A dual-channel fluorescent probe for detecting viscosity and pH, and a preparation method and application thereof

Abstract

The application discloses a fluorescence probe for detecting viscosity and pH in a double channel, and a preparation method and application thereof, and has a structural formula of TPE-PH-KD. The application further specifically discloses a preparation method of the fluorescence probe for detecting viscosity and pH in the double channel, and application of the fluorescence probe in selective dynamic visual detection of viscosity and pH in a biological cell system and preparation of a tumor cell imaging detection preparation. The prepared fluorescence probe has the characteristics of high specificity, good selectivity, good light stability, excellent double-targeting capability of mitochondria and lysosomes and the like.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probes and their detection applications, specifically relating to a dual-channel fluorescent probe for detecting viscosity and pH, its preparation method, and its application. Background Technology

[0002] With the increasing incidence and mortality rates of cancer, it now poses a serious threat to human life and health. Early diagnosis and treatment can significantly improve the survival rate of cancer patients. Therefore, the identification of tumor markers is crucial for achieving early cancer diagnosis and for human health and socio-economic development. In addition to traditional tumor markers, including the expression of cell surface receptors, oncogenes, and circulating nucleic acids, cancer cells also exhibit specific intracellular microenvironments such as polarity, viscosity, pH, and hypoxia. Studies have shown that changes in viscosity, a microenvironmental parameter, play a key role in many biological processes, including intracellular substance transport, chemical signal transduction, protein-protein interactions, and apoptosis. Furthermore, pH balance is essential for maintaining healthy bodily functions, including endocytosis, apoptosis, and proliferation. Cancer cells preferentially take up large amounts of glucose, converting it into lactic acid through non-oxidative catabolism via the Warburg effect, thereby increasing extracellular pH. + The body's pH level is low. Therefore, the detection of solid tumors in vivo often relies on an acidic tumor microenvironment. Recent studies have shown that cancer cells have higher viscosity and lower pH levels than normal cells. Therefore, pH and viscosity together have become novel biomarkers for cancer detection.

[0003] Compared with traditional detection methods, fluorescent probes have advantages such as cell permeability, strong biocompatibility, and non-invasive detection, and have been widely used in in vivo imaging. To detect dynamic viscosity or pH changes in living cells, researchers have designed many small-molecule fluorescent probes, among which molecular rotors are commonly used to construct viscosity-responsive fluorescent probes. The high emissivity of AIE dyes is closely related to the restriction of intramolecular rotation, exhibiting excellent photostability and a large Stokes shift, and is widely used in biomedical imaging. By linking AIE dyes to molecular rotors, high-performance viscosity-responsive fluorescent probes can be prepared. Furthermore, various pH-sensitive fluorescent probes have been developed based on rhodamine, hemicyanine, and BODIPY dye backbones. However, some of these fluorescent probes suffer from drawbacks such as insufficient fluorescence emission wavelength, small Stokes shift, poor water solubility, significant background interference from liver signals, and complex synthesis. Fluorescent probes capable of detecting viscosity and pH fluctuations in different channels are rarely reported. Therefore, it is essential to develop and design a simple and efficient microenvironment-responsive fluorescent probe to simultaneously detect viscosity and pH in different channels, thereby achieving cancer diagnosis.

[0004] In recent years, the research and development of optical fluorescent probes for detecting viscosity / pH has been extremely rapid. Most of the reported fluorescent probes have good analytical performance under tumor microenvironment conditions and avoid some problems caused by the use of multiple probes (such as cumbersome operation procedures, spectral crosstalk, and inaccurate positioning). Thus, they provide an important detection method for the determination of viscosity / pH in the tumor microenvironment and for the imaging study of cells and solid tumors. Summary of the Invention

[0005] To address the current problems and limitations of fluorescent probes for detecting viscosity / pH, this invention provides a dual-channel fluorescent probe for detecting viscosity and pH, along with its preparation method. When viscosity increases, the double bond rotation between the quinoline group and the TPE group in this fluorescent probe is restricted, thereby enhancing the fluorescence intensity. Furthermore, catechol groups that undergo protonation or deprotonation at different pH values ​​are introduced, achieving pH sensitivity. This fluorescent probe exhibits high specificity, good selectivity, good photostability, and excellent dual-targeting capability to mitochondria and lysosomes.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: a dual-channel fluorescent probe for detecting viscosity and pH, characterized in that the fluorescent probe TPE-PH-KD has the following structural formula:

[0007]

[0008] The method for preparing a dual-channel fluorescent probe for detecting viscosity and pH according to the present invention is characterized by the following specific steps:

[0009] Step S1: 1-(4-Pinnayl phenylboronic acid ester)-1,2,2-tristyrene, 4-bromo-2-hydroxybenzaldehyde, Pd(PPh3)4, and K2CO3 were added to a THF / H2O mixed solvent. The mixture was degassed and deoxygenated under a N2 atmosphere, and then stirred at 60°C. After the reaction was complete, the reaction mixture was cooled to room temperature, extracted with CH2Cl2, dried over Na2SO4, and the organic phase was concentrated under vacuum to obtain the crude product. Purification using silica gel column chromatography yielded a yellow solid, compound 1. The corresponding synthetic route is as follows:

[0010]

[0011] Step S2: Compound 1, 1,2-dimethylquinoline iodide, and piperidine were added to anhydrous ethanol and heated to reflux. After the reaction was completed, the reaction mixture was concentrated under vacuum to obtain a crude compound, which was then purified by silica gel column chromatography to obtain a reddish-purple solid, i.e., the fluorescent probe TPE-PH-KD. The corresponding synthetic route is as follows:

[0012]

[0013] The present invention relates to the application of a dual-channel fluorescent probe for detecting viscosity and pH in the selective detection of viscosity and pH.

[0014] The present invention relates to the application of a dual-channel fluorescent probe for detecting viscosity and pH in the dynamic visualization detection of viscosity and pH selectivity in biological cell systems.

[0015] The present invention relates to the application of a dual-channel fluorescent probe for detecting viscosity and pH in the preparation of tumor cell imaging detection agents.

[0016] This invention designs a novel fluorescent probe, TPE-PH-KD, based on the AIE fluorophore and quinoline group. In this probe, the tetraphenylethylene group acts as both the fluorophore and the donor, while the quinoline group acts as the acceptor. As viscosity increases, the double bond rotation between the quinoline group and the TPE group is restricted, thereby enhancing the fluorescence intensity. This change makes it possible to visualize viscosity. Furthermore, the quinoline group assists the fluorescent probe TPE-PH-KD in crossing the cell membrane and remaining within the target organelle. A catechol group, which undergoes protonation or deprotonation at different pH values, is introduced, achieving pH sensitivity. In neutral or alkaline solutions, the fluorescent probe TPE-PH-KD mainly exists in a non-fluorescent deprotonated form. With increasing environmental acidity, the deprotonated form transforms into a protonated structure via a fluorescent "OFF-ON" transition. The fluorescent probe TPE-PH-KD is used to monitor changes in pH or viscosity in a dual-channel system after treatment with CCCP, chloroquine, and nystatin in real time. Because cancer cells exist in a high-viscosity, low-pH environment, this invention successfully utilizes the fluorescent probe TPE-PH-KD to selectively detect tumor cells and tissues without interference from normal tissues. Therefore, this fluorescent probe TPE-PH-KD not only provides a potential method for monitoring viscosity and pH, but also offers a detection tool to guide precise cancer diagnosis.

[0017] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) The synthesis process of the fluorescent probe in the present invention is relatively easy and the post-processing process is relatively simple; (2) The fluorescent probe in the present invention achieves high selectivity detection of viscosity and pH and has excellent selectivity; (3) The fluorescent probe in the present invention has good dual targeting ability of mitochondria and lysosomes, excellent photostability, and can be applied to the real-time dynamic visualization detection of viscosity and pH in cells.

[0018] The fluorescent probe in this invention achieves more accurate and stable optical signals and imaging effects by reducing background interference from autofluorescence in living organisms, minimizing photodamage to biological samples, and improving photostability. Therefore, the fluorescent probe in this invention not only provides a potential method for detecting viscosity and pH, but also offers a detection tool to guide precise cancer diagnosis, which is of great significance for the diagnosis and treatment of diseases caused by abnormal tumor microenvironments. Attached Figure Description

[0019] Figure 1 In Figure A, the fluorescence spectrum of the fluorescent probe TPE-PH-KD (10 μM) in a glycerol-water mixture is shown, with the glycerol content increasing from 0% to 100%. Figure B shows the linear relationship between LogI (I: fluorescence intensity at 576 nm) and logη (η: viscosity).

[0020] Figure 2 In Figure A, the absorption spectrum of the fluorescent probe TPE-PH-KD in PBS buffer solution changes as pH increases from 2 to 12; in Figure B, the fluorescence spectrum of the probe TPE-PH-KD in PBS buffer solution changes as pH increases from 2 to 12.

[0021] Figure 3 In Figure A, the fluorescence intensity response of the fluorescent probe (10 μM) at 651 nm to different analytes (200 μM) and a pH=2 system is shown; in Figure B, the fluorescence intensity response of the fluorescent probe (10 μM) at 576 nm to various analytes (200 μM) and a glycerol system is shown; 1-5: Fluorescent probe, K + Na + Ca 2+ Ni 2+ ;6-10: Mg 2+ Zn 2+ Cr 3+ , Br - NO3 - 11-15: CO3 2- ClO - , H2O2, Hcy, Cys; 16: GSH.

[0022] Figure 4 The fluorescence spectra of the fluorescent probe TPE-PH-KD after irradiation with 365 nm light for 0.5 hours in the PBS (pH=2.08) buffer system in Figure A and the glycerol system in Figure B are shown.

[0023] Figure 5 Colocalization fluorescence imaging of the fluorescent probe TPE-PH-KD in HeLa cells co-incubated with Mitto-tracker Green and Lyso-tracker Green.

[0024] Figure 6 Fluorescence images of HeLa cells incubated with the fluorescent probe TPE-PH-KD (10 μM); 10 μM Nigerian styracin was present at pH 3-7.

[0025] Figure 7Fluorescence imaging of HeLa cells incubated with the fluorescent probe TPE-PH-KD was performed by induction with CCCP (10 μM) or chloroquine (100 μM) for 30 minutes.

[0026] Figure 8 Fluorescence images of HeLa cells incubated with the fluorescent probe TPE-PH-KD (10 μM) after induction with nystatin (10 μM) for 30 minutes.

[0027] Figure 9 Tumor cell lines and normal cell lines HeLa cells (a1-a3), HepG2 cells (b1-b3), A549 cells (c1-c3), BT474 cells (d1-d3), 4T1 cells (e1-e3), Beas-2B cells (f1-f3), and LO2 cells (g1-g3) were incubated with the fluorescent probe TPE-PH-KD (10 μM) for 0.5 hours.

[0028] Figure 10 Fluorescence signal images of ex vivo organs and tumor tissues after tail vein injection of the fluorescent probe TPE-PH-KD (100 μM, 100 μL). Detailed Implementation

[0029] The following examples further illustrate the above-described content of the present invention, but it should not be construed as limiting the scope of the subject matter of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0030] Example 1

[0031] Synthesis of the fluorescent probe TPE-PH-KD:

[0032] (1) Synthesis of Compound 1

[0033] 1-(4-Pinacolyl phenylboronic acid)-1,2,2-triphenylene (457 mg), 4-bromo-2-hydroxybenzaldehyde (301 mg), Pd(PPh3)4 (346 mg), and K2CO3 (414 mg) were added to a THF / H2O mixed solvent (80 mL). The mixture was degassed and deoxygenated under a nitrogen atmosphere, and then stirred at 60 °C for 24 hours. After the reaction was complete, the reaction mixture was cooled to room temperature, extracted with CH2Cl2, dried over Na2SO4, and the organic phase was concentrated under vacuum to obtain the crude product. Purification using silica gel column chromatography yielded a yellow solid, compound 1. The corresponding synthetic route is as follows:

[0034]

[0035] (2) Synthesis of fluorescent probe TPE-PH-KD

[0036] Compound 1 (0.09 g, 0.2 mmol), 1,2-dimethylquinoline iodide (0.038 g, 0.24 mmol), and piperidine (30 μL) were added to anhydrous ethanol (15 mL), and the mixture was heated to reflux for 12 hours. After the reaction was completed, the reaction mixture was concentrated under vacuum to obtain the crude compound, which was then purified by silica gel column chromatography to obtain the reddish-purple solid, i.e., the fluorescent probe TPE-PH-KD. The corresponding synthetic route is as follows:

[0037]

[0038] Example 2

[0039] Determination of fluorescence spectra of fluorescent probe TPE-PH-KD under different viscosity conditions:

[0040] To accurately analyze the correlation between viscosity and fluorescence signal intensity, mixed solvents of water and glycerol in different proportions were used as the detection system. Figure 1 As shown, with increasing glycerol volume ratio, the fluorescence signal of the fluorescent probe TPE-PH-KD at 576 nm gradually increased (by 252 times), which is attributed to the suppression of molecular rotors in the fluorescent probe TPE-PH-KD due to increased viscosity. Furthermore, according to... -Hoffmann equation, Log I576nm The good linear relationship between the value and Logη in the range of 1.20 to 945 cP indicates that the fluorescent probe TPE-PH-KD can quantitatively detect changes in viscosity.

[0041] Example 3

[0042] Determination of fluorescence spectra of fluorescent probe TPE-PH-KD under different pH conditions:

[0043] like Figure 2 As shown, as the pH increases from 2 to 12, the absorption band at 428 nm gradually narrows, a new peak appears at 528 nm, and an isoabsorption point is formed at 492 nm. A noticeable color change from yellow to purple is also observed; this phenomenon can be attributed to the intramolecular rearrangement of the ketone structure caused by the deprotonation of the hydroxyl group. The corresponding fluorescent probe TPE-PH-KD shows no fluorescence emission in the pH range of 7-12, while in the acidic range, the fluorescence emission at 651 nm continuously increases with decreasing pH (e.g., ...). Figure 2 (As shown in B).

[0044] Example 4

[0045] Selectivity analysis of the fluorescent probe TPE-PH-KD:

[0046] Biological organisms are complex systems containing various reactive oxygen species (ROS), reactive sulfur species (RSS), different amino acids, and metal ions. Related analytes that selectively interfere with the fluorescent probe TPE-PH-KD include K... + Na + Ca 2+ Ni 2+ Mg 2+ Zn 2+ Cr 3+ , Br - NO3 - CO3 2- ClO - H2O2, Hcy, Cys, GSH (e.g.) Figure 3 (As shown in A and B). Different interfering substances did not cause significant changes in fluorescence signal, indicating that the fluorescent probe TPE-PH-KD has high selectivity and can measure viscosity and pH changes of different emission channels in complex biological environments.

[0047] Example 5

[0048] Determination of the photostability of the fluorescent probe TPE-PH-KD:

[0049] The photostability of the fluorescent probe TPE-PH-KD was investigated. Figure 4 As shown, after 30 minutes of continuous monitoring, the fluorescence intensity of the fluorescent probe TPE-PH-KD remained almost unchanged. This result indicates that the fluorescent probe TPE-PH-KD has good photostability, which can meet the requirements of long-term in vivo imaging and obtain a stable fluorescence signal.

[0050] Example 6

[0051] Fluorescent probe TPE-PH-KD mitochondrial and lysosomal colocalization assay:

[0052] To determine the cellular colocalization of the fluorescent probe TPE-PH-KD, colocalization experiments were performed in HeLa cells using commercially available organelle dyes (Mito-tracker Green, Lyso-tracker Green) and the fluorescent probe TPE-PH-KD. Figure 5 As observed, the fluorescent signals of the TPE-PH-KD fluorescent probe and Mito-tracker Green showed good overlap in the red channel, with a Pearson correlation coefficient of 0.89. Furthermore, the TPE-PH-KD fluorescent probe and Lyso-tracker Green also showed partial overlap, with a Pearson correlation coefficient of 0.79. These results indicate that the TPE-PH-KD fluorescent probe can dual-target mitochondrial / lysosomal organelles.

[0053] Example 7

[0054] Imaging analysis of intracellular pH under different pH buffer solutions using the fluorescent probe TPE-PH-KD:

[0055] In the presence of nigramycin, intracellular pH ranges of 3-7 were established using PBS buffer solutions of different pH values, and the cells were labeled with the fluorescent probe TPE-PH-KD. As the cell pH decreased from 7 to 3, the fluorescence signal intensity of the TPE-PH-KD probe in the red channel gradually increased, indicating that the TPE-PH-KD probe can track changes in cell pH in real time (e.g., ...). Figure 6 (As shown). The activation of fluorescence signal by the fluorescent probe TPE-PH-KD at low pH indicates that the fluorescent probe TPE-PH-KD can be applied to tumor cell imaging detection.

[0056] Example 8

[0057] Analysis of organelle pH changes induced by the fluorescent probe TPE-PH-KD and the drugs chloroquine and CCCP:

[0058] The effects of pH fluctuations on mitochondrial / lysosomal metabolism were examined by inducing HeLa cells with different drugs. For example... Figure 7 As shown, after treating cells with 100 μM chloroquine (an alkaline drug that increases lysosomal pH) for 30 minutes, the red channel fluorescence signal significantly decreased, confirming that chloroquine can induce intracellular pH changes. Then, further induction of cells with N-(3-chlorophenyl)carbonylhydrazine dicyandicyanate (CCCP), a typical membrane potential uncoupling agent associated with mitochondrial pathophysiology and pharmacology, resulted in a significant increase in the intensity of the red channel fluorescence signal, indicating that CCCP stimulated mitochondrial membrane potential dissociation, leading to mitochondrial acidification.

[0059] Example 9

[0060] Analysis of changes in intracellular viscosity induced by nystatin using the fluorescent probe TPE-PH-KD:

[0061] The performance of the fluorescent probe TPE-PH-KD in detecting changes in viable cell viscosity was demonstrated in this invention. Nystatin was used to induce cells, a drug that alters intracellular viscosity. Confocal fluorescent images of cells induced with nystatin (10 μM) are shown below. Figure 8 As shown, the fluorescence signal in the green channel increased significantly, indicating that the viscosity increased under the action of nystatin, demonstrating that the fluorescent probe TPE-PH-KD can successfully monitor changes in cell viscosity.

[0062] Example 10

[0063] Imaging performance of the fluorescent probe TPE-PH-KD on normal and cancer cell lines:

[0064] Compared to normal cells, cancer cells have a lower pH and higher viscosity. Therefore, different cancer cells (HeLa cells, HepG2 cells, A549 cells, BT474 cells, 4T1 cells) and normal cells (Beas-2B cells, LO2 cells) were incubated with the fluorescent probe TPE-PH-KD. Under the same imaging conditions in both channels, the fluorescence signal of cancer cells was significantly higher than that of normal cells, indicating that the fluorescent probe TPE-PH-KD can successfully distinguish cancer cells from normal cells. Figure 9 Since the fluorescence signal of the fluorescent probe TPE-PH-KD is affected by viscosity and pH, the changes in fluorescence signal intensity indicate that the viscosity and pH are not uniform among different cancer cell lines. Compared with other cancer cell lines, the BT474 cell line has the strongest fluorescence signal, indicating that the fluorescent probe TPE-PH-KD is more enriched in BT474 cells and is more easily activated.

[0065] Example 11

[0066] The application of the fluorescent probe TPE-PH-KD in the diagnosis of solid tumors was analyzed and tested.

[0067] Tumor-bearing mice inoculated with BT474 cells were injected via the tail vein with the fluorescent probe TPE-PH-KD. Sixty minutes later, the organs were separated from the tumor for fluorescence imaging. Figure 10 As shown, the fluorescence signal intensity of tumor tissue is higher than that of other organ tissues, especially unaffected by interference from the liver and kidneys. By comparing the fluorescence signal intensity, tumors can be directly distinguished from other organ tissues. This high selectivity for tumor tissue may be the result of the synergistic effect of acid activation and viscosity response. These results further indicate that the fluorescent probe TPE-PH-KD can serve as a high-contrast visualization tool for tumor cell imaging detection.

[0068] The above embodiments describe the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are only illustrative of the principles of the present invention. Various changes and modifications can be made to the present invention without departing from the scope of the principles of the present invention, and all such changes and modifications fall within the protection scope of the present invention.

Claims

1. A fluorescent probe for dual-channel detection of viscosity and pH, characterized in that... The structural formula of the fluorescent probe TPE-PH-KD is as follows:

2. The method for preparing a dual-channel fluorescent probe for detecting viscosity and pH according to claim 1, characterized in that... The specific steps are as follows: Step S1: 1-(4-Pinnayl phenylboronic acid ester)-1,2,2-tristyrene, 4-bromo-2-hydroxybenzaldehyde, Pd(PPh3)4, and K2CO3 were added to a THF / H2O mixed solvent. The mixture was degassed and deoxygenated under a N2 atmosphere, and then stirred at 60°C. After the reaction was complete, the reaction mixture was cooled to room temperature, extracted with CH2Cl2, dried over Na2SO4, and the organic phase was concentrated under vacuum to obtain the crude product. Purification using silica gel column chromatography yielded a yellow solid, compound 1. The corresponding synthetic route is as follows: Step S2: Compound 1, 1,2-dimethylquinoline iodide, and piperidine were added to anhydrous ethanol and heated to reflux. After the reaction was completed, the reaction mixture was concentrated under vacuum to obtain a crude compound, which was then purified by silica gel column chromatography to obtain a reddish-purple solid, i.e., the fluorescent probe TPE-PH-KD. The corresponding synthetic route is as follows:

3. The application of the dual-channel fluorescent probe for detecting viscosity and pH as described in claim 1 in the selective detection of viscosity and pH.

4. The application of the dual-channel fluorescent probe for detecting viscosity and pH as described in claim 1 in the dynamic visualization detection of viscosity and pH selectivity in biological cell systems.

5. The application of the dual-channel fluorescent probe for detecting viscosity and pH as described in claim 1 in the preparation of tumor cell imaging detection agents.

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