A double-driving force fluorescent probe targeting lipid droplets and a preparation method and application thereof

Abstract

The application discloses a kind of double driving force fluorescent probes targeting lipid droplet and its preparation method and application, belong to fluorescent probe technical field, by applying probe ENT and super-resolution microscope, the application is first discovered in copper death early stage lipophagy is promoted, and in this process, lipid droplet first presents homogenization tendency and shows higher viscosity.The application provides a new marking strategy for designing lipid droplet probe, realizes long-time, high signal-to-noise ratio and more accurate visualization of lipid droplet, which will help to promote the application of copper death in anticancer therapy and related mechanism research.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe technology, and in particular relates to a dual-drive fluorescent probe for targeting lipid droplets, its preparation method, and its application. Background Technology

[0002] Copper death is a programmed cell death process characterized by excessive accumulation of copper ions. Unlike classical apoptosis, this process is not affected by conventional apoptosis inhibitors. Current research suggests that copper death primarily exerts its cytotoxic effects by interfering with amino acid metabolic pathways. Elevated intracellular copper ion levels lead to decreased cystathionine β-synthetase activity, which in turn inhibits protein synthesis, ultimately triggering cell death. As a novel form of cell death, copper death has attracted widespread attention in cancer research. Studies have shown that downregulating specific amino acid transporters in tumor cells using gene editing technology can enhance their sensitivity to copper ions, thereby inducing copper death and inhibiting tumor growth. Although research on the relationship between copper death and cancer is still in its early stages, based on the fact that both are independent biological processes, it can be inferred that there may be some overlap between them. Therefore, in-depth exploration of these overlaps is expected to clarify the precise relationship between copper death and cancer.

[0003] Lipophage is a process in which lipid droplets are selectively degraded via autophagic vesicles. Under normal physiological conditions, lipophage is crucial for maintaining cellular energy homeostasis and fatty acid metabolism stability. When intracellular copper levels are abnormally elevated, a series of signaling pathways that induce lipophage are activated. For example, Cu... 2+ Copper can directly interact with the LC3 protein, promoting its binding to lipid droplet surfaces and accelerating the entry of lipid droplets into autolysosomes for degradation. Furthermore, copper can indirectly affect lipophage activity by regulating the AMPK / mTOR signaling pathway. Moderate lipophage can alleviate copper-induced oxidative stress, while excessive lipophage may exacerbate intracellular lipid peroxidation and accelerate copper death. However, during tumor development, lipid metabolism is often reprogrammed to meet the demands of rapid proliferation, which may lead to resistance in tumor cells to copper-induced lipid peroxidation, thus hindering copper death. Therefore, a deeper understanding of the complex and subtle role of lipophage in copper death and cancer holds promise as a significant breakthrough in cancer treatment.

[0004] Currently, lipophage research often relies on electron microscopy to observe the co-localization of autophagosomes and lipid droplets after sample fixation, indirectly inferring its occurrence. While this method allows for qualitative analysis, it cannot monitor the dynamic changes of lipophage in real time. Of particular note is that changes in intracellular viscosity directly affect the dynamic behavior of lipid droplets, thus impacting lipophage efficiency. Confocal microscopy or super-resolution microscopy platforms offer the possibility of real-time observation of dynamic changes in intracellular events, providing a technological foundation for the visualization of lipophage. Commercial lipid droplet dyes, such as BODIPY and Oil Red O, have been widely used in lipid droplet research. However, these dyes have several problems in practical applications: (i) uneven staining, affecting the accurate assessment of lipid droplet distribution and size; (ii) poor photostability, making long-term observation difficult and affecting the reliability of results; (iii) potential cell damage at high concentrations, affecting normal cell function; and (iv) a lack of detection capabilities beyond staining, resulting in an inability to comprehensively acquire information on the dynamic changes of lipid droplets. Therefore, there is an urgent need to upgrade lipid droplet labeling strategies and develop novel targeted visualization tools to further explore the association between copper death and cancer. Summary of the Invention

[0005] To address the above problems, this invention provides a dual-drive fluorescent probe for targeting lipid droplets, its preparation method, and its application.

[0006] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a dual-drive fluorescent probe (ENT) for targeting lipid droplets, the chemical structure of which is as follows: .

[0008] Secondly, the present invention provides a method for preparing the above-mentioned dual-driving force fluorescent probe targeting lipid droplets, comprising the following steps:

[0009] S1. Dissolve 3-bromo-9-ethylcarbazole, 4-formylphenylboronic acid, potassium carbonate and tetra(triphenylphosphine)platinum in an organic solvent, and heat under nitrogen protection to react and obtain an intermediate;

[0010] S2. Under nitrogen protection, the intermediate, 3-methylbenzo[d]thiazole-3-onium salt and catalyst are added to an organic solvent, heated to react, the solvent is removed, and the mixture is washed to obtain the dual-drive force probe targeting lipid droplets.

[0011] The chemical structural formula of the intermediate is: .

[0012] Further, in step S1, the molar ratio of 3-bromo-9-ethylcarbazole, 4-formylphenylboronic acid, potassium carbonate and tetrakis(triphenylphosphine)palladium is 1:1.2:3.23:0.03.

[0013] Furthermore, the heating reaction is carried out at a temperature of 60 °C for 12 hours.

[0014] Further, in step S2, the molar ratio of the intermediate to 3-methylbenzo[d]thiazole-3-onium salt is 5:6.

[0015] Furthermore, in step S2, the heating reaction is carried out at a temperature of 80 °C for 12 hours.

[0016] Furthermore, step S1, after the heating reaction, also includes a purification step by silica gel column chromatography.

[0017] Furthermore, the eluent used in the silica gel column chromatography purification is a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 10:1.

[0018] The design strategy of the dual-drive fluorescent probe for targeting lipid droplets provided by this invention is as follows: Lipid droplets mainly possess two key characteristics: First, their internal core mainly stores neutral lipids, which endows the lipid droplets with hydrophobic properties; Second, the surface of the lipid droplets is covered by a monolayer of phospholipids and embedded with negatively charged proteins (such as proteins containing acidic amino acids) and phospholipids (such as phosphatidylserine and phosphatidylinositol phosphate), which together endow their surface with negative charge. Based on the above two characteristics, this invention proposes a method for preparing a dual-drive fluorescent probe for targeting lipid droplets by combining a dual-drive targeting strategy of hydrophobic and electrostatic interactions. The fluorescent probe ENT is designed according to the following principles: (1) Hydrophobicity: The probe must be strongly hydrophobic to ensure effective hydrophobic interaction with the lipid droplets; (2) Positive charge: The probe needs to introduce positively charged groups to enhance its electrostatic interaction with the negatively charged lipid droplet surface; (3) Stable fluorescence platform: The probe needs to have excellent fluorescence performance to facilitate subsequent imaging analysis.

[0019] To achieve the above design goals, this invention constructs a probe ENT composed of three parts: carbazole, a benzene ring, and a positively charged benzothiazole salt. The planar conjugated structure of carbazole provides the probe ENT with excellent fluorescence properties, while the introduction of the benzene ring enhances the hydrophobicity of the molecule. The hydrophobic portion composed of 9-ethyl-9H-carbazole and the benzene ring can interact strongly with the hydrophobic tail of the lipid layer, thereby promoting the aggregation of the probe ENT in the nonpolar lipid droplet environment. The benzothiazole salt portion in the probe ENT provides a specific positive charge distribution. The strong electrostatic attraction between the positively charged molecule and the electronegative groups on the lipid droplet surface further enhances the anchoring stability of the probe ENT within the lipid droplet. The enhanced hydrophobicity of carbazole and the benzene ring, combined with the positive charge distribution of the benzothiazole salt, jointly endows the probe with the ability to target lipid droplets. In addition, the benzothiazole salt unit can act as a "molecular rotor," enabling the probe to be sensitive to the viscosity of the microenvironment.

[0020] Thirdly, the present invention provides an application of the dual-driving force fluorescent probe targeting lipid droplets in the preparation of products for monitoring the dynamic changes of lipid droplets during the lipophagy of cancer cells.

[0021] The dual-drive probe for targeting lipid droplets provided by this invention is expected to achieve long-term, high signal-to-noise ratio and high-precision tracking of the dynamic changes of lipid droplets (including size, division, fusion and microenvironment viscosity changes) during the lipophagy of cancer cells.

[0022] Compared with the prior art, the present invention has the following advantages and technical effects:

[0023] This invention proposes a targeting strategy based on the dual driving forces of hydrophobic and electrostatic interactions, and develops a dual-driving force fluorescent probe targeting lipid droplets to study the dynamic changes of lipid droplets during lipogenesis in copper-dead cancer cells. In the above design concept, three main modules—carbazole, a benzene ring, and a benzothiazole salt cation—are rationally connected to form the probe ENT. Carbazole and the benzene ring, as lipophilic units, enhance the lipophilicity of the compound, promoting probe aggregation in the nonpolar lipid droplet environment. The positively charged benzothiazole salt, through strong electrostatic attraction with negatively charged groups on the lipid droplet surface, further anchors the probe to the droplet surface, improving targeting stability. Simultaneously, the free-rotation structure of the benzothiazole salt enables the probe ENT to sense lipid droplet viscosity. By applying the probe ENT with super-resolution microscopy, this invention reveals for the first time that lipogenesis is promoted in the early stages of copper death, and that during this process, lipid droplets initially exhibit a homogenization trend and show higher viscosity. This invention provides a novel labeling strategy for designing lipid droplet probes, enabling long-term, high signal-to-noise ratio, and more accurate visualization of lipid droplets, which will help promote the application of copper death in anticancer therapy and related mechanism research. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 1H NMR spectrum of DMSO-d6 solution of the dual-driving force fluorescent probe for targeting lipid droplets prepared in Example 1.

[0026] Figure 2 The carbon NMR spectrum of the dual-driving force fluorescent probe for targeting lipid droplets prepared in Example 1 in deuterated chloroform ( 13 (C NMR).

[0027] Figure 3The 1H NMR spectrum of the intermediate prepared in Example 1.

[0028] Figure 4 The carbon NMR spectrum of the intermediate prepared in Example 1 ( 13 (C NMR).

[0029] Figure 5 The fluorescence emission spectra of the fluorescent probe ENT prepared in Example 1 in different ratios of glycerol / methanol mixed solvents;

[0030] Figure 6 The results of cytotoxicity assays of different concentrations of the fluorescent probe ENT on HepG2 cells;

[0031] Figure 7 The results show the permeability of different concentrations of the fluorescent probe ENT in HepG2 cells.

[0032] Figure 8 The results of ultra-high resolution microscopy images of the probe ENT in HepG2 cells under different treatment conditions;

[0033] Figure 9 The results of standardized analysis of the fluorescence intensity of the probe ENT in HepG2 cells under different conditions;

[0034] Figure 10 In Figure A, the co-localization of the fluorescent probe ENT prepared in Example 1 with each organelle probe is shown in ultra-high resolution imaging. In Figure B, the quantitative analysis results of the degree of co-localization between the probe ENT and each probe are shown.

[0035] Figure 11 Image A shows the co-localization ultra-high resolution imaging of fluorescent probes ENT and LD-R; image B shows the quantitative analysis results of the signal-to-background ratio of lipid droplets labeled by probes ENT and LD-R; image C shows the analysis results of the photobleaching of probes ENT and LD-R under continuous laser irradiation.

[0036] Figure 12 In the figure, A is the SIM imaging of lipid droplets in HepG2 cells under normal conditions (control group), B is the SIM imaging of lipid droplets in HepG2 cells treated with ilismo (copper death group), C is the statistical analysis results of lipid droplet fluorescence intensity in the control group and copper death group, D is the statistical analysis results of lipid droplet size in the control group and copper death group, and E is the quantitative analysis results of lipid droplet number in cells of the control group and copper death group.

[0037] Figure 13A shows SIM imaging of probes ENT and DAPR in HepG2 cells treated with normal (control group) and ilismo (copper death group); B shows the quantitative analysis results of the co-localization degree of autophagosome fluorescent probe DAPREped-Autophagy Detection (DAPR) and fluorescent probe ENT; C shows the long-term imaging results of fluorescent probes ENT and DAPR and the analysis results of lipid droplet numbers in ilismo-treated HepG2 cells; and D shows the fluorescence intensity distribution of lipid droplets in cells. Detailed Implementation

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0040] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0041] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0042] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0043] Precise visualization of lipid droplet dynamics during copper death in cancer cells can provide new insights for developing copper death-based anti-cancer strategies. However, existing visualization tools suffer from high signal-to-noise ratios and photobleaching issues, making it difficult to track the long-term dynamic behavior of lipid droplets using super-resolution structured light illumination microscopy (SIM). To address this, this invention proposes a dual-drive (DDF) targeting strategy involving hydrophobic and electrostatic interactions and synthesizes a dual-drive fluorescent probe targeting lipid droplets. Using probe ENT combined with SIM imaging, it was observed that after early treatment of different cancer cell lines with ilisimole (a copper death inducer), the interaction between autophagosomes and lipid droplets increased (indicating elevated lipophage levels), lipid droplet viscosity increased, and lipid droplet homogenization occurred. Subsequently, lipid droplets tended to undergo fission, leading to an increase in their number and a decrease in their size. This invention reveals for the first time the dynamic changes and mechanisms of lipid droplets during early copper death in cancer cells and provides an excellent lipid droplet targeting probe based on a DDF targeting strategy.

[0044] The room temperature in this invention refers to 25±2℃.

[0045] Unless otherwise specified, all materials used in this invention are commercially available products.

[0046] Example 1: A method for preparing a dual-drive fluorescent probe targeting lipid droplets.

[0047] The synthesis route is shown below:

[0048] .

[0049] The specific synthesis method includes the following steps:

[0050] S1. 3-Bromo-9-ethylcarbazole (274.1 mg, 1.0 mmol), 4-formylphenylboronic acid (180 mg, 1.2 mmol), potassium carbonate (447 mg, 3.23 mmol), and tetra(triphenylphosphine)palladium (37 mg, 0.03 mmol) were dissolved in 10 mL of tetrahydrofuran and reacted at 60 °C for 12 hours under nitrogen protection. After the reaction was completed, the solvent was removed by vacuum distillation. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate, v / v ratio 10:1) to give 4-(9-ethyl-9H-carbazole-3-yl)benzaldehyde, the intermediate, in 74% yield.

[0051] S2. Under nitrogen protection, the intermediate (0.5 g, 0.5 mmol) and 3-methylbenzo[d]thiazol-3-onium iodide (0.167 g, 0.6 mmol) were dissolved in 15 mL of ethanol, and 3 drops of glacial acetic acid were added as a catalyst. The mixture was then refluxed at 80 °C for 12 hours. The reaction progress was monitored by thin-layer chromatography (developing solvent: dichloromethane / petroleum ether, volume ratio 1:1). After the reaction was completed, the solvent was removed by rotary evaporation under reduced pressure. The crude product was washed three times with 40 mL of diethyl ether to finally obtain the dual-drive fluorescent probe (ENT) targeting lipid droplets as a red solid with a yield of 70%.

[0052] Figure 1 The 1H NMR spectrum (¹H NMR) of the DMSO-d6 solution of the dual-driving force fluorescent probe for targeting lipid droplets prepared in Example 1 is as follows: ¹H NMR (400 MHz, DMSO-d6) δ 8.60 (d, J = 11.5 Hz, 1H), 8.20 (m, 3H), 7.74 (m, 3H), 7.51 (m, 7H), 7.33 (t, J = 11.9 Hz, 1H), 6.95 (d, J = 24.2 Hz, 1H), 6.21 (d, J = 24.2 Hz, 1H), 4.53 (q, J = 10.0 Hz, 2H), 4.39 (s, 1H), 1.37 (t, J = 10.1 Hz, 3H).

[0053] Figure 2 The carbon NMR spectrum of the dual-driving force fluorescent probe for targeting lipid droplets prepared in Example 1 in deuterated chloroform ( 13 ¹³C NMR, nuclear magnetic resonance characterization data are as follows: ¹³C NMR (101 MHz, DMSO-d6) δ 175.38, 141.44, 140.79, 139.77, 137.83, 137.60, 130.92, 130.66, 130.49, 128.78, 127.62, 127.08, 126.66, 125.32, 125.25, 125.00, 123.18, 120.36, 119.89, 117.78, 116.65, 113.28, 110.47, 108.77, 42.77, 37.94, 13.32.

[0054] Figure 3 The ¹H NMR spectrum of the intermediate prepared in Example 1 is as follows: 1HNMR (400 MHz, DMSO-d6) δ 10.05 (s, 1H), 8.65 (d, J = 1.6 Hz, 1H), 8.28 (d, J= 7.6 Hz, 1H), 8.03 (q, J = 8.8 Hz, 4H), 7.90 (dd, J1=8.4 Hz, J2= 2, 1H),7.73 (d, J = 8.8 Hz, 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.49 (m, 1H), 7.24 (t, J= 7.4 Hz, 1H), 4.48 (q, J = 7.0 Hz, 2H), 1.33 (t, J = 7.0 Hz, 3H).

[0055] Figure 4 The carbon NMR spectrum of the intermediate prepared in Example 1 ( 13 C NMR), nuclear magnetic resonance characterization data are: 13 CNMR (101 MHz, DMSO-d6) δ 192.34, 146.71, 139.85, 139.52, 134.00, 129.96,129.18, 126.79, 125.91, 124.73, 122.72, 122.14, 120.50, 118.97, 118.88,109.47, 109.14, 36.88, 13.49.

[0056] Performance testing experiments: 1. Investigation of the optical properties, cytotoxicity, and uptake mechanism of the fluorescent probe ENT.

[0057] Figure 5 The fluorescence emission spectra of the fluorescent probe ENT prepared in Example 1 in different ratios of glycerol / methanol mixed solvents; from Figure 5 As can be seen, the fluorescence emission peak of the probe ENT at 500 nm is significantly enhanced with the increase of solution viscosity, which confirms its feasibility for viscosity detection.

[0058] The cytotoxicity of the probe ENT against HepG2 cells was assessed using the standard MTT assay. Cells were prepared at a density of 2 × 10⁻⁶ cells / cells. 4HepG2 cells / mL were seeded in 96-well plates and co-incubated with different concentrations (0, 0.1, 0.5, 1.0, 5.0, 10, and 50 μM) of ENT for 24 hours. Then, 10 μL of MTT solution (5 mg / mL) was added to each well, and incubation continued for 4.0 hours. The supernatant was discarded, and 100 μL of DMSO was added to each well to dissolve the generated formazan crystals. After shaking the 96-well plate for approximately 10 minutes, the absorbance of each well was measured at 490 nm using a microplate reader. Cell viability was calculated using the following formula:

[0059] Cell viability (%) = (OD) 样品 -OD 空白 ) / (OD 对照 -OD 空白 )×100%.

[0060] Among them, OD 样品 OD 对照 and OD 空白 The absorbance values ​​represent those measured for cells treated with different concentrations of probe, cells without probe, and wells containing only culture medium, respectively.

[0061] The results are as follows Figure 6 As shown, even with a probe ENT concentration as high as 50 μM and incubation for 12 hours, no significant cytotoxicity was observed in HepG2 cells, indicating that the probe has low cytotoxicity.

[0062] To evaluate the photophysical properties of the fluorescent probe ENT prepared in Example 1 in living cells, its ability to enter cells was first detected by flow cytometry. Figure 7 The permeability assay of different concentrations of the fluorescent probe ENT in HepG2 cells showed that even at a low concentration (0.1 μM), probe ENT could effectively penetrate the cell membrane and enter the cell interior, exhibiting excellent cell membrane permeability. Then, the uptake pattern of probe ENT by cells was studied by simulating different conditions, including different energy levels (4℃ and 37℃), metabolic levels (using the mitochondrial inhibitor, MI), and endocytosis levels (using the endocytosis inhibitor NH4Cl). The results are shown below. Figure 8 Ultra-high resolution microscopic images of the probe ENT in HepG2 cells under different treatment conditions and Figure 9 The results of normalized analysis of the fluorescence intensity of the probe ENT in HepG2 cells under different conditions showed that cells incubated at 37°C exhibited the highest probe uptake, suggesting that the probe ENT mainly enters cells through an energy-dependent endocytosis pathway.

[0063] 2. Co-localization analysis of fluorescent probe ENT and lipid droplets

[0064] To clarify the subcellular localization of the fluorescent probe ENT prepared in Example 1 in fluorescence imaging, this invention uses the probe ENT to co-stain hepatocytes with a series of commercial organelle probes, including the commercial lipid droplet probe LD-R, the lysosomal fluorescent probe Lyso-tracker Deep Red (LTDR), and the autophagosome fluorescent probe DAPR.

[0065] Figure 10 Image A shows the ultra-high resolution imaging of co-localization between the fluorescent probe ENT prepared in Example 1 and various organelle probes; image B shows the quantitative analysis results of the degree of co-localization between probe ENT and various probes. Figure 10 As shown in Figure A, the probe ENT aggregates in hepatocyte lipid droplets, exhibiting green punctate fluorescence. Its fluorescence signal highly overlaps with LD-R, with an average Pearson colocalization coefficient of 0.8. However, its colocalization coefficients with LTDR and DAPR are only 0.158 and 0.1, respectively. Figure 10 (B) This indicates that the fluorescent probe ENT prepared in this invention can achieve specific imaging of lipid droplets.

[0066] 3. Evaluation of the imaging performance of the fluorescent probe ENT on lipid droplets

[0067] This invention explores the applicability of the fluorescent probe ENT prepared in Example 1 for high-quality fluorescence imaging at the subcellular level.

[0068] Figure 11 Image A shows a magnified image of the co-localization of fluorescent probes ENT and LD-R in ultra-high resolution imaging, with the signal and background regions of each channel marked. Image B shows the quantitative analysis results of the signal-to-background ratio of lipid droplets labeled by probes ENT and LD-R. Image C shows the analysis results of the photobleaching of probes ENT and LD-R under continuous laser irradiation.

[0069] like Figure 11 As shown in Figures A and B, compared to commercial LD-Rs, the probe ENT exhibits brighter and clearer images with a superior signal-to-background ratio, demonstrating its excellent imaging quality. This invention evaluates the photobleaching resistance of the ENT using LD-Rs as a reference. Figure 11 As shown in Figure C, after 160 seconds of continuous irradiation, the commercial LD-R exhibited significant fluorescence signal attenuation; while the fluorescent probe ENT prepared in this invention still displayed a clear lipid droplet morphology after irradiation for up to 280 seconds, indicating that the fluorescent probe ENT prepared in this invention is more suitable for long-term imaging studies than the commercial LD-R. These results collectively demonstrate that the fluorescent probe ENT can achieve long-term, high signal-to-background ratio imaging of lipid droplets. These superior performances are attributed to its dual-drive targeting strategy, which significantly enhances the stability of probe localization within lipid droplets.

[0070] 4. Visualization of lipid droplet dynamics during early copper death

[0071] Considering that the liver is a vital organ for the body's absorption and metabolism of copper, this invention selected human liver cancer cells as the model cell type, as this cell type is more susceptible to the effects of copper. Subsequently, the cells were treated with the copper-induced cell death agent ilismo.

[0072] Figure 12 In the figure, A shows SIM imaging of lipid droplets in HepG2 cells under normal conditions (control group); B shows SIM imaging of lipid droplets in HepG2 cells treated with ilismo (copper death group); C shows the statistical analysis results of lipid droplet fluorescence intensity in the control group and copper death group; D shows the statistical analysis results of lipid droplet size in the control group and copper death group; and E shows the quantitative analysis results of lipid droplet number in cells of the control group and copper death group.

[0073] like Figure 12 As shown in the AC diagram, the untreated control group cells exhibited extremely weak green fluorescence after staining with the fluorescent probe ENT, while the copper death group showed a significantly enhanced fluorescence signal, indicating that lipid droplet viscosity increased during copper death. Further statistical analysis showed that the number of intracellular lipid droplets significantly increased during copper death (e.g., ...). Figure 12 As shown in D), its particle size is significantly reduced (e.g., Figure 12 (As shown in E).

[0074] 5. An investigation into the regulation of lipid droplet dynamics through copper death driving lipophage.

[0075] By observing the dynamic changes in lipid droplets using the fluorescent probe ENT, it was inferred that the changes induced by copper death are closely related to lipophagy. To further verify the increased autophagy level in cancer cells during copper death, this invention used the probe ENT and the autophagosome probe DAPR to detect the spatial distribution of lipid droplets and autophagosomes.

[0076] Figure 13 In the middle section, A shows the SIM imaging of probes ENT and DAPR in HepG2 cells treated with normal (control group) and ilismo (copper death group); B shows the quantitative analysis results of the co-localization degree of DAPR and fluorescent probe ENT; C shows the long-term imaging results of fluorescent probes ENT and DAPR and the analysis results of lipid droplet numbers in ilismo-treated HepG2 cells; and D shows the fluorescence intensity distribution of lipid droplets in cells.

[0077] like Figure 13 As shown in Figures A and B, in cells induced by ilismo, lipid droplets and autophagosomes exhibit more significant fusion, indicating an intensified autophagy process during copper death. During the fusion of lipid droplets and autophagosomes, the lipid droplets undergo fission, increasing in number while decreasing in size. Figure 13 (C, D), this result is consistent with the aforementioned findings ( Figure 12The results are consistent with those in D and E. It is worth noting that during this process, the viscosity distribution within a single lipid droplet is non-uniform, and there are also differences in viscosity between different lipid droplets, which may be related to the maturation state of the lipid droplets.

[0078] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of a dual-drive fluorescent probe targeting lipid droplets in the preparation of products for monitoring the dynamic changes of lipid droplets during lipophage in cancer cells, characterized in that, The chemical structure of the dual-drive fluorescent probe targeting lipid droplets is as follows: ； The preparation method of the dual-driving force fluorescent probe targeting lipid droplets includes the following steps: S1. Dissolve 3-bromo-9-ethylcarbazole, 4-formylphenylboronic acid, potassium carbonate, and tetrakis(triphenylphosphine)palladium in an organic solvent, and react under nitrogen protection with heating to obtain an intermediate. ; S2. Under nitrogen protection, the intermediate, 3-methylbenzo[d]thiazole-3-onium salt and catalyst are added to an organic solvent, heated to react, the solvent is removed, and the mixture is washed to obtain the dual-drive fluorescent probe targeting lipid droplets.

2. The application according to claim 1, characterized in that, In step S1, the molar ratio of 3-bromo-9-ethylcarbazole, 4-formylphenylboronic acid, potassium carbonate and tetra(triphenylphosphine)palladium is 1:1.2:3.23:0.

03.

3. The application according to claim 1, characterized in that, In step S1, the heating reaction is carried out at a temperature of 60 °C for 12 hours.

4. The application according to claim 1, characterized in that, In step S2, the molar ratio of the intermediate to 3-methylbenzo[d]thiazole-3-onium salt is 5:

6.

5. The application according to claim 1, characterized in that, In step S2, the heating reaction is carried out at a temperature of 80 °C for 12 hours.

6. The application according to claim 1, characterized in that, Step S1, after the heating reaction, also includes a purification step by silica gel column chromatography.

7. The application according to claim 6, characterized in that, The eluent used in the silica gel column chromatography purification is a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 10:1.

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