A fluorescent probe for detecting cholesterol fluctuation during plasma membrane damage repair, and a preparation method and application thereof

Abstract

The application discloses a fluorescent probe for detecting fluctuation of cholesterol during plasma membrane damage repair, and a preparation method and application thereof, and belongs to the technical field of fluorescent probes. 26 H 35 N3O2 2+ , and a structural formula is as follows: The application utilizes the fact that a slight change in the CL level in the plasma membrane can also significantly change the permeability of the membrane, designs a spatial distribution-controllable fluorescent probe, detects the change in the CL level in the plasma membrane when the state of the plasma membrane changes, and realizes dynamic visual detection of the fluctuation of cholesterol in the plasma membrane. The obtained fluorescent probe has high sensitivity, causes minimum damage to a biological sample, realizes in-situ and real-time observation, and is convenient and fast.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of fluorescent probes, and particularly relates to a fluorescent probe for detecting fluctuations in cholesterol during plasma membrane damage repair and a preparation method and application thereof. BACKGROUND

[0002] Plasma membrane damage can lead to impaired cell function, cell death, inflammation and immune response, affecting cell-cell interactions and communication, and imbalance of the intracellular and extracellular environment. Therefore, maintaining the integrity and function of the cell membrane is crucial for the normal function and health of cells and the whole organism.

[0003] Cholesterol (CL) is one of the important components of the plasma membrane, which forms a bilayer lipid structure together with phospholipids. The presence of cholesterol helps to stabilize the plasma membrane and endows it with certain rigidity and elasticity, regulating the fluidity of the cell membrane so that it can adapt to different environments and cell needs. Moreover, cholesterol regulates the permeability of the plasma membrane, and the presence of cholesterol in the plasma membrane can reduce the permeation of water and other solutes, thereby maintaining the osmotic balance between the inside and outside of the cell, and also regulating the function of membrane proteins and partitioning the membrane micro-region. These functions are crucial for maintaining the normal physiological processes and signal transduction of cells.

[0004] Cholesterol disorders can lead to abnormal fluidity of the cell membrane and permeability of the cell membrane, affecting the exchange of substances between the inside and outside of the cell and the movement of proteins on the cell membrane, thereby limiting the exchange of substances. Moreover, cholesterol disorders can affect the function of proteins on the cell membrane, change their conformation and function, interfere with the signal transduction process in the cell, and cause changes in the polarity of the cell membrane. High cholesterol levels can increase the non-polar characteristics of the cell membrane, making it more "rigid", while low cholesterol levels can lead to an increase in the polarity of the cell membrane.

[0005] Many diseases are closely related to abnormal levels of cholesterol CL. A decrease in the level of CL content is closely related to depression, cancer, and cerebral hemorrhage. An increase in the concentration of CL can induce atherosclerosis, increasing the risk of heart attack, stroke, and peripheral vascular disease. Therefore, selective detection of CL levels in the plasma membrane is an important task for in-depth pathological and biological research.

[0006] So far, various traditional methods, such as high-resolution mass spectrometry, enzyme analysis and electrochemical analysis method have been used to detect the CL level in the plasma membrane. However, destructive operation is a necessary prerequisite for CL analysis based on these methods, which can cause distortion and inaccuracy of information. In contrast, probe-based fluorescence imaging technology has obvious advantages, such as significant sensitivity, minimal damage to biological samples, and in situ and real-time observation. However, there are few reports on fluorescent probes for detecting CL in the plasma membrane. And due to the limited CL-induced polarity change of the plasma membrane, the sensitivity of the probe to the CL fluctuation in the plasma membrane is not high. It is still a difficult and important task to develop a high-contrast fluorescent probe for detecting the CL level in the plasma membrane. SUMMARY

[0007] The present application provides a fluorescent probe for visualizing and detecting cholesterol fluctuation in the plasma membrane, and further provides a preparation method and application of the probe. The target probe of the present application is replaced by PCFP unless otherwise specified.

[0008] To achieve the above technical purposes, the technical scheme adopted by the present application is:

[0009] A fluorescent probe for detecting cholesterol fluctuation during plasma membrane damage repair, the molecular formula of the probe is C 26 H 35 N3O2 2+ , and the structural formula is as follows:

[0010]

[0011] A preparation method of a fluorescent probe for detecting cholesterol fluctuation during plasma membrane damage repair, comprising the following preparation steps:

[0012] (1) 200 mg of 4-methylpyridine and 477 mg of (5-bromopentyl)-trimethylammonium bromide were placed in a 50 mL flask, dissolved in 5 mL of ethanol, and refluxed at 80°C under nitrogen protection for 5 h. After the reaction solution was cooled to room temperature, the reaction solution was concentrated by reduced pressure distillation, and then washed with petroleum ether to obtain white solid compound 2, and the structural formula of compound 2 is as follows:

[0013]

[0014]

[0015] (2) 100 mg of compound 2 and 98 mg of 7-(dimethylamino) coumarin-3-carbaldehyde were dissolved in 5 mL of ethanol, and 1 d of piperidine was added dropwise. The mixture was refluxed at 80°C under nitrogen protection for 3 h. The solvent was concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography to obtain the target probe PCFP, and the structural formula of PCFP is as follows:

[0016] ​The eluent of CH2Cl2 / CH3OH=5:1 was used for chromatographic purification to obtain the red solid powder, which was the target probe PCFP.

[0017] The synthesis route of the probe PCFP of the present application is as follows:

[0018]

[0019] The application of a fluorescence probe for detecting the fluctuation of cholesterol when the plasma membrane is damaged and repaired, which is used for detecting the dynamic change of the content of cholesterol in the plasma membrane when the plasma membrane is damaged and repaired in an in-vitro living cell environment to obtain intermediate information. The probe detects the change of fluorescence spectrum when the polarity of cholesterol concentration in a non-polar environment changes by using a fluorescence spectrometer, and detects the dynamic change of the content of cholesterol when the plasma membrane is damaged in an in-vitro living cell environment by using a confocal fluorescence microscope to obtain intermediate information.

[0020] The fluorescence probe PCFP of the present application has weak fluorescence in a 1,4-dioxane system, and exhibits 12-fold fluorescence enhancement at 575 nm after reacting with cholesterol. Moreover, the fluorescence probe exhibits good selectivity for cholesterol and is not affected by other interferents.

[0021] When the cell is in a normal state, the probe mainly locates in the plasma membrane and emits red fluorescence; if the content of cholesterol in the plasma membrane increases, the fluorescence signal of the probe in the plasma membrane is significantly enhanced; however, when the content of cholesterol in the plasma membrane decreases, the fluorescence signal of the probe in the plasma membrane is obviously weakened and the probe penetrates into the mitochondria through the plasma membrane. These experimental results show that the probe can detect cholesterol in the plasma membrane. In addition, after the cell is subjected to ultrasound-induced damage of the plasma membrane, a large amount of cholesterol is required for the repair process of the plasma membrane, and the fluorescence signal of the plasma membrane is enhanced by fluorescence imaging of the probe PCFP, which confirms that the content of cholesterol in the plasma membrane indeed increases. Through the controllable spatial distribution of the fluorescence signal of the probe PCFP in the plasma membrane-mitochondria, the dynamic change of the content of cholesterol on the plasma membrane when the plasma membrane is damaged and repaired can be explored.

[0022] Beneficial effects:

[0023] The present application utilizes the fact that a slight change in the level of CL in the plasma membrane can significantly change the permeability of the membrane, and designs a spatial distribution-controllable fluorescence probe to selectively detect the change of the level of CL in the plasma membrane when the state of the plasma membrane changes, so as to realize dynamic visual detection of the fluctuation of cholesterol in the plasma membrane. The obtained fluorescence probe has high sensitivity and causes minimal damage to biological samples, realizes in-situ and real-time observation, and is convenient and fast. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The 1HNMR spectrum of the probe PCFP in Example 1 of the present application;

[0025] Figure 2 13C NMR spectrum of probe PCFP in Example 1 of the present application;

[0026] Figure 3 HRMS spectrum of probe PCFP in Example 1 of the present application;

[0027] Figure 4 Fluorescence spectrum of probe PCFP in Example 2 of the present application in different solvents;

[0028] Figure 5 UV absorption spectrum of probe PCFP in Example 2 of the present application in different solvents;

[0029] Figure 6 Concentration titration graph of fluorescence enhancement of probe PCFP in Example 3 of the present application at 480 nm excitation and 575 nm emission after response to different concentrations of cholesterol in 1,4-dioxane system;

[0030] Figure 7 Columnar fluorescence data graph of selectivity of probe PCFP in Example 4 of the present application to different interfering analytes;

[0031] Figure 8 Fluorescence response curve graph of probe PCFP in Example 5 of the present application to different concentrations of cholesterol in the time range of 0-14 s;

[0032] Figure 9 Confocal laser fluorescence microscope image of probe PCFP in Example 6 of the present application after addition of exogenous cholesterol and β-cyclodextrin in HeLa cells and treatment with probe PCFP;

[0033] Figure 10 Confocal laser fluorescence microscope image of HeLa cells treated with DIO-tracker green and Mito-tracker green after addition of exogenous cholesterol and β-cyclodextrin in Example 7 of the present application and treatment with probe PCFP;

[0034] Figure 11 Confocal laser fluorescence microscope image of probe PCFP in Example 8 of the present application in HeLa cells treated under different ultrasonic conditions;

[0035] Figure 12 Confocal laser fluorescence microscope image of probe PCFP in Example 9 of the present application after ultrasonic treatment and addition of different concentrations of β-cyclodextrin to HeLa cells. DETAILED DESCRIPTION

[0036] The technical solutions of the present application are further described below in combination with specific embodiments, but are not limited thereto.

[0037] Example 1

[0038] Synthesis of compound 2:

[0039] Put 200 mg 4-methylpyridine, 477 mg (5-bromopentyl)-trimethylammonium bromide into a 50 mL flask, dissolve with 5 mL ethanol, reflux at 80 °C for 5 h under nitrogen protection; after the reaction solution is cooled to room temperature, the reaction solution is concentrated by distillation under reduced pressure, then washed with petroleum ether to obtain white solid compound 2, the synthesis route is as follows:

[0040]

[0041] Synthesis of probe PCFP:

[0042] Put 100 mg compound 2, 98 mg 7-(dimethylamino)coumarin-3-carboxaldehyde into 5 mL ethanol, drop 1 d piperidine, reflux at 80 °C for 3 h under nitrogen protection; the solvent is concentrated under reduced pressure to obtain the crude product, and the crude product is purified by chromatography with CH2Cl2 / CH3OH=5:1 eluent to obtain red solid powder PCFP (32 mg, yield 16.8%).1H NMR (500 MHz, MeOD) δ 8.83-8.81 (d, J = 6.8 Hz, 2H), 8.24 (s, 1H), 8.13-8.11 (d, J = 6.8 Hz, 2H), 7.87-7.77 (q, J = 16.0 Hz, 2H), 7.55-7.54 (d, J = 9.0 Hz, 1H), 6.85-6.83 (dd, J = 8.9, 2.4 Hz, 1H), 6.59-6.59 (d, J = 2.3 Hz, 1H), 4.60-4.57 (d, J = 7.4 Hz, 2H), 3.43–3.40 (m, 2H), 3.17 (s, 9H), 3.15 (s, 6H), 2.15-2.09 (dt, J = 15.4, 7.7 Hz, 2H), 1.95–1.88 (m, 2H), 1.52–1.46 (m, 2H).13C NMR (126 MHz, DMSO) δ 160.07 (s), 156.40 (s), 154.61 (s), 153.83 (s), 146.14 (s), 144.53 (s), 137.44 (s), 130.93 (s), 130.12 (s), 123.90 (s), 123.38 (s), 114.60 (s), 110.80 (s), 109.11 (s), 97.24 (s), 65.25 (s), 59.41 (s), 55.42 (s), 52.66 (s), 42.58 (s), 40.88 (s), 30.38 (s), 22.68 (s), 21.97 (s). HRMS [M] 2+Found, 210.6359, calculated for C 26 H 35 N3O2 2 + ,210.6360.

[0043] The synthetic route of PCFP is as follows:

[0044]

[0045] Example 2

[0046] Preparation of PCFP solution, determination of fluorescence spectrum and UV-Vis absorption spectrum properties

[0047] The probe PCFP prepared in Example 1 was dissolved in DMSO to prepare a probe stock solution with a concentration of 10 mM; the probe PCFP was dissolved in 2 mL of organic solvents with different polarities, and the final concentration of the probe PCFP was 10 μM. The fluorescence spectrum and UV absorption spectrum of the probe PCFP in different polar organic solvents were determined. As shown in Figure 4 : the fluorescence spectrum of different organic solvents was measured with wavelength as abscissa and normalized fluorescence intensity as ordinate, and the fluorescence measuring instrument used was Hitachi / F-7100 fluorescence spectrophotometer. As shown in Figure 5 : the UV-Vis absorption spectrum properties of different organic solvents were measured with wavelength as abscissa and normalized UV-Vis absorption intensity as ordinate, and the instrument used for UV-Vis absorption spectrum determination was Tianmei / UV2600 UV-Vis spectrophotometer.

[0048] Example 3

[0049] Concentration titration fluorescence spectrum test of the interaction of the fluorescence probe PCFP with cholesterol

[0050] Different concentrations (0-14 mg / mL) of cholesterol were added to 2 mL of 1,4-Dioxane solution with a probe concentration of 10 μM, and the fluorescence spectrum changes of the probe with different concentrations of cholesterol reaction solution were tested by fluorescence spectrometer (excitation wavelength was 480 nm, and the fluorescence emission peak at 500-900 nm was collected), and the fluorescence spectrum changes are shown in Figure 6 : with the addition of different concentrations of cholesterol, the fluorescence intensity value at 575 nm gradually increased. Figure 6 : wavelength as abscissa and fluorescence intensity as ordinate. From bottom to top, the concentrations of cholesterol were 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 mg / mL, and the fluorescence measuring instrument used was Hitachi / F-7100 fluorescence spectrophotometer.

[0051] Example 4

[0052] Selective test of fluorescent probe PCFP to different interfering analytes

[0053] 2 mL of 1,4-Dioxane test solution with probe concentration of 10 μM was respectively prepared into 14 mg / mL analyte or maximum solubility limit analyte: 1. Blank, 2. CL, 3. O2 - , 4. Na + , 5. Cl - , 6. GSH, 7. L-Histidine, 8. Val, 9. Ca 2+ , 10. Cu 2+ , 11. H2O2, 12. Sarcosine, 13. DNA, 14. SO4 2- , 15. Hcy, 16. Boc-L-glutamic acid 1-tert-butyl ester, 17. glyceryl trioleate, 18. Cys, 19. Oleic acid, 20. Sunflowerseed oil, 21. RNA, 22. Phosphatidyl cholines, 23. DMPC, after 14 s of reaction, the fluorescence spectrum change of the test solution was detected. As shown in Figure 7 : the fluorescence intensity of the test solution with added cholesterol was significantly enhanced, and the fluorescence intensity of the other test solutions did not change significantly, indicating that the probe PCFP has good selectivity to cholesterol. The fluorescence measuring instrument used was Hitachi / F-7100 fluorescence spectrophotometer.

[0054] Example 5

[0055] Time-dependent fluorescence intensity of fluorescent probe PCFP and cholesterol

[0056] 2 mL of 1,4-Dioxane test solution with probe concentration of 10 μM was respectively prepared into 4, 8, 12 mg / mL cholesterol analyte, and the time-dependent fluorescence intensity of the probe and different concentrations of cholesterol was tested. As shown in Figure 8 : with the increase of time, the fluorescence intensity at 575 nm gradually increased, and the fluorescence intensity reached a peak after 10 s. The fluorescence measuring instrument used was Hitachi / F-7100 fluorescence spectrophotometer.

[0057] Example 6

[0058] Confocal laser scanning fluorescence microscope images of probe PCFP in HeLa cells treated with exogenous cholesterol, β-cyclodextrin (cholesterol scavenger)

[0059] HeLa cells were incubated with saturated CL solution for 40 min, and then co-stained with 10 μΜ PCFP and DIO-tracker green (2 μΜ) for 30 min, or incubated with β-cyclodextrin (1 mM) for 1 h, and then co-stained with 10 μΜ PCFP and Mito-tracker green (2 μΜ) for 30 min. After washing with PBS, the cells were imaged. The microscopic imaging was performed using confocal laser scanning microscope (CLSM, Zeiss LMS880). The excitation wavelength of DIO-tracker green and Mito-tracker green was 488 nm, and the emission wavelength range was 500-550 nm; the excitation wavelength of probe PCFP was 561 nm, and the emission wavelength range was 570-616 nm. Figure 9 As shown in FIG. 6, the red channel fluorescence image of PCFP and the green channel fluorescence image of Mito-tracker green and DIO-tracker green all have good overlap effect, and the Pearson correlation coefficients are 0.90 and 0.91, respectively, indicating that the probe is mainly distributed in the plasma membrane and mitochondria, and the spatial distribution is controlled by the cholesterol content in the plasma membrane.

[0060] Example 7

[0061] Confocal laser scanning fluorescence microscope images of probe PCFP in HeLa cells treated with different ultrasound conditions

[0062] HeLa cells were incubated with saturated CL solution for 40 min, and then co-stained with 10 μΜ PCFP and DIO-tracker green (2 μΜ) for 30 min, or incubated with β-cyclodextrin (1 mM) for 1 h, and then co-stained with 10 μΜ PCFP and Mito-tracker green (2 μΜ) for 30 min. After washing with PBS, the cells were imaged. The microscopic imaging was performed using confocal laser scanning microscope (CLSM, Zeiss LMS880). The excitation wavelength of DIO-tracker green and Mito-tracker green was 488 nm, and the emission wavelength range was 500-550 nm; the excitation wavelength of probe PCFP was 561 nm, and the emission wavelength range was 570-616 nm. Figure 10 As shown in FIG. 6, the red channel fluorescence image of PCFP and the green channel fluorescence image of Mito-tracker green and DIO-tracker green all have good overlap effect, and the Pearson correlation coefficients are 0.90 and 0.91, respectively, indicating that the probe is mainly distributed in the plasma membrane and mitochondria, and the spatial distribution is controlled by the cholesterol content in the plasma membrane.

[0063] Example 8

[0064] Confocal laser scanning fluorescence microscope images of probe PCFP in HeLa cells treated with different ultrasound conditions

[0065] HeLa cells were treated with 1 W ultrasonic for 0, 0.5, 1, 2 min, respectively, and then 10 μM PCFP was added for 30 min. Microscopic imaging was performed using confocal laser scanning microscope (CLSM, Zeiss LMS880) with the probe PCFP excitation wavelength of 561 nm and the emission wavelength range of 570-616 nm. As shown in FIG. 2: Figure 11 It can be observed that the fluorescence of the probe PCFP on the plasma membrane is enhanced with the increase of the degree of cell damage, indicating that the content of cholesterol in the plasma membrane repair process is increased and depends on the degree of damage.

[0066] Example 9

[0067] Confocal laser fluorescence microscope images of the probe PCFP and HeLa cells treated with ultrasonic and then added with different concentrations of β-cyclodextrin

[0068] HeLa cells were treated with 1 W ultrasonic for 2 min, and then 0.25, 0.5, 1 mM β-cyclodextrin was added for 1 h, and then 10 μM PCFP was added for 30 min. Microscopic imaging was performed using confocal laser scanning microscope (CLSM, Zeiss LMS880) with the probe PCFP excitation wavelength of 561 nm and the emission wavelength range of 570-616 nm. As shown in FIG. 4: Figure 12 It can be observed that the fluorescence of the probe PCFP on the plasma membrane is gradually weakened with the decrease of the content of cholesterol in the plasma membrane caused by the increase of β-cyclodextrin.

[0069] It should be noted that the above examples are only part of the preferred modes of implementing the present application, but not all. Obviously, based on the above examples of the present application, all other examples obtained by those skilled in the art without creative labor should belong to the scope of protection of the present application.

Claims

1. A fluorescent probe for detecting cholesterol fluctuations during plasma membrane damage and repair, characterized in that: The molecular formula of the probe is C 26 H 35 N3O2 2+ , the structural formula is as follows: 。 2. A method for preparing a fluorescent probe for detecting cholesterol fluctuations during plasma membrane damage and repair according to claim 1, characterized in that: The method comprises the following preparation steps: (1) 200 mg of 4-methylpyridine and 477 mg of (5-bromopentyl)-trimethylammonium bromide were placed in a 50 mL flask, 5 mL of ethanol was added to dissolve, and the mixture was refluxed at 80°C for 5 h under nitrogen protection. After the reaction solution was cooled to room temperature, it was concentrated by vacuum distillation and then washed with petroleum ether to obtain compound 2 as a white solid. The structural formula of compound 2 is: ； (2) 100 mg of compound 2 and 98 mg of 7-(dimethylamino)coumarin-3-carboxaldehyde were dissolved in 5 mL of ethanol, 1d piperidine was added dropwise, and the mixture was refluxed at 80°C for 3 h under nitrogen protection; the solvent was concentrated under reduced pressure to obtain a crude product, which was purified by chromatography using an eluent of CH2Cl2 / CH3OH=5:1 to obtain a red solid powder, which was the target probe PCFP.

3. Use of the fluorescent probe for detecting cholesterol fluctuations during plasma membrane damage and repair according to claim 1 in non-disease diagnosis and treatment, characterized in that: The probe is used to detect the dynamic changes of cholesterol content in the plasma membrane during plasma membrane damage repair in an in vitro living cell environment, so as to obtain intermediate information.

Images