A lipid droplet fluorescent probe Lipi-om and a preparation method and application thereof

Abstract

This invention discloses a lipid droplet fluorescent probe, Lipi-om, its preparation method, and its applications, belonging to the field of bioimaging and fluorescent probe technology. The technical solution includes a lipid droplet fluorescent probe, Lipi-om, with a molecular structure of electron donor-electron acceptor-electron donor. This lipid droplet fluorescent probe can perform high-resolution imaging of intracellular lipid droplets under SIM conditions, enabling the tracking of lipid droplet distribution and dynamic behavior, including lipid droplet fusion and division processes. Simultaneously, this probe can be used in conjunction with the mitochondrial probe PK Mito Deep Red to visualize the dynamic and stable contact processes between lipid droplets and mitochondria. This allows for the study of organelle interactions and the distribution and dynamic behavior of lipid droplets in living cells, and can be used for the visual analysis of lipid droplet-mitochondrial interactions, possessing significant application value in cell biology and disease mechanism research.

Description

Technical Field

[0001] This invention belongs to the field of bioimaging and fluorescent probe technology, and particularly relates to a lipid droplet fluorescent probe Lipi-om, its preparation method and application. Background Technology

[0002] Lipid droplets are a class of spherical organelles widely found in eukaryotic cells, consisting of a neutral lipid core (such as triglycerides and cholesterol esters) and an outer phospholipid monolayer membrane. Recent studies have shown that lipid droplets not only serve as energy reservoirs but also participate in various biological processes, including membrane synthesis and transport, protein regulation, inflammatory responses, and cellular stress. Notably, lipid droplets often form close and dynamic interactions with mitochondria, establishing functional contact sites and promoting fatty acid transport. β -Oxidation and energy production. This bidirectional interaction is particularly crucial in cancer cells, where lipid droplet-mitochondrial coupling can drive metabolic reprogramming and enhance cellular adaptability under stress. Therefore, precise visualization of lipid droplet dynamics and their interaction with mitochondria is essential for elucidating their roles in cellular physiology and pathology.

[0003] Although the importance of lipid droplet-mitochondrial interactions is widely recognized, their spatial organization, dynamic behavior, and regulatory mechanisms remain poorly understood. Traditional electron microscopy offers high spatial resolution but is only suitable for stationary samples, unable to observe the dynamics of live cells. While confocal laser scanning microscopy (CLSM) is suitable for live cell imaging, it is limited by the optical diffraction limit (approximately 200 nm), making it difficult to resolve nanoscale contacts and transient separation events between lipid droplets and mitochondria. Against this backdrop, super-resolution fluorescence microscopy has emerged as a powerful tool to overcome these limitations, including stimulated emission depletion microscopy (STED), photoactivated localization microscopy (PALM / STORM), and structured illumination microscopy (SIM). Among these, SIM is particularly suitable for studying lipid droplet-mitochondrial interactions due to its fast imaging speed, low phototoxicity, and good live-cell compatibility. However, the limitations of existing lipid droplet fluorescent probes still restrict super-resolution imaging of lipid droplets and mitochondria in live cells. Patent CN 115536669 A discloses an electron donor-electron acceptor-electron acceptor (DA) type near-infrared luminescent cell lipid droplet fluorescence imaging probe and its application. It has ultra-high photostability, can be used for SIM imaging to dynamically track lipid droplets, and has discovered the movement of small lipid droplets.

[0004] Although the electron donor-electron acceptor-electron acceptor (DA) type near-infrared luminescent cellular lipid droplet fluorescence imaging probe disclosed in patent CN 115536669 A can be used for dynamic tracking of lipid droplets in SIM imaging, it still has some problems: This probe, based on an electron donor-electron acceptor-electron acceptor structure, has a strong ICT effect, but this excessively strong ICT effect makes it prone to fluorescence quenching or luminescence reduction in highly polar cellular environments. Because the polarity of lipid droplets varies significantly across different cell lines—cancer cell lipid droplets are generally less polar than normal cells—the luminescence intensity of the lipid droplet fluorescence probe disclosed in patent CN 115536669 A varies considerably in lipid droplets across different cell lines. Therefore, it is impossible to study the dynamic behavior of lipid droplets in different cell lines. Therefore, the research in this patent is limited to lipid droplet imaging in HeLa cells; in addition, the probe is limited to tracking lipid droplet distribution and dynamic fusion, and does not capture the key dynamic process of lipid droplet division; when further tracking the dynamic process of lipid droplets and mitochondria in HeLa cells with the mitochondrial probe MitoTracker Green in SIM mode, the mitochondrial probe MitoTracker Green showed obvious photobleaching, which led to a significant attenuation of the mitochondrial signal, thus making it impossible to monitor the dynamic contact, separation and interaction process between lipid droplets and mitochondria. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention aims to solve the technical problem that existing lipid droplet fluorescence imaging probes are unable to capture key dynamic processes such as lipid droplet fusion and division, and cannot monitor the dynamic contact, separation, and interaction processes between lipid droplets and mitochondria. This invention proposes a lipid droplet fluorescence probe, Lipi-om, with red light emission, high staining selectivity, and excellent photostability, along with its preparation method and applications. The Lipi-om probe can perform high-resolution imaging of intracellular lipid droplets under SIM conditions, enabling the tracking of lipid droplet distribution and dynamic behavior, including lipid droplet fusion and division processes. Simultaneously, this probe can be used in conjunction with the mitochondrial probe PK Mito Deep Red to visualize the dynamic and stable contact processes between lipid droplets and mitochondria, thereby facilitating the study of organelle interactions.

[0006] To solve the aforementioned technical problem, the technical solution adopted by the present invention is as follows: This invention provides a lipid droplet fluorescent probe, Lipi-om, whose molecular structure is an electron donor-electron acceptor-electron donor structure, and whose chemical structure is shown in formula (I): (I).

[0007] In a second aspect, the present invention provides a method for preparing the above-mentioned lipid droplet fluorescent probe Lipi-om, comprising the following steps: 1) Add 2,6-dibromo-4,8-dipropoxybenzo[1,2-b:4,5-b′]dithiophene-1,1,5,5-tetraoxide, N,N-diethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)aniline, potassium carbonate and tetratriphenylphosphine palladium to the reaction vessel, then add a mixed solvent to remove oxygen. After the reaction, extract and evaporate the filtrate to obtain reaction intermediate 2. 2) The reaction intermediates 2,4-methoxyphenylboronic acid, potassium carbonate and tetra-triphenylphosphine palladium obtained in step 1) were added to the reaction vessel, and then a mixed solvent to remove oxygen was added. After the reaction, the lipid droplet fluorescent probe Lipi-om was obtained by extraction and column chromatography purification.

[0008] Preferably, in step 1), the ratio of the amounts of 2,6-dibromo-4,8-dipropoxybenzo[1,2-b:4,5-b′]dithiophene-1,1,5,5-tetraoxide, N,N-diethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)aniline, potassium carbonate, and tetraphenylphosphine palladium is 0.35-0.45 mmol : 0.35-0.40 mmol : 3-5 mmol : 0.025-0.075 mmol; In step 2), the ratio of 4-methoxyphenylboronic acid, potassium carbonate and tetra-triphenylphosphine palladium added is 1-1.5 mmol: 3-5 mmol: 0.025-0.075 mmol.

[0009] Preferably, the mixed solvent in steps 1) and 2) is composed of toluene, ethanol and water in a volume ratio of 7-9:0.5-1.5:0.5-1.5.

[0010] Preferably, in step 1), the reaction temperature is 85-95 °C and the mixture is stirred for 0.5-1.5 hours. In step 2), the reaction is stirred at 85-95 ℃ for 10-14 hours.

[0011] In a third aspect, the present invention provides the application of the above-mentioned lipid droplet fluorescent probe Lipi-om in intracellular lipid droplet super-resolution imaging.

[0012] Preferably, the lipid droplet fluorescent probe Lipi-om is able to track the distribution and dynamic behavior of intracellular lipid droplets under structured light illumination microscopy.

[0013] Preferably, the lipid droplet fluorescent probe Lipi-om is able to track the fusion and division process of intracellular lipid droplets under structured light illumination microscopy.

[0014] Preferably, the cells include HeLa cells, SKOV3 cells, and CAL27 cells.

[0015] In a fourth aspect, the present invention provides the application of the above-mentioned lipid droplet fluorescent probe Lipi-om in monitoring the interaction between intracellular lipid droplets and mitochondria.

[0016] Preferably, the cells include HeLa cells, SKOV3 cells, and CAL27 cells.

[0017] A fifth aspect of the present invention provides a method for monitoring the interaction between intracellular lipid droplets and mitochondria, comprising the following steps: The cells to be tested were co-incubated with the above-mentioned lipid droplet fluorescent probe Lipi-om and mitochondrial probe PK Mito Deep Red. After washing, they were imaged by structured light illumination microscopy, and the interaction between lipid droplets and mitochondria was monitored in real time by acquiring continuous frame images.

[0018] Preferably, the cells include HeLa cells, SKOV3 cells, and CAL27 cells.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a lipid droplet fluorescent probe, Lipi-om, with an asymmetric electron donor-electron acceptor-electron donor (DAD) structure. The strong donor enhances charge transfer capability to achieve long-wavelength emission, while the weak donor regulates electron distribution, helping to maintain high fluorescence brightness. Furthermore, the benzodithiophene tetraoxide unit has strong electron-withdrawing capability, contributing to improved photostability. The DAD structure effectively reduces excessive intramolecular charge transfer (ICT) effects while maintaining long-wavelength emission characteristics, thereby improving fluorescence brightness and imaging signal stability in the aggregated state. It is suitable for lipid droplet fluorescent labeling and imaging studies in different cell types. The application in multi-cell systems helps to more comprehensively and objectively reveal the dynamic interaction between lipid droplets and mitochondria, effectively avoiding the accidental or exceptional results that may arise from single-cell models, and improving the universality and reliability of research results. The fluorescent probe Lipi-om prepared in this invention exhibits excellent photophysical properties, including a large Stokes shift and obvious environmentally responsive fluorescence characteristics. In fluorescence imaging applications, the large Stokes shift can effectively avoid the overlap between the absorption spectrum and the emission spectrum, thereby improving the imaging signal-to-noise ratio; red light emission is beneficial to reduce cell autofluorescence interference and improve imaging contrast; at the same time, the molecule has suitable hydrophobicity, which is beneficial to selectively enriching in the lipid droplet microenvironment, thereby achieving specific labeling of lipid droplets. The lipid droplet fluorescent probe Lipi-om of this invention can perform high-resolution imaging of intracellular lipid droplets under SIM conditions, enabling the tracking of lipid droplet distribution and dynamic behavior, including the fusion and division processes of lipid droplets. Simultaneously, this probe can be used in conjunction with the mitochondrial probe PK Mito Deep Red to visualize the dynamic and stable contact processes between lipid droplets and mitochondria, thereby facilitating the study of organelle interactions. Therefore, the lipid droplet fluorescent probe Lipi-om of this invention can serve as a high-performance lipid droplet fluorescent probe for studying the distribution and dynamic behavior of lipid droplets in living cells, and can be used for the visualization analysis of lipid droplet-mitochondrial interactions, possessing significant application value in cell biology and disease mechanism research. Compared with existing lipid droplet fluorescent probes, it has advantages such as high imaging signal-to-noise ratio, high lipid droplet selectivity, excellent photostability, and low cytotoxicity, showing broad application prospects in the field of lipid droplet super-resolution imaging. Attached Figure Description

[0020] Figure 1 : The 1H NMR spectrum of the lipid droplet fluorescent probe Lipi-om prepared in Example 1 of this invention; Figure 2 The single-crystal X-ray diffraction analysis crystal structure diagram of the lipid droplet fluorescent probe Lipi-om prepared in Example 1 of this invention; Figure 3 The absorption-emission spectrum of the lipid droplet fluorescent probe Lipi-om prepared in Example 1 of this invention in toluene; wherein, the dashed line on the left is the absorption spectrum and the solid line on the right is the emission spectrum; Figure 4 The graph shows the cell viability of HeLa cells stained with the lipid droplet fluorescent probe Lipi-om prepared in Example 1 of this invention for 24 hours under different concentrations. Figure 5 The images show the co-localization of the lipid droplet fluorescent probes Lipi-om and BODIPY493 / 503 prepared in Example 1 of this invention in HeLa cells; where a) is a fluorescence image of BODIPY493 / 503 in the 488-510 nm imaging channel under 470 nm laser excitation; b) is a fluorescence image of Lipi-om in the 650-680 nm imaging channel under 530 nm laser excitation; c) is a superposition of the first two fluorescence images and the bright-field image using ImageJ software; d) is the Pearson correlation coefficient of the first two fluorescence channels. R =0.80; Scale bar: 2 μm; Figure 6The following is a quantitative diagram of the photostability of the lipid droplet fluorescent probes Lipi-om and Nile Red prepared in Example 1 of this invention in HeLa cells; wherein, a) is the first and 120th frames of 120 consecutive images of the same region after staining HeLa cells with fluorescent probes Lipi-om and Nile Red; scale bar: 5 μm; b) is the data processing of the relative fluorescence intensity of Lipi-om and Nile Red in 120 consecutive images as a function of the number of images, respectively, using Origin software; scale bar: 5 μm; Figure 7 The lipid droplet fluorescent probe Lipi-om prepared in Example 1 of this invention tracks the lipid droplet dynamics process in HeLa cells in SIM mode; wherein, the four images from left to right are the SIM mode images of frame 1, frame 400, frame 800 and frame 1200 respectively; scale bar: 3 μm; Figure 8 The lipid droplet fluorescent probe Lipi-om prepared in Example 1 of this invention tracks the lipid droplet fusion process in HeLa cells in SIM mode; the six images from left to right are SIM mode photos of frames 411, 412, 413, 414, 415 and 416 respectively; scale bar: 1 μm; Figure 9 The lipid droplet fluorescent probe Lipi-om prepared in Example 1 of this invention tracks the lipid droplet division process in SKOV3 cells in SIM mode; the six images from left to right are SIM mode photos of frame 1, frame 572, frame 891, frame 1166, frame 1571 and frame 1740 respectively; scale bar: 1 μm; Figure 10 The lipid droplet fluorescent probe Lipi-om and mitochondrial probe PK Mito DeepRed prepared in Example 1 of this invention were used to track the dynamics of lipid droplets and mitochondria in CAL27 cells in SIM mode; the two images from left to right are the 1st and 100th frames of SIM mode images respectively; scale bar: 5 μm. Detailed Implementation

[0021] The technical solutions in specific embodiments of the present invention will now be described in detail and completely with reference to the accompanying drawings. Obviously, the described embodiments are merely some specific implementations of the overall technical solution of the present invention, and not all implementations. Based on the overall concept of the present invention, all other embodiments obtained by those skilled in the art fall within the protection scope of the present invention.

[0022] This invention provides a lipid droplet fluorescent probe, Lipi-om, whose molecular structure is an electron donor-electron acceptor-electron donor structure, and whose chemical structure is shown in formula (I): (I).

[0023] It should be noted that although the importance of lipid droplet-mitochondrial interactions is widely recognized, their spatial organization, dynamic behavior, and regulatory mechanisms remain incompletely understood. Structured illumination microscopy (SIM) is particularly suitable for studying dynamic organelle interactions due to its fast imaging speed, low phototoxicity, and good live-cell compatibility. However, the limitations of existing lipid droplet fluorescent probes still restrict super-resolution imaging of lipid droplets-mitochondria in live cells. Existing probes such as BODIPY 493 / 503 and Nile Red can label neutral lipids, but they generally have poor photostability and limited selectivity, making them unsuitable for long-term imaging under high-intensity illumination. In addition, most existing probes are designed for traditional confocal microscopy and lack the photophysical properties required for SIM imaging, such as high brightness, low background, stable emission, and excellent photostability. A near-infrared luminescent cell lipid droplet fluorescent imaging probe of the electron donor-electron acceptor-electron acceptor (DA) type is disclosed in the prior art, but it exhibits a strong ICT effect, resulting in weak fluorescence in some highly polar cell environments. Furthermore, its application is limited to tracking the distribution and dynamic behavior of HeLa lipid droplets, making it difficult to capture the key dynamic processes of lipid droplet division.

[0024] To address the above problems, this invention provides a lipid droplet fluorescent probe, Lipi-om, chemically named 2-(4-(diethylamino)phenyl)-6-(4-methoxyphenyl)-4,8-dipropoxybenzo[1,2-b:4,5-b′]dithiophene-1,1,5,5-tetraoxide, abbreviated as Lipi-om. It uses 1,1,5,5-tetraoxide benzo[1,2-b:4,5-b′]dithiophene as the central electron acceptor, with one end connected to the strong electron donor diethylaminophenyl and the other end connected to the weak electron donor methoxyphenyl, thereby constructing an asymmetric electron donor-electron acceptor-electron donor (DAD) structure. This structural design enhances charge transfer capability through a strong donor to achieve long-wavelength emission, while a weak donor regulates electron distribution, helping to maintain high fluorescence brightness. Meanwhile, the benzodithiophene tetraoxide unit has a strong electron-withdrawing ability, which helps to improve the photostability of the molecule. Based on this, the probe can effectively suppress excessive intramolecular charge transfer (ICT) effects while maintaining long-wavelength emission characteristics, thereby improving fluorescence brightness and imaging signal stability in the aggregated state. This makes it more suitable for lipid droplet fluorescence labeling and imaging studies in different types of cells, as it has better environmental adaptability and imaging brightness.

[0025] Another aspect of the present invention provides a method for preparing the above-mentioned lipid droplet fluorescent probe Lipi-om, comprising the following steps: 1) Add 2,6-dibromo-4,8-dipropoxybenzo[1,2-b:4,5-b′]dithiophene-1,1,5,5-tetraoxide, N,N-diethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)aniline, potassium carbonate and tetratriphenylphosphine palladium to the reaction vessel, then add a mixed solvent to remove oxygen. After the reaction, extract and evaporate the filtrate to obtain reaction intermediate 2. 2) The reaction intermediates 2,4-methoxyphenylboronic acid, potassium carbonate and tetra-triphenylphosphine palladium obtained in step 1) were added to the reaction vessel, and then a mixed solvent to remove oxygen was added. After the reaction, the lipid droplet fluorescent probe Lipi-om was obtained by extraction and column chromatography purification.

[0026] By employing the above-described technical solution, a two-step sequential coupling method, rather than a one-step simultaneous coupling, can effectively improve the regioselectivity and reaction controllability of the target product. Due to the different electronic properties and reactivity of the two boric acid / boronic acid ester substrates, a one-step direct reaction can easily generate disubstituted byproducts or cross-coupling side reactions, leading to reduced yields and difficulties in subsequent separation. This invention, by first constructing the key intermediate and then performing a second-step functionalization modification, can effectively reduce the probability of side reactions and improve the purity and overall yield of the target product.

[0027] In a preferred embodiment, in step 1), the ratio of the amounts of 2,6-dibromo-4,8-dipropoxybenzo[1,2-b:4,5-b′]dithiophene-1,1,5,5-tetraoxide, N,N-diethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)aniline, potassium carbonate, and tetratriphenylphosphine palladium added is 0.35-0.45 mmol : 0.35-0.40 mmol : 3-5 mmol : 0.025-0.075 mmol; In step 2), the ratio of 4-methoxyphenylboronic acid, potassium carbonate and tetra-triphenylphosphine palladium added is 1-1.5 mmol: 3-5 mmol: 0.025-0.075 mmol.

[0028] In a preferred embodiment, the mixed solvent in steps 1) and 2) is composed of toluene, ethanol and water in a volume ratio of 7-9:0.5-1.5:0.5-1.5.

[0029] In a preferred embodiment, in step 1), the reaction is carried out at a temperature of 85-95 °C with stirring for 0.5-1.5 hours; In step 2), the reaction is stirred at 85-95 ℃ for 10-14 hours.

[0030] Furthermore, the preparation reaction formula for the fluorescent probe Lipi-om is as follows: .

[0031] A third aspect of the present invention provides the application of the above-mentioned lipid droplet fluorescent probe Lipi-om in intracellular lipid droplet super-resolution imaging.

[0032] The fluorescent probe Lipi-om prepared in this invention exhibits excellent photophysical properties, including a large Stokes shift and significant environmentally responsive fluorescence characteristics. In fluorescence imaging applications, the large Stokes shift effectively avoids overlap between absorption and emission spectra, thereby improving the imaging signal-to-noise ratio; red emission helps reduce interference from cell autofluorescence, improving imaging contrast. Simultaneously, the molecule possesses suitable hydrophobicity, which facilitates selective enrichment in the lipid droplet microenvironment, thus achieving specific labeling of lipid droplets. Under the same staining and imaging conditions, its staining specificity is significantly superior to that of the lipid droplet fluorescent probe Nile Red, and cell viability experiments show that Lipi-om has low cytotoxicity. Compared with existing lipid droplet fluorescent probes, the fluorescent probe Lipi-om of this invention has advantages such as high imaging signal-to-noise ratio, high lipid droplet selectivity, excellent photostability, and low cytotoxicity, and has broad application prospects in the field of lipid droplet super-resolution imaging.

[0033] In a preferred embodiment, the lipid droplet fluorescent probe Lipi-om is able to track the distribution and dynamic behavior of intracellular lipid droplets under structured light illumination microscopy.

[0034] In a preferred embodiment, the lipid droplet fluorescent probe Lipi-om is able to track the fusion and division of intracellular lipid droplets under structured light illumination microscopy.

[0035] The lipid droplet fluorescent probe Lipi-om described in this invention can perform high-resolution imaging of intracellular lipid droplets under SIM conditions, enabling the tracking of lipid droplet distribution and dynamic behavior, including the fusion and division processes of lipid droplets.

[0036] In a preferred embodiment, the cells include HeLa cells, SKOV3 cells, and CAL27 cells.

[0037] In a fourth aspect, the present invention provides the application of the above-mentioned lipid droplet fluorescent probe Lipi-om in monitoring the interaction between intracellular lipid droplets and mitochondria.

[0038] The lipid droplet fluorescent probe Lipi-om described in this invention can be used in conjunction with the mitochondrial probe PK Mito Deep Red. More importantly, Lipi-om still exhibits excellent photostability under strong excitation light conditions, enabling long-term continuous imaging (more than a thousand frames), which is suitable for real-time monitoring of dynamic processes. This allows for visualization of the dynamic and stable contact processes between lipid droplets and mitochondria, thus enabling the study of organelle interactions.

[0039] In a preferred embodiment, the cells include HeLa cells, SKOV3 cells, and CAL27 cells.

[0040] In summary, the fluorescent probe Lipi-om described in this invention can serve as a high-performance lipid droplet fluorescent probe for studying the distribution and dynamic behavior of lipid droplets in living cells, and can also be used for the visualization analysis of lipid droplet-mitochondrial interactions, thus having significant application value in cell biology and disease mechanism research.

[0041] A fifth aspect of the present invention provides a method for monitoring the interaction between intracellular lipid droplets and mitochondria, comprising the following steps: The cells to be tested were co-incubated with the above-mentioned lipid droplet fluorescent probe Lipi-om and mitochondrial probe PK Mito Deep Red. After washing, they were imaged by structured light illumination microscopy, and the interaction between lipid droplets and mitochondria was monitored in real time by acquiring continuous frame images.

[0042] In a preferred embodiment, the cells include HeLa cells, SKOV3 cells, and CAL27 cells.

[0043] The lipid droplet fluorescent probe Lipi-om of this invention is suitable for lipid droplet fluorescent labeling and imaging studies in different cell types. The application of multi-cell systems helps to reveal the dynamic interaction between lipid droplets and mitochondria more comprehensively and objectively, effectively avoiding the accidental or exceptional results that may be brought about by single-cell models, and improving the universality and reliability of research results.

[0044] To provide a clearer and more detailed description of the lipid droplet fluorescent probe Lipi-om, its preparation method, and its applications provided in the embodiments of the present invention, the following description will be based on specific embodiments.

[0045] In the following embodiments of the present invention, the mitochondrial probe PK Mito Deep Red was purchased from Hangzhou Baimai Technology Co., Ltd., and the model is PKMDR-2; The lipid droplet fluorescent probe BODIPY493 / 503 used was purchased from Shanghai Beyotime Biotechnology Co., Ltd. The lipid droplet fluorescent probe Nile Red used was purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0046] In the following embodiments of the present invention, the compound 2,6-dibromo-4,8-dipropoxybenzo[1,2-b:4,5-b′]dithiophene-1,1,5,5-tetraoxide has the CAS number 920304-57-0.

[0047] The preparation of the compound N,N-diethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)aniline was carried out in accordance with the literature “Liu, G.; Peng, G.; Dai, J.; Zhou, R.; Wang, C.; Yan, X.; Jia, X.; Liu, X.; Gao, Y.; Wang, L.; et al. STED Nanoscopy Imaging of CellularLipid Droplets Employing a Superior Organic Fluorescent Probe. AnalyticalChemistry 2021, 93 (44), 14784-14791. DOI: 10.1021 / acs.analchem.1c03474.” The specific preparation steps are described in the supporting information of that literature.

[0048] Example 1: Preparation of the lipid droplet fluorescent probe Lipi-om: Synthesis of 2-(4-(diethylamino)phenyl)-6-(4-methoxyphenyl)-4,8-dipropoxybenzo[1,2-b:4,5-b′]dithiophene-1,1,5,5-tetraoxide: 1) To a reaction flask containing 0.200 g (0.397 mmol) of 2,6-dibromo-4,8-dipropoxybenzo[1,2-b:4,5-b′]dithiophene-1,1,5,5-tetraoxide, 0.104 g (0.379 mmol) of N,N-diethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)aniline, 553 mg (4.00 mmol) of potassium carbonate and 0.058 g (0.050 mmol) of tetratriphenylphosphine palladium, 50 mL of a mixed solvent of deoxygenated toluene, ethanol and water (V toluene:V ethanol:V water = 8:1:1) was added. The mixture was stirred at 90 °C for 1 hour. After extraction and rotary evaporation, unpurified reaction intermediate 2 was obtained. 2) The reaction intermediate 2 obtained in step 1), 0.173 g (1.14 mmol) of 4-methoxyphenylboronic acid, 553 mg (4.00 mmol) of potassium carbonate and 0.058 g (0.050 mmol) of tetrakis(triphenylphosphine) palladium were added to a reaction flask. Then, 50 mL of a mixed solvent of toluene, ethanol and water (V toluene:V ethanol:V water = 8:1:1) was added to the reaction flask. The mixture was stirred at 90 °C for 12 hours. After extraction and column chromatography purification, 100 mg (0.160 mmol, 42%) of red powder Lipi-om was obtained.

[0049] The reaction formula for preparing the fluorescent probe Lipi-om is as follows: .

[0050] 1 H NMR (400 MHz, CDCl3): δ 7.83 – 7.64 (m, 4H), 7.29 (s, 1H), 7.15 (s,1H), 7.05 – 6.95 (m, 2H), 6.76 (s, 2H), 4.53 – 4.34 (m, 4H), 3.87 (s, 3H), 3.44 (d, J = 8.8 Hz, 4H), 2.06 – 1.85 (m, 4H), 1.30 – 1.18 (m, 6H), 1.18 –1.06 (m, 6H). Figure 1 The above is the 1H NMR spectrum of the lipid droplet fluorescent probe Lipi-om synthesized in Example 1. Figure 2 The image shows the crystal structure of the lipid droplet fluorescent probe Lipi-om synthesized in Example 1, indicating that the target product Lipi-om was successfully prepared.

[0051] Example 2: Determination of the absorption-emission spectrum of the fluorescent probe Lipi-om prepared in Example 1: The fluorescent probe Lipi-om synthesized in Example 1 was prepared into a 10 μM solution (total volume 10 mL) using toluene as solvent. At room temperature, its absorption spectrum was scanned in the wavelength range of 300-800 nm using a UV-Vis spectrophotometer; simultaneously, its fluorescence emission spectrum was acquired using a fiber optic fluorescence spectrometer at an excitation wavelength of 405 nm. The obtained spectral data were processed using Origin software, and the results are as follows. Figure 3 As shown in the figure. The dashed line on the left represents the absorption spectrum, and the solid line on the right represents the fluorescence emission spectrum.

[0052] The results showed that Lipi-om exhibited clear absorption and emission characteristics in toluene solution and had a significant Stokes shift, indicating that the fluorescent probe has good photophysical separation properties, which is beneficial for reducing excitation light interference and improving the signal-to-noise ratio during imaging.

[0053] Example 3: Culture of HeLa, SKOV3, and CAL27 cells: In this embodiment, all percentages are volume fractions.

[0054] HeLa, SKOV3, and CAL27 cell lines were cultured in a cell culture incubator at 37 ℃ and 5% CO2. The culture medium used was high-glucose DMEM containing a mixture of 10% fetal bovine serum and 1% penicillin-streptomycin (antibiotic). The fetal bovine serum, antibiotic, and high-glucose DMEM were all purchased directly from commercial kits.

[0055] When the cells reach the logarithmic growth phase, they are passaged. First, discard the original 5 mL of culture medium in the cell culture flask, add 2 mL of high-glucose DMEM culture medium (containing 1% penicillin and antibiotics) without fetal bovine serum to wash the cells, and discard the washing solution. Then, add 0.5 mL of trypsin digestion solution and digest at 37 °C for approximately 2 min. After most cells have detached from the cell wall, add 2 mL of high-glucose DMEM culture medium containing 10% fetal bovine serum and 1% penicillin and antibiotics to stop the digestion, and gently pipette to ensure the cells are fully dispersed. Subsequently, take an appropriate amount of the cell suspension and seed it into new cell culture flasks and culture dishes, and continue culturing in a 37 °C, 5% CO2 incubator. Once the cells in the culture dishes have grown to a suitable density, they can be used for subsequent confocal or SIM imaging experiments.

[0056] Example 4: Test of cytotoxicity of the fluorescent probe Lipi-om prepared in Example 1: The cytotoxicity of the fluorescent probe Lipi-om was evaluated using the 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT) method.

[0057] HeLa cells were used at a rate of 1×10 4 Cells were seeded at a density of [number] cells / well in 96-well plates and cultured in a cell culture incubator containing 5% CO2 for 24 h to allow for full cell adhesion and growth. Subsequently, the culture medium in the middle 60 wells was removed, and culture medium containing different concentrations of Lipi-om (0.5 μM, 1.0 μM, 2.0 μM, 5.0 μM, and 10.0 μM) and 1% (v / v) DMSO was added, with 10 parallel wells for each concentration. A control group without the probe was also included. After culturing for another 24 h, 10 μM MTT solution was added to each well, and the plates were incubated for 4 h to allow viable cells to metabolize and formazan crystals. The supernatant was then discarded, and 100 μL of DMSO was added to each well to dissolve the formazan crystals. After standing at room temperature for 30 min, the absorbance of each well was measured at 490 nm using a microplate reader. The cell viability of each concentration group was calculated with the average absorbance of the control group (without the probe) as 100%.

[0058] The results are as follows Figure 4As shown, Lipi-om has little effect on HeLa cell viability in the range of 0.5-10.0 μM. Even after incubation at a concentration of 10.0 μM for 24 h, the cells still maintain a high survival rate, indicating that the fluorescent probe has good biocompatibility and low cytotoxicity.

[0059] Example 5: Co-staining experiment of the fluorescent probe Lipi-om and the lipid droplet fluorescent probe BODIPY493 / 503 prepared in Example 1 in HeLa cells: The HeLa cells cultured in Example 3 were seeded into 20 mm diameter glass-bottomed culture dishes and cultured in a cell culture incubator containing 5% CO2 for 2 days to achieve good adhesion. Subsequently, the culture dishes were removed from the incubator, the original DMEM culture medium was discarded, and 1 mL of DMEM culture medium containing Lipi-om (2 μM), lipid droplet fluorescent probe BODIPY 493 / 503 (2 μM), and 1% (v / v) DMSO was added. The cells were then incubated in the cell culture incubator for another 2 h. After incubation, the cells were washed three times with HBSS solution to remove free probes, and fluorescence imaging was performed in HBSS solution.

[0060] The results are as follows Figure 5 As shown, the fluorescent probe Lipi-om prepared in Example 1 and the lipid droplet fluorescent probe BODIPY 493 / 503 showed good fluorescence signal overlap in HeLa cells, indicating significant co-localization. This suggests that Lipi-om can highly selectively label cellular lipid droplets and has excellent lipid droplet targeting ability.

[0061] Example 6: Photostability test of the fluorescent probe Lipi-om prepared in Example 1 The HeLa cells cultured in Example 3 were seeded into two 20 mm diameter glass-bottomed culture dishes and cultured in a cell culture incubator containing 5% CO2 for 2 days to achieve good adhesion. Subsequently, the culture dishes were removed from the incubator, the original DMEM culture medium was discarded, and DMEM culture medium containing 2 μM Lipi-om and 1% DMSO was added, or DMEM culture medium containing 2 μM Nile Red lipid droplet fluorescent probe and 1% DMSO was added. The dishes were then incubated at 37 °C and 5% CO2 for another 2 h. The culture dishes were then removed and washed three times with HBSS solution to remove unbound probes, followed by fluorescence imaging experiments. Under the same imaging conditions, 120 consecutive frames of confocal imaging were performed on both groups of samples.

[0062] like Figure 6As shown, the results indicate that Nile Red undergoes significant photobleaching during continuous imaging, with its fluorescence intensity decreasing to 63% of its initial value. In contrast, the fluorescent probe Lipi-om prepared in Example 1 retains 96% of its initial fluorescence intensity under the same conditions. This result demonstrates that Lipi-om exhibits significantly superior photostability compared to Nile Red, meeting the requirements for long-term dynamic imaging.

[0063] Example 7: The fluorescent probe Lipi-om prepared in Example 1 was used to track the lipid droplet dynamics in HeLa cells in SIM mode. The HeLa cells cultured in Example 3 were seeded in 20 mm diameter glass-bottomed culture dishes and cultured in a cell culture incubator containing 5% CO2 for 2 days to achieve good adhesion. Subsequently, the culture dishes were removed from the incubator, and DMEM culture medium containing 2 μM Lipi-om and 1% DMSO was added. The cells were then incubated at 37 °C and 5% CO2 for another 2 h. After incubation, the cells were washed three times with HBSS solution to remove unbound probes, followed by SIM imaging.

[0064] like Figure 7 As shown, the results indicate that after selecting the lipid droplet-rich region, continuous dynamic imaging was performed in SIM mode, acquiring a total of 1200 images. Under prolonged high-intensity excitation conditions, the images still maintained clear structural information and identifiable fluorescence signals, without significant signal collapse or severe bleaching. Simultaneously, the rapid movement and dynamic changes of small lipid droplets could be clearly observed during this continuous imaging process.

[0065] The above results demonstrate that Lipi-om possesses excellent photostability, enabling it to support long-term SIM super-resolution dynamic imaging and to be used for real-time tracking of the dynamic behavior of lipid droplets in living cells, showcasing its significant application potential as a super-resolution imaging fluorescent probe for lipid droplets in organelle dynamics research.

[0066] Example 8: The fluorescent probe Lipi-om prepared in Example 1 was used to track the lipid droplet fusion process in HeLa cells in SIM mode. The HeLa cells cultured in Example 3 were seeded into 20 mm diameter glass-bottomed culture dishes and cultured in a cell culture incubator containing 5% CO2 for 2 days to achieve good adhesion. Subsequently, the culture dishes were removed from the incubator, the original DMEM culture medium was discarded, and DMEM culture medium containing 2 μM Lipi-om and 1% DMSO was added. The cells were then incubated at 37 °C and 5% CO2 for another 2 hours. After incubation, the cells were washed three times with HBSS solution to remove unbound probes, followed by SIM imaging.

[0067] like Figure 8As shown, the results indicate that after selecting a lipid droplet-rich region, 1200 frames of images were continuously acquired in SIM mode for dynamic observation. In this continuous imaging sequence, the dynamic process of two small lipid droplets gradually approaching and eventually fusing was clearly observed from frames 411 to 416. These results demonstrate that Lipi-om can achieve real-time tracking of lipid droplet fusion behavior under SIM super-resolution imaging conditions, showing its application potential in revealing lipid droplet dynamic processes and organelle behavior mechanisms.

[0068] Example 9: The fluorescent probe Lipi-om prepared in Example 1 was used to track the lipid droplet division process in SKOV3 cells in SIM mode. SKOV3 cells cultured in Example 3 were seeded in 20 mm diameter glass-bottomed culture dishes and cultured in a cell culture incubator containing 5% CO2 for 2 days to achieve good adhesion. Subsequently, the culture dishes were removed from the incubator, the original DMEM culture medium was discarded, and DMEM culture medium containing 2 μM Lipi-om and 1% DMSO was added. The cells were then incubated at 37 °C and 5% CO2 for another 2 h. After incubation, the cells were washed three times with HBSS solution to remove unbound probes, followed by SIM imaging.

[0069] like Figure 9 As shown, the results indicate that after selecting the lipid droplet-rich region, continuous dynamic imaging was performed in SIM mode, acquiring a total of 1740 images. During this continuous imaging process, the dynamic process of a large lipid droplet gradually undergoing morphological changes and eventually splitting into multiple smaller lipid droplets was clearly observed. These results demonstrate that Lipi-om can stably support long-term dynamic observation under SIM super-resolution imaging conditions and can achieve real-time tracking of the lipid droplet splitting process, further proving its application potential in analyzing the dynamic behavior of lipid droplets and related cell biological mechanisms.

[0070] Example 10: The fluorescent probe Lipi-om and the mitochondrial probe PK Mito Deep Red prepared in Example 1 were used to track intracellular lipid droplet and mitochondrial dynamics in CAL27 cells in SIM mode. CAL27 cells cultured in Example 3 were seeded in 20 mm diameter glass-bottomed culture dishes and cultured in a cell culture incubator containing 5% CO2 for 2 days to achieve good adhesion. Subsequently, the culture dishes were removed from the incubator, the original DMEM culture medium was discarded, and DMEM culture medium containing 2 μM Lipi-om, 1 μM mitochondrial probe PK Mito Deep Red, and 1% (v / v) DMSO was added. The cells were then incubated at 37 °C and 5% CO2 for 2 h. After incubation, the cells were washed three times with HBSS solution to remove unbound dye, followed by SIM imaging.

[0071] like Figure 10 As shown, after selecting the region where lipid droplets and mitochondria coexist, 100 frames of images were continuously acquired in SIM mode for dynamic observation. Throughout the imaging process, both the lipid droplet fluorescent probe Lipi-om and the mitochondrial probe PK Mito DeepRed maintained stable and clear fluorescence signals without significant photobleaching, indirectly indicating that Lipi-om possesses excellent photostability. Simultaneously, the dynamic changes in the relative spatial positions between lipid droplets and mitochondria could be observed in this continuous imaging sequence. These results demonstrate that Lipi-om not only possesses excellent photostability but can also be used for two-color imaging analysis under SIM super-resolution conditions, enabling real-time monitoring of lipid droplet-mitochondrial interactions, and has promising application potential.

Claims

1. A lipid droplet fluorescent probe, Lipi-om, characterized in that, Its molecular structure is an electron donor-electron acceptor-electron donor structure, and its chemical structure is shown in formula (I): （I）。 2. The method for preparing the lipid droplet fluorescent probe Lipi-om according to claim 1, characterized in that, Includes the following steps: 1) Add 2,6-dibromo-4,8-dipropoxybenzo[1,2-b:4,5-b′]dithiophene-1,1,5,5-tetraoxide, N,N-diethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)aniline, potassium carbonate and tetratriphenylphosphine palladium to the reaction vessel, then add a mixed solvent to remove oxygen. After the reaction, extract and evaporate the filtrate to obtain reaction intermediate 2. 2) The reaction intermediates 2,4-methoxyphenylboronic acid, potassium carbonate and tetra-triphenylphosphine palladium obtained in step 1) were added to the reaction vessel, and then a mixed solvent to remove oxygen was added. After the reaction, the lipid droplet fluorescent probe Lipi-om was obtained by extraction and column chromatography purification.

3. The preparation method according to claim 2, characterized in that, In step 1), the ratio of the amounts of 2,6-dibromo-4,8-dipropoxybenzo[1,2-b:4,5-b′]dithiophene-1,1,5,5-tetraoxide, N,N-diethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)aniline, potassium carbonate, and tetraphenylphosphine palladium added is 0.35-0.45 mmol : 0.35-0.40 mmol : 3-5 mmol : 0.025-0.075 mmol; In step 2), the ratio of 4-methoxyphenylboronic acid, potassium carbonate and tetra-triphenylphosphine palladium added is 1-1.5 mmol: 3-5 mmol: 0.025-0.075 mmol.

4. The preparation method according to claim 2, characterized in that, The mixed solvents in steps 1) and 2) are composed of toluene, ethanol and water in a volume ratio of 7-9:0.5-1.5:0.5-1.

5.

5. The application of the lipid droplet fluorescent probe Lipi-om as described in claim 1 in intracellular lipid droplet super-resolution imaging.

6. The application according to claim 5, characterized in that, The lipid droplet fluorescent probe Lipi-om can track the distribution and dynamic behavior of intracellular lipid droplets under structured light illumination microscopy.

7. The application according to claim 5, characterized in that, The lipid droplet fluorescent probe Lipi-om can track the fusion and division of intracellular lipid droplets under structured light illumination microscopy.

8. The application according to any one of claims 5-7, characterized in that, The cells include HeLa cells, SKOV3 cells, and CAL27 cells.

9. The application of the lipid droplet fluorescent probe Lipi-om according to claim 1 in monitoring intracellular lipid droplet-mitochondrial interactions, characterized in that, The cells include HeLa cells, SKOV3 cells, and CAL27 cells.

10. A method for monitoring the interaction between intracellular lipid droplets and mitochondria, characterized in that, Includes the following steps: The cells to be tested were co-incubated with the lipid droplet fluorescent probe Lipi-om and the mitochondrial probe PK Mito DeepRed as described in claim 1. After washing, they were imaged by structured light illumination microscopy. The interaction between lipid droplets and mitochondria was monitored in real time by acquiring continuous frame images. The cells included HeLa cells, SKOV3 cells and CAL27 cells.

Images