CN116284147B - Fluorescent probe for selectively detecting hNQO1 with high expression in tumor cells and application thereof - Google Patents
Fluorescent probe for selectively detecting hNQO1 with high expression in tumor cells and application thereof Download PDFInfo
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- CN116284147B CN116284147B CN202310135408.9A CN202310135408A CN116284147B CN 116284147 B CN116284147 B CN 116284147B CN 202310135408 A CN202310135408 A CN 202310135408A CN 116284147 B CN116284147 B CN 116284147B
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a fluorescent probe for selectively detecting hNQO1 in tumor cells and application thereof, wherein the fluorescent probe can start red fluorescent emission at 640nm of a complex after reacting with hNQO1 enzyme, and the fluorescent probe has the capability of effectively recognizing hNQO enzyme in phosphate buffer solution, the detection limit is 0.4239ng/ml, and the fluorescent probe is not interfered by other biological related metal ions, anions and biomolecules. The probe has high selectivity and sensitivity, excellent water solubility and membrane permeability and low cytotoxicity, can be used as a tool for visually monitoring the over-expressed hNQO enzyme in living cells, can effectively distinguish NQO1 positive cancer cells from NQO1 negative cells, can be applied to pathological diagnosis in hNQO1 over-expressed tumor tissue section operation, realizes hNQO1 in micron-scale high-resolution real-time imaging tumor, and is favorable for early diagnosis and timely treatment of hNQO over-expressed tumor.
Description
Technical Field
The invention relates to a fluorescent probe, in particular to a fluorescent probe for selectively detecting highly expressed NAD (P) H: quinone oxidoreductase (hNQO 1) in tumor cells and application thereof.
Background
Cancer has been one of the most serious public health problems, and the overall survival rate of cancer patients is very low and is closely related to the stage of early diagnosis of tumors. Therefore, accurate identification of cancer cells is critical for early diagnosis and timely treatment of malignant tumors. The photoluminescence imaging technology based on the probe not only has higher selectivity, sensitivity and space-time resolution, but also can carry out real-time noninvasive monitoring on the micro-environment of the pathological tissue cells, and is an excellent choice for identifying cancer cells. However, it is critical to have probes that target tumor markers (enzymes, genes, proteins, and small molecules) and that rapidly initiate the ability to accurately recognize cancer cells. The human NAD (P) H is closely related to cancer, and is highly expressed (5-200 times) in various tumor cells such as lung cancer, colon cancer, liver cancer, breast cancer, melanoma, pancreatic cancer, gastric cancer and the like compared with normal cells, plays an important role in the development of the cancer, and can be used as an ideal target point of biosensor design. In recent years, a series of fluorescent probes containing various fluorophores capable of activating hNQO1 based on the trimethylbenzoquinone locking effect have been developed for tumor imaging. However, most reported fluorophores have some limitations, for example, ultraviolet excitation is prone to damage to biological samples; small stokes shifts can lead to reduced sensitivity and accuracy; photobleaching results in fluorescence quenching of the probe; the low water solubility also prevents its practical use in the biological field. Therefore, it remains critical to construct a novel fluorescent probe that efficiently recognizes hNQO enzymes within tumor cells.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a fluorescent probe for detecting the NAD (P) H: quinone oxidoreductase (hNQO 1) with high expression in tumor cells.
The fluorescent probe for detecting the NAD (P) H with high expression in tumor cells comprises a quinone oxidoreductase (hNQO) 1, wherein the structure of a phosphorescence probe [ Ru (TLQ-PIPY) (bipy) 2]2+ of the ruthenium (II) complex is shown as (I):
The preparation method of the fluorescent probe compound for detecting the NAD (P) H with high expression in tumor cells, namely quinone oxidoreductase (hNQO 1), is summarized as follows:
To increase the water solubility of the probe and to obtain relatively good photophysical properties at the same time as the practicality under physiological environment, the inventors dissolved 2, 5-dibromopyrazine and N-Boc-piperazine in N-methylpyrrolidone (NMP), reacted for 24 hours under reflux to obtain compound 1 (BRPY), and then added to a toluene solution containing 2-tributyltin-based pyridine to react for 10 hours under stirring at room temperature to obtain compound 2 (PIPY). Subsequently, the resulting compound 2 (PIPY) was dissolved in methylene chloride to react with trimethylquinone acid, and 2- (7-azobenzotriazol-1-yl) -N, N' -tetramethylammonium Hexafluorophosphate (HATU) and N, N-Diisopropylethylamine (DIPEA) were added to react for 24 hours to synthesize a novel ligand TLQ-PIPY. The synthesis of the final ruthenium (II) complex fluorescent probe requires that cis-bis (2, 2' -bipyridine) ruthenium (II) dichloride dihydrate, silver trifluoromethane sulfonate and ligand TLQ-PIPY (II) are subjected to reflux reaction in methanol for 4-8 hours under the protection of inert gas, cooled to room temperature, filtered and precipitated, and diethyl ether is added to obtain red powder [ Ru (TLQ-PIPY) (bipy) 2]2+, wherein the specific preparation reaction formula is as follows:
The probe of the invention detects the optical properties of hNQO enzyme in phosphate buffer solution.
The probe can start red fluorescence emission at 640nm of the complex after reacting with hNQO enzyme; to evaluate the sensing ability of probe [ Ru (TLQ-PIPY) (bipy) 2]2+ to hNQO1, the change in absorbance and emission spectra at 25℃was measured in PBS buffer (0.01 mol/L, pH 7.4). In the absorption spectrum of [ Ru (TLQ-PIPY) (bipy) 2]2+ (FIG. 1 a), the absorption peak around 450nm belongs to the metal-to-ligand charge transfer transition (MLCT). The strong absorption band around 286nm is caused by pi-pi transitions of the ligand groups, while the shoulder at 300-350nm results from transitions between ligands (L PIPYLTLQ CT and L bipyLTLQ CT). As can be seen, the [ Ru (TLQ-PIPY) (bipy) 2]2+ and NADH (100. Mu. Mol/L) mixed solution had a strong absorbance at 260nm and a medium absorbance at 337 nm. The probe [ Ru (TLQ-PIPY) (bipy) 2]2+ shows weak fluorescence at 640nm under excitation at 450nm (FIG. 1 b). The reaction of probe [ Ru (TLQ-PIPY) (bipy) 2]2+ with hNQO1 in the presence of NADH causes strong fluorescence emission at 640nm with a large Stokes shift (190 nm). As shown in FIGS. 2a and 2d, the fluorescence intensity of probe [ Ru (TLQ-PIPY) (bipy) 2]2+ increased rapidly over 20min at 640nm in the presence of NADH and hNQO1, indicating that [ Ru (TLQ-PIPY) (bipy) 2]2+ had a strong reactivity and rapid response capacity to hNQO1, followed by a gradual decrease in the rate of increase of fluorescence intensity. In FIGS. 3a and 3b, with the addition of hNQO a gradual increase in fluorescence intensity, eventually about an 8-fold increase, is achieved at a hNQO1 concentration of 0.4 μg/mL. The limit of detection (LOD) for probe pair hNQO1 is 0.4239ng/ml, indicating that probe [ Ru (TLQ-PIPY) (bipy) 2]2+ is highly sensitive to hNQO1, with the potential to monitor trace hNQO1 in cells.
The probe detects the kinetic parameters of the enzymatic reaction of hNQO enzymes in phosphate buffer solution.
The reaction rate V (. Mu. Mol min -1mghNQO1-1) was obtained by plotting the fluorescence intensity of solutions of different concentrations [ Ru (TLQ-PIPY) (bipy) 2]2+ over time (4 a). By Michaelis-Menten analysis, the apparent kinetic parameters were determined to be Michaelis constant (K m) =1.66.+ -. 0.24. Mu. Mol, maximum velocity (V max)＝2.83±0.15μmol min-1mghNQO1-1, catalytic constant (K cat)＝56.63S-1, specificity constant (K cat/Km)＝34.07μmol-1S-1).
The probe of the invention is selective for hNQO enzyme.
The fluorescence intensity of the probe [ Ru (TLQ-PIPY) (bipy) 2]2+ was unchanged when added to a sample containing equimolar amounts of Na+、K+、Mg2+、Zn2+、Cu2+、Co2+、Ni2+、Ca2+、Cd2+、Mn2+、Pb2+、Fe3+、Cr3+、F-、Cl-、Br-、I-、AC-、SO3 2-、S2-、SO4 2-、HCO3 -、CO3 2-、NO3 -、NO2 -、PO4 3-、H2PO2 -、S2O3 2-、 cysteine, glutathione, lysine, alanine, threonine, asparagine, serine, glutamic acid, tryptophan, histidine, respectively. As shown in FIG. 4b, fluorescence enhancement at 640nm was evident when only NADH and hNQO1 were present in the probe [ Ru (TLQ-PIPY) (bipy) 2]2+ solution, while fluorescence response by other interfering species was negligible, indicating good selectivity of probe [ Ru (TLQ-PIPY) (bipy) 2]2+ to hNQO 1.
Cytotoxicity assays of the probes of the invention
Wherein: the cells were A549 and MDA-MB-231 cells.
Cytotoxicity experiments on A549 cells by [ Ru (TLQ-PIPY) (bipy) 2]2+ (FIG. 5) showed that [ Ru (TLQ-PIPY) (bipy) 2]2+ (10, 20, 50, 100, 500. Mu.M) had little toxicity on A549 cells, with cell viability >0.95. As [ Ru (TLQ-PIPY) (bipy) 2]2+ concentration increased to 1000. Mu.m, cell viability was slightly lower but more than 90% of cells survived. MDA-MB-231 cells were also almost unaffected by [ Ru (TLQ-PIPY) (bipy) 2]2+, and cell viability was >0.90. These data indicate that [ Ru (TLQ-PIPY) (bipy) 2]2+ does not have any significant cytotoxicity.
Application of probe in hNQO detection imaging in living cells
Wherein: the cells were A549 and MDA-MB-231 cells.
Experimental results confirm that confocal images labeled with fluorescent probes of the ruthenium (II) complexes of the invention showed that probe [ Ru (TLQ-PIPY) (bipy) 2]2+ -treated NQO 1-positive A549 cells showed bright red fluorescence due to intracellular hNQO1 activity, while treated NQO 1-negative MDA-MB-231 cells showed only weak fluorescence (FIG. 6). In addition, fluorescence imaging of NQO1 positive a549 cells after pretreatment with the biscoumarin (hNQO inhibitor) and incubation with the probe [ Ru (TLQ-PIPY) (bipy) 2]2+ showed very weak fluorescence, further confirming specific recognition of hNQO1 by [ Ru (TLQ-PIPY) (bipy) 2]2+. These results indicate that [ Ru (TLQ-PIPY) (bipy) 2]2+ has good membrane permeability and low cytotoxicity, can be used as a tool for visually monitoring the over-expressed hNQO enzyme in living cells, can effectively distinguish NQO1 positive cancer cells from NQO1 negative cells, and is beneficial to early diagnosis and timely treatment of malignant tumors.
Application of probe in hNQO1 high-expression living tumor detection imaging
As shown in FIG. 7a, after intratumoral injection of [ Ru (TLQ-PIPY) (bipy) 2]2+ min, a clear fluorescent signal was seen at 670nm at the tumor site. Under the action of hNQO < 1 >, the fluorescence intensity of the tumor area is gradually enhanced within 1-50 minutes. The fluorescence reaches a maximum value after 40-50 minutes of intratumoral injection, and then slowly decays in 50-90 minutes. The result shows that [ Ru (TLQ-PIPY) (bipy) 2]2+ has the capability of being rapidly activated by NQO1 enzyme in vivo, can be further used for detecting the level of NQO1 in vivo tumor, and is beneficial to early diagnosis and timely treatment of malignant tumor.
The probe is applied to hNQO in vivo tumor section detection imaging application of high expression of1
Under confocal microscopy, a549 tumor sections showed clear red fluorescence with resolution reaching 7-9 μm (fig. 7 b). Under a Transmission Electron Microscope (TEM), the [ Ru (TLQ-PIPY) (bipy) 2]2+ presents irregular black spherical distribution in an A549 tumor slice (figure 7 c), and the [ Ru (TLQ-PIPY) (bipy) 2]2+ can be applied to the intraoperative pathological diagnosis of hNQO1 over-expression tumor tissue slices, has considerable potential in the aspect of hNQO1 activity in micron-level high-resolution real-time imaging tumor, and is beneficial to early diagnosis and timely treatment of hNQO1 over-expression tumor.
Drawings
Fig. 1: absorption (a) and fluorescence (b) spectra of probe [ Ru (TLQ-PIPY) (bipy) 2]2+.
Fig. 2: fluorescence intensity change with time of probe [ Ru (TLQ-PIPY) (bipy) 2]2+ after hNQO/NADPH addition (a, b).
Fig. 3: probe fluorescence spectra (a) after addition of different concentrations hNQO1 in the presence of probe [ Ru (TLQ-PIPY) (bipy) 2]2+ and NADH; linear relationship of fluorescence intensity to hNQO concentration (b).
Fig. 4: (a) The enzymatic kinetics of probe [ Ru (TLQ-PIPY) (bipy) 2]2+ against hNQO1 in PBS buffer containing NADH at pH7.4,0.01 mol/L; (b) The fluorescence intensity of the probe [ Ru (TLQ-PIPY) (bipy) 2]2+ varied in the presence of hNQO, NADH and different interfering factors (metal ion (Na+、K+、Mg2+、Zn2+、Cu2+、Co2+、Ni2+、Ca2+、Cd2+、Mn2+、Pb2+、Fe3+、Cr3+), anion (F-、Cl-、Br-、I-、AC-、SO3 2-、S2-、SO4 2-、HCO3 -、CO3 2-、NO3 -、NO2 -、PO4 3-、H2PO2 -、S2O3 2-)、 biomolecules (cysteine, glutathione, lysine, alanine, threonine, aspartic acid, serine, glutamic acid, tryptophan, histidine), NADH, NADH+ hNQO 1).
Fig. 5: cytotoxicity test of probes.
Fig. 6: (a) Fluorescence images of A549 cells, dicoumarol pretreated A549 cells and MDA-MB-231 cells incubated with [ Ru (TLQ-PIPY) (bipy) 2]2+ at 37 ℃. (b) relative fluorescence intensity of group a cell imaging.
Fig. 7: (a) In vivo fluorescence imaging of endogenous hNQO a549 tumor-bearing mice at various times (0-90 min) following intratumoral administration of [ Ru (TLQ-PIPY) (bipy) 2]2+ (1000 μΜ In,50 μl, λex=460 nm, λem=670 nm). (b) Tumor tissue frozen section fluorescence imaging of a549 tumor-bearing mice 40-50min after intratumoral administration of [ Ru (TLQ-PIPY) (bipy) 2]2+ (1000 μM in,50 μL, λex=488 nm, λem=550-690 nm). (c) Tumor tissue transmission electron micrographs of A549 tumor-bearing mice 40-50min after intratumoral administration of [ Ru (TLQ-PIPY) (bipy) 2]2+ (1000. Mu.M, 50. Mu.L).
Detailed Description
The present invention will be described in detail with reference to specific examples.
Example 1: synthesis of 2-bromo-5- (piperazin-1-yl) pyrazine (1) (BRPY)
2, 5-Dibromopyrazine (1.0 g,4.20 mmol) and N-Boc-piperazine (0.93 g,5.0 mmol) were dissolved in 50mL NMP and stirred at 110deg.C, then DIPEA was added dropwise and refluxed for 2h, the reaction mixture was cooled to room temperature and filtered to give a pale yellow solid, which was dissolved in a trifluoroacetic acid/dichloromethane mixture (1:1, v/v). The reaction mixture was stirred at room temperature for 6 hours, and the solvent was removed to give a pale yellow solid, yield :0.8g(78.7％).1H NMR(600MHz,Chloroform-d)δ8.12(d,J＝1.4Hz,1H),7.86(d,J＝1.5Hz,1H),3.56–3.47(m,4H),3.04-2.94(m,4H),1.88(s,1H).13C NMR(151MHz,Chloroform-d)δ154.16,143.84,130.17,125.80,45.70,45.66.
Example 2: synthesis of 2- (piperazin-1-yl) -5- (pyridin-2-yl) pyrazine (2) (PIPY)
2-Bromo-5- (piperazin-1-yl) pyrazine (2.1 g,8.67 mmol) was added to a solution of 2-tributyltin-pyridine (3.8 g,10.4 mmol) in toluene (100 ml) with stirring at 110℃under reflux for 24h under nitrogen. After cooling the reaction mixture to room temperature, the mixture was concentrated and purified by silica gel column chromatography to give a pale yellow solid product. Yield of products :1.25g(60％).1H NMR(600MHz,DMSO-d6)δ9.00(s,1H),8.62-8.59(m,1H),8.38(s,1H),8.12(d,J＝8.3Hz,1H),7.88(dd,J＝7.6,2.0Hz,1H),7.34(d,J＝5.0Hz,1H),3.65-3.62(m,4H),2.91-2.84(m,4H),1.24(t,J＝7.2Hz,1H).13C NMR(151MHz,DMSO-d6)δ155.04,154.84,149.66,139.89,139.00,137.67,130.30,123.28,119.36,52.55,45.37,45.11,7.72.
Example 3: synthesis of ligand TLQ-PIPY (3)
2- (Piperazin-1-yl) -5- (pyridin-2-yl) pyrazine (0.48 g,2.0 mmol) was mixed ammonium 2- (7-azobenzotriazol-1-yl) -N, N, N ', N' -tetramethylhexafluorophosphate (HATU) (0.91 g) and N, N-Diisopropylethylamine (DIPEA) (0.52 g) in dichloromethane (20 mL) under nitrogen and stirred for 30min. Then trimethylquinone acid was added to the mixture and stirred at room temperature for 24h. And purifying by silica gel column chromatography after the reaction is finished to obtain a pale yellow solid product. Yield of products :0.57g(60％)1H NMR(600MHz,Chloroform-d)δ9.10(d,J＝1.6Hz,1H),8.64-8.60(m,1H),8.15-8.11(m,2H),7.78-7.73(m,1H),7.24-7.20(m,1H),3.75(dd,J＝4.5,2.4Hz,2H),3.70-3.59(m,6H),3.04(s,2H),2.13(s,3H),1.93(t,J＝1.2Hz,3H),1.89(q,J＝1.1Hz,3H),1.45(s,6H).13C NMR(151MHz,Chloroform-d)δ191.47,187.72,170.88,155.01,154.32,154.23,149.30,143.18,140.76,140.58,138.24,136.91,136.72,129.08,122.81,119.87,47.10,45.11,44.70,44.51,40.87,37.84,28.95,14.29,12.75,12.21.
Example 4: synthesis of Probe [ Ru (TLQ-PIPY) (bipy) 2]2+ (4)
The cis-bis (2, 2' -bipyridine) ruthenium (II) dichloride dihydrate (0.06 g,0.12 mmol), silver trifluoromethane sulfonate (0.144 mmol) and ligand TLQ-PIPY were placed in 5ml methanol under reflux for 4 hours under nitrogen protection, the reaction was concentrated and diethyl ether was added to give [ Ru (TLQ-PIPY) (bipy) 2]2+ as a red powder, yield :0.064g(36％).1H NMR(600MHz,DMSO-d6)δ9.39(s,1H),8.85(t,J＝8.1Hz,3H),8.81(d,J＝8.1Hz,1H),8.61(d,J＝8.3Hz,1H),8.26-8.14(m,4H),8.12-8.02(m,2H),7.77(d,J＝5.6Hz,1H),7.70(d,J＝5.7Hz,1H),7.66(d,J＝5.6Hz,1H),7.58(q,J＝7.0Hz,2H),7.53(p,J＝6.2,5.0Hz,3H),7.35(t,J＝6.7Hz,1H),6.96(s,1H),3.57(s,2H),3.49-3.37(m,6H),2.95(s,2H),2.02(s,3H),1.86(s,3H),1.78(s,3H),1.34(s,6H).13C NMR(151MHz,DMSO-d6)δ190.96,187.42,170.94,157.24,157.17,156.84,156.80,156.55,155.68,154.84,152.25,152.08,151.55,151.47,150.85,143.80,143.76,139.45,138.69,138.55,138.14,137.18,135.46,131.45,128.47,128.42,128.30,128.18,126.02,125.26,125.01,124.93,124.85,121.75,46.01,44.15,43.99,43.54,37.92,28.55,14.29,13.05,12.23.
Example 5: ultraviolet absorption and fluorescence emission spectra of the Probe [ Ru (TLQ-PIPY) (bipy) 2]2+
Ru (TLQ-PIPY) (bipy) 2]2+ was dissolved in PBS buffer (pH 7.4,0.01 mol/L). The test concentrations were: [ Ru (TLQ-PIPY) (bipy) 2]2+ (5. Mu. Mol/L), hNQO/NADPH (0.4. Mu.g/mL, 100. Mu. Mol/L). As shown in FIG. 1a, the characteristic absorption peak of the metal-to-ligand charge transfer transition (MLCT) appears at around 450 nm. As shown in FIG. 1b, the reaction of probe [ Ru (TLQ-PIPY) (bipy) 2]2+ with hNQO1 in the presence of NADH causes strong fluorescence emission at 640nm with a large Stokes shift (190 nm).
Example 6: probe [ Ru (TLQ-PIPY) (bipy) 2]2+ vs. hNQO enzyme response time assay
Ru (TLQ-PIPY) (bipy) 2]2+ was dissolved in PBS buffer (pH 7.4,0.01 mol/L). The test concentrations were: [ Ru (TLQ-PIPY) (bipy) 2]2+ (5. Mu. Mol/L), hNQO/NADPH (0.2. Mu.g/mL, 100. Mu. Mol/L). The fluorescence intensities at different time points (0-120 min) were measured continuously at room temperature using 450nm as excitation wavelength. As shown in FIG. 2, [ Ru (TLQ-PIPY) (bipy) 2]2+ has strong reactivity and rapid response to hNQO 1.
Example 7: fluorescence titration experiment of Probe [ Ru (TLQ-PIPY) (bipy) 2]2+
Different concentrations hNQO (0-0.5. Mu.g/mL) were added to (5. Mu. Mol/L) PBS buffer (0.01 mol/L, pH 7.4) containing NADH (100. Mu. Mol/L) and [ Ru (TLQ-PIPY) (bipy) 2]2+, and the change in fluorescence intensity of the solution was measured at room temperature using 450nm as excitation wavelength. As shown in FIG. 3, with the addition of hNQO1, the fluorescence intensity gradually increased, eventually reaching saturation at 0.4. Mu.g/mL NQO1, and the fluorescence intensity increased approximately 8-fold. Moreover, in the range of 0-0.4 μg/mL, the hNQO concentration in solution showed a good linear relationship with the change in fluorescence intensity of [ Ru (TLQ-PIPY) (bipy) 2]2+, which had a higher signal-to-noise ratio and sensitivity for [ Ru (TLQ-PIPY) (bipy) 2]2+ to hNQO1 for the hNQO1 detection line lod= 0.4239 ng/mL.
Example 8: enzymatic kinetic analysis of Probe [ Ru (TLQ-PIPY) (bipy) 2]2+
The enzyme kinetic experiments of [ Ru (TLQ-PIPY) (bipy) 2]2+ were performed using a fluorescence spectrophotometer at 25 ℃. Different concentrations of [ Ru (TLQ-PIPY) (bipy) 2]2+ (0.5-6. Mu. Mol/L) were added to PBS buffer (0.01 mol/L, pH 7.4) containing NADH (100. Mu. Mol/L) and hNQO1 (0.25. Mu.g/mL). The fluorescence intensity at 640nm was measured at intervals of 1min for 10min. As shown in FIG. 4a, michaelis constant (K m), maximum reaction rate (V max), catalytic constant (K cat) and specificity constant (K cat/Km) were obtained by Michaelis-Menten analysis.
Example 9: selectivity of the probe [ Ru (TLQ-PIPY) (bipy) 2]2+ to the hNQO enzyme
The metal ion (100equiv.hNQO1)(Na+、K+、Mg2+、Zn2+、Cu2+、Fe2+、Co2+、Ni2+、Ca2+、Cd2+、Mn2+、Pb2+、Fe3+、Cr3+、Al3+)、 anion (F-、Cl-、Br-、I-、AC-、SO3 2-、S2-、SO4 2-、HCO3 -、CO3 2-、NO3 -、NO2 -、PO4 3-、H2PO2 -、S2O3 2-) and biomolecules (cysteine, glutathione, lysine, alanine, threonine, aspartic acid, serine, glutamic acid, tryptophan, histidine) were added to PBS buffer (0.01 mol/L, pH 7.4) containing [ Ru (TLQ-PIPY) (bipy) 2]2+ (5. Mu. Mol/L), NADH (100. Mu. Mol/L) and hNQO1 (0.5. Mu.g/mL) and tested for fluorescence intensity, fluorescence spectra recorded at 500-900nm, excitation at 450nm. As shown in FIG. 4b, probe [ Ru (TLQ-PIPY) (bipy) 2]2+ has good selectivity to hNQO.
Example 10: culture of A549 and MDA-MB-231 cells
A549 and MDA-MB-231 cells were first injected into F12K and L-15 medium containing diabody (penicillin and streptomycin) and 10% calf serum, respectively, and then placed in a constant temperature (37 ℃) cell incubator containing 5% CO 2% for culture. When cells were grown to log phase in flasks, then plated: soaking the cover glass for half an hour by using chromic acid washing liquid, flushing the cover glass with clean water and ultrapure water for three times, drying, sterilizing and placing the cover glass in a sterile disposable culture dish. After pouring out the culture solution in the culture flask, flushing the cells with PBS buffer solution for three times, digesting for 3 minutes with 1 milliliter of pancreatin with the concentration of 0.25 percent, pouring out pancreatin solution, adding a small amount of culture medium solution, blowing uniformly with a gas of a pipetting gun, counting the cells, taking cells with proper density, adding the culture medium, blowing uniformly again, adding the cells into a culture dish which is prepared in advance and contains a glass slide, putting the inoculated culture dish into a cell culture box, and growing until the cells cling to the wall. Culturing in a CO 2 constant temperature cell incubator for 12-36 hours until the cell density reaches 50% -70%, and then using the cell in a cell staining experiment.
Example 11: cell MTT assay of Probe pair A549 and MDA-MB-231 cells
Firstly, a cell counting plate is used for detecting the cell concentration, one drop of cell culture solution is dripped on the counting plate containing four large squares of 16 grids of 1mm multiplied by 1mm, the number of cells on each square is counted, and the average value n (the cell concentration is n/mm 3) is obtained. The concentration of the cells is controlled to be 5000-50000/mm 3, then the cell culture solution is diluted and then placed into a 96-well plate for culture. After one day of cell growth in 96-well plates, 10, 20, 50, 100, 500, 1000. Mu.M probes were added to the plates, respectively, and then 20. Mu.L of 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyltetrazolium bromide at a concentration of 5mg/ml was added to the plates, followed by further culturing for 24 hours. The culture solution in the well plate was sequentially aspirated, 150. Mu.L of DMSO was added to each well, and the well plate was placed on a shaker and oscillated at low speed to sufficiently dissolve the crystals. Finally, the absorbance (OD 490 nm) of each well was read on a microplate reader for calculation. Control wells (culture, cells, drug dissolution medium at the same concentration, dimethyl sulfoxide and MTT) were zeroed out wells (dimethyl sulfoxide, culture medium and MTT). As shown in fig. 5, the cytotoxicity test results can be seen: the viability of the A549 and MDA-MB-231 cells after 24h incubation in the medium containing 1000. Mu.M dye was above 90%, which was considered to be no significant toxicity to the cells.
Example 12: probe pair A549 and MDA-MB-231 cell hNQO imaging detection
A549 cells were stained with a PBS buffer solution (0.01 mol/L, pH 7.4) of probe [ Ru (TLQ-PIPY) (bipy) 2]2+ (1000 μm) for 5h at 37 ℃ and mda-MB-231 cells were incubated under the same conditions. A hNQO enzyme inhibition test was also performed. A549 cells were first incubated with biscoumarin (100 μm) at 37 ℃ for 12h, and then treated with probe [ Ru (TLQ-PIPY) (bipy) 2]2+ (1000 μm) in PBS buffer (0.01 mol/L, pH 7.4) at 37 ℃ for 4h. As shown in FIG. 6, the confocal image marked by the fluorescent probe of the ruthenium (II) complex of the invention shows that the probe can well penetrate through the cell membranes of living A549 and MDA-MB-231 cells to enter the cells, and the hNQO enzyme in the cells can be intuitively monitored, so that NQO1 positive cancer cells and NQO1 negative cells can be effectively distinguished.
Example 13: probe for hNQO1 high expression living tumor detection imaging
A549 cells in logarithmic growth phase were taken and 5×10 6 cells were subcutaneously injected into mice. To examine endogenous hNQO1 activity, [ Ru (TLQ-PIPY) (bipy) 2]2+ was dissolved in PBS buffer (0.01 mol/L, pH 7.4) and injected into tumors (1000. Mu. Mol/L, 50. Mu.L). Animals were imaged at different time intervals using PERKINELMER IVIS imaging systems. As shown in FIG. 7a, imaging of the probe in mice, after intratumoral injection of [ Ru (TLQ-PIPY) (bipy) 2]2+ min, a clear fluorescent signal was seen at 670nm at the tumor site. Under the action of hNQO < 1 >, the fluorescence intensity of the tumor area is gradually enhanced within 1-50 minutes. The fluorescence reaches a maximum value after 40-50 minutes of intratumoral injection, and then slowly decays in 50-90 minutes. Tumor tissue frozen sections of A549 tumor-bearing mice 40-50min after intratumoral administration of [ Ru (TLQ-PIPY) (bipy) 2]2+ (1000. Mu.M in, 50. Mu.L) were cut into 15 μm thick sections in a cryostat (Leical 1950 cryomicrotome, germany) at-20 ℃. Fluorescence imaging was performed using a nikon A1R HD25 confocal microscope (λex=488 nm, λem=550-690 nm) (fig. 7 b). And A549 tumor-bearing mouse tissue sections (1 mm 3) were taken, rapidly fixed at 2.5% glutaraldehyde 4℃for 2h, then fixed in 0.1mol/L PBS containing 1% osmium acid for 2h at room temperature. After dehydration, infiltration, embedding, sectioning, staining, the samples were photographed under a transmission electron microscope (HITACHI, HT7800/HT 7700) at 80kV (FIG. 7 c). The result shows that [ Ru (TLQ-PIPY) (bipy) 2]2+ can be applied to the intraoperative pathological diagnosis of hNQO1 over-expression tumor tissue sections, has great potential in hNQO1 activity in micron-scale high-resolution real-time imaging tumor, and is beneficial to early diagnosis and timely treatment of hNQO1 over-expression tumor.
It will be understood that modifications and variations will be apparent to those skilled in the art from the foregoing description, and it is intended that all such modifications and variations be included within the scope of the following claims.
Claims (5)
1. A fluorescent probe for selectively detecting hNQO1 highly expressed in tumor cells, characterized in that: the structural general formula of the fluorescent probe is shown in (I):
2. The method for preparing a fluorescent probe according to claim 1, wherein: the method comprises the following steps: dissolving 2, 5-dibromopyrazine and N-Boc-piperazine in N-methylpyrrolidone (NMP), carrying out reflux reaction for 24 hours to obtain a compound 1 (BRPY), then adding the compound 1 into a toluene solution containing 2-tributyltin-based pyridine, and carrying out stirring reaction at room temperature for 10 hours to obtain a compound 2 (PIPY); subsequently, the obtained compound 2 (PIPY) is dissolved in methylene chloride to react with trimethylquinone acid, and 2- (7-azobenzotriazol-1-yl) -N, N, N ', N' -tetramethyl ammonium Hexafluorophosphate (HATU) and N, N-Diisopropylethylamine (DIPEA) are added to react for 24 hours to synthesize a novel ligand TLQ-PIPY; the synthesis of the final ruthenium (II) complex fluorescent probe requires that cis-bis (2, 2' -bipyridine) ruthenium (II) dichloride dihydrate and silver trifluoromethane sulfonate and ligand TLQ-PIPY (II) are subjected to reflux reaction in methanol for 4-8 hours under the protection of inert gas, cooled to room temperature, filtered and precipitated, and diethyl ether is added to obtain red powder Ru (TLQ-PIPY) (bipy) 2, wherein the specific preparation reaction formula is as follows:
3. the use of the fluorescent probe according to claim 1, wherein the fluorescent probe is used for preparing a detection reagent and a detection kit for NQO1 enzyme.
4. The use of claim 3, wherein NQO1 positive cancer cells are effectively distinguished from NQO1 negative cells by selective detection hNQO enzyme imaging.
5. The use of claim 4, wherein the NQO1 positive cells are a549 cells and the NQO1 negative cells are MDA-MB-231 cells.
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| CN110967326A (en) * | 2019-12-12 | 2020-04-07 | 北京师范大学 | Near-infrared light-emitting binuclear ruthenium complex as tumor cell recognition and imaging reagent |
| CN113045599A (en) * | 2021-03-17 | 2021-06-29 | 山西大学 | Method for distinguishing cancer cells/tissues with high contrast and preparation of fluorescent probe |
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| CN110967326A (en) * | 2019-12-12 | 2020-04-07 | 北京师范大学 | Near-infrared light-emitting binuclear ruthenium complex as tumor cell recognition and imaging reagent |
| CN113045599A (en) * | 2021-03-17 | 2021-06-29 | 山西大学 | Method for distinguishing cancer cells/tissues with high contrast and preparation of fluorescent probe |
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