Targeted lipid droplet fluorescent probe with long-term retention capacity as well as preparation method and application of targeted lipid droplet fluorescent probe

Abstract

The invention relates to a fluorescent probe for targeting lipid droplets with long-term retention capacity, a preparation method and application thereof, and belongs to the technical field of fluorescent probes. The structural formula of the fluorescent probe is shown in the specification. The ClogP value of the fluorescent probe is greater than 8, high-specificity targeting to lipid droplets can be realized, and the fluorescent probe can be stably retained in living cells for 72 hours. The fluorescent probe not only can trace abnormal accumulation of lipid droplets in a Parkinson's disease model on a cellular level, but also can realize visual detection of lipid droplet pathological accumulation in a nigra area of a living animal model through intracerebral in-situ injection. The fluorescent probe is obtained by carrying out a Knoevenagel reaction on 4, 4 '-diformyl triphenylamine and benzothiazole-2-acetonitrile, and has the characteristics of simplicity and convenience in synthesis, good biocompatibility, high signal-to-noise ratio and long cell retention time.

Description

Technical Field

[0001] This invention relates to a fluorescent probe for targeting lipid droplets with long-term retention capability, its preparation method, and its application, belonging to the field of fluorescent probe technology. Background Technology

[0002] Parkinson's disease (PD) is a neurodegenerative disease with a prevalence of 2% to 3% in people over 65 years of age. With the accelerating aging of the global population, more and more people are suffering from PD. Initially, PD patients exhibit pathological changes in the olfactory bulb and brainstem regions, including the loss of dopaminergic neurons, α-synuclein aggregation, and neuroinflammation. These lesions then gradually spread to the substantia nigra and other brain regions. The loss of dopaminergic neurons in these areas (especially the substantia nigra) leads to clinical symptoms such as resting tremor, bradykinesia, and rigidity. Research indicates that lipids play a crucial role in the pathology of PD: Xicoy et al. found that PD-related gene variants can regulate the levels of specific lipid classes in the blood of patients. Furthermore, studies have confirmed that lysophosphatidylcholine can bind to α-synuclein, a key pathogenic protein in PD, maintaining its compact conformation and inhibiting its aggregation. Certain lipids, such as phosphatidylcholine (40:7) and phosphatidylcholine (40:6p), have been shown to have the potential to serve as diagnostic biomarkers for Parkinson's disease. However, the detection and analysis of these lipids primarily rely on metabolomics analysis of blood samples, a technique that is complex to perform and lacks spatial information about lesion sites (such as the substantia nigra). Therefore, developing simpler and more intuitive detection and analysis methods is of great significance for the clinical diagnosis of Parkinson's disease.

[0003] Fluorescent probes are sophisticated molecular tools that can convert invisible cellular events into detectable optical signals. Fluorescent probe-based bioimaging technologies have shown great potential in clinical disease diagnosis due to their high sensitivity, high specificity, real-time visualization capabilities, and relative safety. Monitoring changes in organelle structure and function often reflects disease-related pathological states more accurately than detecting single biomarkers. For example, Huang et al. constructed a fluorescent probe called TPA-BT-SCP targeting mitochondria, which can be used for the early diagnosis and disease progression monitoring of Parkinson's disease (PD). Zhou et al. reported a method for tracking Fe³⁺ in lysosomes. + A fluorescent probe that can effectively detect Fe³⁺ induced by lysosomal ferritin autophagy in Parkinson's disease cells and animal models. + Overload. Therefore, utilizing bioimaging technology to detect pathological changes in organelles provides a new strategy for the development of diagnostic techniques for Parkinson's disease.

[0004] Lipid droplets are highly dynamic organelles within cells, crucial for cellular lipid metabolism. They participate in the synthesis, storage, and lipolysis of intracellular lipids, maintaining cellular lipid homeostasis. Disruptions in lipid droplet homeostasis can lead to neuronal dysfunction, metabolic abnormalities, and nervous system diseases. Brekk et al. found abnormal lipid droplet aggregation in dopaminergic neurons and surrounding microglia in Parkinson's disease (PD) patients and mouse models. Subsequent studies confirmed that lipid droplets and α-synuclein co-localize in neurons. This abnormal aggregation enhances the resistance of α-synuclein to proteolytic digestion, indicating that the combination of the two exacerbates the pathological misfolding and aggregation of α-synuclein. Several fluorescent probes targeting lipid droplets are currently available for detecting lipid droplet accumulation in neurons in PD models. For example, Li et al. constructed the polymerization-induced emission probe 2-DPAN to observe the dynamic changes in lipid droplet levels in neurons treated with 6-OHDA. Ma et al. reported that the F-675 chemical toolkit can measure lipid droplet accumulation in neuronal, *Caenorhabditis elegans*, and Drosophila PD models with extremely low biofluorescence background. However, due to limitations in retention time, these probes have significant limitations in in vivo animal imaging applications, especially in imaging lesions such as the substantia nigra. Therefore, designing a fluorescent probe that can remain in live animals for extended periods and target lipid droplets remains a significant challenge. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a fluorescent probe for targeting lipid droplets with long-term retention capability, its preparation method, and its application. The fluorescent probe has a ClogP value greater than 8, enabling highly specific targeting of lipid droplets and stable retention in living cells for up to 72 hours. This fluorescent probe can not only trace the abnormal accumulation of lipid droplets in a Parkinson's disease model at the cellular level, but also, through in situ intracerebral injection, achieve visualized detection of pathological lipid droplet accumulation in the substantia nigra region of a live animal model, providing an effective tool for the diagnosis and research of Parkinson's disease.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows.

[0007] A fluorescent probe for targeting lipid droplets with long-term retention capability, the structural formula of the fluorescent probe is as follows:

[0008] .

[0009] Preferably, the ClogP value of the fluorescent probe is greater than 8.

[0010] Preferably, the fluorescent probe exhibits specific targeting to lipid droplets and does not stain other membrane structures.

[0011] Preferably, the fluorescent probe remains stably in the cell for more than 72 hours.

[0012] Preferably, the excitation wavelength of the fluorescent probe is 488 nm, and the acquisition wavelength is 570~670 nm.

[0013] A method for preparing a fluorescent probe for targeting lipid droplets with long-term retention capability, as described in this invention, includes the following steps: 4,4'-Dicarboxytriphenylamine and benzothiazol-2-acetonitrile are reacted via a Knoevenagel reaction to yield (2E,2'E)-3,3'-{[phenyl(aza)diyl]bis(4,1-phenylene)}bis[2-(1,3-benzothiazol-2-yl)prop-2-enonitrile], a fluorescent probe targeting lipid droplets with long-term retention capability.

[0014] Preferably, 4,4'-dicarboxytriphenylamine and benzothiazole-2-acetonitrile are dissolved in ethanol, then piperidine is added as a catalyst, and the mixture is heated under reflux for more than 24 hours. After the reaction is completed, column chromatography is used to separate the solid and collect it to obtain a fluorescent probe for targeting lipid droplets with retention capacity.

[0015] The application of a fluorescent probe for targeting lipid droplets with long-term retention capability as described in this invention in the preparation of lipid droplet fluorescence imaging products or lipid droplet-specific labeling products for non-therapeutic and diagnostic purposes.

[0016] Preferably, the fluorescent probe is used to detect lipid droplets in cells, tissues, and living organisms.

[0017] The application of a fluorescent probe for targeting lipid droplets with long-term retention capability as described in this invention in the preparation of a long-term lipid droplet tracking product for Parkinson's disease cell models for non-therapeutic and non-diagnostic purposes.

[0018] The application of a fluorescent probe for targeting lipid droplets with long-term retention capability as described in this invention in a visualization detection product for pathological accumulation of lipid droplets in the substantia nigra region in the preparation of an animal model of Parkinson's disease for non-therapeutic and diagnostic purposes.

[0019] Beneficial effects This invention provides a fluorescent probe for targeting lipid droplets with long-term retention capability. The fluorescent probe can specifically label lipid droplets in living cells, isolated tissues, and living animals, enabling long-term tracking of abnormal accumulation of lipid droplets associated with Parkinson's disease. Its high lipophilicity makes the fluorescent probe difficult to efflux from living cells, exhibiting stable retention in cells for up to 72 hours. Therefore, the fluorescent probe can target lipid droplets and remain in nerve cells and animals for extended periods, making it suitable for the diagnosis of Parkinson's disease.

[0020] This invention provides an application of a fluorescent probe targeting lipid droplets with long-term retention capability. The fluorescent probe can label lipid droplets in dopamine neurons with high specificity and high signal-to-noise ratio at the cellular level, and has no significant cytotoxicity. Based on its excellent cell retention capability, the probe can track the abnormal accumulation of lipid droplets in damaged dopamine neurons over a long period. In animal models of Parkinson's disease, the probe can not only clearly display the pathological accumulation of lipid droplets in dopamine neurons in fresh isolated substantia nigra tissue and fixed brain tissue sections, but more importantly, through in situ intracerebral injection, it can achieve in situ visualization of abnormal lipid droplet accumulation in the substantia nigra region of live Parkinson's disease model mice. The fluorescent probe of this invention is characterized by simple synthesis, good biocompatibility, high signal-to-noise ratio, and long cell retention time. It is particularly suitable for real-time, in situ, and long-term tracking of pathological changes in Parkinson's disease-related lipid droplets under confocal fluorescence microscopy and small animal in vivo imaging systems, and has broad application prospects in the study of the mechanism of Parkinson's disease, early diagnosis, and drug evaluation. Attached Figure Description

[0021] Figure 1 This is a schematic diagram illustrating the mechanism by which the fluorescent probe described in this invention targets lipid droplets and has long retention capability.

[0022] Figure 2 These are confocal fluorescence images of HeLa cells stained with the fluorescent probes s-CBTA, b-CBTA, and LD-b-PBTA described in Example 2. The excitation wavelength of s-CBTA was 405 nm, and the acquisition wavelength was 500-550 nm; the excitation wavelength of b-CBTA was 405 nm, and the acquisition wavelength was 500-550 nm; the excitation wavelength of LD-b-PBTA was 488 nm, and the acquisition wavelength was 570-670 nm.

[0023] Figure 3 This is a confocal fluorescence image from Example 4 showing the co-localization experiment of the fluorescent probe LD-b-PBTA and the commercial lipid droplet probe BODIPY on dopaminergic neurons SH-SY5Y and MES23.5. The excitation wavelength of LD-b-PBTA was 488 nm, and the acquisition band was 570-670 nm; the excitation wavelength of BODIPY was 488 nm, and the acquisition band was 500-540 nm. The scale bar is 20 µm.

[0024] Figure 4 This is a confocal fluorescence image from a long-term cell tracking experiment using the fluorescent probe LD-b-PBTA described in Example 5 and the commercial lipid droplet probe BODIPY. The excitation wavelength of LD-b-PBTA was 488 nm, and the acquisition band was 570-670 nm; the excitation wavelength of BODIPY was 488 nm, and the acquisition band was 500-540 nm. The scale bar is 20 µm.

[0025] Figure 5 This is a confocal fluorescence image of the dopaminergic neuronal SH-SY5Y and MES23.5 cells after 24 h of rotenone treatment, monitored by the fluorescent probe LD-b-PBTA described in Example 6.

[0026] Figure 6 The results are behavioral test results of the Parkinson's disease model mice in Example 7.

[0027] Figure 7 These are confocal fluorescence images of fresh and fixed tissues from a Parkinson's disease model mouse stained with LD-b-PBTA as described in Example 8. The excitation wavelength of LD-b-PBTA was 488 nm, and the acquisition band was 570-670 nm.

[0028] Figure 8 The image shown in Example 9 is a confocal fluorescence image of the substantia nigra stained with LD-b-PBTA and used to verify the stereotactic coordinates of the substantia nigra. The excitation wavelength of rhodamine was 488 nm, and the acquisition wavelength was 540-560 nm. The excitation wavelength of LD-b-PBTA was 488 nm, and the acquisition wavelength was 570-670 nm.

[0029] Figure 9 The image shows a confocal fluorescence image of the substantia nigra of a live Parkinson's disease model mouse stained with LD-b-PBTA as described in Example 9. The excitation wavelength of LD-b-PBTA was 488 nm, and the acquisition band was 570-670 nm. Detailed Implementation

[0030] The present invention will be further described in detail below with reference to specific embodiments.

[0031] Preparation of s-CBTA (Comparative Example 1) N-ethylcarbazole-3-carboxaldehyde (0.22 g, 1 mmol) and benzothiazolium-2-acetonitrile (0.17 g, 1 mmol) were dissolved in 10 mL of ethanol, and 100 μL of piperidine was added. The mixture was heated to reflux at 76 °C for 24 h. The mixture was then purified by silica gel column chromatography to give a yellow solid in 73% yield.

[0032] The preparation reaction formula is as follows:

[0033] The 1H NMR spectrum of the yellow solid is as follows: 1 H NMR (500 MHz, CDCl3), δ(ppm): 8.80 (s, 1H), 8.48 (s, 1H), 8.29 (d, J =8.6 Hz, 1H), 8.19 (d, J = 7.8 Hz, 1H), 8.10 (d, J = 8.2 Hz, 1H), 7.92 (d, J = 8.0Hz, 1H), 7.55-7.45 (m, 4H), 7.43 (t, J = 7.6 Hz, 1H), 7.34 (t, J = 7.4 Hz, 1H), 4.42 (q, J = 7.3 Hz, 2H), 1.49 (t, J = 7.2 Hz, 3H). 13 C NMR (101 MHz, CDCl3), δ (ppm): 164.10, 153.66, 148.25, 142.25, 140.63, 134.74, 128.37, 126.82,126.75, 125.49, 124.44, 123.66, 123.50, 123.17, 122.87, 121.58, 120.95,120.39, 117.71, 109.17, 109.15, 100.80, 37.95, 13.89. The results showed that the yellow solid was (E)-2-(benzo[d]thiazolyl)-3-(9-ethyl-9H-carbazole-3-yl)acrylonitrile, denoted as s-CBTA.

[0034] Comparative Example 2: Preparation of β-CBTA N-ethylcarbazole-3,6-dicarboxaldehyde (0.25 g, 1 mmol) and benzothiazolium-2-acetonitrile (0.17 g, 1 mmol) were dissolved in 10 mL of ethanol, and 100 μL of piperidine was added. The mixture was heated to reflux at 76 °C for 24 h. The mixture was then purified by silica gel column chromatography to give a yellow solid in 64% yield.

[0035] The preparation reaction formula is as follows:

[0036] The 1H NMR spectrum of the yellow solid is as follows: 1 H NMR (400 MHz, CDCl3), δ(ppm): 8.72 (d, J = 1.7 Hz, 2H), 8.45-8.28 (m,4H), 8.09 (dd, J = 8.2, 1.0 Hz, 2H), 7.97-7.85 (m, 2H), 7.61-7.48 (m, 4H), 7.42(ddd, J = 8.2, 7.2, 1.2 Hz, 2H), 4.44 (q, J = 7.3 Hz, 2H), 1.53 (t, J = 7.2 Hz, 3H). 13 C NMR (151 MHz, CDCl3), δ (ppm): 163.63, 153.65, 147.54, 142.75, 134.81,129.93, 128.77, 126.87, 125.74, 124.92, 124.88, 123.48, 123.38, 121.65,117.40, 109.99, 102.33, 38.44, 14.02. The results showed that the yellow solid was (2E,2'E)-3,3'-(9-ethyl-9H-carbazole-3,6-diyl)bis(2-(benzo[d]thiazo-2-yl)acrylonitrile), denoted as b-CBTA.

[0037] Example 1: Preparation of fluorescent probes for long-term retention targeting lipid droplets 4,4'-dicarboxytriphenylamine (0.31 g, 1 mmol) and benzothiazol-2-acetonitrile (0.17 g, 1 mmol) were dissolved in 10 mL of ethanol, and 100 μL of piperidine was added. The mixture was heated to reflux at 76 °C for 24 h. The mixture was then purified by silica gel column chromatography to give a yellow solid in 69% yield.

[0038] The preparation reaction formula is as follows:

[0039] The 1H NMR spectrum of the yellow solid is as follows: 1 H NMR (400 MHz, CDCl3), δ (ppm): 8.19 (s, 2H), 8.07 (d, J = 8.2 Hz, 2H),8.02-7.95 (m, 4H), 7.94-7.88 (m, 2H), 7.53 (ddd,J = 8.3, 7.2, 1.3 Hz, 2H),7.43 (t, J = 7.8 Hz, 4H), 7.33-7.28 (m, 1H), 7.22 (td, J = 6.4, 1.7 Hz, 6H). 13 CNMR (100 MHz, CDCl3), δ (ppm): 163.29, 153.72, 149.91, 145.63, 145.24, 134.94,132.12, 130.20, 127.11, 127.03, 126.89, 126.42, 125.77, 123.38, 122.97,121.65, 117.08, 102.89. The results showed that the yellow solid was (2E,2'E)-3,3'-{[phenyl(aza)diyl]bis(4,1-phenylene)}bis[2-(1,3-benzothiazo-2-yl)prop-2-enonitrile], denoted as LD-b-PBTA.

[0040] The ClogP values ​​of s-CBTA, b-CBTA, and LD-b-PBTA were determined, and the results are as follows: Figure 1 As shown, the values ​​are 5.76, 7.02, and 8.24, respectively, indicating that LD-b-PBTA has the strongest lipophilicity, which is consistent with its expectation of long-term tracking of lipid droplets. The results demonstrate that LD-b-PBTA, with its ultra-high lipophilicity, can achieve single, highly specific targeting of lipid droplets without staining other membrane structures.

[0041] Example 2 Imaging experiments of s-CBTA, b-CBTA and LD-b-PBTA The confocal culture dishes inoculated with HeLa cells were divided into three groups. The first group was stained with 5 μM s-CBTA for 1 h; the second group was stained with 5 μM b-CBTA for 1 h; and the third group was stained with 5 μM LD-b-PBTA for 1 h.

[0042] After staining, the stained areas of the cells were observed using a laser scanning confocal microscope, and the results are as follows: Figure 2 As shown, all three molecules exhibited a speckled staining pattern in HeLa cells (indicated by red arrows), and their distribution highly overlapped with that observed in the differential interference contrast (DIC) images, confirming their staining ability for lipid droplets. However, s-CBTA and b-CBTA additionally stained other membrane structures (marked by yellow arrows). In contrast, LD-b-PBTA showed specific targeting to lipid droplets and did not stain other membrane structures.

[0043] Example 3: Culture of cancer cells (HeLa) and dopaminergic neurons (SH-SY5Y and MES23.5) All cell lines were cultured in a 37°C, 5% CO2 saturated humidity incubator. HeLa and SH-SY5Y cell lines were cultured in DMEM / F12 medium containing 10% fetal bovine serum (FBS) (containing 1% penicillin-dip antibiotics), while MES23.5 cell lines were cultured in H-DMEM medium containing 10% FBS (containing 1% penicillin-dip antibiotics). Once cells reached the logarithmic growth phase, the cells in 100 mL cell culture flasks were washed three times with PBS, digested with 1 mL of 0.25% trypsin for 3-5 min, carefully poured out the culture medium, added a small amount of fresh culture medium and agitated thoroughly. After cell counting, cells at an appropriate density were retained, and the culture medium was added to the desired volume (controlling the final cell concentration to 1×10⁶ cells / mL). 4 The cells were seeded into confocal glass-bottomed culture dishes and placed in a CO2 incubator to allow them to adhere to the glass bottom and grow.

[0044] Example 4: Co-localization experiment of LD-b-PBTA and commercial lipid droplet probe BODIPY SH-SY5Y cells and MES23.5 cells were seeded in confocal glass culture dishes. The cells were first stained with 1 µM BODIPY for 30 min, followed by 2 µM LD-b-PBTA staining for 1 h.

[0045] After staining, the stained areas of the cells were observed using a laser scanning confocal microscope, and the results are as follows: Figure 3 As shown, the fluorescence signals of LD-b-PBTA and BODIPY have good overlap, indicating that LD-b-PBTA stains lipid droplets.

[0046] Example 5: Long-term cell tracking experiment of LD-b-PBTA The confocal culture dishes seeded with SH-SY5Y cells were divided into two groups. One group was stained with 2µM LD-b-PBTA for 1 hour; the other group was stained with BODIPY for 1 hour.

[0047] After staining, the stained areas of the cells were observed using a laser scanning confocal microscope, and images were acquired at different time points (0, 24, 48, and 72 hours). The results are as follows: Figure 4 As shown, the fluorescence intensity of BODIPY decreased significantly after 24 hours and continued to decay over time; while the fluorescence of LD-b-PBTA showed almost no decay over 72 hours.

[0048] Example 6 Imaging experiment with rotenone, a drug that induces damage to dopaminergic neurons. Confocal culture dishes seeded with SH-SY5Y or MES23.5 cells were divided into two groups. One group was stained with 2 µM LD-b-PBTA for 1 h; the other group was first treated with 1 µM rotenone for 24 h to induce dopaminergic neuron damage, and then stained with 2 µM LD-b-PBTA for 1 h.

[0049] After staining, the stained areas of the cells were observed using a laser scanning confocal microscope, and the results are as follows: Figure 5 As shown, 24-hour rotenone treatment significantly increased the number and area of ​​lipid droplets in SH-SY5Y and MES23.5 cells. These results indicate that rotenone leads to the accumulation of lipid droplets in dopaminergic neurons. Furthermore, this demonstrates that LD-b-PBTA can effectively characterize lipid droplet accumulation in a Parkinson's disease cell model.

[0050] Example 7: MPTP drug induces key clinical features of Parkinson's disease in mice. A C57BL / 6J mouse model was used, with MPTP (25 mg / kg) administered intraperitoneally daily for 7 consecutive days. Subsequently, a series of behavioral tests were conducted to assess the anxiety-depression-like behaviors and motor function of the MPTP-treated mice. Results are as follows: Figure 6 As shown, the open-field test revealed that anxious mice tended to move along the edges, and compared to the control group, the MPTP-treated group exhibited a significantly reduced proportion of distance traveled in the central area. The elevated cross maze test yielded consistent results. MPTP-treated mice rarely entered the open arms, and the proportion of distance traveled in the open arms was significantly lower than that in the control group. These results indicate that MPTP treatment induces anxiety-like behavior in mice.

[0051] Example 8: Imaging experiment of LD-b-PBTA on lipid droplets in the substantia nigra of mouse brain. Mice were euthanized with an overdose of pentobarbital after MPTP treatment, and substantia nigra tissue was rapidly collected for staining experiments. Substantia nigra samples were rinsed with physiological saline and stained with 5 μM LD-b-PBTA for 30 minutes. Results are as follows: Figure 7 As shown, LD-b-PBTA can specifically locate lipid droplets in fresh substantia nigra. Compared with the control group, the number and area of ​​lipid droplets in the MPTP-treated group of mice were significantly increased.

[0052] To more accurately image lipid droplets within dopaminergic neurons in the substantia nigra, immunofluorescence staining was used to stain fixed tissue sections for tyrosine hydroxylase (a biomarker of dopaminergic neurons), followed by LD-b-PBTA staining. The results are as follows: Figure 7As shown, the fluorescence signal of tyrosine hydroxylase (green) is mainly localized in the substantia nigra region. The LD-b-PBTA signal (red) significantly overlaps with tyrosine hydroxylase-positive neurons, indicating that this dye specifically localizes to dopaminergic neurons in the substantia nigra. MPTP treatment significantly downregulated tyrosine hydroxylase expression, suggesting the loss of dopaminergic neurons in the substantia nigra. Furthermore, the results showed that MPTP treatment significantly increased the number and area of ​​lipid droplets within dopaminergic neurons in the substantia nigra. These results demonstrate that LD-b-PBTA can effectively localize lipid droplets in both fresh and fixed tissues and visualize the accumulation of lipid droplets within dopaminergic neurons in the substantia nigra of a PD animal model.

[0053] Example 9: Imaging experiment of LD-b-PBTA on lipid droplets in in situ of live mouse brain The stereotactic coordinates of the substantia nigra were verified by intracranial injection of rhodamine, and the results were as follows: Figure 8 As shown, significant rhodamine (green) fluorescence was detected in the substantia nigra region, confirming the accuracy of the coordinates in subsequent studies. LD-b-PBTA was then administered to mice via intracranial injection, and the fluorescence signal was measured using a small animal imaging system. Results are as follows... Figure 8 As shown, no fluorescence signal was detected in the blank control group. However, significant fluorescence signal was detected in the LD-b-PBTA treated group, indicating that this reagent is suitable for in situ imaging of live animals.

[0054] Finally, the LD-b-PBTA fluorescence signal in the brains of mice in the control group and the MPTP-treated group was measured. Figure 9 As shown, the fluorescence signal of LD-b-PBTA in the brains of mice treated with MPTP was significantly increased compared with that in the control group. This confirms that LD-b-PBTA is also applicable to in vivo mouse models of Parkinson's disease.

[0055] In summary, the invention includes, but is not limited to, the above embodiments. Any equivalent substitutions or partial improvements made under the spirit and principles of this invention shall be considered to be within the protection scope of this invention.

Claims

1. A fluorescent probe for targeting lipid droplets with long-term retention capability, characterized in that: The structural formula of the fluorescent probe is as follows: 。 2. The fluorescent probe for targeting lipid droplets with long-term retention capability as described in claim 1, characterized in that: The ClogP value of the fluorescent probe is greater than 8.

3. The fluorescent probe for targeting lipid droplets with long-term retention capability as described in claim 1, characterized in that: The fluorescent probe remains stably in cells for more than 72 hours.

4. A fluorescent probe for targeting lipid droplets with long-term retention capability as described in claim 1, characterized in that: The excitation wavelength of the fluorescent probe is 488 nm, and the acquisition wavelength is 570~670 nm.

5. A method for preparing a fluorescent probe for targeting lipid droplets with long-term retention capability as described in any one of claims 1 to 4, characterized in that: The method steps include: 4,4'-Dicarboxytriphenylamine and benzothiazol-2-acetonitrile are reacted via a Brainwell reaction to yield (2E,2'E)-3,3'-{[phenyl(aza)diyl]bis(4,1-phenylene)}bis[2-(1,3-benzothiazol-2-yl)prop-2-enonitrile], a fluorescent probe targeting lipid droplets with long-term retention capability.

6. The method for preparing a fluorescent probe for targeting lipid droplets with long-term retention capability as described in claim 5, characterized in that: 4,4'-dicarboxytriphenylamine and benzothiazole-2-acetonitrile were dissolved in ethanol, and then piperidine was added as a catalyst. The mixture was heated under reflux for more than 24 hours. After the reaction was completed, the mixture was separated by column chromatography, and the solid was collected to obtain a fluorescent probe for targeting lipid droplets with retention ability.

7. The use of a fluorescent probe for targeting lipid droplets with long-term retention capability as described in any one of claims 1 to 4 in the preparation of lipid droplet fluorescence imaging products or lipid droplet-specific labeling products for non-therapeutic and diagnostic purposes.

8. The application as described in claim 7, characterized in that: The fluorescent probe is used to detect lipid droplets in cells, tissues, and living organisms.

9. The use of a fluorescent probe for targeting lipid droplets with long-term retention capability as described in any one of claims 1 to 4 in a long-term lipid droplet tracking product for the preparation of a Parkinson's disease cell model for non-therapeutic and non-diagnostic purposes.

10. The application of a fluorescent probe for targeting lipid droplets with long-term retention capability as described in any one of claims 1 to 4 in a visualization detection product for pathological accumulation of lipid droplets in the substantia nigra region in the preparation of an animal model of Parkinson's disease for non-therapeutic and diagnostic purposes.

Images