Lysosome-nucleolus migration type fluorescent probe, preparation method and application

Abstract

This invention relates to the field of molecular probe technology, specifically disclosing a lysosome-nucleolus migration fluorescent probe, its preparation method, and its application. This fluorescent probe is a PPCs series probe with a novel structural design and ultra-high hydrophilicity. It does not enter normal cells but can stain lysosomes during cell apoptosis and nucleoli during cell necrosis, achieving migration from lysosomes to nucleoli. It provides real-time, in-situ visualization of the transition from healthy cells to apoptosis and necrosis.

Description

Technical Field

[0001] This invention relates to the field of molecular probe technology, and in particular to a lysosome-nucleolar migration fluorescent probe, its preparation method, and its application. Background Technology

[0002] In eukaryotic cells, multiple biological processes maintain orderly metabolic homeostasis through a dynamic, coordinated process. Accurately tracking these naturally occurring cellular processes is crucial for studying life activities and drug development. Apoptosis is a programmed cell death pathway that removes abnormal and senescent cells from the body. Cells undergoing apoptosis are characterized by phosphatidylserine exposure in the outer leaf of the plasma membrane, plasma membrane bubbling, and nuclear condensation. However, when apoptotic cells are not promptly eliminated, secondary necrosis begins, and cells exhibit necrotic cellular characteristics such as loss of cell membrane integrity and organelle rupture. Notably, previous reports have shown that this important shift in cell death patterns is closely related to in vivo microenvironment homeostasis, making it a promising target for new drug development.

[0003] Currently, several methods have been used to study apoptosis and necrosis, such as electrochemical analysis, transmission electron microscopy, and scanning electron microscopy. However, these methods require pretreatment of cells, such as fixation and freezing, and cannot achieve in-situ real-time visualization in living cells. Based on the advantages of high resolution and non-destructive imaging in confocal microscopy using fluorescent probes, significant progress has been made in the development of small-molecule fluorescent probes. Furthermore, some fluorescent probes can monitor apoptosis or necrosis by enhancing or weakening fluorescence; however, these probes are susceptible to interference from the microenvironment, detection instruments, and dye concentrations. Notably, organelle migration fluorescent probes can report signals based on the temporal and spatial differences in fluorescence of different organelles, thus possessing the advantage of being unaffected by staining uniformity and fluctuations in excitation intensity. However, single-molecule organelle migration probes capable of in-situ real-time tracking of the cell process from health to apoptosis and then to necrosis have not yet been reported, which will hinder research into the transition mechanisms and interactions between these two closely related processes.

[0004] As is well known, cell membrane integrity is a crucial indicator for distinguishing between apoptotic and necrotic cells. Due to the significant spatial difference between lysosomes and the nucleolus, lysosomes are easily distinguishable from the nucleolus, and lysosomes play a vital role in both apoptosis and necrosis. To achieve real-time, in-situ visualization and differentiation of healthy, apoptotic, and necrotic cells, we aim to develop a single-molecule probe to observe apoptosis through changes in cell membrane permeability and to observe necrosis by moving the probe from the lysosome to the nucleolus. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention proposes a lysosome-nucleolus migration fluorescent probe, its preparation method, and its applications. The novel structural design of this fluorescent probe gives it ultra-high hydrophilicity, preventing it from entering normal cells. It stains lysosomes during cell apoptosis and nucleoli during cell necrosis, achieving migration from lysosomes to nucleoli. This provides real-time, in-situ visualization of the transition from healthy cells to apoptosis and necrosis, solving the problems existing in existing technologies.

[0006] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0007] A lysosome-nucleolar migration fluorescent probe, the chemical structure of which is shown in formula (I): in,

[0008] R1 = H, R2 = H3; or, R1 = H, R2 = N + H3Br - Alternatively, R1 = C4H9, R2 = N + H3Br - .

[0009] This series of probes is collectively referred to as PPCs.

[0010] Furthermore, the chemical structure of the representative fluorescent probe of the above-mentioned lysosome-nucleolar migration fluorescent probe is shown in formula (II), and its chemical name is (E)-1-(3-aminopropyl)-4-(4-(piperazin-1-yl)styryl)pyridine-1-onium; abbreviated as PPCN;

[0011] The present invention also provides the following technical solutions:

[0012] A method for preparing a lysosome-nucleolar migration fluorescent probe includes the following steps:

[0013] (1) 4-methylpyridine and bromopropane, 4-methylpyridine and bromopropane and 3-bromo-1-propanamine hydrochloride were dissolved in acetonitrile and added to a round-bottom flask. The mixture was heated under reflux at 80℃-85℃ to obtain compounds 1a and 1b.

[0014] (2) N-Butylpiperazine, p-fluorobenzaldehyde and potassium carbonate were dissolved in acetonitrile and added to a round-bottom flask. The mixture was heated under reflux at 120°C to obtain compound 3.

[0015] (3) After thoroughly stirring 4-(4-formylphenyl)piperazine-1-carboxylic acid tert-butyl ester, 1a, 1b, and compounds 3 and 1b in ethanol, a small amount of piperidine was added and reacted at 85°C to obtain a series of bright orange PPCs.

[0016] The preparation reaction formula is as follows:

[0017]

[0018] Further, in step (3), after 4-(4-formylphenyl)piperazine-1-carboxylic acid tert-butyl ester reacts with 1a and 1b to obtain a bright orange solid, HCl and MeOH, and HBr and MeOH are added at 25°C to obtain an orange solid; the reaction time in step (3) is 12h.

[0019] Furthermore, in step (1), the mixture is heated and refluxed at 85°C for 24 hours.

[0020] Furthermore, the orange solid obtained in 1a above is PPC, i.e., a compound with the chemical structure corresponding to R1 = H and R2 = H3; the orange solid obtained in 1b above is PPCN, i.e., R1 = H and R2 = N. + H3Br - The corresponding chemical structural formula of the compound; the above 1b and compound 3 were used to prepare CPCCN, i.e., R1 = C4H9, R2 = N + H3Br - The corresponding chemical structural formulas of the compounds. This series of fluorescent probes all possess excellent photophysical properties.

[0021] Furthermore, in vitro spectroscopy revealed that the absorption and emission peaks of PPCN were located near 405 nm and 575 nm, respectively. The significant Stokes shift (approximately 170 nm) greatly reduced crosstalk between excitation and emission, enabling clean-free imaging of the probe. A slight blue shift in the emission peak of PPCN was observed with increasing solvent polarity. Other molecules (PPC and CPCCN) exhibited similar photophysical properties in solvents of varying polarities.

[0022] Furthermore, the aforementioned fluorescent probes PPCs exhibit good water solubility. Compared to PPC (Log Po / w = -0.84), the Log Po / w value of PPCN decreased to -2.94, indicating a significant increase in probe hydrophilicity due to the introduction of the quaternary ammonium salt. The Log Po / w value of the probe CPCCN, with added side chains, increased to -1.64. This demonstrates that constructing a dicationic pyridine structure and introducing a piperazine group can significantly improve the probe's hydrophilicity. The hydrophilicity of PPCN suggests that absorption by cells under normal conditions may be challenging.

[0023] Furthermore, the fluorescent probe PPCN exhibits good RNA affinity. With the continuous addition of RNA (0-3 mg / mL), the fluorescence emission intensity of PPCN at 568 nm significantly increases (approximately 17.7-fold). Theoretical calculations indicate that PPCN inserts into the groove of RNA, and the pyridine ring unit stably binds to the six-membered ring in the nucleic acid base via π-π interactions. The three hydrogen atoms on the pyridine cation quaternary ammonium salt form hydrogen bonds with the oxygen atoms of the hydroxyl groups and phosphate units on the bases. The hydrogen atoms of the piperazine also form hydrogen bonds with the oxygen atoms of the hydroxyl groups on the bases. Based on these molecular docking results, PPCN demonstrates a strong binding to nucleic acids, laying the foundation for subsequent nucleolar-targeted imaging within cells.

[0024] This invention also provides the following applications of the above-mentioned lysosome-nucleolar migration fluorescent probe:

[0025] The lysosome-nucleolar migration fluorescent probe is used in the preparation of products that are not taken up by cells and can distinguish healthy cells. In this application: when normal MCF-7 cells are incubated with the probe PPCN for 50 min, the probe does not enter the cells, and no fluorescent signal is detected inside the cells.

[0026] The lysosome-nucleolar migration fluorescent probe described herein is used in the preparation of detection products for real-time in situ monitoring of cell health, apoptosis, and necrosis dynamics for non-diagnostic purposes. In this application: the fluorescent probe PPCN shows no fluorescence signal in cells not treated with hydrogen peroxide, but accumulates at the lysosomes of cells that have entered an apoptotic state after treatment. Furthermore, as the treatment time increases, the cells become completely necrotic, and PPCN accumulates at the nucleolus.

[0027] Furthermore, the lysosome-nucleolar migration fluorescent probe is applied in real-time visual monitoring of cells transitioning from a healthy state to an apoptotic state and then to a necrotic state under various non-diagnostic and therapeutic conditions. In this application: the fluorescent probe PPCN showed the following in MCF-7 cells treated with rotenone and paclitaxel for different time periods: no fluorescence signal was detected in untreated cells; PPCN was enriched at the lysosomes of cells that entered an apoptotic state after short-term treatment; and at the nucleolus after long-term treatment when the cells were completely necrotic. In MCF-7 cells treated with different concentrations of copper and nickel ions, no fluorescence signal was detected in the treated cells; at low concentrations, PPCN was enriched at the lysosomes of cells that entered an apoptotic state; and at high concentrations, the cells were completely necrotic, and PPCN was enriched at the nucleolus.

[0028] The application of the described lysosome-nucleolar migration fluorescent probe in the preparation of reagents for staining apoptotic cell lysosomes. In this application, apoptotic cells are treated with hydrogen peroxide, incubated with PPCN for 20 min, and then stained with the commercially available probe Lyso Tracker. TM When co-stained with Deep Red (LTDR), a common fluorescent enrichment region with good fluorescence overlap can be found in MCF-7 cells.

[0029] The application of the described lysosome-nucleolar migration fluorescent probe in the preparation of reagents for staining necrotic cell nucleoli. In this application, when cells were fixed with 4% paraformaldehyde and apoptotic cells were incubated with PPCN for 10 min and co-stained with the commercial probe Hoechst33342, it was found that the fluorescent enrichment areas in MCF-7 cells did not overlap, indicating that PPCN specifically targeted the nucleoli.

[0030] The above-described solution of the present invention can bring the following beneficial effects:

[0031] The fluorescent probes (PPCs) described in this invention are designed based on the push-pull structure of styrene-pyridinium salt, with the introduction of bisquaternary ammonium salt and piperazine to regulate hydrophilicity. On the one hand, piperazine is a good lysosomal targeting unit, and the salt formed by pyridine and amino groups has excellent nucleolar affinity; on the other hand, both piperazine and bisquaternary ammonium salt are highly hydrophilic structures. Thus, the molecule cleverly possesses both good hydrophilicity and potential targeting of two organelles.

[0032] This invention combines a bisquaternary ammonium cation salt and a diamino structure to synthesize a series of small-molecule fluorescent probes for monitoring the progression from apoptosis to necrosis. By introducing a bisquaternary ammonium cation salt into the classic D-π-A structure, the probe's water solubility is significantly improved, making it difficult to cross the lipid membrane of normal cells. Furthermore, the bisquaternary ammonium salt can stably bind to nucleic acids through electrostatic / hydrogen bonding, equipping the probe with a lysosome-targeting piperazine unit. When apoptosis begins, cell membrane permeability changes, allowing the probe to penetrate the cell membrane and preferentially accumulate in lysosomes. As the cell transitions from apoptosis to necrosis, leading to lysosomal damage and rupture, the probe continuously enters the cell nucleus, illuminating nucleic acids. Therefore, under normal cellular conditions, the probe is not absorbed, avoiding cell damage, thus enabling the differentiation between normal and apoptotic cells. Therefore, apoptosis and necrosis can be distinguished by the migration of the fluorescent probe in lysosomes / nucleoli. Simultaneously, healthy cells, apoptotic cells, and necrotic cells can be distinguished by the fluorescence images of the fluorescent probe at three different locations within the cell.

[0033] This invention features a representative fluorescent probe—a large Stokes shift fluorescent probe (PPCN)—which can be used for in situ real-time monitoring of the progression of cells from apoptosis to necrosis. Due to its hydrophilic nature, PPCN cannot cross the membrane and invade normal cells, thus significantly reducing the potential risk to normal cells. Because of its strong nucleic acid binding ability and good lysosomal targeting, PPCN can migrate into lysosomes and neurolysosomes as cells progress from apoptosis to necrosis. Therefore, based on the localization of PPCN in fluorescence images, the three states of cells (normal, apoptotic, and necrotic) can be distinguished and observed. More importantly, as an application example, PPCN has been successfully used to comprehensively evaluate the state of cells after treatment with hydrogen peroxide, rotenone, paclitaxel, and different heavy metal ions. PPCN can serve as a powerful tool for in situ real-time monitoring of cell apoptosis to necrosis, promoting research on the cell death process. Attached Figure Description

[0034] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings:

[0035] Figure 1 The figures show the 1H NMR spectrum (a), 1C NMR spectrum (b), and high-resolution mass spectrometry spectrum (c) of the probe PPCN of this invention; the data in the figures can indicate the correct structure and purity of the compound.

[0036] Figure 2 The normalized ultraviolet absorption spectrum and normalized fluorescence emission spectrum of the probe PPCN in different polar solvents, and the oil-water partition coefficient of the tested PPCs are shown.

[0037] Figure 3 The fluorescence emission spectra and molecular docking calculations of the probe PPCN of this invention in Tris-HCl buffer at different concentrations of RNA are shown.

[0038] Figure 4 Fluorescence images and fluorescence signal images of the cytoplasm and nucleolus were obtained by incubating live MCF-7 cells and fixed MCF-7 cells with PPCN under the same laser conditions.

[0039] Figure 5 Fluorescence images and fluorescence intensity distribution maps were obtained for MCF-7 cells treated with hydrogen peroxide and co-incubated with PPCN (10 μM), followed by co-staining with the commercial lysosomal probe LTDR (100 nM); and for fixed MCF-7 cells co-incubated with PPCN (10 μM) and then co-staining with the commercial lysosomal probe Hoechst 33342 (100 μM).

[0040] Figure 6 The survival rate of MCF-7 cells after incubation with different concentrations of probe PPCN for 24 hours;

[0041] Figure 7 Confocal images of the entire process from normal state to apoptosis to necrosis, recorded in situ in real time, for MCF-7 cells induced by hydrogen peroxide and co-incubated with PPCN (10 μM).

[0042] Figure 8 Confocal images of the entire process from normal state to apoptosis to necrosis, recorded in situ in real time, to induce cell treatment with different drugs and co-incubate with PPCN (10 μM).

[0043] Figure 9 The figures show the 1H NMR spectrum (a), 1C NMR spectrum (b), and high-resolution mass spectrometry spectrum (c) of the probe PPC of this invention; the data in the figures can indicate the correct structure and purity of the compound.

[0044] Figure 10 The normalized UV absorption spectra and normalized fluorescence emission spectra of the probe PPC of this invention in different polar solvents are shown.

[0045] Figure 11 The survival rate of MCF-7 cells after incubation with different concentrations of probe PPC for 24 hours;

[0046] Figure 12 The figures show the 1H NMR spectrum (Figure a), 1C NMR spectrum (Figure b), and high-resolution mass spectrometry (Figure c) of the probe CPCCN of this invention; the data in the figures can indicate the correct structure and purity of the compound.

[0047] Figure 13 The normalized UV absorption spectrum and normalized fluorescence emission spectrum of the probe CPCCN of this invention in different polar solvents are shown.

[0048] Figure 14 The survival rate of MCF-7 cells after incubation with different concentrations of the probe CPCN for 24 hours;

[0049] in, Figure 2 a) shows the normalized UV absorption spectrum and normalized fluorescence emission spectrum of the fluorescent probe PPCN in different polar solvents and under 488 nm excitation; the results show that the probe PPCN has a large Stokes shift; b) is a comparison of the oil-water partition coefficients of PPCs, and PPCN has the best hydrophilicity among the comparison.

[0050] Figure 3a) shows the fluorescence emission spectra of the fluorescent probe PPCN in Tris-HCl buffer at different concentrations of RNA; b) shows the molecular docking calculation diagram of the fluorescent probe PPCN. As can be seen from a), with the increase of RNA concentration, the fluorescence intensity of probe PPCN at 575 nm increased by about 17.7 times, which indicates that probe PPCN is relatively sensitive to RNA. From the molecular docking results in b), it can be concluded that PPCN binds to the base sequence through electrostatic interactions, π-π interactions and hydrogen bonding interactions, which gives the probe good RNA targeting ability.

[0051] Figure 4 a) shows fluorescence images of live MCF-7 cells and fixed MCF-7 cells incubated with PPCN under the same laser conditions; b) shows fluorescence signals collected from the cytoplasm and nucleolus; the excitation wavelength of PPCN was 473 nm, and the absorption range was 550 nm-650 nm; the results show that the probe PPCN cannot enter live cells, but it can stain the RNA of fixed cells.

[0052] Figure 5 a) shows the fluorescence image obtained by treating MCF-7 cells with hydrogen peroxide and co-incubating them with PPCN (10 μM), followed by staining with the commercial lysosomal probe LTDR (100 nM); b) shows the fluorescence intensity distribution of PPCN and LTDR collected based on a); c) shows the fluorescence image obtained by fixing MCF-7 cells, co-incubating them with PPCN (10 μM), and co-staining them with the commercial lysosomal probe Hoechst 33342 (100 μM); d) shows the fluorescence intensity distribution of PPCN and Hoechst collected based on c). The excitation wavelengths of Hoechst 33342, PPCN, and LTDR are 405 nm, 473 nm, and 633 nm, respectively, and the absorption ranges are 410 nm-450 nm, 550 nm-650 nm, and 650 nm-750 nm, respectively. The experimental results show that probes PPCN and LTDR have a common enrichment region and good fluorescence overlap, while PPCN and Hoechst... 33342 has almost no common enrichment regions and its fluorescence signals hardly overlap;

[0053] Figure 8 In the middle, a), b), c), and d) are confocal images of cells in three states (normal, apoptosis, and necrosis) recorded in real time in situ after being treated with paclitaxel, rotenone, copper ions, and nickel ions and co-incubated with 10 μM PPCN. Detailed Implementation

[0054] To more clearly illustrate the overall concept of the present invention, a detailed description is provided below with reference to the accompanying drawings. The examples described below are merely preferred embodiments of the present invention. It should be noted that the following description is solely for explaining the present invention and does not constitute any limitation thereof. Any simple modifications, equivalent variations, or alterations made to the embodiments based on the technical essence of the present invention fall within the scope of the present invention.

[0055] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0056] Example 1

[0057] I. Synthesis of probe PPCN

[0058] (1) A solution of 4-methylpyridine (0.94 g, 10 mM) and 3-bromopropylamine hydrobromide (2.19 g, 10 mM) in EtOH (20 mL) was heated at 85 °C for 24 hours. The mixture was washed with dichloromethane and dried under vacuum to obtain a white solid, which is 1-(3-aminopropyl)-4-methylpyridine-1-onium (1b).

[0059] (2) Add one drop of piperidine to a solution of compound 1b (0.62 g, 2 mM) and tert-butyl piperazine-1-carboxylate (0.53 g, 2 mM) in EtOH (10 mL). Stir the solution at 85 °C for 12 hours, cool to room temperature, remove the solution under reduced pressure, and subject the residue to rapid column chromatography to obtain an orange solid, which is (E)-1-(3-aminopropyl)-4-(4-(4-(tert-butyloxycarbonyl)piperazin-1-yl)styryl)pyridin-1-onium (2b).

[0060] (3) At 25°C, 40% HBr (2 mL) and MeOH (3 mL) were added to 2b (0.2 g, 0.3 mM) and left overnight. The precipitate was filtered, washed with dichloromethane, and dried under vacuum. The resulting PPCN was an orange solid with a yield of approximately 44%.

[0061] The experimental results are shown in Figure 1 , Figure 1 Figure 1 shows the 1H NMR spectrum of PPCN (Figure 1a); the 1C NMR spectrum (Figure 2b); and the high-resolution mass spectrometry spectrum (Figure 3c).

[0062] 1H NMR(400MHz,D2O)δ8.40(d,J=6.8Hz,2H),7.78(d,J=6.8Hz,2H),7.51-7.43(m,3H),6.98(d,J=8.8Hz,2H),6.93(d,J =16.2Hz,1H),4.37(t,J=7.6Hz,2H),3.51-3.45(m,4H),3.34-3.27(m,4H),3.02-2.95(m,2H),2.22(p,J=8.0Hz,2H). 13 HRMS: calculated for C 20 H 28 N4 2+ m / z 162.1152, found 162.1158.

[0063] II. Sensitivity test of probe PPCN to solvent polarity and lipid-water partition coefficient of PPCs

[0064] 10 μM PPCN stock solutions were prepared using solvents of different polarities (tetrahydrofuran, acetone, acetonitrile, and water). Absorption spectra of PPCN in different solvents were obtained using a HITACH U-2910 UV spectrophotometer, and fluorescence emission spectra were obtained using a HITACH F-2700 fluorescence spectrophotometer equipped with a 450W Xe lamp at 473 nm excitation. Figure 2 (Figure a)

[0065] A 1:1 mixture of n-octanol and water was pre-saturated on a shaker for 24 hours. The resulting two-phase solution was then added in equal volumes to a centrifuge tube containing the complex, and the mixture was shaken for 48 hours. After standing and separation, the two-phase solution of the complex was obtained, and the precipitate was removed. The two-phase solution was diluted with methanol to the same concentration, and the UV-Vis absorbance was measured using a HITACH U-29 UV spectrophotometer. (logP) o / w =log 10 (A o / A w ) to calculate and test the oil-water partition coefficient of PPCs ( Figure 2 (Figure b)

[0066] Depend on Figure 2The results show that the peak absorption spectrum of the probe PPCN is at 405 nm, the peak emission spectrum is at 575 nm, and the Stokes shift is as high as 170 nm. This large Stokes shift avoids self-emission crosstalk and is insensitive to changes in solvent polarity. The Log Po / w of PPCN is -2.94, indicating a significant increase in hydrophilicity due to the introduction of the quaternary ammonium salt. Constructing a dicationic pyridine structure and introducing a piperazine group can significantly improve the probe's hydrophilicity. The hydrophilicity of PPCN means that this probe is difficult to be taken up by normal cells.

[0067] III. Response of probe PPCN to in vitro RNA and theoretical calculations.

[0068] In RNA titration experiments, stock RNA solutions of different concentrations (3 mg / mL) were prepared in Tris-HCl buffer (pH = 7.2). To calculate the molar concentration of the stock solutions, they were diluted to 0.5%, and then the absorption spectra were measured. The molar concentration of the RNA stock solutions was calculated using the following formula:

[0069]

[0070] The RNA stock solution was diluted with Tris-HCl buffer to prepare 10 μM PPCN solutions containing different concentrations of RNA. The fluorescence emission spectra of the PPCN were obtained using a HITACH F-2700 fluorescence spectrophotometer equipped with a 450W Xe lamp at 473 nm excitation.

[0071] Nucleic acid preparation: The initial structure of RNA was downloaded from the RCSB protein database (PDB code: 5T2C). PyMOL was used to remove the original ligands and water for docking studies.

[0072] Preparation of the ligand (PPCN): Using the optimized compound structure, partial atomic charges were calculated at the HF / 6-31g* level using confined electrostatic potentials (RESPs) in Gauss09. Except for the number of GA runs (GA run = 50) and the number of maximum energy evaluations (GA num evals = 2,500,000), all other parameters were obtained using the default Lamarckian genetic algorithm. Images were rendered using PyMOL.

[0073] The experimental results are shown in Figure 3 As the RNA concentration (0-3 mg / mL) increased, the fluorescence emission intensity of the probe at 568 nm increased significantly (approximately 17.7 times). Figure 3(Figure a) Theoretical calculations show that when the probe inserts into the groove of the RNA, the pyridine ring stably binds to the six-membered ring in the nucleic acid base via π-π interactions. The three hydrogen atoms on the pyridine cation quaternary ammonium salt form hydrogen bonds with the oxygen atoms of the hydroxyl groups and phosphate units on the bases. The hydrogen atoms of the piperazine also form hydrogen bonds with the oxygen atoms of the hydroxyl groups on the bases. Based on these molecular docking results, PPCN shows a strong binding to nucleic acids, laying the foundation for subsequent nucleolar-targeted imaging within cells. Figure 3 (Figure b)

[0074] IV. Preparation of live cell samples for testing

[0075] MCF-7 cells were maintained in DMEM containing 10% FBS (Gibco BRL), 100 μg / mL streptomycin (Gibco BRL), and 100 U / mL penicillin (Gibco BRL). Cells were cultured in a humidified incubator with a 5% CO2 atmosphere at a constant temperature of 37°C, and passaged every 2-3 days.

[0076] When MCF-7 cells reach the logarithmic growth phase, slide culture is performed as follows: ① Soak coverslips in ethanol for 30 min, dry them with an alcohol lamp, and then place them in a disposable 35 mm confocal culture dish for later use; ② Wash the cells in the cell culture flask three times with PBS, and digest them with 1 mL of 0.25% trypsin for 1-2 min. Then, pipette out the trypsin, add DMEM, mix well, and count the cells. Control the cell density by adding culture medium to achieve a final cell concentration of 1 × 10⁶ cells / mL. 5 Then, the cells were seeded into culture dishes containing coverslips and incubated at 37°C in a 5% CO2 incubator to allow them to grow on the coverslips. Once the cells had confined the coverslips, the MCF-7 cells obtained from this confined growth were obtained for experiments.

[0077] V. Staining live and fixed cell samples with PPCN probe.

[0078] Live cells: MCF-7 cells were seeded in 35 mm culture dishes and cultured for 24 h. After incubation with PPCN (10 μM) at 37 °C for 50 min, the cells were observed directly under a confocal microscope without washing.

[0079] Cell fixation: MCF-7 cells were seeded in 35 mm culture dishes and cultured for 24 h. They were then fixed at 37 °C for 1 h with 4% paraformaldehyde and incubated with PPCN (10 μM) at 37 °C for 10 min. No washing was required, and the cells were directly imaged and observed under a confocal microscope.

[0080] The experimental results are shown in Figure 4 The excitation wavelength and collection range of PPCN are λ. ex =473nm, λem =550-650nm.

[0081] Depend on Figure 4 The experimental results show that the probe PPCN cannot enter living cells, but it can stain and fix the RNA in cells.

[0082] VI. Colocalization experiments validated the staining results of the PPCN probe on apoptotic and fixed MCF-7 cells.

[0083] Prepare a DMSO solution containing 10 mM PPCN probe as a stock solution. After the cells in the confocal microscope are confluent, treat the cells with 3 mM hydrogen peroxide for 40 min, then stain the cells with 10 μM PPCN, and then add 100 nM LTDR to the culture dish for co-incubation. After treating with 4% paraformaldehyde for 1 h, stain the cells with 10 μM PPCN, and then add 10 μM Hoechst 33342 to the culture dish for co-incubation. Observe the staining under a laser confocal microscope.

[0084] See results Figure 5 The excitation wavelengths of Hoechst 33342, PPCN, and LTDR are 405nm, 473nm, and 633nm, respectively, and the light-gathering ranges are 410nm-450nm, 550nm-650nm, and 650nm-750nm, respectively.

[0085] The experimental results show that in apoptotic cells, the PPCN-stained areas overlap well with the LTDR-stained areas, with a Pearson correlation coefficient (PCC) of 0.83. On the other hand, in fixed cells, the PPCN-stained areas hardly overlap with the Hoechst 33342-stained areas. These experimental results indicate that the PPCN probe can stain the lysosomes of apoptotic cells and the nucleoli of necrotic cells, allowing for the differentiation of different cell states through probe localization within the cell.

[0086] VII. Cytotoxicity test of probe PPCN

[0087] The cytotoxicity of PPCN against the MCF-7 cell line was determined using the standard MTT assay. Cells in the logarithmic growth phase were seeded into 96-well plates and incubated for 24 h. Different concentrations (0 μM, 1 μM, 5 μM, 10 μM, 20 μM, 30 μM) of the probe PPCN were added to the wells of the treatment group and incubated for 24 h. Then, 20 μL of MTT (5 mg / mL) was added to each well. After incubation for 4 h, the culture medium in each well was removed, and DMSO (100 μL) was added to dissolve the purple crystals. After 20 min, the absorbance was measured at 570 nm using a microplate reader. The cytotoxicity assay was repeated three times.

[0088] The experimental results are shown in Figure 6 The experimental results show that the cytotoxicity of the PPCN probe is negligible at incubation concentrations of 0-30 μM. At working concentrations of up to 30 μM PPCN, the survival rate of MCF-7 cells can reach over 90%. These data indicate that the PPCN probe has low cytotoxicity and has the potential for application in live-cell imaging.

[0089] 8. Visualization of cellular state changes from normal cells to necrosis to apoptosis induced by hydrogen peroxide using the PPCN cell imaging probe.

[0090] Prepare a DMSO solution containing 5 mM PPCN probe as a stock solution. After the cells have grown to a confocal depth in the confocal dish, label the cells with 10 μM Hoechst 33342 and 100 nM LTDR, then treat the cells with 3 mM hydrogen peroxide and stain them with 10 μM PPCN. Observe the staining under a laser confocal microscope.

[0091] See results Figure 7 The experimental results show that when the cells are in a normal state, no fluorescence signal is observed in the green fluorescent channel of PPCN staining. As the treatment time reaches 20 min, the lysosomes in the green channel are illuminated, indicating apoptosis. With further treatment time, the signal in the green channel migrates from the lysosomes to the nucleolus, indicating complete cell necrosis.

[0092] 9. The PPCN cell imaging probe enables real-time monitoring of apoptosis and necrosis induced by different drugs.

[0093] To simulate a model of normal cells undergoing apoptosis and necrosis induced by different drugs, MCF-7 cells from the same batch were treated with rotenone, paclitaxel, and Nitroglycerin, respectively. 2+ and Cu 2+ Incubate the cells.

[0094] The experimental results are shown in Figure 8 The results showed that MCF-7 cells were treated with rotenone and paclitaxel (5 μM) for different time periods, followed by cell staining with PPCN. Cells not treated with paclitaxel showed no fluorescence signal in the green channel, indicating that PPCN was not absorbed in this state; after 6 hours of drug incubation, PPCN entered the lysosomes and stained, indicating that the cells were in the apoptotic stage. With further extension of treatment time, the cells gradually died, and PPCN migrated to the nucleolus. In cells treated with rotenone, the probe also showed similar migration changes. Different concentrations of Ni... 2+ and Cu 2+MCF-7 cells were treated with these two ions for 40 minutes and stained with PPCN. Cells not treated with heavy metal ions showed no fluorescence signal in the green channel, indicating that PPCN was not absorbed under these conditions. Ni... 2+ (1mM) or Cu 2+ After treatment with (0.5 mM) for 40 minutes, PPCN entered lysosomes and stained them, indicating that the cells were in the apoptotic stage. With increasing treatment concentration, the cells gradually died, and PPCN migrated into the nucleolus. The experimental results show that the probe PPCN can be used for real-time, in-situ, and precise monitoring of various drug-induced cell damage through cascade imaging of lysosomes and nucleoli.

[0095] Example 2

[0096] I. Synthesis of Probe PPC

[0097] (1) 4-Methylpyridine (0.94 g, 10 mM) and 1-bromopropane (1.23 g, 10 mM) were dissolved in dry ethanol (20 mL) and the mixture was heated to 85 °C and reacted for 24 hours. After the solution cooled to room temperature, the solvent was removed under reduced pressure. The resulting residue was subjected to rapid column chromatography. 1a was given in approximately 80% yield as a yellow oil.

[0098] (2) One drop of pyridine was added dropwise to a solution of EtOH (10 mL) containing compound 1a (0.58 g, 2 mM) and tert-butyl-4-(4-formylphenyl)piperazine-1-carboxylic acid ester (0.53 g, 2 mM). The reaction was stirred at 85 °C for 12 hours. After the solution cooled to room temperature, the solvent was removed under reduced pressure. 2a was obtained without purification. 40% HBr (2 mL) and MeOH (3 mL) were added to the mixture of 2a at 25 °C, and the reaction was allowed to proceed overnight. The precipitate was filtered, washed with dichloromethane, and dried under reduced pressure. PPC was obtained as an orange solid in approximately 70% yield.

[0099] The experimental results are shown in Figure 9 , Figure 9 Figure 1 shows the 1H NMR spectrum of PPC (Figure 1a); the 1C NMR spectrum (Figure 2b); and the high-resolution mass spectrometry spectrum (Figure 3c).

[0100] 1H NMR(400MHz,D2O)δ8.43(dd,J=10.0,6.9Hz,2H),7.86(dd,J=10.4,6.9Hz,2H),7.66-7.55(m,3H),7.13-7.03(m,3H),4.30(t,J=7.3Hz ,2H),3.47-3.43(m,1H),3.31(dd,J=6.2,4.1Hz,3H),3.16-3.12(m,1H),3.10-3.06(m,3H),1.91-1.85(m,2H),0.84(t,J=7.4Hz,3H). 13 HRMS: calculated for C 20 H 26 N3 + m / z 308.2122,found308.2109.

[0101] II. Sensitivity test of probe PPC to solvent polarity

[0102] 10 μM PPCN stock solutions were prepared using solvents of different polarities (tetrahydrofuran, acetone, acetonitrile, and water). Absorption spectra of PPC in different solvents were obtained using a HITACH U-2910 UV spectrophotometer, and fluorescence emission spectra were obtained using a HITACH F-2700 fluorescence spectrophotometer equipped with a 450W Xe lamp at 473 nm excitation. (See [link to relevant documentation]). Figure 10 .

[0103] Depend on Figure 10 The results show that the peak absorption spectrum of the probe PPC is at 398 nm, the peak emission spectrum is at 571 nm, and the Stokes shift is as high as 167 nm. This large Stokes shift avoids self-emission crosstalk and is insensitive to changes in solvent polarity. The Log Po / w of PPC is -0.84, indicating a significant increase in hydrophilicity due to the introduction of the quaternary ammonium salt. Constructing a dicationic pyridine structure and introducing a piperazine group can significantly improve the probe's hydrophilicity. The hydrophilicity of PPC means that this probe is difficult to be taken up by normal cells.

[0104] III. Cytotoxicity test of probe PPC

[0105] The test method is described in Example 1, and the experimental results are shown in [example 1]. Figure 11The experimental results show that the cytotoxicity of the probe PPC is negligible at incubation concentrations of 0-30 μM. At working concentrations of up to 30 μM PPC, the survival rate of MCF-7 cells can reach over 90%. These data indicate that the probe PPC has low cytotoxicity and has the potential for application in live-cell imaging.

[0106] Example 3

[0107] I. Synthesis of the probe CPCCN

[0108] (1) 4-Methylpyridine (0.94 g, 10 mmol) and 1-bromopropane (1.23 g, 10 mmol) were dissolved in dry ethanol (20 mL) and the mixture was heated to 85°C and reacted for 24 hours. After the solution cooled to room temperature, the solvent was removed under reduced pressure. The resulting residue was subjected to rapid column chromatography. 1a was given in approximately 80% yield as a yellow oil.

[0109] (2) K₂CO₃ (1.44 g, 12 mM) was added to a solution of 1-butylpiperazine (1.42 g, 10 mM) and 4-fluorobenzaldehyde (1.24 g, 10 mM) in acetonitrile (20 mL). The reaction was stirred at 120 °C for 24 hours. After the solution cooled to room temperature, the solvent was removed by vacuum. The residue was subjected to rapid column chromatography. Compound 3 was given in approximately 90% yield as a yellow oil.

[0110] (3) One drop of pyridine was added dropwise to an ethanol (10 mL) solution containing compound 3 (0.59 g, 2 mM) and compound 1b (0.62 g, 2 mM). The reaction was stirred at 85°C for 12 hours. After the solution cooled to room temperature, the solvent was removed under reduced pressure. The resulting residue was subjected to rapid column chromatography. CPCCN was obtained as an orange solid with a yield of approximately 70%.

[0111] The experimental results are shown in Figure 12 , Figure 12 Figure 1 shows the proton NMR spectrum of CPCCN (Figure 1a); carbon NMR spectrum (Figure 2b); and high-resolution mass spectrometry spectrum (Figure 3c).

[0112] 1H NMR (400MHz, DMSO-d6) δ8.92(d,J=6.8Hz,2H),8.18(d,J=6.8Hz,2H),8.07(s,3H),7.98(d,J=16.2H z,1H),7.67(d,J=8.8Hz,2H),7.35(d,J=16.2Hz,1H),7.12(d,J=8.9Hz,2H),4.59(t,J=6.9Hz,2H), 4.03(d,J=12.8Hz,2H),3.58(d,J=11.4Hz,2H),3.24(t,J=12.2Hz,3H),3.12(t,J=10.5Hz,4H),2.9 0-2.80(m,2H),2.21(p,J=6.9Hz,2H),1.74-1.67(m,2H),1.38-1.30(m,2H),0.93(t,J=7.4Hz,3H). 13 C HRMS: calculated for C 24 H 36 N4 2+ m / z 190.1463, found 190.1465.

[0113] II. Sensitivity test of probe CPCCN to solvent polarity

[0114] 10 μM PPCN stock solutions were prepared using solvents of different polarities (tetrahydrofuran, acetone, acetonitrile, and water). Absorption spectra of CPCCN in different solvents were obtained using a HITACH U-2910 UV spectrophotometer, and fluorescence emission spectra were obtained using a HITACH F-2700 fluorescence spectrophotometer equipped with a 450W Xe lamp at 473 nm excitation. (See [link to relevant documentation]). Figure 13 .

[0115] Depend on Figure 13The results show that the peak absorption spectrum of the probe CPCCN is at 395 nm, the peak emission spectrum is at 575 nm, and the Stokes shift is as high as 170 nm. This large Stokes shift avoids self-emission crosstalk and is insensitive to changes in solvent polarity. The Log Po / w of CPPCN is -1.64, indicating a significant increase in hydrophilicity due to the introduction of the quaternary ammonium salt. Constructing a dicationic pyridine structure and introducing a piperazine group can significantly improve the probe's hydrophilicity. The hydrophilicity of CPPCN means that this probe is difficult to be taken up by cells under normal cellular conditions.

[0116] III. Cytotoxicity test of probe CPCCN

[0117] The test method is described in Example 1, and the experimental results are shown in [example 1]. Figure 14 The experimental results show that the cytotoxicity of the probe CPCCN is negligible at incubation concentrations of 0-10 μM. At a working concentration of 10 μM CPCCN, the survival rate of MCF-7 cells can reach over 90%.

[0118] The above description is merely an embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of the present invention should be included within the scope of the claims of the present invention.

Claims

1. A lysosome-nucleolar migration fluorescent probe, characterized in that, Its chemical structural formula is shown in formula (I): (I); where, R1=H, R2=H3; or, R1=H, R2=N + H3Br - Alternatively, R1 = C4H9, R2 = N + H3Br - .

2. The lysosome-nucleolar migration fluorescent probe according to claim 1, characterized in that, Its chemical structural formula is shown in formula (II), and its chemical name is: (E)-1-(3-aminopropyl)-4-(4-(piperazin-1-yl)styryl)pyridine-1-onium; (II).

3. The method for preparing the lysosome-nucleolar migration fluorescent probe as described in claim 1, characterized in that, Includes the following steps: (1) Dissolve 4-methylpyridine and bromopropane in acetonitrile and add them to a round-bottom flask. Heat the mixture under reflux at 80℃-85℃ to give compound 1a; dissolve 4-methylpyridine and 3-bromo-1-propanamine hydrobromide in acetonitrile and add them to a round-bottom flask. Heat the mixture under reflux at 80℃-85℃ to give compound 1b; (2) N-butylpiperazine, p-fluorobenzaldehyde and potassium carbonate were dissolved in acetonitrile and added to a round-bottom flask. The mixture was heated under reflux at 120 °C to obtain compound 3. (3) After stirring 4-(4-formylphenyl)piperazine-1-carboxylic acid tert-butyl ester and 1a in ethanol, a small amount of piperidine was added and reacted at 85 °C to obtain a bright orange solid 2a. HCl and MeOH were added at 25 °C to obtain an orange solid PPC. After thoroughly stirring 4-(4-formylphenyl)piperazine-1-carboxylic acid tert-butyl ester and 1b in ethanol, a small amount of piperidine was added, and the reaction was carried out at 85 °C to obtain a bright orange solid 2b. HBr and MeOH were then added at 25 °C to obtain an orange solid PPCN. After thoroughly stirring compounds 3 and 1b in ethanol, a small amount of piperidine was added, and the mixture was reacted at 85 °C to give the bright orange product CPCCN. The above preparation reaction formula is as follows: 。 4. The method for preparing the lysosome-nucleolar migration fluorescent probe according to claim 3, characterized in that, The reaction time in step (3) is 12 hours.

5. The method for preparing the lysosome-nucleolar migration fluorescent probe according to claim 3, characterized in that, In step (1), the mixture is heated and refluxed at 85°C for 24 hours.

6. The application of the lysosome-nucleolar migration fluorescent probe as described in claim 1 or 2 in the preparation of detection products for real-time in situ monitoring of cell health status, apoptosis and necrosis dynamics for non-diagnostic purposes.

7. The application of the lysosome-nucleolar migration fluorescent probe as described in claim 1 or 2 in real-time in situ monitoring of healthy cells from apoptosis to necrosis induced by various drugs or heavy metal ions for non-diagnostic purposes.

8. The use of the lysosome-nucleolar migration fluorescent probe as described in claim 1 or 2 in reagents for preparing staining lysosomes of apoptotic cells or reagents for staining nucleoli of necrotic cells.

9. The application according to claim 8, characterized in that, After treating apoptosis with hydrogen peroxide, apoptotic cells were incubated with a lysosome-nucleolar migration fluorescent probe for 20 min. When co-stained with commercial probe lysosome tracking deep red, a common fluorescent enrichment region and fluorescence overlap were observed in MCF-7 cells. Cells were fixed with 4% paraformaldehyde. When apoptotic cells were incubated with a lysosome-nucleolar migration fluorescent probe for 10 min and then co-stained with the commercial probe Hoechst33342, the fluorescence enrichment areas in MCF-7 cells did not overlap, and the lysosome-nucleolar migration fluorescent probe specifically targeted the nucleolus.

10. The application according to claim 8 or 9, characterized in that, The lysosome-nucleolar migration fluorescent probe is PPCN, and its structural formula is as follows: 。

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