Blue fluorescent probe targeting mitochondria and application thereof

Abstract

The application belongs to the technical field of fluorescent dye preparation, and particularly relates to a blue fluorescent probe targeting mitochondria and application, wherein the fluorescent probe is aristolane I, the distribution of the probe in cells is related to the membrane potential of mitochondria, the probe can enter cells by passive diffusion, can exist in cells for a long time, can target mitochondria, and provides a self-stable and long-lasting blue fluorescent reagent for mitochondrial imaging, and the reagent can be prepared into a kit, the preparation process is simple, the equipment cost is low, and the reagent has good application potential in the field of mitochondrial imaging.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of fluorescent dye preparation, and particularly relates to a blue fluorescent probe targeting mitochondria and application thereof. BACKGROUND

[0002] Mitochondrion is a subcellular organelle with a double-membrane structure, which exists in most eukaryotic cells and is the main place for ATP production in cells. In recent years, it has been found that mitochondria are closely related to cell growth and differentiation, cell information transmission and cell apoptosis, in addition to providing energy for cells. Mitochondria are related to human diseases such as tissue fibrosis, neurodegenerative diseases (Alzheimer's disease and Parkinson's disease), heart dysfunction, cancer, immunity and aging. Mitochondria have become a research hotspot in the field of biology and life science and technology, and provide strategies for disease prevention and treatment.

[0003] Fluorescent staining is the most important method for realizing mitochondrial imaging, and therefore the development of a corresponding mitochondrial fluorescent kit is a current research hotspot. Commercially available mitochondrial fluorescent probes are mainly divided into two categories: one is a large molecule protein, and the other is a small molecule fluorescent dye. Among them, the large molecule protein has the disadvantages of poor light stability, large structure size, difficulty in entering cells and mitochondria, and easy immunoreaction, although it has strong specificity. The small molecule fluorescent dye can enter cells and be distributed in mitochondria by simple incubation, and has uniform staining and bright fluorescence. Therefore, in addition to the classic mitochondrial tracking staining reagent such as rhodamine 123 and MitoTracker series dyes, many new mitochondrial dyes have appeared, for example, the application of HQO as a fluorescent probe for monitoring the process of mitochondrial autophagy in living cells is disclosed in the invention patent CN105838354B; a flavone fluorescent probe targeting mitochondria and a preparation method and application thereof are disclosed in the invention patent CN107043372B; however, no research has shown that aristolochic lactam I can be specifically and long-term enriched in mitochondria and can be used for labeling mitochondria in living cells.

[0004] The inventors accidentally discovered a compound aristolochic lactam I which can spontaneously emit blue fluorescence during the research process. The compound can quickly enter living cells and be specifically and long-term enriched in mitochondria, and is prepared into a mitochondrial staining kit (Ex / Em: 393 / 474 nm). The staining kit can label mitochondria in living cells to make them emit blue fluorescence and be applied to mitochondrial imaging. SUMMARY

[0005] The primary object of the present application is to provide a blue fluorescent probe targeting mitochondria. The fluorescent probe is aristolochic lactam I.

[0006] Preferably, the fluorescent probe is targeted and distributed in mitochondria.

[0007] The second object of the present application is to provide a reagent for mitochondrion imaging of living cells, which is prepared by the following method: weighing Aristolochic lactam I and dissolving it in DMSO.

[0008] The third object of the present application is to provide a preparation method of the reagent for mitochondrion imaging of living cells, which comprises the following steps: weighing Aristolochic lactam I and dissolving it in DMSO.

[0009] Preferably, the reagent is obtained by dissolving 0.5-1 mg of Aristolochic lactam I in 100 μl of DMSO.

[0010] The fourth object of the present application is to provide the application of the blue fluorescent probe targeting mitochondrion in the preparation of a reagent for mitochondrion imaging of living cells.

[0011] The fifth object of the present application is to provide the application of the blue fluorescent probe targeting mitochondrion in mitochondrion labeling.

[0012] The present application provides a blue fluorescent probe targeting mitochondrion, which is Aristolochic lactam I. The distribution of the probe in cells is related to the membrane potential of mitochondrion. The probe enters cells by passive diffusion and can exist in cells for a long time. The probe can target mitochondrion and provide a self-stable and long-lasting blue fluorescent reagent for mitochondrion imaging. The reagent can be prepared into a kit. The preparation process is simple, the equipment cost is low, and the reagent has good application potential in the field of mitochondrion imaging. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 Laser confocal imaging of HK-2 cells dyed by the present mitochondrion dyeing kit Note: A-D are the distribution of Aristolochic lactam I in cells; E-H are the distribution of mitochondrion dye rhodamine 123 (green) and Aristolochic lactam I (blue) in cells, respectively; I-L are the distribution of mitochondrion dye Mito-Tracker (red) and Aristolochic lactam I (blue) in cells, respectively

[0014] Figure 2 Cell uptake of Aristolochic lactam I by HK-2 cells

[0015] Note: A: relationship between cell uptake amount of Aristolochic lactam I and incubation time; B: relationship between Aristolochic lactam I concentration and cell uptake amount thereof; C and D: effect of mitochondrion membrane potential interfering agent FCCP on cell uptake of 0.1 and 0.2 μM Aristolochic lactam I

[0016] Figure 3 Effect of temperature on cell uptake of Aristolochic lactam I

[0017] Figure 4 Amount of aristolochic acid I in cells at different times of aristolochic acid release

[0018] Figure 5 Uptake of aristolochic acid I by different amounts of mitochondria DETAILED DESCRIPTION

[0019] The present application will be described in detail below with specific reference being made to the drawings and embodiments. The following examples are illustrative only of the best modes of practicing the application and are not intended to limit the scope of the application in any way. Any simple modifications of the embodiments, equivalent variations, and equivalent modifications that are based on the technical essence of the present application are within the scope of the present application.

[0020] In the following examples, the materials, reagents, etc. used are obtained from commercial sources unless otherwise specified.

[0021] Example 1: Preparation of a blue fluorescent probe with mitochondrial targeting function

[0022] Dye diluent: 118 mM NaCl, 23.8 mM NaHCO3, 4.8 mM KCl, 1.0 mM KH2PO4, 1.2 mM MgSO4, 12.5 mM HEPES, 5 mM glucose and 1.5 mM CaCl2(pH 7.4).

[0023] Mitochondrial dye: 1 mg of aristolochic acid I (CAS No.: 13395-02-3) was accurately weighed and dissolved in 100 μl of DMSO to obtain a mitochondrial dye stock solution. Storage method: -20°C, avoid light, shelf life 1 year.

[0024] Table 1 Kit composition

[0025] Composition Amount Mitochondrial dye 100 μl (DMSO stock solution) Dye dilution 100 mL

[0026] Example 2: Application of a blue fluorescent probe with mitochondrial targeting function

[0027] 1. Method for use:

[0028] (1) Mitochondrial staining solution

[0029] Take 4.2 μL mitochondrial dye stock solution (packaged and stored in the dark to avoid repeated freeze-thawing), dissolved in 1.4 mL dye diluent, to obtain a 100 μM mitochondrial dye solution, then dilute the mitochondrial dye solution with basal medium or dye diluent to obtain a 0.2-2 μM mitochondrial staining working solution for cell mitochondrial staining. The concentration can be optimized according to the experimental requirements.

[0030] (2) Cell staining

[0031] Adherent cells: HK-2 cells (human renal cortical proximal tubular epithelial cells, Chinese Academy of Sciences) reached 60-70% confluence, the culture medium was removed, PBS was washed twice, mitochondrial staining working solution was added, and it was incubated in a cell incubator for 5 min, the dye solution was removed, and the dye diluent was washed 5 times, and observed under a fluorescence microscope or a laser confocal microscope.

[0032] Suspension cells: centrifuge the cell suspension at 1000 rpm for 5 min, discard the supernatant, suspend the cells with the mitochondrial staining working solution, incubate in a cell incubator for 5 min, centrifuge the cells at 1000 rpm for 5 min, discard the dye solution, wash with dye diluent 3 times, and observe under a fluorescence microscope or a laser confocal microscope.

[0033] 2. Results

[0034] By Figure 1 It can be seen that the mitochondrial staining kit can excite blue fluorescence, which has excellent overlap with the classic mitochondrial dye rhodamine 123 (green) and the mitochondrial dye Mito-Tracker (red), indicating that the kit is a good reagent for mitochondrial staining. (Rhodamine 123 and MiTo-Tracker are purchased from Biyun Tian Biotechnology Co., Ltd.)

[0035] Example Three, Cell Translational Kinetics of a Blue Fluorescent Probe with Mitochondrial Targeting Function

[0036] 1. Experimental method:

[0037] HK-2 cells (human renal cortical proximal tubular epithelial cells, Chinese Academy of Sciences) were seeded in a 12-well plate (5×10 5 The culture medium was removed, and different concentrations of oxymatrine I were added to the culture medium, which was incubated at different temperatures for different times. After the appropriate incubation time, the culture medium was collected, and then washed twice with ice-cold buffer. Add 200 μL of distilled water to each well, collect the cells and break them by ultrasonic. Measure the protein concentration by BCA kit (purchased from Thermo), and determine the oxymatrine I concentration by HPLC-FLD.

[0038] HK-2 cells (human kidney proximal tubular epithelial cells, CAS) were seeded in 12-well plates (5 x 10 5 The culture medium was removed and pre-incubated with 10 mM mitochondrial membrane potential disrupter FCCP (purchased from MedChemExpress) for 15 min, then incubated with 0.1 or 0.2 mM oxymatrine I (purchased from MedChemExpress) for 15 min. The culture medium was collected and then washed twice with ice-cold buffer. 200 mL of distilled water was added to each well, and the cells were collected and disrupted by ultrasonication. The protein concentration was measured by a BCA kit (Thermo), and the oxymatrine I concentration was determined by HPLC-FLD.

[0039] HK-2 cells were seeded in 12-well plates (5 x 10 5 The culture medium was removed and pre-incubated with 10 mM mitochondrial membrane potential disrupter FCCP (purchased from MedChemExpress) for 15 min, then incubated with 0.1 or 0.2 mM oxymatrine I (purchased from MedChemExpress) for 15 min. The culture medium was collected and then washed twice with ice-cold buffer. 200 mL of distilled water was added to each well, and the cells were collected and disrupted by ultrasonication. The protein concentration was measured by a BCA kit (Thermo), and the oxymatrine I concentration was determined by HPLC-FLD.

[0040] 2. Results

[0041] From Figure 2 It can be seen that oxymatrine I can quickly distribute in cells, and mitochondrial disrupter FCCP can reduce the uptake of oxymatrine I by cells, further proving that the distribution of oxymatrine I in cells is related to the mitochondrial membrane potential.

[0042] From Figure 3 It can be seen that oxymatrine I can enter cells at 0 and 37°C, suggesting that oxymatrine I can enter cells by passive diffusion.

[0043] From Figure 4 It can be seen that after 30 min of oxymatrine I uptake, the extracellular incubation solution containing oxymatrine I was removed, and blank incubation solution was added to release oxymatrine I in the cells. After 180 min of release, only about 2 / 3 of the oxymatrine I in the cells was released, indicating that oxymatrine I has the characteristic of persisting in cells.

[0044] Example Four, Mitochondrial Uptake of a Blue Fluorescent Probe with Mitochondrial Targeting Function

[0045] 1. Experimental method:

[0046] HK-2 cells (human kidney proximal tubular epithelial cells, CAS) were seeded in 12-well plates (5 x 10 5Cells were grown to 80-90% in the wells, trypsinized, centrifuged to collect cells, resuspended in ice PBS, counted and centrifuged at 600g, the supernatant was discarded and 1.5ml mitochondrial separation reagent (purchased from Biyun Tian Biotechnology Co., Ltd.) was added, and the cells were transferred to a grinder and ground until the cell survival rate was below 50%, then centrifuged at 4°C, 600g for 10min, the supernatant was collected, and the supernatant was centrifuged at 4°C, 11,000g for 10min to obtain mitochondria. The obtained mitochondria were suspended with mitochondrial storage solution and mixed.

[0047] Different volumes of mitochondrial storage solution were added to the incubation solution, and the same concentration of aristolochic lactam I was added, and after incubation for 15min, 11,000g centrifugation for 10min, the mitochondria and the incubation solution outside the mitochondria were collected, and the amount of aristolochic lactam I in the mitochondria and the incubation solution was determined by HPLC-FLD.

[0048] 2. Results

[0049] From Figure 5 It can be seen that with the increase of the concentration of mitochondria, the amount of aristolochic lactam I in mitochondria increases, and the amount of aristolochic lactam I in the incubation solution decreases, which further indicates that the distribution of aristolochic lactam I in cells is related to mitochondria.

[0050] In summary, the present application provides a blue fluorescent probe targeting mitochondria, which is aristolochic lactam I, the distribution of the probe in cells is related to the mitochondrial membrane potential, the probe enters cells in a passive diffusion manner and can persist in cells, can target mitochondria, provides a spontaneous stable and persistent blue fluorescent reagent for mitochondrial imaging, and the reagent can be prepared into a kit, the preparation process is simple, the equipment cost is low, and the reagent has good application potential in the field of mitochondrial imaging.

Claims

1. Use of aristolane I in the preparation of a reagent for imaging mitochondria in living cells.

2. A method for the preparation of a reagent for live cell mitochondrial imaging, characterized in that, The reagent is prepared by dissolving 0.5-1 mg of aristolane I in 100 μl of DMSO.

3. The method for preparing the reagent for live-cell mitochondrial imaging as described in claim 2, characterized in that, The reagent is prepared by dissolving 0.5-1 mg of aristolane I in 100 μl of DMSO.

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