Ratio-type fluorescent probe for detecting polarity of lipid droplet as well as preparation method and application of ratio-type fluorescent probe

Abstract

The invention belongs to the technical field of fluorescent polymer synthesis, and provides a ratio-type fluorescent probe for detecting the polarity of a lipid droplet, and a preparation method and application of the ratio-type fluorescent probe. The structural formula of the fluorescent probe is shown in the specification. The compound can be prepared into a fat droplet tracer for detecting cells or organisms or a reagent for detecting polarity change of fat droplets. The probe is strong in water solubility and good in light stability, can be developed into a good biocompatibility lipid droplet tracing and polarity detection reagent, and has wide application value in medical diagnosis and clinic.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of fluorescent polymer synthesis, and particularly relates to a fluorescent probe for detecting lipid droplet polarity. BACKGROUND

[0002] Lipid droplets (LDs), the key organelles for intracellular lipid storage, play an irreplaceable role in many life processes, including energy metabolism, membrane lipid synthesis and signal transduction. However, abnormal states of LDs, such as changes in size, number and composition, have been proved to be closely related to metabolic diseases (such as obesity and diabetes), neurodegenerative diseases (such as Alzheimer's disease and Parkinson's disease) and cardiovascular diseases (such as atherosclerosis). With the in-depth study of lipid droplet-related diseases, it is found that the abnormal function of lipid droplets is often accompanied by changes in the microenvironment, such as polarity or viscosity. Polarity is a key factor affecting cell function and regulatory mechanisms, and is involved in various physiological processes in vivo. Changes in the polarity of LDs are related to many diseases. Therefore, tracking the changes in the polarity of lipid droplets helps better study the physiological and pathological effects related to lysosomes.

[0003] In recent years, polymer probes have attracted the attention of researchers due to their good light stability, low biological toxicity and structure-adjustable advantages. As a kind of functional polymer, polyethylene glycol methyl ether methacrylate has been widely used in many fields, but its application as a fluorescent probe substrate is rare. They can be synthesized by atom transfer radical polymerization (ATRP), and the molecular weight is controllable. The polymer chain can emit visible fluorescence, and has potential application prospects in fluorescence imaging. SUMMARY

[0004] In view of the problems in the prior art, the application provides a fluorescent probe for detecting the polarity of lipid droplets, which has good stability, is less affected by background and has good biocompatibility.

[0005] Another object of the application is to provide an application of the above fluorescent probe in detecting the polarity changes of lipid droplets in cells, tissues or biological cells.

[0006] To achieve the above object, the application adopts the following technical solutions.

[0007] A ratio-type fluorescent probe for detecting the polarity of lipid droplets, referred to as NP-PEGMA, has the chemical structural formula: ; In the formula, m is 5 on average, and n is 10-20.

[0008] The preparation method of the fluorescent probe comprises the following steps: (1) using CuBr as a catalyst, The intermediate NapBr-PEGMA is obtained by initiating polymerization of polyethylene glycol methyl ether methacrylate in dichloromethane and isopropyl alcohol under an oxygen isolation condition, using (NapBr) as an initiator and 2,2'-dipyridyl as a ligand. The product NP-PEGMA is obtained by heating and reacting the intermediate NapBr-PEGMA, 4-boronic aniline, potassium carbonate and tetrakis-(triphenylphosphine)palladium in tetrahydrofuran, and then isolating and purifying.

[0009] The average molecular weight of the polyethylene glycol methyl ether methacrylate is 300.

[0010] The volume ratio of the dichloromethane and isopropyl alcohol is 3:1.

[0011] The molar ratio of the NapBr and polyethylene glycol methyl ether methacrylate is 1:10.

[0012] The mass ratio of the intermediate NapBr-PEGMA and 4-boronic aniline is 10:1.

[0013] The above-mentioned fluorescent probe can be prepared into a lipid droplet tracer or a reagent for detecting the polarity change of lipid droplets in cells or organisms.

[0014] The mechanism of the present application is as follows: In the NP-PEGMA molecule, the two amide groups in the naphthalimide are electron-withdrawing groups and can also act as electron acceptors. Diphenylamine acts as an excellent donor, which helps to extend the conjugated structure of the compound. At the same time, the benzyl group is used to connect the electron donor and acceptor parts, and its rigidity is conducive to inhibiting non-radiative energy loss. The probe emits strong green fluorescence in a lower polarity environment, and emits weak fluorescence in a higher polarity environment due to its solvent kinetic effect. The introduction of the atypical fluorescent polymer makes the probe itself have good lipophilicity, which is convenient for penetrating the cell membrane into the cell; after entering the cell, the probe actively accumulates in the lipid droplets; the blue and green fluorescence emitted by the atypical fluorescent polymer and the naphthalimide fluorophore realizes the ratio imaging of the polarity change of the lipid droplets.

[0015] The present application has the following advantages: The existing polarity probes are mostly small molecule fluorescent probes, which have poor stability and are easily removed, and usually use a single fluorescent signal which is easily affected by the concentration of the probe and the intracellular environment. The present application provides a new polymer ratio fluorescent probe NP-PEGMA for monitoring the polarity change of LDs with high selectivity and high stability, which can be developed into a good biocompatible polymer lipid droplet polarity ratio fluorescent probe, and has wide application value and significance in medical diagnosis and clinical application. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1is the intermediate NapBr-PEGMA 1 H NMR spectrum; Figure 2 is the fluorescent probe NP-PEGMA 1 H NMR spectrum; Figure 3 is the response of the fluorescent probe NP-PEGMA to different proportions of water and 1,4-dioxane mixed solvents Figure 4 is the response of the fluorescent probe NP-PEGMA to 1,4-dioxane solution in different interferents Figure 5 is the fluorescent imaging image of the co-localization of the fluorescent probe NP-PEGMA and Nile Red with lipid droplets in HeLa cells Figure 6 is the fluorescent image of the fluorescent probe NP-PEGMA on oleic acid pretreated HeLa cells Figure 7 is the fluorescence intensity ratio of the fluorescent probe NP-PEGMA on different channels of oleic acid pretreated HeLa cells Figure 8 is the fluorescent image of the fluorescent probe on lipopolysaccharide pretreated HeLa cells Figure 9 is the fluorescence intensity ratio of the fluorescent probe NP-PEGMA on different channels of lipopolysaccharide pretreated HeLa cells DETAILED DESCRIPTION

[0017] The present application will be further described below in conjunction with examples and drawings, but the present application is not limited by the following examples.

[0018] Example 1 Synthesis of fluorescent probe 1. Synthesis of polymer NapBr-PEGMA Synthesis of NapBr according to the method of Lin W. et al., 2016 (Zhang H., Kong X., Tang Y., Lin W. Hydrogen Sulfide Triggered Charge-Reversal Micelles for Cancer Targeted Drug Delivery and Imaging [J]. ACS Appl. Mater. Interfaces 2016, 8 (25), 16227-16239); NapBr (182 mg, 0.38 mmol), polyethylene glycol methyl ether methacrylate (1.2 g, 4 mmol, average molecular weight 300) and 2,2'-bipyridine (80 mg) were dispersed in 2.8 mL of a solvent mixture of dichloromethane / isopropyl alcohol (3:1, v:v) and subjected to three freeze-vacuum-thaw processes under N2protection; then CuBr (25 mg, 0.17 mmol) was added and, after stirring to homogeneity, the three freeze-vacuum-thaw processes were repeated again; after thawing, stirring to homogeneity was performed and the mixture was left to react at room temperature for 24 h; after the reaction was completed, 3 mL of dichloromethane (CH2Cl2) was added to terminate the reaction and a neutral alumina column was used to remove the copper salt; the filtrate was precipitated in petroleum ether, stirred to homogeneity, filtered and dried under vacuum to obtain NapBr-PHGMA as a colorless viscous oil; its 1 H NMR spectrum is shown in Figure 1 According to the hydrogen on the naphthalene ring (labeled as a) and the hydrogen on the end methyl group of the polymer (labeled as l), the value of n is calculated to be about 11.

[0019] 2. NP-PEGMA synthesis NapBr-PHGMA (50 mg), 4-boronic acid triphenylamine (43 mg), potassium carbonate (61 mg) and tetrakis-(triphenylphosphine)palladium (5 mg) were dissolved in a round-bottom flask with an appropriate amount of tetrahydrofuran (THF) and stirred in an oil bath at 60°C under N2protection for 12 h; after the reaction was completed, the mixture was added dropwise to a large amount of petroleum ether to precipitate; the resulting product was dried under vacuum at 25°C for 24 h to obtain NP-PEGMA as a yellow solid, which 1 H NMR spectrum is shown in Figure 2 .

[0020] Example 2. Response of fluorescent probe to different polarities A stock solution of the fluorescent probe NP-PEGMA prepared in Example 1 was prepared at a concentration of 6 mg / mL for standby. The probe was prepared at a concentration of 60 µg / mL and added to different proportions of water and 1,4-dioxane mixed solvents, respectively, and subjected to fluorescence detection (λ ex ex= 350 and 440 nm).

[0021] The fluorescence emission spectrum of NP-PEGMA in different proportions of 1,4-dioxane and water mixed solvents under excitation at a wavelength of 350 nm is shown in Figure 3 a: NP-PEGMA shows a maximum emission peak at a wavelength of 417 nm, and the fluorescence intensity of the probe changes little with the increase of water content in the mixed solvent Figure 3a). Under excitation at 440 nm, NP-PEGMA exhibits a maximum emission peak with a redshift from 578 to 609 nm. The fluorescence intensity decreases significantly as the water content in the mixed solvent increases from 0% to 20%. Figure 3 b). The ratio of fluorescence intensity at 578 nm to fluorescence intensity at 417 nm decreases with increasing polarity. 578 / I 417 The ratio decreased from 1.317 to 0.016, a ratio response of approximately 82.3 times, indicating that the probe has a sensitive ratio response to polarity. Figure 3 c). Furthermore, the fluorescence intensity ratio (I... 578 / I 417 There is a good linear relationship between the water constant (0%-2%) and the water constant (R). 2 = 0.9806)( Figure 3 c).

[0022] Example 3: Selectivity of fluorescent probes for different ions Prepare 5 mL of PBS aqueous solution with a concentration of 100 mM for various common ions, thiols and reactive oxygen species, and NP-PEGMA stock solution with a concentration of 6 mg / mL for later use.

[0023] Add 30 μL of probe stock solution and 10 equivalents of ions, thiols, and reactive oxygen species to 3 mL of a mixed solvent of 1,4-dioxane and water (8:2, V:V), shake well, and then perform fluorescence detection (λ). ex (350 and 440 nm), establish the fluorescence intensity ratio of NP-PEGMA with different analytes (I 578 / I 417 (a bar chart of ).

[0024] The results are as follows Figure 4 As shown, after adding various substances, the fluorescence intensity ratio of NP-PEGMA (IF) was [not specified]. 578 / I 417 The extremely small fluctuations indicate that NP-PEGMA has a negligible response to various interfering substances, and the probe has good selectivity.

[0025] Example 4: Co-localization of fluorescent probes and commercial probes HeLa cells were placed in culture medium (90% DMEM medium and 10% fetal bovine serum) and incubated in an incubator with conditions of 37°C, 5% CO2 and 20% O2 for 24-48 h. The fluorescent probe described in Example 1 (concentration of 60 μg / mL) and commercial lipid droplet localization dye Nile Red were added to the HeLa cells, and after incubation for 30 min, laser confocal imaging was performed. The excitation wavelength of the green channel was 488 nm. The wavelength range collected was 500-550 nm; the excitation wavelength of the red channel was 488 nm, and the wavelength range collected was 570-620 nm.

[0026] The imaging results are shown in Figure 5 It can be seen that the fluorescence signal overlap coefficient of the fluorescent probe of the present application and the commercial lipid droplet dye in cells is as high as 0.96, indicating that the fluorescent probe can localize lipid droplets.

[0027] Example 5 Imaging application of fluorescent probe in living cells 1. Oleic acid treatment HeLa cells were pretreated with different concentrations of oleic acid OA (0 μM, 25 μM and 50 μM) for 7 h, then NP-PEGMA (60 μg / mL) was added and incubated for 30 min before imaging. The excitation wavelengths were 405 and 488 nm, and the emission wavelengths were 425-475 and 500-550 nm.

[0028] It can be seen from Figure 6 and Figure 7 that the fluorescence intensity ratio increases with the increase of the concentration of OA. This result indicates that the stimulation of OA increases the number of LDs in cells, and at the same time, the polarity of LDs is slightly reduced due to the change of oil components.

[0029] 2. Lipopolysaccharide treatment HeLa cells were treated with 15 μg / mL lipopolysaccharide (LPS) for 1 h and 3 h, then NP-PEGMA (60 μg / mL) was added, and after incubation for 30 min, imaging was performed. The excitation wavelengths were 405 and 488 nm, and the emission wavelengths were 425-475 and 500-550 nm.

[0030] The results are shown in Figure 8 and Figure 9 It can be seen that the fluorescence ratio of the LPS treatment group is greater than that of the normal group, and the ratio increases with the extension of the treatment time, indicating that the polarity of LDs decreases after LPS treatment.

Claims

1. A ratiometric fluorescent probe for detecting lipid droplet polarity, the chemical structural formula of which is: ； in, The average value of m is 5, and the average value of n is 10-20.

2. A method for preparing a fluorescent probe as described in claim 1, characterized in that, Includes the following steps: (1) Using CuBr as a catalyst, Using 2,2'-bipyridine as an initiator and oxygen-free conditions, polyethylene glycol methyl ether methacrylate was polymerized in a mixture of dichloromethane and isopropanol. The intermediate NapBr-PEGMA was obtained by separation and purification. (2) The intermediate NapBr-PEGMA, 4-boronic acid triphenylamine, potassium carbonate and tetra-(triphenylphosphine)palladium were heated and reacted in tetrahydrofuran, and the product NP-PEGMA was obtained by separation and purification.

3. The preparation method according to claim 2, characterized in that, The average molecular weight of the polyethylene glycol methyl ether methacrylate is 300; The volume ratio of dichloromethane to isopropanol is 3:1; The The molar ratio of polyethylene glycol methyl ether methacrylate to polyethylene glycol methyl ether methacrylate is 1:10; The mass ratio of the intermediate NapBr-PEGMA to triphenylamine 4-boronic acid is 10:

1.

4. A reagent for preparing lipid droplet tracers or detecting changes in lipid droplet polarity in cells using the fluorescent probe as described in claim 1.

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