was dried to obtain an intermediate. The intermediate was redissolved in a mixed solvent of dichloromethane (50 mL) and concentrated hydrochloric acid (15 mL) and the reaction was continued at 50° C. until the reaction of the raw materials was complete as monitored by TLC. After the reaction, the dichloromethane was dried to obtain the remaining acid solution, and the pH of the remaining acid solution was adjusted to 6-7 with a Na2CO3 aqueous solution, and then the crude product was extracted and dried to obtain the crude product. The crude product was separated and purified by column chromatography (developing solvent: CH2Cl2:MeOH:TFA=400:10:1, V / V / V) to obtain the target compound CRCHC (0.270 g, yield 52.0%). Figure 2 NMR and HRMS charts of compound CRCHC.

[0035] 1 H NMR(600MHz,CD3OD)8.30(s,1H),8.27(d,J=8.4Hz,2H),7.80(d,J=2.4Hz,1H),7.7 3(d,J=8.4Hz,1H),6.91(d,J=7.8Hz,1H),6.62(s,1H),5.69(t,J=4.8,6.0Hz,1H), 4.76(d,J=14.4Hz,1H),4.35(t,J=13.8,13.2Hz,1H),3.61(d,J=6.0Hz,4H),3.07( d,J=12.0Hz,1H),2.71(t,J=4.2,7.2Hz,1H),1.84(d,J=16.8Hz,6H),1.28(s,6H); 13CNMR(150MHz,CD3OD)176.2,167.2,167.0,160.2,158.5,155.4,144.2,142.7,140.7,131.0,130.6,126.6 ,123.6,112.8,111.0,108.6,101.0,100.1,97.0,72.5,51.2,45.1,42.3,26.4,25.0,24.8,11.4; ESI-MS[M + ]: calculated for 485.2071, found 485.2076.

[0036] Example 2

[0037] 1. Test solution preparation

[0038] The dye CRCHC was prepared into a 2 mM stock solution in DMSO (containing 2% acetic acid) and then diluted to the test concentration with the solvent to be tested.

[0039] 2. Study on photophysical properties

[0040] Table 1 Photophysical properties of CRCHC

[0041]

[0042] First, we studied the photophysical properties of CRCHC in dichloromethane, acetonitrile and PBS. As shown in Table 1, the maximum absorption and emission wavelengths of CRCHC in the above three solvents are 628 / 661nm, 591 / 652nm and 582 / 651nm, respectively. Compared with the classic rhodamine, Bodipy and cyanine dyes (<20nm), the probe has a large Stokes shift (>30nm in DCM, >60nm in acetonitrile and PBS), which indicates that it can effectively avoid the interference of autofluorescence and excitation light scattering in biological imaging. In addition, CRCHC has a large molar absorption coefficient (ε) and fluorescence quantum yield (Φ) in different solvents, indicating that it has a large fluorescence brightness (ε×Φ), so it has more advantages in biological imaging.

[0043] 3. Cell Imaging Studies

[0044] All cell lines were purchased from GeneFull Biotechnology Co., Ltd. (China). A549 cells and HepG2 cells were cultured in DMEM medium containing 10% FBS (fetal bovine serum), 100 U / mL penicillin G sodium, and 100 μg / mL streptomycin at 37°C in a 5% carbon dioxide humidified environment, and LO2 cells and BEAS-2B cells were cultured in RPMI 1640 medium containing 10% FBS (fetal bovine serum), 100 U / mL penicillin G sodium, and 100 μg / mL streptomycin.

[0045] Before conducting the cell imaging experiment, the CCK-8 cell proliferation assay was first used to evaluate the cytotoxicity of CRCHC. Figure 3 As shown, when A549 cells were treated with different concentrations (0 μM, 1.0 μM, 2.0 μM, 3.0 μM, 4.0 μM and 5.0 μM) of CRCHC for 24 hours, the cell survival rate was greater than 85%, indicating that it has good biocompatibility.

[0046] Subsequently, the cell imaging ability of CRCHC was investigated using a Ceiss LMS 710 confocal microscope. A549 cells were incubated in DMEM medium containing CRCHC (0.5 μM) for 20 min, and then images were acquired at the indicated time points, e.g. Figure 4 As shown in the figure, CRCHC can accumulate rapidly on the cell membrane and distribute in a scattered pattern, and its fluorescence signal cannot overlap with the commercial cell membrane dye DiO ( Figure 5 A), which indicates that CRCHC only targets a certain area of ​​the cell membrane. Further co-localization experiments show that the fluorescence signal of CRCHC can overlap well with the commercial ester raft probe CLS-PEG-FITC ( Figure 5 B), indicating that it can specifically target lipid rafts on the cell membrane.

[0047] Considering that lipid rafts are overexpressed on the membranes of many cancer cells, CRCHC is expected to be used to distinguish cancer cells from normal cells by fluorescence, e.g. Figure 6 As shown, the cell membranes of cancer cells (A549 cells and HepG2 cells) loaded with CRCHC showed strong fluorescence signals, while the cell membranes of normal cells (BEAS-2B cells and LO2 cells) loaded with CRCHC showed almost no fluorescence signals, indicating that CRCHC can distinguish cancer cells from normal cells based on the differences in lipid raft expression levels on cancer / normal cell membranes.

[0048] 4. Tissue Slice Imaging Studies

[0049] All animal experiments were conducted in accordance with the relevant laws and guidelines issued by the Ethics Committee of Shanxi University. BALB / c male nude mice (6-8 weeks old) were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. SKOV3 cells (1×10 6 10 cells) were injected subcutaneously into the base of the left leg of nude mice. 15 days after inoculation, tumor-bearing mice were obtained. The tumor-bearing mice were killed by excessive anesthesia, and the five internal organs and tumors were removed and sliced ​​into 10 μm thick sections on a freezing microtome. The slices of different tissues were treated with CRCHC (1 μM) for 5 minutes and imaged on a laser confocal microscope. Figure 7 As shown, the fluorescence intensity of cancer tissues was significantly higher than that of heart, liver, spleen, lung and kidney tissues. The above results indicate that CRCHC can distinguish tumor tissues from normal tissues of tumor-bearing mice.

[0050] 5. Fluorescence navigation-guided tumor resection

[0051] Finally, we evaluated the potential of CRCHC as a fluorescent surgical navigation agent to guide surgical resection of tumors in SKOV3-bearing mice using a Bruker multimodal in vivo imaging system. Figure 8 As shown, Figure 8 A in the middle is a tumor-bearing mouse before dissection, and the tumor and normal tissues were exposed after dissection ( Figure 8 Middle B), the imaging results showed that no fluorescent signal was seen in any area ( Figure 8 Middle C); Images 5 minutes after spraying CRCHC (2 μM) on the tumor and surrounding normal tissues ( Figure 8 Middle D), only the tumor site showed strong fluorescence signal; after the tumor was completely removed, the image was taken again ( Figure 8 In the middle (E), there is almost no fluorescence signal in the remaining tissue. The results show that CRCHC has a good application prospect in fluorescence navigation-guided surgical tumor resection.

[0052] In summary, the present invention has developed a specific targeted lipid raft fluorescent probe (CRCHC), which can specifically target lipid rafts overexpressed on the cell membrane of cancer cells, and thus can be used to distinguish cancer cells / cancer tissues from normal cells / normal tissues; in addition, the probe can also be used as a fluorescent surgical navigation agent to guide the removal of tumors in SKOV3 tumor-bearing mice.

[0053] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A specific targeting lipid raft fluorescent probe, characterized in that: The structural formula of the probe is:

2. A method for preparing a specific targeting lipid raft fluorescent probe according to claim 1, characterized in that: The following steps are involved: (1) Under nitrogen protection, methyl 2,3,3-trimethyl-3H-indole-5-carboxylate, 2-(2-bromoethyl)-1,3-dioxane, NaHCO3 and NaI were dissolved in anhydrous acetonitrile, stirred for reaction, and after the reaction, the reaction solution was cooled to room temperature, the solvent was dried by spin drying, and the crude product was separated and purified by column chromatography to obtain compound 3; (2) Compound 3, 3-formyl-4-chlorocoumarin, triethylamine hydrochloride and piperidine are dissolved in acetonitrile in sequence, and refluxed under vigorous stirring. After the reaction, the reaction solution is cooled to room temperature, and the solvent is dried to obtain an intermediate; the intermediate is redissolved in a mixed solvent of dichloromethane and concentrated hydrochloric acid and the reaction is continued until the reaction of the raw materials is complete as monitored by TLC. After the reaction, the dichloromethane is dried to obtain the remaining acid solution, and the pH is adjusted to 6-7 with a Na2CO3 aqueous solution, and then extracted and dried to obtain a crude product, and the crude product is separated and purified by column chromatography to obtain the target compound; The structural formula of the compound 3 is: The structural formula of the intermediate is:

3. The method for preparing a specific targeting lipid raft fluorescent probe according to claim 2, characterized in that: In the step (1), the molar ratio of 2,3,3-trimethyl-3H-indole-5-carboxylic acid methyl ester, 2-(2-bromoethyl)-1,3-dioxane, NaHCO3 and NaI is 2:3:4:

3.

4. The method for preparing a specific targeting lipid raft fluorescent probe according to claim 2, characterized in that: The stirring reaction temperature in step (1) is 95° C. and the stirring reaction time is 18 h.

5. The method for preparing a specific targeting lipid raft fluorescent probe according to claim 2, characterized in that: The developing solvent for column chromatography separation and purification in step (1) is petroleum ether:ethyl acetate=5:1 (v / v).

6. The method for preparing a specific targeting lipid raft fluorescent probe according to claim 2, characterized in that: In the step (2), the molar ratio of compound 3, 3-formyl-4-chlorocoumarin and triethylamine hydrochloride is 1:1:1, and the volume ratio of dichloromethane and concentrated hydrochloric acid is 50 mL:15 mL.

7. The method for preparing a specific targeting lipid raft fluorescent probe according to claim 2, characterized in that: The temperature of the reflux reaction in step (2) is 82°C, the time is 12h, and the temperature of the continued reaction is 50°C.

8. The method for preparing a specific targeting lipid raft fluorescent probe according to claim 2, characterized in that: The developing solvent for column chromatography separation and purification in step (2) is CH2Cl2:MeOH:TFA=400:10:1 (v / v / v) 9. An application of the specific targeting lipid raft fluorescent probe according to claim 1, characterized in that: Used to prepare reagents to distinguish cancer cells / tissues from normal cells / tissues.

10. An application of the specific targeting lipid raft fluorescent probe according to claim 1, characterized in that: Used to prepare fluorescent surgical navigation reagents for guided surgery to remove tumors in SKOV3 tumor-bearing mice.

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