A fluorescent probe for detecting lysosomal viscosity, and a preparation method and application thereof

Abstract

The application belongs to the technical field of polymer fluorescent probes, and provides a fluorescent probe for detecting lysosome viscosity, wherein n is 10-30. The probe is used for tracing the change of lysosome viscosity in cells by a fluorescence imaging technology, and can be used for evaluating and researching physiological and pathological processes related to lysosome viscosity.

Description

Technical Field

[0001] This invention belongs to the field of polymer fluorescent probe technology, specifically relating to a fluorescent probe for detecting lysosomal viscosity and its application. Background Technology

[0002] Lysosomes are organelles in eukaryotic cells, containing various hydrolytic enzymes that break down large molecules from both exogenous and endogenous sources, playing a crucial role in various physiological processes within organisms. A stable lysosomal microenvironment is closely related to lysosomal function; microenvironmental disturbances can lead to lysosomal dysfunction and consequently metabolic diseases. A stable lysosomal microenvironment is beneficial for maintaining normal physiological function, and key parameters include viscosity, polarity, temperature, and pH. Viscosity plays a significant role at both the subcellular and cellular levels. Abnormal changes in viscosity are associated with many diseases, including Alzheimer's disease, atherosclerosis, diabetes, and even cancer. Therefore, tracking changes in lysosomal viscosity can help in better studying the physiological and pathological functions of lysosomes.

[0003] In recent years, fluorescent probes have become effective tools for studying lysosomal viscosity changes due to their advantages such as non-destructiveness, high sensitivity, real-time and in-situ detection. As a class of functional polymers, methacrylate polymers have been widely used in many fields, but their application as fluorescent probe matrices is rare. Methacrylate polymers can be synthesized through atom transfer radical polymerization (ATRP), and their polymer chains can emit atypical fluorescence, showing potential applications in fluorescence imaging. Therefore, we constructed a novel polymeric fluorescent probe with dual-response sites for detecting viscosity changes. Existing viscosity probes are mostly small-molecule fluorescent probes, which have poor stability and are easily cleared. Developing biocompatible polymeric lysosomal viscosity probes is of great significance in order to improve the water solubility and photostability of probes. Summary of the Invention

[0004] To address the problems in the prior art, the present invention provides a fluorescent probe for detecting lysosomal viscosity, which is stable in cells and has good biocompatibility.

[0005] Another object of the present invention is to provide an application of the above-mentioned fluorescent probe in detecting and locating lysosomes and detecting their viscosity.

[0006] To achieve the above objectives, the present invention adopts the following technical solution.

[0007] A fluorescent probe for detecting lysosomal viscosity, abbreviated as In-PHEM, has the chemical structure shown in formula (I):

[0008]

[0009] Where n = 10-30

[0010] Formula (I).

[0011] The preparation method of the above-mentioned fluorescent probe includes the following steps:

[0012] (1) Using CuBr as catalyst, CHOBr as initiator, and 2,2'-bipyridine as ligand, 2-hydroxyethyl methacrylate (HEM) was polymerized in isopropanol under oxygen-free conditions, and the intermediate CHOBr-PHEM was obtained by separation and purification.

[0013] (2) The intermediate CHOBr-PHEM and 1,2,3,3-tetramethyl-3H-indole iodide were heated in ethanol and reacted, and the product In-PHEM was obtained by separation and purification.

[0014] Preferably, step (1) includes the following steps: dispersing CHOBr, 2-hydroxyethyl methacrylate, CuBr and 2,2'-bipyridine in isopropanol, and completing three freezing-vacuuming-thawing processes in a protective atmosphere; then adding CuBr and repeating the freezing-vacuuming-thawing process three more times in a protective atmosphere; and reacting at 30°C after thawing.

[0015] Preferably, the separation and purification in step (1) includes the following steps: after the reaction is completed, dichloromethane is added to the system and passed through a neutral alumina column, the effluent is precipitated in petroleum ether, and the precipitate is dried.

[0016] In step (2), the molar ratio of CHOBr-PHEM polymer to 1,2,3,3-tetramethyl-3H-indole iodide is 1:5.

[0017] Preferably, in step (2), the heating temperature is 90°C.

[0018] Preferably, the separation and purification in step (2) includes the following steps: after the reaction is completed, the solvent is removed, and the residue is dripped into petroleum ether to precipitate; the precipitate is redispersed with methanol, then filtered, and the solvent is removed from the filtrate.

[0019] An application of the above-mentioned fluorescent probe in locating lysosomes or detecting lysosomal viscosity.

[0020] The mechanism of this invention is as follows:

[0021] The polymer chains on the In-PHEM probe enable it to effectively locate lysosomes. In a non-viscous environment, the polymer chains emit weak blue fluorescence, while the fluorophore emits very weak light because the rotor portion can rotate freely, resulting in energy loss through non-radiative transitions. In a viscous environment, due to the increased rigidity of the polymer chains and the suppression of molecular rotor rotation, the probe emits strong blue and green fluorescence.

[0022] The present invention has the following advantages:

[0023] The probe of this invention can be used to track changes in lysosomal viscosity in cells using fluorescence imaging technology. It is stable in cells and has good biocompatibility, and can be used to evaluate and study physiological and pathological processes related to lysosomal viscosity. Attached Figure Description

[0024] Figure 1 It is an In-PHEM fluorescent probe. 1 H NMR spectrum;

[0025] Figure 2 The fluorescence spectra of the fluorescent probe In-PHEM in mixed solvents of methanol and glycerol at different volume fractions are shown; the excitation wavelengths are 350 nm (a) and 440 nm (b); the probe concentration is 60 µg / mL.

[0026] Figure 3 The selectivity of the fluorescent probe In-PHEM is as follows: excitation wavelengths are 350 nm (a) and 440 nm (b); probe concentration: 60 µg / mL, analyte concentration: 100 mM;

[0027] Figure 4 This is a co-localization imaging of the fluorescent probes In-PHEM and Lyso-Tracker Red in HeLa cells; the excitation wavelengths were 488 nm (In-PHEM) and 561 nm (Lyso-Tracker Red); the probe concentration was 60 µg / mL.

[0028] Figure 5 The cytotoxicity of the fluorescent probe In-PHEM; the relative cell viability after incubation of cells with different concentrations (0, 6, 12, 30, 60 and 120 μg / mL) of In-PHEM for 24 h;

[0029] Figure 6 This is an imaging study of HeLa cells and cells treated with a viscosity inducer using the fluorescent probe In-PHEM; the excitation wavelengths are 405 nm and 488 nm; the probe concentration is 60 µg / mL. Detailed Implementation

[0030] The present invention will be further described below with reference to the embodiments and accompanying drawings, but the present invention is not limited to the following embodiments.

[0031] Example 1 Synthesis of fluorescent probes

[0032]

[0033] (1) Synthesis of polymer CHO-PHEM:

[0034] CHOBr was synthesized according to the method described by Narain R. et al., 2003 (Narain R, Armes S P. Synthesis and Aqueous Solution Properties of Novel Sugar Methacrylate-Based Homopolymers and BlockCopolymers[J]. Macromolecules, 2003, 4(6): 1746-1758).

[0035] CHOBr (104 mg, 0.39 mmol), 2-hydroxyethyl methacrylate (520 mg, 4 mmol), CuBr (20 mg), and 2,2'-bipyridine (40 mg) were dispersed in 2.5 mL of isopropanol. Under N2 protection, the mixture underwent three freeze-vacuum-thawing cycles. Then, 10 mg (0.1 mmol) of CuBr was added, and the mixture was stirred until homogeneous. Under N2 protection, the freeze-vacuum-thawing cycle was repeated three times. After thawing, the mixture was stirred until homogeneous and reacted in an oil bath at 30 °C for 12 h. After the reaction was completed, 3 mL of dichloromethane (CH2Cl2) was added, and the copper salt was removed using a neutral alumina column. The effluent was precipitated in petroleum ether, allowed to stand for 1 h, and the supernatant was poured off. The precipitate was dried under vacuum to obtain a white solid, namely CHO-PHEM.

[0036] (2) Synthesis of polymer In-PHEM:

[0037] CHO-PHEM (40 mg) and 1,2,3,3-tetramethyl-3H-indole iodide (40 mg, 0.94 mmol) were placed in a round-bottom flask and dissolved in ethanol (10 mL). The mixture was stirred at 90 °C for 24 h. After the reaction was complete, the solvent was removed by rotary evaporation, and the residue was added dropwise to a large amount of petroleum ether to precipitate. The precipitate was redispersed with methanol, then filtered under reduced pressure. The filtrate was evaporated by rotary evaporation to remove the solvent, yielding an orange viscous solid. 1 H NMR spectrum as follows Figure 1 .

[0038] Example 2: Response of fluorescent probe to methanol-glycerol mixed solvent

[0039] A stock solution of the lysosomal viscosity fluorescent probe In-PHEM described in this invention with a concentration of 6 mg / mL was prepared for later use. A probe concentration of 60 µg / mL was prepared by adding different volume fractions (0:10, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, and 10:0) of methanol and glycerol mixed solvents, and fluorescence detection (λ) was performed. ex(At 350 nm and 440 nm), its fluorescence spectra are as follows: Figure 2 As shown. By Figure 2 It can be observed that the fluorescence of the In-PHEM probe gradually increases with increasing viscosity.

[0040] Example 3: Selectivity of fluorescent probes for different ions

[0041] Prepare 5 mL of PBS aqueous solutions containing various common ions, thiols, and reactive oxygen species at a concentration of 100 mM, and an In-PHEM fluorescent probe stock solution with a concentration of 6 mg / mL as described in this invention, based on lysosomal viscosity, for later use. Add 20 μL of probe stock solution, 200 μL of DMSO, and 10 equivalents of ions, thiols, and reactive oxygen species, and bring the volume to 2 mL with phosphate-buffered saline (PBS) (pH 7.4). After mixing, perform fluorescence detection (λ). ex (At 350 nm and 440 nm), construct a bar chart of fluorescence intensity versus each ion (or reactive oxygen species). Figure 3 (The list includes ingredients 1-24, which are: PBS solution, calcium chloride, cobalt chloride, copper chloride, ferric chloride, mercuric chloride, potassium chloride, magnesium chloride, nickel chloride, tin chloride, glucose, glycine, glutathione, cysteine, sodium thiosulfate, sodium sulfite, sodium hypochlorite, sodium bisulfite, sodium nitrite, sodium acetate, hydroxide ions, peroxynitrite ions, hydrogen peroxide, and glycerol.) Figure 3 It can be observed that conventional ions (thiols or reactive oxygen species) have almost no effect on the fluorescence of polymer In-PHEM.

[0042] Example 4: Co-localization of fluorescent probes and commercial probes

[0043] HeLa cells were placed in culture medium (DMEM and 10% fetal bovine serum) and cultured in an incubator at 37°C, 5% CO2, and 20% O2 for 24-48 hours. The fluorescent probe described in Example 1 (60 µg / mL) and the commercially available lysosomal localization dye Lyso-Tracker Red (1 μM) were added to the HeLa cells. After culturing for 30 minutes, laser confocal imaging was performed. The excitation wavelength for the green channel was 488 nm, and the collection wavelength range was 500-550 nm; the excitation wavelength for the red channel was 561 nm, and the collection wavelength range was 570-620 nm. The imaging results are as follows: Figure 4 As shown in the figure, ad is the In-PHEM fluorescence image of HeLa cells co-stained with the commercial dye Lyso-Tracker Red; e is the ROI intensity distribution in the green and red channels; and F is the intensity scatter plot of the two channels. As can be seen from the figure, the fluorescence signal overlap coefficient between the fluorescent probe of this invention and commercial lysosomes in cells is as high as 0.90, indicating that the fluorescent probe can locate lysosomes.

[0044] Example 5: Cell toxicity of fluorescent probes

[0045] The MTT assay was used to detect the cytotoxicity of different concentrations (0, 6, 12, 30, 60, and 120 μg / mL) of the In-PHEM probe against HeLa live cells. Figure 5 As shown, when the concentration was increased to 120 μg / mL, the cell survival rate was as high as 90%, indicating that In-PHEM has low cytotoxicity to live cells.

[0046] Application Example 1: Application of fluorescent probes in imaging viscosity changes in HeLa cells

[0047] Prepare 1 mL of PBS solution (pH 7.2) containing the In-PHEM probe at a concentration of 60 µg / mL. Add several HeLa cell lines to PBS solution or 10 µM lipopolysaccharide (LPS), monensin (Mon), or nystatin (Nys), respectively, and stimulate for 30 min. Then add the probe and incubate for another 30 min before imaging. Excitation wavelengths were 405 nm and 488 nm, and emission wavelengths were 425-475 nm and 500-550 nm. Results are as follows: Figure 6 As shown, ad represents HeLa cells, eh represents cells after lipopolysaccharide (LPS) stimulation, il represents cells after monensin (Mon) stimulation, and mp represents cells after nystatin (Nys) stimulation; a, e, i, and m represent bright field; b, f, j, and n represent blue fluorescence field after incubation with a 60 μg / mL probe for 30 min; c, g, k, and o represent green fluorescence field after incubation with a 60 μg / mL probe for 30 min; d, h, l, and p represent superimposed fields. Figure 5 As can be seen, compared with PBS solution, HeLa cells pretreated with LPS, Mon, or Nys showed significant enhancement of blue and green fluorescence, which is attributed to the increase in lysosomal viscosity; this indicates that the fluorescent probe In-PHEM can respond to changes in lysosomal viscosity in cells.

Claims

1. A fluorescent probe for detecting lysosomal viscosity, the chemical structure of which is shown in formula (I): Where n = 10-30 Formula (I).

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, CHOBr as an initiator, and 2,2'-bipyridine as a ligand, 2-hydroxyethyl methacrylate was polymerized in isopropanol under oxygen-free conditions. The intermediate CHOBr-PHEM was obtained by separation and purification. ; (2) The intermediate CHOBr-PHEM and 1,2,3,3-tetramethyl-3H-indole iodide were heated in ethanol and reacted. The product was then separated and purified to obtain the fluorescent probe for detecting lysosomal viscosity. The structural formula of CHOBr is as follows: .

3. The preparation method according to claim 2, characterized in that, Step (1) includes the following steps: CHOBr, 2-hydroxyethyl methacrylate, CuBr and 2,2'-bipyridine are dispersed in isopropanol and the freezing-vacuuming-thawing process is completed three times in a protective atmosphere; CuBr is then added and the freezing-vacuuming-thawing process is repeated three times in a protective atmosphere; after thawing, the reaction is carried out at 30°C.

4. The preparation method according to claim 2, characterized in that, The separation and purification in step (1) includes the following steps: after the reaction is completed, dichloromethane is added to the system and passed through a neutral alumina column, the effluent is precipitated in petroleum ether, and the precipitate is dried.

5. The preparation method according to claim 2, characterized in that, In step (2), the molar ratio of CHOBr-PHEM polymer to 1,2,3,3-tetramethyl-3H-indole iodide is 1:

5.

6. The preparation method according to claim 2, characterized in that, In step (2), the heating temperature is 90°C.

7. The preparation method according to claim 2, characterized in that, The separation and purification in step (2) includes the following steps: after the reaction is complete, remove the solvent and drop the residue into petroleum ether to precipitate; redisperse the precipitate with methanol and then filter it, removing the solvent from the filtrate.

8. The use of a fluorescent probe as described in claim 1 in locating lysosomes or detecting lysosomal viscosity.

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