Fluorescent probe as well as preparation method and application thereof

Abstract

The invention discloses a fluorescent probe as well as a preparation method and application thereof. The structural formula of the fluorescent probe is # imgabs0 #, wherein X <-> is one of Cl <->, Br <-> and I <->. According to the fluorescent probe disclosed by the invention, a biological orthogonal shearing reaction can be quickly carried out to specifically activate fluorescence at a tumor part, so that the imaging specificity of a target part is improved; the probe can be anchored on tumor cell membrane glycoprotein for long-time fluorescence imaging, and is suitable for noninvasive detection of tumors and surgical excision of tumors.

Description

Technical Field

[0001] The present invention relates to the field of fluorescence imaging technology, and in particular to a fluorescent probe and a preparation method and application thereof. Background Art

[0002] Fluorescence imaging is an important tumor imaging method with the advantages of visualization, non-invasiveness and real-time imaging, and is widely used. However, the fluorescent probes currently used in clinical practice (such as indocyanine green, methylene blue, etc.) are all non-specific probes with low signal-to-noise ratio and short retention time in tumor tissue. Although researchers have long been committed to developing fluorescent probes that are specifically activated or coupled to tumor tissue to improve imaging specificity and prolong imaging time, it is still a huge challenge to simultaneously achieve precise activation and long-term retention of fluorescent probes in complex physiological environments.

[0003] Therefore, it is of great significance to develop a fluorescent probe that can achieve tumor-specific activation and retention in a complex physiological environment, synergistically improve the fluorescence imaging signal-to-noise ratio, and prolong the imaging time. Summary of the invention

[0004] The purpose of the present invention is to provide a fluorescent probe and a preparation method and application thereof.

[0005] The technical solution adopted by the present invention is:

[0006] A fluorescent probe, the structural formula of which is:

[0007]

[0008] A sort of.

[0009] A method for preparing the fluorescent probe as described above comprises the following steps:

[0010] 1) performing a nucleophilic addition reaction of 2,3,3-trimethyl-3H-indole and ethane halide to obtain 1-ethyl-2,3,3-trimethyl-3H-indol-1-ium halide, and performing an amide addition reaction of cyclohexanone and N,N-dimethylformamide to obtain 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carboxaldehyde;

[0011] 2) reacting 1-ethyl-2,3,3-trimethyl-3H-indol-1-ium halide with 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carboxaldehyde to obtain

[0012] 3) Conduct and 3,5-dihydroxybenzoic acid to obtain

[0013]

[0014] 4) Conduct and (2E)-trans-cyclooctene-(4-nitrophenyl) carbonate to obtain

[0015]

[0016] 5) Conduct The fluorescent probe is obtained by reacting with cyclopropanecyclooctyne polyethylene glycol.

[0017] Preferably, a method for preparing the fluorescent probe as described above comprises the following steps:

[0018] 1) dispersing 2,3,3-trimethyl-3H-indole and ethane halide in a solvent for nucleophilic addition reaction, and then separating and purifying the product to obtain 1-ethyl-2,3,3-trimethyl-3H-indol-1-ium halide; dispersing cyclohexanone, N,N-dimethylformamide and a catalyst in a solvent for amide addition reaction, and then separating and purifying the product to obtain 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carboxaldehyde;

[0019] 2) dispersing 1-ethyl-2,3,3-trimethyl-3H-indol-1-ium halide, 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carboxaldehyde and a catalyst in a solvent for reaction, and then separating and purifying the product to obtain

[0020] 3) 3,5-Dihydroxybenzoic acid and a catalyst are reacted in a solvent, and then the product is separated and purified to obtain

[0021] 4) (2E)-trans-cyclooctene-(4-nitrophenyl) carbonate and a catalyst are dispersed in a solvent for reaction, and then the product is separated and purified to obtain

[0022] 5) Cyclopropanecyclooctyne polyethylene glycol and a catalyst are dispersed in a solvent for reaction, and then the product is separated and purified to obtain a fluorescent probe.

[0023] Preferably, in step 1), the molar ratio of 2,3,3-trimethyl-3H-indole to ethane halides is 1:1-2.

[0024] Preferably, in step 1), the usage ratio of 2,3,3-trimethyl-3H-indole to solvent is 1 g: 20 mL to 50 mL.

[0025] Preferably, the halogenated ethane in step 1) is one of ethyl chloride, ethyl bromide and ethyl iodide.

[0026] Preferably, the solvent for the nucleophilic addition reaction in step 1) is acetonitrile.

[0027] Preferably, the nucleophilic addition reaction in step 1) is carried out at a temperature of 60° C. to 100° C., and the reaction time is 12 h to 48 h.

[0028] Preferably, the product separation and purification process of the nucleophilic addition reaction in step 1) comprises the following operations: adding the reaction product dropwise into anhydrous ether for precipitation, filtering, and drying the solid.

[0029] Preferably, in step 1), the molar ratio of cyclohexanone to N,N-dimethylformamide is 1:3-4.

[0030] Preferably, in step 1), the molar ratio of N,N-dimethylformamide to the catalyst is 1:1-2.

[0031] Preferably, the catalyst in step 1) is phosphorus oxychloride.

[0032] Preferably, the solvent for the amide addition reaction in step 1) is dichloromethane.

[0033] Preferably, the amide addition reaction in step 1) is carried out at a temperature of 40° C. to 60° C., and the reaction time is 4 h to 6 h.

[0034] Preferably, the separation and purification process of the product of the amide addition reaction in step 1) comprises the following operations: dropping the reaction product onto ice, crystallizing overnight, filtering, and drying the solid.

[0035] Preferably, in step 2), the molar ratio of the 1-ethyl-2,3,3-trimethyl-3H-indol-1-ium halide to 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carbaldehyde is 1 to 3:1.

[0036] Preferably, in step 2), the molar ratio of 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carbaldehyde to the catalyst is 1:1-3.

[0037] Preferably, the catalyst in step 2) is sodium acetate.

[0038] Preferably, in step 2), the usage ratio of the 1-ethyl-2,3,3-trimethyl-3H-indol-1-ium halide and the solvent is 1 g: 4 mL to 15 mL.

[0039] Preferably, the solvent in step 2) is acetic anhydride.

[0040] Preferably, the reaction in step 2) is carried out at a temperature of 120° C. to 1600° C., and the reaction time is 1 h to 2 h.

[0041] Preferably, the product separation and purification process in step 2) comprises the following operations: adding the reaction product dropwise into anhydrous ether for precipitation, filtering, and drying the solid.

[0042] Preferably, step 3) The molar ratio of 3,5-dihydroxybenzoic acid is 1:3-4.

[0043] Preferably, in step 3), the molar ratio of the compound 3,5-dihydroxybenzoic acid to the catalyst is 1:3-4.

[0044] Preferably, the catalyst in step 3) is triethylamine.

[0045] Preferably, step 3) The dosage ratio of the solvent is 1g:20mL~50mL.

[0046] Preferably, the solvent in step 3) is acetonitrile.

[0047] Preferably, the reaction in step 3) is carried out at room temperature (20°C to 25°C) and the reaction time is 12h to 24h.

[0048] Preferably, the product separation and purification process in step 3) comprises the following operations: purifying the reaction product by column chromatography.

[0049] Preferably, step 4) The molar ratio of (2E)-trans-cyclooctene-(4-nitrophenyl) carbonate is 1:1-2.

[0050] Preferably, in step 4), the molar ratio of the (2E)-trans-cyclooctene-(4-nitrophenyl) carbonate to the catalyst is 1:1-2.

[0051] Preferably, the catalyst in step 4) is triethylamine.

[0052] Preferably, in step 4), the ratio of the (2E)-trans-cyclooctene-(4-nitrophenyl) carbonate to the solvent is 1 g: 100 mL to 150 mL.

[0053] Preferably, the solvent in step 4) is N,N-dimethylformamide.

[0054] Preferably, the reaction in step 4) is carried out at room temperature for 48 h to 72 h.

[0055] Preferably, the product separation and purification process in step 4) comprises the following operations: purifying the reaction product by column chromatography.

[0056] Preferably, step 5) The molar ratio of cyclopropanecyclooctyne polyethylene glycol is 1:1-2.

[0057] Preferably, step 5) The molar ratio of the catalyst is 1:1-3.

[0058] Preferably, the catalyst in step 5) is at least one of N,N-diisopropylethylamine (DIPEA) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU).

[0059] Preferably, step 5) The dosage ratio of the solvent is 1g:150mL~250mL.

[0060] Preferably, the solvent in step 5) is at least one of N,N-dimethylformamide, dimethyl sulfoxide and dichloromethane.

[0061] Preferably, the reaction in step 5) is carried out at room temperature for 12 h to 24 h.

[0062] Preferably, the product separation and purification process in step 5) comprises the following operations: purifying the reaction product by column chromatography.

[0063] A tumor detection reagent comprises the above fluorescent probe.

[0064] A detection reagent for guiding surgical excision of cancer tissue, comprising the fluorescent probe.

[0065] The beneficial effects of the present invention are as follows: the fluorescent probe of the present invention can rapidly undergo a bio-orthogonal shearing reaction to specifically activate fluorescence at the tumor site, thereby improving the imaging specificity of the target site; at the same time, by utilizing the bio-orthogonal coupling group of the probe to undergo a bio-coupling effect with the cell, the probe can be anchored on the tumor cell membrane glycoprotein for long-term fluorescence imaging, and is suitable for non-invasive detection of tumors and surgical resection of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 is the compound 1 in the example 1 H NMR spectra.

[0067] Figure 2 is the compound 2 in the example 1 H NMR spectra.

[0068] Figure 3 is the compound 3 in the embodiment 1 H NMR spectra.

[0069] Figure 4 is the compound CyOH in the example 1 H NMR spectra.

[0070] Figure 5 For the compound CyT in the example 1 H NMR spectra.

[0071] Figure 6 The fluorescent probe CyTB in the embodiment 1 H NMR spectra.

[0072] Figure 7 is the fluorescent probe CyB in the comparative example 1 H NMR spectra.

[0073] Figure 8 The figure is a reaction route diagram of the click reaction between the fluorescent probe CyTB and Tz in the embodiment.

[0074] Fig. 9 The UV spectra and fluorescence spectra of the fluorescent probe CyTB in the example reacting with different concentrations of Tz are shown.

[0075] Fig.10 This is a diagram showing the selectivity test results of the fluorescent probe CyTB in the example.

[0076] Fig.11 This is the reaction rate constant test curve of the fluorescent probe CyTB in the example.

[0077] Fig.12 This is a laser confocal image of the fluorescence signal activation and retention of the fluorescent probe CyTB on the cell surface in the example.

[0078] Fig.13 This is a laser confocal image of the specific imaging of the fluorescent probe CyTB on the cell surface in the example.

[0079] Fig.14 This is a laser confocal image of the fluorescent probe CyTB in the example being captured and activated in the mobile phase.

[0080] Fig.15 The figure is a specific fluorescence imaging image of the fluorescent probe CyTB in the example at the tumor site in living mice.

[0081] Fig.16 The figure shows the fluorescence imaging and H&E staining results of the fluorescent probe CyTB in the example to guide the resection in mouse isolated organs.

[0082] Fig.17 This is a diagram showing the safety test analysis results of the fluorescent probe CyTB in the example in mice. DETAILED DESCRIPTION

[0083] The present invention will be further explained and illustrated below in conjunction with specific embodiments.

[0084] Example:

[0085] A fluorescent probe, the preparation method of which is as follows:

[0086] 1) 4.93 g (31 mmol) of 2,3,3-trimethyl-3H-indole and 7.25 g (46.5 mmol) of iodoethane were dissolved in 100 mL of acetonitrile, refluxed at 90°C for 24 h, cooled naturally to room temperature, concentrated by rotary evaporation, and the reaction product was added dropwise to anhydrous ether for precipitation, filtered, and the filter residue was washed with ether three times, and then placed in a vacuum drying oven for 24 h to obtain 8.78 g (27.9 mmol) of 1-ethyl-2,3,3-trimethyl-3H-indol-1-ium iodide (off-white block solid, recorded as compound 1; yield 90%); 40 mL (324 mmol) of N,N-dimethylformamide was dissolved in 40 mL of anhydrous dichloromethane, and the mixture was stirred under ice bath. After stirring for 10 min, 36 mL of a dichloromethane solution of phosphorus oxychloride (containing 383.4 mmol of phosphorus oxychloride and 30 mL of dichloromethane) was added dropwise under nitrogen protection. After the addition was completed, stirring was continued for 15 h. Then 10.6 mL (102.4 mmol) of cyclohexanone precooled to 0° C. was added, stirring was continued for 30 min, and then heated to 55° C. for reaction for 5 h. The mixture was naturally cooled to room temperature, and then the reaction solution was dropped onto ice surface for overnight crystallization. The mixture was filtered, and the filter residue was washed with water 3 times, and then dried in a vacuum drying oven for 24 h to obtain 10.57 g (61.44 mmol) of 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carbaldehyde (bright yellow solid, recorded as compound 2; yield was 60%).

[0087] 2) 2.40 g (13.95 mmol) of 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carboxaldehyde was dissolved in 36 mL of acetic anhydride, and 8.78 g (27.9 mmol) of 1-ethyl-2,3,3-trimethyl-3H-indol-1-ium iodide and 2.29 g (27.9 mmol) of sodium acetate were added under nitrogen atmosphere, and the mixture was heated to 130° C. for reaction for 1 h, and filtered with suction. The filtrate was added dropwise to anhydrous ether and precipitated with ether 3 times, filtered, and the solid was placed in a vacuum drying oven and dried for 24 h to obtain 8.0 g (12.6 mmol) of (dark green solid with metallic luster, recorded as compound 3; yield 90%);

[0088] 3) 1.0 g (1.57 mmol) of Dissolved in 40 mL of acetonitrile, then slowly added 0.73 g (4.73 mmol) of 3,5-dihydroxybenzoic acid and 1.59 g (15.75 mmol) of triethylamine under a nitrogen atmosphere, stirred at room temperature for 12 h, monitored the completion of the reaction by thin layer chromatography, and the solvent was removed by rotary evaporation, and then purified by silica gel column chromatography, the eluent of the column chromatography was composed of dichloromethane and methanol in a volume ratio of 10:1, to obtain 0.27 g (0.47 mmol) of (dark green solid, recorded as compound CyOH; yield 30%);

[0089] 4) Dissolve 40 mg (0.14 mmol) of (2E)-trans-cyclooctene-(4-nitrophenyl) carbonate in 5 mL of N,N-dimethylformamide, and add 14 mg (0.26 mmol) of triethylamine and 40 mg (0.07 mmol) of The mixture was then placed in the dark at room temperature for 72 h. The reaction was monitored by thin layer chromatography to complete. The solvent was removed by rotary evaporation and then purified by silica gel column chromatography. The eluent of the column chromatography consisted of dichloromethane and methanol in a volume ratio of 15:1 to obtain 29 mg (0.046 mmol) of (dark blue solid, denoted as fluorescent probe CyT; yield 33%);

[0090] 5) 29 mg (0.046 mmol) of The mixture was dissolved in 5 mL of N,N-dimethylformamide, and then 7.1 mg (0.055 mmol) of DIPEA and 20.9 mg (0.055 mmol) of HATU were added, and the mixture was stirred for 5 min. Then 16.9 mg (0.046 mmol) of cyclopropanecyclooctyne polyethylene glycol was added, and the mixture was allowed to react for 24 h in the dark at room temperature. The reaction was monitored by thin layer chromatography to complete, and the solvent was removed by rotary evaporation. The mixture was purified by silica gel column chromatography, and the eluent of the column chromatography was composed of dichloromethane and methanol in a volume ratio of 20:1, to obtain 27 mg (0.025 mmol) of the fluorescent probe (dark blue solid, referred to as probe CyTB; the yield was 55%).

[0091] The synthesis reaction of the fluorescent probe CyTB in this example is as follows:

[0092]

[0093] Comparative Example:

[0094] A fluorescent probe, the preparation method of which is as follows:

[0095] 24 mg (0.042 mmol) of The mixture was dissolved in 5 mL of N,N-dimethylformamide, and then 6.5 mg (0.05 mmol) of DIPEA and 19 mg (0.05 mmol) of HATU were added, and the mixture was stirred for 5 min. Then 15.5 mg (0.042 mmol) of cyclopropanecyclooctyne polyethylene glycol was added, and the mixture was allowed to react for 24 h in the dark at room temperature. The reaction was monitored by thin layer chromatography to complete, and the solvent was removed by rotary evaporation. The mixture was purified by silica gel column chromatography, and the eluent of the column chromatography was composed of dichloromethane and methanol in a volume ratio of 20:1, to obtain 24 mg (0.026 mmol) of the fluorescent probe (dark blue solid, referred to as probe CyB; the yield was 63%).

[0096] The synthesis reaction of the fluorescent probe CyB in this comparative example is as follows:

[0097]

[0098] Performance Test:

[0099] 1) Structural characterization of fluorescent probes:

[0100] The H NMR spectrum of compound 1 in the example ( 1 H NMR) Figure 1 As shown, compound 2 1 H NMR images Figure 2 As shown, compound 3 1 H NMR images Figure 3 As shown, the compound CyOH 1 H NMR images Figure 4 As shown, compound CyT 1 HNMR images Figure 5 As shown, the fluorescent probe CyTB 1 H NMR images Figure 6 shown.

[0101] The fluorescent probe CyB in the comparative example 1 H NMR images Figure 7 shown.

[0102] Depend on Figures 1 to 7 It can be seen that: the bioorthogonal fluorescent probe having two click groups, TCO and BCN, was successfully synthesized in the embodiment.

[0103] 2) UV and fluorescence spectra of the fluorescent probe CyTB reacting with different concentrations of Tz:

[0104] The fluorescent probe CyTB was dissolved in a PBS buffer containing 10% (volume percentage) DMSO to prepare a solution with a concentration of 100 μM, and then different concentrations (0-100 μM) of 6-methyl-1,2,4,5-tetrazine-3-thioglycolic acid (Tz) were added and incubated in a shaker at 37°C for 30 min (the reaction route of the click reaction between the fluorescent probe CyTB and Tz is shown in Figure 8 The UV spectra and fluorescence spectra of the reaction solution were measured by UV spectrophotometer and fluorescence spectrophotometer. The test results are shown in Fig. 9 As shown (a is UV spectrum, b is fluorescence spectrum).

[0105] Depend on Fig. 9 From a in the figure, we can see that with the addition of Tz to the reaction, the maximum ultraviolet absorption of the fluorescent probe CyTB undergoes an obvious red shift.

[0106] Depend on Fig. 9 As shown in b, with the increase of Tz concentration (0-100 μM), the fluorescence signal of the fluorescent probe CyTB showed a 6-fold enhancement, and this fluorescence intensity enhancement was positively correlated with the Tz concentration.

[0107] 3) Selectivity test of fluorescent probe CyTB:

[0108] Different fluorescent probes (control group: fluorescent probe CyT / CyB; experimental group: fluorescent probe CyTB) were reacted with Tz respectively, and then the reactivity of fluorescent probes with Tz was detected by high performance liquid chromatography. The fluorescent probe CyTB was mixed with different interfering substances, and then the fluorescence intensity was tested by small animal in vivo imaging. The selectivity test results of the fluorescent probe CyTB were as follows: Fig.10 (a is a high performance liquid chromatogram, b is a fluorescence imaging diagram and a fluorescence intensity test result diagram).

[0109] Depend on Fig.10 From a in the figure, we can see that different fluorescent probes (containing TCO or BCN groups) can undergo click reactions with Tz with good reactivity.

[0110] Depend on Fig.10 From b, we can see that when the fluorescent probe CyTB is mixed with other interfering substances, other substances except Tz fail to cause changes in the fluorescence intensity of the fluorescent probe CyTB, indicating that the fluorescent probe CyTB has good reaction selectivity.

[0111] 4) Reaction rate constant test of fluorescent probe CyTB:

[0112] The fluorescence intensity of the fluorescent probe CyT and different concentrations of Tz (0-50 μM) incubated for 5 min in 10% (volume percentage) DMSO PBS buffer and 10% cell lysate was measured by a microplate reader, and its secondary reaction rate constant was calculated by the fluorescence change. For the bioorthogonal coupling reaction of the fluorescent probe CyB and Tz, Tz was incubated with different concentrations of BCN (0-5 μM) in 10% (volume percentage) DMSO PBS buffer and 10% cell lysate, and the consumption of Tz was detected by ultraviolet absorption and its secondary reaction rate constant was calculated. The reaction rate constant curve of the fluorescent probe CyTB is shown in the figure. Fig.11 (a is the reaction rate constant curve of the bioorthogonal cleavage reaction between CyT and Tz, and b is the reaction rate constant curve of the bioorthogonal coupling reaction between the fluorescent probe CyB and Tz) as shown (the fluorescent probe CyT and Tz will undergo a bioorthogonal cleavage reaction, and the fluorescent probe CyB and Tz will undergo a bioorthogonal coupling reaction. The reaction rates of the two reactions are detected to infer the reaction between the two groups in the fluorescent probe CyTB and Tz).

[0113] Depend on Fig.11 It can be seen that in the cell lysate reaction system, the reaction rate of the bioorthogonal shearing between the fluorescent probe CyT and Tz is 11.8M -1 ·s -1 ±0.45M -1 ·s -1 The bioorthogonal coupling reaction rate of fluorescent probe CyB and Tz is 9.73M -1 ·s -1 ±0.56M -1 ·s -1 The reaction rate constants of the two reactions differ by less than one order of magnitude, which provides a basis for the simultaneous occurrence of two reactions in the fluorescent probe CyTB.

[0114] 5) Laser confocal imaging test of fluorescence signal activation and retention of fluorescent probe CyTB on the cell surface:

[0115] KPC cells were inoculated in a confocal dish. After culturing for 12 hours, the adherent KPC cells were incubated with the pre-targeting molecule tetrazine sugar (MTz; 200nM; prepared by reference to Acta Pharm Sin B. 2023Jun; 13(6): 2736-2746) for 24 hours to label the cell surface with Tz molecules. The cells were then incubated with different fluorescent probes for 20 minutes, and then Hoechst33342 was used to stain the nucleus and fluorescence imaging was performed by laser confocal microscopy (channels: DAPI, Cy5). The test results are shown in Figure 2. Fig.12 shown.

[0116] Depend on Fig.12 It can be seen that: when only the fluorescent probe CyTB exists in the culture medium, no red fluorescence appears within 72 hours, indicating that the prepared fluorescent probe CyTB has good stability and will not spontaneously activate fluorescence under the cell metabolism environment; the MTz+CyT group showed obvious red fluorescence after 5 minutes of incubation, which lasted until 24 hours. After 48 hours, the red fluorescence was significantly weakened and was almost invisible after 72 hours; in the MTz+CyTB group, the fluorescence intensity first increased and then slowly decreased. After 72 hours, the red fluorescence still existed, showing good fluorescence activation and retention effects.

[0117] 6) Laser confocal imaging test of fluorescent probe CyTB on cell surface:

[0118] MTz was coupled to cetuximab (Cet) (binding to EGFR receptor) to prepare Cet-MTz as a pre-targeting molecule for high expression of EGFR, and then KPC cells (high expression of EGFR) and MEF cells (low expression of EGFR) were selected as verification cells. KPC cells were pre-stained with CFSE (green fluorescence) in advance, and then KPC cells and MEF were co-cultured in a confocal dish. After Cet-MTz pretreatment and replacement of supernatant, CyTB probe was added and incubated for 30 minutes. After replacing the supernatant, Hoechst33342 was added to stain the nucleus and the activation of the probe in the cell was detected by confocal microscopy (channels: DAPI, Cy5, FITC). The test results are as follows: Fig.13 shown.

[0119] Depend on Fig.13 It can be seen that the fluorescent probe CyTB can activate imaging in tumor cells with high EGFR expression, but cannot be activated in cells with low EGFR expression, and has specific imaging function.

[0120] 7) Laser confocal imaging test of fluorescent probe CyTB being captured and activated in the mobile phase:

[0121] The microfluidic chip device ibidi (μ-Slide I 0.4Luer, 80176, Germany) was used to simulate the process of the probe being captured and activated in the blood flow. The chip was 50 mm long, 5.0 mm wide, and 0.4 mm high. 100 μL of cell suspension (2×10 6 mL -1 ) and incubate the cells. After the cells are stable, the fluorescent probe CyTB (20 μM) or fluorescent probe CyT (20 μM) solution is prepared into a 50 mL syringe as the mobile phase and injected into the chip at a constant speed through a syringe pump. The flow rate is controlled at 1 mL / min. After different time periods, the cells on the chip are stained for nuclear staining and the activation of the probe is detected by confocal microscopy (channels: DAPI, Cy5). The test results are shown in Fig.14 shown.

[0122] Depend on Fig.14 It can be seen that the fluorescent probe CyT, which can only perform bioorthogonal shear activation, has a poor retention effect in the simulated vascular environment, while the fluorescent probe CyTB, which has both bioorthogonal activation and coupling functions, has a significantly prolonged retention time.

[0123] 8) Specific fluorescence imaging test of fluorescent probe CyTB in tumor sites of living mice:

[0124] Fifteen C57BL / 6 mice were orthotopically implanted with pancreatic cancer. One week later, the successful implantation was confirmed by bioluminescence, and then the tumor-bearing mice were randomly divided into five groups, with three mice in each group, marked as G1: blank group, G2: CyOH group, G3: Cet-MTz+CyT group, G4: Cet-MTz+CyB group, G5: Cet-MTz+CyTB group. G3, G4 and G5 groups were continuously injected with Cet-MTz (20 mg / kg) for 3 days for pre-targeting (once a day), and on the 4th day, the Cet-MTz (20 mg / kg) was injected into the Cet-MTz group. On the first three days, the fluorescent probes CyT, CyB and CyTB (1 mg / kg) were injected through the tail vein of the mice, while the G1 group was injected with the same volume of saline at the same time point each time. The G2 group was injected with the same volume of saline for the first three days and CyOH (1 mg / kg) on ​​the fourth day. The distribution of the probes in the mice was observed by taking pictures with a small animal in vivo imaging device at different time points after injection, and the changes in the fluorescence intensity of each group and the tumor tissue / normal tissue fluorescence intensity ratio (TNR) were further quantified. The test results are shown in Fig.15 (a is a fluorescence imaging diagram, b is a curve showing the relationship between the average radiation efficiency of the tumor and the injection time, and c is a curve showing the relationship between TNR and time).

[0125] Depend on Fig.15 It can be seen that: except for the blank group, the fluorescence intensity of the other groups first increased and then decreased with time, but the fluorescence decrease rate of the two groups injected with coupled fluorescent probes (CyB, CyTB) was significantly slower than that of G2 and G3 groups, verifying that the probes had a good retention effect; secondly, quantitative analysis showed that the G5 group had the highest signal-to-noise ratio at each time point, and the signal-to-noise ratio increased with time and reached more than 15; the above results all indicate that in the mouse in situ pancreatic cancer model, the fluorescent probe CyTB has the advantages of tumor-specific activation, high signal-to-noise ratio (TNR>15) and long-term imaging (>130h).

[0126] 9) Testing the performance of the fluorescent probe CyTB in guiding excision in isolated mouse organs:

[0127] As above, mice were implanted with pancreatic cancer orthotopically and given drugs in groups. The mice were euthanized 48 hours after drug administration, and the tumor tissues at the pancreatic cancer site were removed according to the guidance of fluorescence imaging. The removed tumor tissues were then stained with H&E for observation. At the same time, the main organs (heart, liver, spleen, lung, kidney) and muscle tissues of each group were obtained, and the fluorescence intensity in each tissue sample was detected by small animal in vivo imaging. The test results are shown in the figure. Fig.16 shown.

[0128] Depend on Fig.16 It can be seen that after the G5 group probe guided the resection of the tumor, no fluorescence was observed in the tissues surrounding the tumor, and there was almost no fluorescence in other ex vivo organs. Combined with the clean tumor resection margins in the G5 group in H&E staining, this indicates that the fluorescent probe CyTB has excellent tumor specificity and can effectively distinguish tumor tissue from normal tissue.

[0129] 10) Safety test of fluorescent probe CyTB in mice:

[0130] Fifteen C57BL / 6 mice were randomly divided into five groups as above. After 24 hours of drug administration, the eyeballs of the mice were removed to collect blood. Blood samples of physiological indicators were collected in EDTA anticoagulant tubes, and blood samples of biochemical indicators were collected in 1.5 mL centrifuge tubes. The physiological and biochemical indicators of each group were measured. The test results are shown in Fig.17 shown.

[0131] Depend on Fig.17 It can be seen that after different drug treatments, the alkaline phosphatase (ALP), alanine aminotransferase (ALT), urea nitrogen (BUN), creatinine (CREN), white blood cells (WBC), red blood cell count (RBC), hemoglobin concentration (HGB) and hematocrit (HCT) indicators of mice were all within the normal range, indicating that the fluorescent probe CyTB has almost no effect on the liver and kidney function and routine blood indicators of mice.

[0132] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A fluorescent probe, characterized in that The structural formula is: In the formula, X - Cl - Br - ,I - One of them.

2. A method for preparing a fluorescent probe as claimed in claim 1, characterized in that: The following steps are involved: 1) performing a nucleophilic addition reaction of 2,3,3-trimethyl-3H-indole and ethane halide to obtain 1-ethyl-2,3,3-trimethyl-3H-indol-1-ium halide, and performing an amide addition reaction of cyclohexanone and N,N-dimethylformamide to obtain 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carboxaldehyde; 2) reacting 1-ethyl-2,3,3-trimethyl-3H-indol-1-ium halide with 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carboxaldehyde to obtain 3) Conduct and 3,5-dihydroxybenzoic acid to obtain 4) Conduct and (2E)-trans-cyclooctene-(4-nitrophenyl) carbonate to obtain 5) Conduct The fluorescent probe is obtained by reacting with cyclopropanecyclooctyne polyethylene glycol.

3. The preparation method according to claim 2, characterized in that: The molar ratio of 2,3,3-trimethyl-3H-indole to ethane halides in step 1) is 1:1-2; the molar ratio of cyclohexanone to N,N-dimethylformamide in step 1) is 1:3-4.

4. The preparation method according to claim 2, characterized in that: In step 2), the molar ratio of the 1-ethyl-2,3,3-trimethyl-3H-indol-1-ium halide to 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carbaldehyde is 1 to 3:

1.

5. The preparation method according to claim 2, characterized in that: Step 3) The molar ratio of 3,5-dihydroxybenzoic acid is 1:3-4.

6. The preparation method according to claim 2, characterized in that: Step 4) The molar ratio of (2E)-trans-cyclooctene-(4-nitrophenyl) carbonate is 1:1-2.

7. The preparation method according to claim 2, characterized in that: Step 5) The molar ratio of cyclopropanecyclooctyne polyethylene glycol is 1:1-2.

8. The preparation method according to any one of claims 2 to 7, characterized in that: The nucleophilic addition reaction in step 1) is carried out at a temperature of 60°C to 100°C, and the reaction time is 12h to 48h; the amide addition reaction in step 1) is carried out at a temperature of 40°C to 60°C, and the reaction time is 4h to 6h; the reaction in step 2) is carried out at a temperature of 120°C to 1600°C, and the reaction time is 1h to 2h; the reaction in step 3) is carried out at room temperature, and the reaction time is 12h to 24h; the reaction in step 4) is carried out at room temperature, and the reaction time is 48h to 72h; the reaction in step 5) is carried out at room temperature, and the reaction time is 12h to 24h.

9. A tumor detection reagent, characterized in that: The fluorescent probe according to claim 1 is included.

10. A detection reagent for guiding surgical resection of cancer tissue, characterized in that: The fluorescent probe according to claim 1 is included.

Images