Lipophilic fluorescent probe with dual response of lipid droplet polarity and pH as well as preparation method and application of lipophilic fluorescent probe
By developing a lipophilic fluorescent probe that is dual-responsive to lipid droplet polarity and pH, the problem of high misdiagnosis rate in the early diagnosis of Parkinson's disease in existing technologies has been solved. This enables efficient and low-interference monitoring of lipid droplet autophagy in living cells and biological tissues, with excellent biocompatibility and penetration depth.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-02
- Publication Date
- 2026-03-27
AI Technical Summary
Existing fluorescent probes have a high misdiagnosis rate when detecting biomarkers in the early stages of Parkinson's disease, and are difficult to meet the needs of early clinical diagnosis and treatment. Furthermore, the application of most viscosity-sensitive fluorescent probes in complex biological systems is limited.
A lipophilic fluorescent probe with both lipid droplet polarity and pH response was developed. It has a near-infrared emission wavelength and can specifically respond to changes in lipid droplet polarity and pH. The synthesis process is simple and the conditions are mild. It can be applied to live cell and biological tissue imaging.
It achieves clear capture of lipid droplet polarity reduction characteristics in living cells and biological tissues, with excellent biological tissue penetration depth and low biological background interference, high signal-to-noise ratio and good biocompatibility, and has been successfully applied to lipid droplet autophagy monitoring in Parkinson's disease models.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of chemical biomaterials, in particular to a lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe and a preparation method and application thereof. BACKGROUND
[0002] Parkinson's disease (PD) is a progressive clinical syndrome induced by multiple causes, with progressive loss of neurons as the core feature. Its typical clinical phenotypes include bradykinesia, muscle stiffness and tremor. As the second most common neurodegenerative disease affecting millions of people worldwide, it has become a major public health challenge. Currently, the diagnosis of PD mainly relies on retrospective clinical feature assessment. The existing detection methods have a high misdiagnosis rate in the early stages of the disease, which cannot meet the needs of early diagnosis and treatment in the clinic.
[0003] In recent years, small molecule fluorescent probes based on fluorescent probe technology have been widely used and rapidly developed in the field of biomarker detection. This technology, with the advantages of small sampling volume, high sensitivity, simple operation, good biocompatibility, etc., can realize real-time dynamic monitoring of biomarkers in vivo, providing a new idea for disease diagnosis. However, most of the reported viscosity-sensitive fluorescent probes have the limitations of short emission wavelength and easy interference by biological background fluorescence, which greatly limits their application in complex biological systems. In contrast, near-infrared emission wavelength fluorescent probes not only effectively reduce biological background interference and significantly improve the signal-to-noise ratio, but also have the outstanding advantages of deep tissue penetration depth and low biological toxicity, showing higher clinical translation value.
[0004] Therefore, the development of a new type of fluorescent probe with both polarity / pH dual-response characteristics and lipid droplet targeting function, and its application in the visualization monitoring of lipid droplet autophagy and PD model research, is expected to provide a precise detection tool for early diagnosis of PD, and lay an important technical foundation for in-depth exploration of lipid autophagy mechanism. SUMMARY
[0005] An object of the present application is to solve at least the above problems and / or disadvantages and to provide at least the advantages described later.
[0006] Another object of the present application is to provide a lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe, which not only has excellent biological tissue penetration depth, but also can effectively eliminate biological background fluorescence interference; with the help of dual-channel response imaging technology, not only can the characteristic of reduced lipid droplet polarity in the PD model group compared with the normal group be clearly captured, but also the probe has good specificity to pH changes, and excellent biocompatibility. The probe is successfully applied to fluorescence imaging in living cells, nematodes and fruit fly brain tissues.
[0007] The application also aims to provide a method for preparing the lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe, which has a simple synthesis process and mild conditions.
[0008] The application also aims to provide an application of the lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe in preparing a lipid droplet polarity and pH dual-responsive probe for a Parkinson's disease model, which can achieve the purpose of lipid droplet polarity and pH dual-response in PC-12 cell level, nematode and fruit fly models.
[0009] In order to achieve the above objects and other advantages according to the application, a lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe is provided, which has the following formula (I): (I).
[0010] The objects of the application can also be further achieved by a preparation method of the lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe, which comprises the following steps: S1, 2-methylbenzothiazole and 2-bromoethanol are dissolved in a solvent to prepare an intermediate formula (II) compound by reflux reaction; S2, the formula (II) compound is dissolved in an organic reagent to react with 4-formyltriphenylamine, and after the reaction, the organic solvent is spun off, and column chromatography purification is performed to obtain the formula (I) compound; (II).
[0011] Preferably, in step S1, the molar ratio of the mixture of 2-methylbenzothiazole and 2-bromoethanol is 1:3.
[0012] Preferably, in step S1, the reflux reaction temperature is 70-80℃, and the reflux reaction time is 5-7 h.
[0013] Preferably, in step S2, the reaction temperature is 75-80℃, and the reaction time is 6 h.
[0014] Preferably, in step S2, the molar ratio of the formula (II) compound to 4-formyltriphenylamine is 1:1.
[0015] Preferably, in step S2, the eluent used in column chromatography is dichloromethane and petroleum ether with a volume ratio of 10:1.
[0016] The objects of the application can also be further achieved by an application of the lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe in preparing a lipid droplet polarity and pH detection probe.
[0017] Preferably, the polar and pH dual-responsive lipophilic fluorescent probe is used for specifically detecting the lipid droplet autophagy process at the cell level.
[0018] Preferably, the polar and pH dual-responsive lipophilic fluorescent probe is used for detecting a Parkinson's disease nematode model or a Parkinson's disease fruit fly model.
[0019] The present application at least includes the following beneficial effects: First, the lipid droplet polar and pH dual-responsive lipophilic fluorescent probe of the present application can specifically detect the biomarker (lipid droplet polarity) in the PD model and respond to the near-infrared fluorescent probe in the acidic environment. The emission wavelength of the probe is in the near-infrared region, which not only has excellent biological tissue penetration depth, but also can effectively eliminate the interference of biological background fluorescence; with the help of dual-channel response imaging technology, not only can the characteristic of the reduced lipid droplet polarity of the PD model group compared with the normal group be clearly captured, but the probe also has good specificity to pH changes and excellent biocompatibility.
[0020] Second, the method for preparing the lipid droplet polar and pH dual-responsive lipophilic fluorescent probe has simple synthesis process and mild conditions.
[0021] Third, the lipid droplet polar and pH dual-responsive lipophilic fluorescent probe of the present application can achieve the purpose of lipid droplet polarity and pH detection in PC-12 cell level, nematode and fruit fly model, and the fluorescence intensity responding to polarity and pH is not affected by other ions or molecules, different viscosity environments, has good specificity and stability to polarity and pH, can be successfully applied to the detection of polarity and pH in PD model cells and can further observe the change of lipid phagocytosis.
[0022] Other advantages, objects, and features of the present application will be apparent from the following description, and will be understood by those skilled in the art. BRIEF DESCRIPTION OF DRAWINGS
[0023] Figure 1 The nuclear magnetic resonance hydrogen spectrum of the lipid droplet polar and pH dual-responsive lipophilic fluorescent probe M6 prepared in Example 1 of the present application; Figure 2 The nuclear magnetic resonance carbon spectrum of the lipid droplet polar and pH dual-responsive lipophilic fluorescent probe M6 prepared in Example 1 of the present application; Figure 3 The mass spectrum of the lipid droplet polar and pH dual-responsive lipophilic fluorescent probe M6 prepared in Example 1 of the present application; Figure 4 The fluorescence emission spectrum of the lipid droplet polar and pH dual-responsive lipophilic fluorescent probe M6 in the 1,4-dioxane and water mixed probe solution under different water contents in Example 2 of the present application; Figure 5 Fig. 6A is the UV normalized spectrum of the lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe M6 in different pH PBS solutions, and Fig. 6B is the fluorescence emission spectrum of the lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe M6 in different pH PBS solutions; Figure 6 Fig. 7A is the fluorescence emission spectrum of the lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe M6 in different glycerol-H2O media, and Fig. 7B is the ion selectivity and interference fluorescence emission spectrum of the lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe M6; Figure 7 Fig. 8 is a graph showing the effect of the lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe M6 on PC12 cell survival rate; Figure 8 Fig. 9 is a graph showing the lipid droplet co-localization specificity bioimaging of the lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe M6 in PC12 cells; Figure 9 Fig. 10A is a graph showing the specificity bioimaging of the lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe M6 on the PC12 cell-induced lipid droplet autophagy model, and Fig. 10B is a fluorescence quantification graph of the PC12 cell-induced lipid droplet autophagy model confocal imaging; Figure 10 Fig. 11 is a fluorescence imaging graph of the lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe M6 on the lipid droplet polarity and pH in a Parkinson's disease nematode model; Figure 11 Fig. 12 is a fluorescence imaging graph of the lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe M6 on the lipid droplet polarity and pH in a Parkinson's disease fruit fly brain model. DETAILED DESCRIPTION
[0024] The present application will be further described below in conjunction with the accompanying drawings, so that those skilled in the art can implement the present application according to the description and drawings.
[0025] It should be understood that the terms such as "have", "contain" and "include" used herein do not exclude the presence or addition of one or more other elements or combinations thereof.
[0026] It should be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.
[0027] Main equipment: UV-visible spectrophotometer (UH-5300, Hitachi, Japan), fluorescence spectrophotometer (F-7100, Hitachi, Japan), rotary evaporator (RE-2000B, Hangzhou Ruigia Precision Scientific Instruments), microplate reader (SpectraMax190, USA), laser confocal microscope (FV-1000, Japan).
[0028] Main chemical reagents: methanol (A.R., Thermo Scientific (China)), dichloromethane (A.R., Tianjin Damao Chemical Reagent Factory), DMEM high-sugar basal medium (Wuhan Punsai Life Science and Technology Co., Ltd.), fetal bovine serum (FBS) (Wuhan Punsai Life Science and Technology Co., Ltd.), penicillin-streptomycin solution (Wuhan Punsai Life Science and Technology Co., Ltd.), 0.25% trypsin solution (Wuhan Punsai Life Science and Technology Co., Ltd.), anhydrous ethanol (A.R., Tianjin Zhiyuan Chemical Reagent), resveratrol (Biyun Tian), rapamycin (Aladdin), chloroquine (Aladdin), 2-methylbenzothiazole (Anjie), 2-bromoethanol (Macklin), 4-formyltriphenylamine (Anjie).
[0029] Example 1 A lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe M6 has the following formula (I): (I) The specific synthesis route is as follows: The specific synthesis steps are as follows: S1, 2-methylbenzothiazole (1.5 mL) and 2-bromoethanol (2.5 mL) were placed in a 25 mL round-bottom flask, and the reaction was condensed and refluxed at 80°C for 6 h. TLC plate was used to monitor whether the reaction was complete. After the reaction was completed, the solvent was removed by rotary evaporation under reduced pressure, recrystallized with ethyl acetate, and white solid was precipitated after filtration, then washed with ethyl acetate (50 mL x 3 times), and finally obtained white solid, which was intermediate 1 (1.192 g, yield 52.4%). 1H NMR (400 MHz, DMSO-d6),δ (ppm): 8.46 (dd, J = 8.1, 1.2 Hz, 1H), 8.33 (dd, J = 8.4, 1.0 Hz, 1H), 7.89(ddd, J = 8.6, 7.3, 1.3 Hz, 1H), 7.81 (ddd, J = 8.3,7.3, 1.1 Hz, 1H), 5.24(s, 1H), 4.87 (dd, J = 6.6, 3.5 Hz, 2H), 3.90 (d, J = 4.6 Hz, 2H), 3.23 (s,3H)。
[0030] S2, Intermediate 1 (200 mg, 0.52 mmol) and 4-formyltriphenylamine (281.48 mg, 0.52 mmol) were dissolved in 8 mL of anhydrous ethanol, and condensed reflux at 78 °C for 6 h. TLC plate was used to monitor whether the reaction was completed. After the reaction was completed, the volatile solvent was removed by vacuum rotary evaporator. The crude product was separated and purified by silica gel column chromatography (eluent: dichloromethane and methanol, v / v = 10:1) to obtain a purple solid. After recrystallization with petroleum ether, centrifugation was performed for 5 minutes, and the supernatant was discarded to obtain the final purified product, which was a purple solid, i.e., the lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe M6 (94.6 mg, yield 20.5%). The hydrogen spectrum of the lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe M6 is shown in Figure 1 , the carbon spectrum is shown in Figure 2 , and the mass spectrum is shown in Figure 3 .
[0031] Example 2 Example 1 prepared lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe M6 for different polarity recognition: The lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe M6 prepared in Example 1 was prepared into a DMSO stock solution, and then different proportions of water and 1,4-dioxane mixed systems were added, and diluted to a concentration of 10 μM for fluorescence spectrum test. Based on the change of fluorescence intensity of the lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe M6 at 453 nm, the influence of different volume ratios of water and 1,4-dioxane mixed systems on the fluorescence emission intensity was explored. The test results are shown in Figure 4 , and Figure 4It can be seen that compared with the high polarity aqueous solution, the polarity of 1,4-dioxane solution is lower, and with the increase of the volume ratio of 1,4-dioxane in the mixed system, the polarity of the lipid droplet and the fluorescence emission intensity of the pH and polarity dual-responsive lipophilic fluorescent probe M6 gradually increase.
[0032] Example 3 The pH and polarity dual-responsive lipophilic fluorescent probe M6 prepared in Example 1 recognizes different pH environments: The pH and polarity dual-responsive lipophilic fluorescent probe M6 prepared in Example 1 was prepared into a DMSO stock solution, and then diluted with PBS buffers with different pH values to a concentration of 10 μM to be tested, and then the ultraviolet-visible absorption spectrum and fluorescence emission spectrum were tested. With the fluorescence intensity at 650 nm as the index, the effect of different pH environments on the fluorescence emission intensity of the pH and polarity dual-responsive lipophilic fluorescent probe M6 was investigated. The test results are shown in Figure 5 A, 5B, it can be seen that Figure 5 It can be seen that in the range of pH = 4-12, with the decrease of the pH value of the system, the ultraviolet absorption intensity and the fluorescence emission intensity of the pH and polarity dual-responsive lipophilic fluorescent probe M6 gradually increase.
[0033] Example 4 The selectivity and interference of the pH and polarity dual-responsive lipophilic fluorescent probe M6 prepared in Example 1 to other microenvironments: Viscosity response test: The pH and polarity dual-responsive lipophilic fluorescent probe M6 prepared in Example 1 was prepared into a DMSO stock solution, and different volume ratios of glycerol-water mixed systems were prepared to construct different viscosity test environments, and then an appropriate amount of probe DMSO stock solution was added, so that the final concentration of the probe was 10 μ0, and three parallel samples were set for each group. Test on the HITACHI F-7100 fluorescence spectrophotometer, set the excitation wavelength to 354 nm, monitor the fluorescence emission at 400 nm, and investigate the fluorescence response characteristics of the pH and polarity dual-responsive lipophilic fluorescent probe M6 under different viscosity conditions. The test results are shown in Figure 6 A, it can be seen that Figure 6 As shown in the middle A, the test system has no interference with the polarity response of the pH and polarity dual-responsive lipophilic fluorescent probe M6.
[0034] Selective test: The lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe M6 prepared in Example 1 was prepared into a DMSO stock solution, and then added into an aqueous solution containing different ions, amino acids, reactive oxygen / active nitrogen (ROS / RNS) and other endogenous substances in the body, and diluted to a final concentration of 10 μ0. The fluorescence spectrum was tested at an excitation wavelength of 354 nm. By comparing the fluorescence intensity at 453 nm, the interference of various exogenous substances on the fluorescence performance of the probe was evaluated. The test results are shown in Figure 6 Fig. 6B, and it can be seen from Figure 6 Fig. 6B that the lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe M6 has almost no response to the above-mentioned substances at 453 nm, and its fluorescence intensity is only controlled by polarity change and is not interfered by other biological related substances, showing excellent selectivity.
[0035] Example 5 Effect of the lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe M6 prepared in Example 1 on PC12 cell survival rate: PC12 cells were inoculated in a 96-well plate, and when the cells grew to about 80%, different concentrations of the lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe M6 (concentration gradient: 0 μM, 2 μM, 4 μM, 6 μM, 8 μM, 10 μM, 15 μM, 20 μM and 25 μM) were added to each well, and the cells were cultured for 24 hours. Then MTT solution was added to each well, and the 96-well plate was incubated in a 37℃, 5% CO2 incubator for 4 hours. The absorbance of each well was measured at the maximum absorption wavelength λ max = 490 nm by an enzyme-labeled instrument, and the cell survival rate was calculated according to the absorbance. The experimental results are shown in Figure 7 Fig. 7B, and it can be seen from Figure 7 that the lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe M6 has low cytotoxicity and good biological safety.
[0036] Example 6 Lipid droplet co-localization specific bioimaging of the lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe M6 prepared in Example 1 in PC12: The triphenylamine group has a lipid droplet targeting property. Since the lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe M6 contains this group, the lipid droplet targeting ability of the probe was further explored. The commercial lipid droplet specific probe Nile Red was used as a positive control, and a cell co-incubation experiment was carried out with the lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe M6. As Figure 8As shown, Nile Red can be specifically located in lipid droplets and emit red fluorescence, and the lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe M6 of the application emits blue-green fluorescence; the fluorescence overlap composite diagram presents a clear white co-localization signal, indicating that the two have good overlap in the lipid droplet area, confirming that the lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe M6 has the ability of lipid droplet targeting. Figure 8 The data further shows that the Pearson correlation coefficient of the co-localization of the lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe M6 and Nile Red is as high as 0.95; in addition, the Pearson correlation coefficients of the co-localization of the lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe M6 and the mitochondrial specific probe Mito-Tracker Red and the lysosome specific probe Lyso-Tracker Red are only 0.30 and 0.38, respectively. In summary, the above experimental results confirm that, compared with other organelles such as mitochondria and lysosomes, the lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe M6 can be selectively located in lipid droplets. At the same time, combined with the verified polarity change detection function of the lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe M6, it has significant application potential in the field of lipid droplet polarity dynamic monitoring, and provides important technical support for subsequent targeted monitoring of lipid droplet autophagy related research.
[0037] Example 7 Specific bioimaging of the lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe M6 prepared in Example 1 on the PC12 cell induced lipid droplet autophagy model: To verify the detection performance of the lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe M6 in visualizing monitoring the lipid droplet autophagy process in living cells, the present application processed PC12 cells by three ways of starvation treatment, rapamycin induction and chloroquine intervention to construct a lipid droplet autophagy induction model. Fluorescence imaging analysis was performed by confocal laser scanning microscope: the blue-green channel of the lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe M6 can specifically respond to the polarity of lipid droplets, and after autophagy induction, benefiting from the acid response characteristics of the lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe M6, it will be targeted to locate in lysosomes, and thus the contact between lipid droplets and lysosomes and the occurrence of lipidophagy process can be observed in real time.
[0038] As Figure 9 As shown in FIG. 8A, compared with the control group treated only with the lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe M6, the lipid droplet signal corresponding to the blue-green channel of the lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe M6 in the starvation induction group and the rapamycin induction group was significantly reduced, and the red channel fluorescence intensity was significantly enhanced; while in the chloroquine treatment group, the lipid droplet signal of the blue-green channel of the lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe M6 was significantly increased, and the red channel fluorescence intensity was significantly reduced.Figure 9 Quantitative analysis of B showed that the red channel fluorescence intensity of the lipophilic fluorescent probe M6, which is responsive to both lipid droplet polarity and pH, was approximately three times that of the control group in the starvation-induced and rapamycin-induced groups.
[0039] The above results indicate that the lipophilic fluorescent probe M6, which is both polar and pH-responsive, can sensitively respond to the dynamic changes in the lipid droplet autophagy process. Although starvation and rapamycin induce lipid droplet autophagy through different mechanisms, both can significantly activate the specific fluorescence signal of the lipophilic fluorescent probe M6, which is both polar and pH-responsive, thus verifying the high sensitivity and reliability of the lipophilic fluorescent probe M6 in detecting the lipid droplet autophagy process in living cells.
[0040] Example 8 Example 1: Fluorescence imaging of lipid droplet polarity and pH in a Parkinson's disease nematode model using the lipophilic fluorescent probe M6, which is dual-responsive to lipid droplet polarity and pH. Wild-type nematodes and Parkinson's disease model nematodes were synchronized to ensure consistent growth cycles. The experimental grouping was as follows: five oviposition-stage nematodes were selected from each of the wild-type and Parkinson's disease model nematode cultures and placed in the corresponding blank control group culture dish; another five oviposition-stage nematodes were selected from the Parkinson's disease model nematode culture dish and placed in the treatment group culture dish containing resveratrol. All culture dishes were then placed in a 20°C constant temperature and humidity incubator for static incubation.
[0041] After 4 days of culture, all nematodes on the surface of the culture medium developed into adults. Five adults were picked from each culture dish and immersed for 5 minutes in PBS buffer (pH=7.4) containing 100 μM of a lipophilic fluorescent probe M6 with both lipid drop polarity and pH dual response. The nematodes were then transferred to a culture medium without OP50 *E. coli*. After removing any remaining food debris and unbound probe from the nematode surface, they were transferred to a slide containing 2% agar for fixation and fluorescence imaging using a confocal laser scanning microscope. The results are shown below. Figure 10 As shown, by Figure 10 It is known that the lipophilic fluorescent probe M6 of the present invention, which is responsive to both lipid droplet polarity and pH, can effectively distinguish between nematodes in a Parkinson's disease model and wild-type nematodes.
[0042] Example 9 Example 1: Fluorescence imaging of lipid droplet polarity and pH in a Drosophila brain model of Parkinson's disease using M6, a lipophilic fluorescent probe prepared with dual lipid droplet polarity and pH response. Firstly, the wild type fruit fly (WT) and Parkinson's disease (PD) model fruit fly are synchronized to ensure the growth cycle of the two types of fruit flies. Three groups are set: wild type control group (WT group), Parkinson's disease model group (PD group) and resveratrol intervention Parkinson's disease treatment group (PD + resveratrol group), wherein the PD + resveratrol group is constructed by transferring part of the PD model fruit fly to a special culture bottle containing resveratrol. All experimental groups are placed in a constant temperature environment of 23 DEG C for 20 days, and then the brain tissue of the fruit fly is peeled off and placed in a 96-well plate containing PBS buffer (pH = 7.4); the concentration of the lipid droplet polarity and pH double-response lipophilic fluorescent probe M6 solution is 100 μM, and the peeled fruit fly brain tissue is transferred to the probe solution for incubation for 30 minutes, and then confocal fluorescence imaging detection is carried out. The test results are shown in Figure 11 As can be seen from Figure 11 The lipid droplet polarity and pH double-response lipophilic fluorescent probe M6 of the present application can effectively distinguish the Parkinson's disease model fruit fly from the wild type fruit fly.
[0043] The lipid droplet polarity and pH double-response lipophilic fluorescent probe M6 provided by the present application has excellent double-response characteristics for the change of lipid droplet polarity and pH. The probe has the core performance of 353 nm and 650 nm double fluorescence emission channels, and can monitor the dynamic change of the polarity and pH level in the biological system through the double channels; at the same time, it has the technical advantages of strong selectivity, low background interference, high detection sensitivity and good biocompatibility, and has been successfully applied to a variety of biological systems such as living cells, Caenorhabditis elegans and fruit flies, and has realized the accurate detection of lipid droplet polarity and pH level by means of confocal microscope.
[0044] In addition, the present application proves that the probe has the ability to visually monitor the autophagy process of lipid droplets, and provides a fluorescent detection method with application prospect for dynamic monitoring of the progression of Parkinson's disease, which can be further applied to the fluorescence imaging research of other disease-related biomarkers, and provides new technical support for disease mechanism exploration and clinical diagnosis.
[0045] Although the embodiments of the present application have been disclosed as above, it is not limited to the application listed in the specification and embodiments. It can be fully applied to various fields suitable for the present application. Those skilled in the art can easily make other modifications. Therefore, the present application is not limited to specific details and figures shown and described herein without departing from the general concept defined by the claims and equivalent scope.
Claims
1. A lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe, characterized in that, It has the following formula (I) structure: （I）。 2. A method for preparing the lipid droplet polarity and pH dual-responsive lipophilic fluorescent probe according to claim 1, characterized in that, It comprises the following steps: S1, 2-methylbenzothiazole and 2-bromoethanol as raw materials are dissolved in a solvent, and an intermediate compound of formula (II) is prepared by refluxing reaction; S2, the compound of formula (II) is dissolved in an organic reagent with 4-formyltriphenylamine, the organic solvent is spun off after the reaction, and column chromatography purification is carried out to obtain the compound of formula (I); (II).
3. The method of claim 2, wherein, In step S1, the molar ratio of 2-methylbenzothiazole and 2-bromoethanol mixed is 1:
3.
4. The method of claim 2, wherein, In step S1, the temperature of the refluxing reaction is 70-80℃, and the refluxing reaction time is 5-7 h.
5. The method of claim 2, wherein, In step S2, the reaction temperature is 75-80℃, and the reaction time is 6 h.
6. The method of claim 2, wherein, In step S2, the molar ratio of the compound of formula (II) to 4-formyltriphenylamine is 1:
1.
7. The method of claim 2, wherein, In step S2, the eluent used in column chromatography is dichloromethane and petroleum ether with a volume ratio of 10:
1.
8. The application of the lipid droplet polarity and pH double-responsive lipophilic fluorescent probe in the preparation of a lipid droplet polarity and pH detection probe according to claim 1.
9. Use according to claim 8, wherein the compound is ###0002### The lipid droplet polarity and pH double-responsive lipophilic fluorescent probe is used for specific detection of the lipid droplet autophagy process at the cell level.
10. The use according to claim 8, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The lipid droplet polarity and pH double-responsive lipophilic fluorescent probe is used for detection of a Parkinson's disease nematode model or a Parkinson's disease fruit fly model on nematodes or fruit flies.