Fluorescent probe and its preparation method and application

Abstract

The present invention discloses a fluorescent probe and a preparation method and application thereof, belonging to the field of fluorescent probe imaging technology. The fluorescent probe, whose structural formula is one or more of the following structural formulas: or In addition, the present invention also proposes a method for preparing a fluorescent probe, mixing a compound, a solvent and piperidine to obtain a mixture, stirring the mixture at 75-85°C to obtain a fluorescent probe; the compound is or In addition, the present invention also proposes an application of the above fluorescent probe or the fluorescent probe obtained by the above preparation method in cell membrane imaging. The fluorescent probe proposed by the present invention can achieve long-term imaging of cell membranes.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent probe imaging, and in particular to a fluorescent probe and a preparation method and application thereof. Background Art

[0002] Cell membrane (CM) viscosity is an important parameter for indicating cell status and development. The development of fluorescent probes that can detect CM viscosity is of great significance for cell research and disease diagnosis. However, most of the developed CM probes are easily internalized by cells and cannot achieve long-term imaging. In addition, the use of CM probes for disease diagnosis (such as diabetes) has not been fully explored.

[0003] The cell membrane is a barrier between the intracellular and extracellular environments. It not only maintains the stability of the cell morphology and internal environment, but also undertakes various important biological functions. The microenvironment of CM (viscosity, polarity, pH, etc.) affects the physical or chemical behavior of many surrounding molecules and is an important parameter indicating the state of the cell. Among them, CM viscosity plays an important role in controlling pathological and physiological processes such as substance transport, cell signal transduction, cell apoptosis, and maintaining a stable intracellular environment. Abnormal CM viscosity may lead to metabolic disorders and various diseases, such as Alzheimer's disease, diabetes, atherosclerosis, and tumors. Therefore, monitoring changes in CM viscosity is crucial to maintaining the balance of the CM microenvironment and understanding disease progression.

[0004] As a metabolic disease, diabetes affects the metabolism of sugars, lipids, and proteins, leading to various complications, including severe kidney disease and retinopathy. In addition, diabetes can cause varying degrees of damage to various organs in the body, most of which can cause inflammation, leading to changes in cell or tissue viscosity. Therefore, viscosity testing is essential for the diagnosis and evaluation of diabetes and its complications. However, traditional viscosity analysis methods, such as viscometers, usually require a large amount of sample, and more importantly, it is difficult to non-invasively detect the viscosity of living samples.

[0005] Therefore, how to achieve long-term imaging of cell membranes is one of the technical problems that the existing technology needs to solve. Summary of the invention

[0006] The purpose of the present invention is to overcome the above technical deficiencies, provide a fluorescent probe and a preparation method and application thereof, and solve the technical problem of how to achieve long-term imaging of cell membranes in the prior art.

[0007] In order to achieve the above technical objectives, the technical solution of the present invention provides a fluorescent probe, the structural formula of which is one or more of the following structural formulas:

[0008]

[0009] Wherein, R1 represents one of C1-C6 alkyl groups; R2 represents or R3 represents one of C1-C6 alkyl groups, and R4 represents one of C1-C6 alkyl groups.

[0010] In any embodiment, the structural formula is one or more of the following structural formulas:

[0011]

[0012] In addition, the present invention also provides a method for preparing a fluorescent probe, comprising the following steps:

[0013] The compound The solvent and piperidine are mixed to obtain a mixture, and the mixture is stirred at 75-85° C. to react to obtain a fluorescent probe; the compound is

[0014] In any embodiment, the reaction time is 5-8 hours under stirring at 75-85°C.

[0015] In addition, the present invention also proposes an application of the fluorescent probe or the fluorescent probe prepared by the preparation method in cell membrane imaging.

[0016] In addition, the present invention also proposes an application of the fluorescent probe or the fluorescent probe prepared by the above preparation method in a fluorescent probe for distinguishing cancer cells from normal cells.

[0017] In addition, the present invention also proposes an application of the fluorescent probe or the fluorescent probe prepared by the preparation method as a viscosity probe.

[0018] In addition, the present invention also proposes an application of the fluorescent probe or the fluorescent probe prepared by the preparation method in detecting cell membrane viscosity.

[0019] In addition, the present invention also proposes an application of the fluorescent probe or the fluorescent probe prepared by the preparation method in detecting changes in cell membrane viscosity caused by hyperglycemia.

[0020] In any embodiment, the cell membrane viscosity change is a cell membrane viscosity change in the kidney.

[0021] Compared with the prior art, the beneficial effects of the present invention include: the fluorescent probe proposed by the present invention can achieve long-term imaging of cell membranes. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 are the absorption spectra and fluorescence spectra of the fluorescent probe of Example 2 of the present invention in different solvents; wherein, Figure 1A-1B is the absorption and fluorescence spectra of LT-M (10 μM) in different solvents; Figure 1 C-1D is the absorption and fluorescence spectra of LT-S (10 μM) in different solvents; Figure 1 E-1F is the absorption and fluorescence spectra of LT-N (10 μM) in different solvents. ex =520nm, slit width: d ex =d em =5nm.

[0023] Figure 2 : is the fluorescence spectrum of the fluorescent probe of Example 2 of the present invention; wherein, (A) the fluorescence spectrum of LT-M, LT-S and LT-N (10 μM each) in a water-glycerol mixture (Gly% from 100% to 0%). ex =520nm, slit width: d em =d ex = 5 nm; (B) Linear relationship between log I641 and logη. The data of each probe are -Hoffmann equation fitting.

[0024] Figure 3 is the fluorescence spectra of the fluorescent probes LT-M, LT-S and LT-N (10 μM each) in Example 2 of the present invention in water and glycerol at 25° C.; Figure 3 A: LT-M, Figure 3 B: LT-S, Figure 3 C: LT-N. ex =520nm, slit width: d em =d ex =5nm.

[0025] Figure 4 1 is the fluorescence spectrum of the fluorescent probe of Example 2 of the present invention at different temperatures. Figure 4 A- Figure 4 C is the fluorescence spectra of LT-M, LT-S and LT-N (10 μM) at different temperatures. ex =520nm. Slit width: d ex =2.5nm,d em =5nm. Figure 4 D- Figure 4 F is the reversibility of the fluorescence response of LT-M, LT-S and LT-N (10 μM) in glycerol alternating between 25°C and 37°C. ex / λ em =520 / 641nm. Slit width: d ex =2.5nm,d em =5nm.

[0026] Figure 5 is the fluorescence intensity of 10 μM LT-M, LT-S and LT-N at different pH values ​​at 641 nm in Example 2 of the present invention; wherein, Figure 5 A:LT-M, Figure 5 B:LT-S, Figure 5 C: LT-S. All experiments were performed in 10 mM PBS buffer containing 75% glycerol. ex =520nm. Slit width: d ex =d em =5nm.

[0027] Figure 6 The fluorescence intensity of LT-M, LT-S and LT-N (10 μM) in the present invention was affected by adding different substances at 25°C; (1) Gly, (2) HClO, (3) ONOO - ,(4)H2O2,(5)HNO,(6)NO2 - ,(7)Cys,(8)GSH(5mM),(9)Hcy(1mM),(10)Arg,(11)Ile,(12)Thr,(13)Trp,(14)ASP,(15)Tyr,(16)SO3 2- ,(17)Mg 2+ ,(18)S2O3 2- ,(19)Na + ,(20)Cl - ,(21)SCN,(22)Al 3+ ,(23)HS - ,(24)SO4 2- ,(25)Fe 3+ ,(26)Cu 2+ ,(27)Zn 2+ ,(28)K + .λ ex =520nm.Slit width:d ex =d em =5nm.

[0028] Figure 7 It is the response of the fluorescent probe of Example 2 of the present invention to the fluorescence intensity when different kinds of substances (100 μM, unless otherwise stated) are added to 641 nm glycerol and 25% PBS (10 mM, pH 7.4). Figure 7 A:LT-M, Figure 7 B:LT-S and Figure 7 C:LT-N. From left to right in the figure are (1) none, (2) HClO, (3) ONOO -,(4)H2O2,(5)HNO,(6)NO2 - ,(7)Cys,(8)GSH(5mM),(9)Hcy(1mM),(10)Arg,(11)Ile,(12)Thr,(13)Trp,(14)ASP,(15)Tyr,(16)SO3 2- ,(17)Mg 2+ ,(18)S2O3 2- ,(19)Na + ,(20)Cl - ,(21)SCN,(22)Al 3+ ,(23)HS - ,(24)SO4 2- ,(25)Fe 3+ ,(26)Cu 2+ ,(27)Zn 2+ ,(28)K + .λ ex =520nm.Slit width:d ex =d em =5nm.

[0029] Figure 8 This is a cytotoxicity study of HeLa cells after incubation for 24 hours with different concentrations of LT-M, LT-S and LT-N (0, 5, 10, 15 and 20 μM) of Example 2 of the present invention. The toxicity study was conducted using the MTT method.

[0030] Fig. 9 is the fluorescent probe cell membrane imaging result of Example 2 of the present invention; wherein, Fig. 9 A is a comparison of the long-term imaging capabilities of LT-M, LT-S and LT-N probes on the cell membrane; HeLa cells were incubated with probes (10 μM) for 0.5, 2, 6, and 12 h. Red channel: λ ex / λ em =561 / 580-670nm; Fig. 9 B is the data of 15 min co-localization imaging of LT-M (10 μM) and DiO (10 μM) in HeLa cells; P: Pearson correlation coefficient, LT-M technology was used to perform three-dimensional imaging of the cell membrane; Fig. 9 C is the image of HeLa cells after co-staining with DiO (10 μM) and LT-M (10 μM) at different times. ex / λ em :DiO,488 / 500-550nm; LT-M,561 / 580-670nm.

[0031] Fig.10These are confocal images of HeLa cells stained with LT-M at different times in Example 2 of the present invention; A: 14h, B: 16h, C: 18h, D: 24h, ex =561nm,λ em =580~670nm.

[0032] Fig.11 This is the long-term imaging of LT-M (10 μM) in Example 2 of the present invention in different cells. (A) HepG2 cells, (B) 4T1 cells. ex =561nm,λ em =580-670 nm. Scale bar = 10 μm.

[0033] Fig.12 The confocal images of HeLa cells stained with different concentrations of LT-M in Example 2 of the present invention are shown in Figure 2. (A) 200nm, (B) 500nm, (C) 1μM, (D) 5μM, (E) 10μM. ex =561nm,λ em =580-670 nm. Scale bar = 10 μm.

[0034] Fig.13 λ is the fluorescence image of HeLa cells after staining with 10 μM LT-M for different time periods (10 s to 30 min) in Example 2 of the present invention. ex =561nm,λ em =580-670nm. Scanning bar =10μm.

[0035] Fig.14 is the result of confocal fluorescence staining of LT-M in Example 2 of the present invention and the intensity distribution diagram; wherein Fig.14 (A) HeLa cells were stained with LT-M (10 μM) for 30 min with confocal fluorescence, followed by comparison of the cells with and without PBS washing. Fig.14 (B) Intensity distribution of ROI-1 and ROI-2. ex =561nm,λ em =580-670nm. Scanning bar =15μm.

[0036] Fig.15 The light stability of LT-M in Example 2 of the present invention in HeLa cells under 561nm continuous laser irradiation (using 20% ​​power, normal imaging usually only requires 1% power). (A) Confocal fluorescence image changing with irradiation time. ex =561nm,λ em = 580-670nm. (B) Merged image of red channel and bright area. (C) Changes of fluorescence intensity over time during irradiation. Scale bar: 10μm.

[0037] Fig.16 : is the confocal image and fluorescence intensity of LT-M of Example 2 of the present invention. Fig.16 (A) Images and relative fluorescence intensity of HeLa cells stained with 10 μM LT-M for 30 min without (control), monensin (Mon), and nystatin (Nys) stimulation. Fig.16 (B) Confocal images and fluorescence intensity of HeLa cells after staining with 10 μM LT-M at different temperatures. Fig.16 (C) Confocal images and fluorescence intensity of HeLa, HepG2, HEK 293T and RAW 264.7 cells after 10 μM LT-M for 30 min. The attached figure is a schematic diagram of LT-M staining of tumor CM and normal CM. Fluorescence image: ex / λ em =561 / 580-670nm. Error bars represent mean deviation (±sd), n=4.

[0038] Fig.17 Figure 2 is the image and relative fluorescence intensity of HEK 293T cells after LT-M staining; (A) HEK 293T cell image after 10 μM LT-M staining for 30 min under stimulation of no (control), monensin (Mon) and nystatin (Nys); (B) Fig.17 Relative fluorescence intensity of the image in (A), λ ex / λ em =561 / 580-670nm. Scale bar = 15μm.

[0039] Fig.18 The confocal images and relative fluorescence intensity of HEK 293T cells treated with different concentrations of glucose for 12 hours and stained with 10 μM LT-M for 30 minutes in Example 2; wherein (A) is the confocal image of HEK 293T cells treated with different concentrations of glucose for 12 hours and stained with 10 μM LT-M for 30 minutes. Red channel: λ ex / λ em =561 / 580-670nm. Scale bar = 20 μM. (B) Relative fluorescence intensity of images at different glucose concentrations.

[0040] Fig.19 The cell images of Example 2. (A) HeLa cell image; (a) 10 μM LT-M staining for 30 min; (bd) 50, 100, 150 mM glucose treatment for 12 h, followed by 10 μM LT-M staining for 30 min. ex / λ em=561 / 580-670nm. (B) Relative fluorescence intensity of ad; (C) Flow cytometry data of ad.

[0041] Fig. 20 The confocal images and relative fluorescence intensity of Example 2; wherein (A) is the confocal image of HEK 293T cells treated with different concentrations of glucose for 12 hours and stained with 10 μM LT-M for 30 minutes. Red channel: λ ex / λ em =561 / 580-670nm. Scale bar = 20 μm. (B) Relative fluorescence intensity of images at different glucose concentrations.

[0042] Fig.21 The confocal images and relative fluorescence intensity of Example 2; (A) Confocal images of HeLa cells after incubation with glucose (150 mM) for different time periods and staining with 10 μM LT-M. (B) Relative fluorescence intensity of the image in (A). Red channel: λ ex / λ em =561 / 580-670nm. Scale bar = 15μm.

[0043] Fig. 22 The in vivo imaging, fluorescence intensity, and visceral images of mice in Example 2. (A) In vivo imaging of normal / diabetic mice. The normal group and the diabetic group of mice were imaged after intraperitoneal injection of LT-M (100 μL, 200 μM PBS). ex / λ em =560 / 670nm; (B) Fluorescence intensity of imaging mice. (C) Images of the heart, liver, spleen, lung, and kidney of mice injected intraperitoneally with LT-M (100μL, 200μM in PBS) 15 minutes later; λ ex / λ em =560 / 670nm; (D) Fluorescence intensity diagram of organ imaging; (E) Kidney tissue images of normal / diabetic mice after H&E staining and fasting blood glucose (FBG) content of normal (5.0mmol / L) and diabetic (24.3mmol / L) mice.

[0044] Fig.23 is the confocal image of Example 2; wherein (A) 10 μM LT-M (λ ex / λ em =561 / 580-670nm) after staining of normal kidney tissue and diabetic kidney tissue sections; (B) Relative fluorescence intensity of normal / diabetic tissues; (C) Confocal images of normal / diabetic kidney tissue sections under high magnification.

[0045] Fig.24 It is the hydrogen spectrum of LT-M prepared in Example 1.

[0046] Fig.25 This is the hydrogen spectrum of LT-S prepared in Example 1.

[0047] Fig.26 It is the hydrogen spectrum of LT-N prepared in Example 1. DETAILED DESCRIPTION

[0048] The "range" disclosed in the present application is defined in the form of a lower limit and an upper limit, and a given range is defined by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundaries of the particular range. The range defined in this way can be inclusive or exclusive of the end values, and can be arbitrarily combined, that is, any lower limit can be combined with any upper limit to form a range. For example, if a range of 60 to 120 and 80 to 110 is listed for a particular parameter, it is understood that a range of 60 to 110 and 80 to 120 is also expected. In addition, if the minimum range values ​​1 and 2 are listed, and if the maximum range values ​​3, 4 and 5 are listed, the following ranges can all be expected: 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4 and 2 to 5. In the present application, unless otherwise specified, the numerical range "a to b" represents an abbreviation of any real number combination between a and b, where a and b are both real numbers. For example, the numerical range "0-5" means that all real numbers between "0-5" are listed in this document, and "0-5" is just an abbreviation of these numerical combinations. In addition, when a parameter is expressed as an integer ≥ 2, it is equivalent to disclosing that the parameter is, for example, an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0049] This specific embodiment provides a fluorescent probe, whose structural formula is one or more of the following structural formulas:

[0050]

[0051] Wherein, R1 represents one of C1-C6 alkyl groups; R2 represents or R3 represents one of C1-C6 alkyl groups, and R4 represents one of C1-C6 alkyl groups.

[0052] In some embodiments, the structural formula of the fluorescent probe is one or more of the following structural formulas:

[0053]

[0054] This specific embodiment also proposes a method for preparing a fluorescent probe, comprising the following steps:

[0055] The compound The solvent and piperidine are mixed to obtain a mixture, and the mixture is stirred at 75-85° C. for 5-8 hours to obtain a fluorescent probe; the compound is

[0056]

[0057] This specific embodiment also proposes an application of the fluorescent probe or the fluorescent probe prepared by the above preparation method in cell membrane imaging.

[0058] This specific embodiment also proposes an application of the fluorescent probe mentioned above or the fluorescent probe prepared by the above preparation method in a fluorescent probe for distinguishing cancer cells from normal cells.

[0059] This specific embodiment also proposes an application of the fluorescent probe or the fluorescent probe prepared by the above preparation method as a viscosity probe.

[0060] This specific embodiment also proposes an application of the fluorescent probe or the fluorescent probe prepared by the above preparation method in detecting cell membrane viscosity.

[0061] This specific embodiment also proposes an application of the fluorescent probe or the fluorescent probe prepared by the preparation method to detect changes in cell membrane viscosity caused by hyperglycemia.

[0062] In some embodiments, the cell membrane viscosity change is a cell membrane viscosity change in the kidney.

[0063] The present invention proposes three new CM probes (LT-M, LT-S and LT-N). Among them, LT-M containing sterically hindered tetraalkylammonium salt can achieve long-term imaging of CM and sensitive detection of CM viscosity. LT-M has a longer emission wavelength (641nm), a larger Stokes shift (121nm), fast non-elution imaging performance and good photostability. By utilizing the sensitivity of LT-M to viscosity, cancer cells can be distinguished from normal cells. At the same time, we found that the viscosity of CM increases under the stimulation of ferroptosis and hyperglycemia. More importantly, in vivo mouse imaging showed that LT-M can target the kidneys, and the renal interstitial viscosity of diabetic patients will increase, which provides a valuable new method for the diagnosis and evaluation of diabetic nephropathy.

[0064] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0065] In the present invention, reference is made to “some embodiments”, “this embodiment”, examples, etc., which describe a subset of all possible embodiments, but it can be understood that “some embodiments” may be the same subset or different subsets of all possible embodiments and may be combined with each other without conflict.

[0066] Hereinafter, the embodiments of the present application will be described. The embodiments described below are exemplary and are only used to explain the present application, and should not be construed as limiting the present application. If no specific techniques or conditions are indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. If the manufacturer is not indicated in the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0067] Unless otherwise stated, reagents and chemicals were purchased from commercial suppliers and used as received. Spectra were recorded on a Varian 600 or 400 NMR instrument. 1 H and 13 C nuclear magnetic resonance spectra. Mass spectra were obtained using an E2695LC-MS system (for low-resolution mass spectrometry) and a Bruker microTOF-Q instrument (for high-resolution mass spectrometry, HR-MS). Fluorescence quantum yields were obtained using rhodamine B in ethanol solution as a reference. UV-visible spectra and fluorescence spectra were obtained using an Agilent Cary-60 UV-visible spectrophotometer and an Agilent Cary Eclipse fluorescence spectrophotometer, respectively. Flow cytometry analysis BD FACS Aria III in FL4, excitation / emission: 488 / 610±20nm. BAlB / c mice 10 weeks (18-20g) were purchased from the Wuhan Center for Disease Control and Prevention, and imaging experiments were performed after anesthesia. The mouse experiment was approved by the Animal Ethics Committee of Central China Normal University, and mouse images were measured on a PerkinElmer IVIS Lumina Series III instrument. Photographs of sections stained with hematoxylin-eosin were taken under an inverted fluorescence microscope.

[0068] Example 1

[0069] The fluorescent probe was synthesized according to the following synthetic route:

[0070]

[0071] Synthesis of compound 1: 4-methylpyridine (465 mg, 5 mmol) and 1,4-dibromobutane (3.2 g, 15 mmol) were dissolved in dry acetonitrile (20 mL), stirred at 45 ° C for 12 h, and the solvent was evaporated under reduced pressure. The crude product was purified by column chromatography (silica gel) to obtain a solid product compound 1 (1.226 g, yield 80%). The hydrogen spectrum data are as follows:

[0072] 1 H NMR(600MHz, CDCl3)δ9.48(m,2H),7.89(d,J=5.8Hz,2H),5.10–5.07(m,2H),3.49(td ,J=6.4,2.3Hz,2H),2.69(d,J=2.5Hz,3H),2.24(t,J=7.6Hz,2H),2.05–2.02(m,2H). 13 C NMR (151MHz, CDCl3) δ158.99,144.38,128.85,59.71,32.78,30.59,28.87,22.37.

[0073] Synthesis of compound 2: Compound 1 (604 mg, 10.2 mmol) and 1,4-diazobicyclo[2.2.2]octane (448 mg, 4 mmol) were dissolved in dry acetonitrile (5 mL). Refluxed under N2 protection for 18 h, then added ether (20 mL). Then stirred at room temperature for 1 hour, the precipitate was filtered out, and washed with dry acetone to obtain compound 2, which was stored under vacuum. Compound 2 was 729 mg of brown solid (yield: 87%). The hydrogen spectrum data are as follows:

[0074] 1 H NMR (600MHz, DMSO-d6) δ9.01(d,J=6.3Hz,2H),8.00(d,J=6.2Hz,2H),4.62(t,J=7.3Hz,2H),3. 30–3.26(m,8H),2.99(t,J=7.5Hz,6H),2.59(s,3H),1.94–1.88(m,2H),1.68(t,J=7.9Hz,2H). 13 C NMR (101MHz, DMSO-d6) δ159.41,144.29,128.94,62.78,59.49,52.02,45.17,27.97,21.93,18.51.

[0075] Compound 3 and compound 4 were prepared according to existing methods, for example, according to the literature Yan, P.; Acker, CD; Loew, LMACS Sens. 2018, 3, 2621–2628; Han, X.; Ma, Y.; Chen, Y.; Wang, X.; Wang, Z. Anal. Chem. 2020, 92, 2830–2838.

[0076] Synthesis of fluorescent probe TM: Compound 2 (210 mg, 0.5 mmol) and (150mg, 0.75mmol) 1 drop of piperidine was added to 10mL of ethanol to obtain a mixture, the mixture was stirred at 80°C for 8h, and the reaction process was monitored by thin layer chromatography (methanol / acetic acid 5:1). After cooling to room temperature, the solvent was removed by evaporation under reduced pressure. Then a mixed solution of ethyl acetate and ethanol (ethyl acetate: ethanol = 100:1) was added, and a large amount of red precipitate appeared in the mixture under the action of ultrasound. The precipitate was recrystallized in methanol to obtain 148mg of red LT-M powder with a yield of 49%. The hydrogen spectrum is as follows Fig.24 The hydrogen spectrum data are as follows:

[0077] 1 H NMR (600MHz, DMSO-d6) δ8.70(s,2H),7.98(d,J=6.2Hz,2H),7.80(d,J=16.0Hz,1H),7.13(s,2H),7.05(d,J=15.7Hz,1 H),4.43(s,2H),3.23(d,J=7.6Hz,12H),3.00(t,J=7.4Hz,6H),2.68(t,J=6.3Hz,4H),1.91–1.81(m,6H),1.65(m,2H).

[0078] Synthesis of LT-S: Compound 3 (108 mg, 0.5 mmol) and The crude product was purified by silica gel column chromatography (dichloromethane / methanol=15:1, v / v) to obtain 124 mg of red solid LT-S in a yield of 61%.

[0079] Hydrogen spectrum Fig.25 The hydrogen spectrum data are as follows:

[0080] 1 H NMR (400MHz, DMSO-d6) δ8.69(d,J=6.7Hz,2H),7.96(d,J=6.6Hz,2H),7.78(d,J=16.0Hz,1H),7.15(s,2H),7.04(d,J=16.0Hz,1H),4 .52(t,J=6.9Hz,2H),3.25(t,J=5.7Hz,4H),2.70(t,J=6.3Hz,4H),2.42(t,J=7.1Hz,2H),2.17(p,J=7.0Hz,2H),1.93-1.82(m,4H).

[0081] Synthesis of LT-N: Compound 4 (174 mg, 0.5 mmol) and (150mg, 0.75mmol) 1 drop of piperidine was added to 4mL of ethanol to obtain a mixture, the mixture was stirred at 80°C for 7h, cooled to room temperature, and 10mL of ethyl acetate was added to precipitate. After standing overnight, the precipitate was filtered and washed with dichloromethane and ethyl acetate respectively to obtain 204mg of black solid LT-N with a yield of 76%. The hydrogen spectrum is as follows Fig.26 The hydrogen spectrum data are as follows:

[0082] 1 H NMR (400MHz, D2O) δ8.34(d,J=6.5Hz,2H),7.72(d,J=6.5Hz,2H),7.47(d,J=15.9Hz,1H),7.04(s,2H),6.77(d,J=15.8Hz,1H),4.39(t, J=7.5Hz,2H),3.45-3.38(m,2H),3.13(t,J=5.8Hz,4H),3.10(s,9H),2.62(t,J=6.4Hz,4H),2.50-2.40(m,2H),1.82(p,J=6.4Hz,4H).

[0083] Example 2

[0084] All spectroscopic measurements were performed at 25 °C with a 10 μM concentration of LT-M, LT-S, and LT-N in solution. For fluorescence measurements, λ ex =520nm.

[0085] Cell culture and cytotoxicity evaluation. HeLa, HepG2, 4T1, HEK293T and RAW264.7 cells were cultured in DMEM (Dulbecco's modified Eagle's medium). All cell culture media were supplemented with 1% penicillin-streptomycin solution and 10% heat-inactivated fetal bovine serum, and all cells were cultured in a 37°C humidified incubator with 5% CO2. The conventional MTT method was used to evaluate the cytotoxicity of the probe LT-M.

[0086] Imaging of normal cells and tumor cells. To investigate the ability of LT-M to distinguish tumor cells from normal cells, tumor cells (HeLa and HepG2 cells) and normal cells (HEK 293T and RAW 264.7 cells) were cultured with 10 μM LT-M for 30 min and confocal imaging was performed under the same imaging conditions. Fluorescence imaging: ex / λ em =561 / 580-670nm.

[0087] Flow cytometry detection. HeLa cells (10 5) were inoculated in 6-well plates and cultured for 24 hours. Then, the cells were treated with different concentrations of glucose (50, 100, 150 mM) for 12 hours and then stained with 10 μM LT-M for 30 minutes. The cells were carefully collected by trypsin digestion, washed three times with PBS centrifugation (1500 rpm, 5 minutes, 4°C), and collected for flow cytometry (λ ex =488nm, PC 5.5-A channel) analysis.

[0088] Cell imaging experiments

[0089] HeLa cells were mainly used as cell models in cell imaging experiments. In order to study the long-term imaging ability of LT-M, LT-S and LT-N probes, cells were imaged after staining with 10 μM probes at 37°C for 0.5, 2, 6 and 12 h. ex / λ em =561 / 570-670nm. To determine the location of the probe, cells were incubated with 10 μM LT-M and 10 μM commercial CM dye (DiO) for 15 min, and confocal images were collected after PBS washing. ex / λ em For DiO: 488 / 500-550nm and LT-M: 561 / 580-670nm.

[0090] CMs imaging viscosity change experiment

[0091] HeLa cells were incubated with 10μM nystatin and 10μM monensin for 30min. Then they were stained with 10μM LT-M for 30min. Cells directly stained with LT-M were used as controls. The same experiment was also performed in HEK 293T cells. In addition, confocal imaging of LT-M (10μM) in HeLa cells at different temperatures (4℃, 25℃, and 37℃) was compared.

[0092] Imaging viscosity changes during hyperglycemia stimulation and ferroptosis

[0093] HeLa cells and HEK 293T cells were used to prepare hyperglycemia injury cell models. The cells were incubated with glucose (50, 100 and 150 mM, respectively) for 12 h and then incubated with LT-M for 30 min. Erastin-induced ferroptosis was prepared in HeLa cells. Cells were pretreated with Erastin (10, 30, 50 μM) for 4 h and then stained with 10 μM LT-M for 30 min.

[0094] Imaging experiments in diabetic mice

[0095] Before imaging, a diabetic mouse model was established: 10-week-old female BALB / c mice were intraperitoneally injected with streptozotocin (STZ, 10mM citrate buffer, pH 4.5) at a dose of 150mg / kg body weight, and normal mice in the control group were injected with saline (100μL) once a day for 3 consecutive days. The blood glucose of diabetic mice was ≥16.7mmol / L by blood glucose meter, confirming hyperglycemia. Mice were imaged by intraperitoneal injection of LT-M (100μL, 200μM in PBS). Afterwards, the main organs were quickly separated from the mice and imaged for comparison. ex / λ em =560 / 670nm. Frozen sections of kidney tissues from diabetic mice and normal mice were taken and confocal fluorescence imaging was performed using 10μM (LT-M) (λ ex / λ em =561 / 580~670nm), staining for 15min. Mouse kidney tissue sections were stained with hematoxylin-eosin (H&E).

[0096] Results and Discussion

[0097] Design of probes. LT-M, LT-S, and LT-N are all designed as viscosity-responsive molecular rotors. The julolidine (electron donor, D) and pyridine (electron acceptor, A) groups are connected by C=C bonds (π bridges) to form conjugated D-π-A structures, which give these probes far-red emission, which is beneficial for bioimaging. However, in low-viscosity environments, these functional groups can rotate freely, causing energy loss in a non-radiative manner, thereby quenching fluorescence. Under high-viscosity conditions, rotation is inhibited and the fluorescence signal is restored. The introduction of 1,4-diazo[2.2.2]octane with high steric hindrance and rigidity into LT-M is expected to enhance its anchoring ability on the cell membrane, reduce its internalization ability, and achieve long-term imaging of the cell membrane. Sulfonate and quaternary ammonium salt were added to LT-S and LT-N, respectively, making them conventional cell membrane probes as controls.

[0098] Spectral properties. Figure 1 As shown in the figure, LT-M, LT-S and LT-N all showed strong absorption around 520nm in different solvents, but only showed strong fluorescence around 641nm in the high viscosity solvent glycerol (Gly), indicating that they are all potential viscosity probes. Then, in the water-glycerol system, as the glycerol content gradually increased, the relationship between their fluorescence intensity and viscosity was studied. Figure 2 As shown in Figure 2, as the solution viscosity increases from 0.893 cP (100% water) to 945 cP (100% Gly), the fluorescence of LT-M, LT-S, and LT-N gradually increases. The fluorescence intensity and viscosity of each probe can be used -Hoffmann equation fitting showed a good linear relationship. It is worth noting that when the viscosity increased from 0.893 cP (100% water) to 945 cP (100% Gly), the fluorescence intensity of LT-M, LT-S and LT-N increased by 130 times, 169 times and 115 times, respectively ( Figure 3 A), indicating that they are all very sensitive to viscosity. As shown in Table 1, the fluorescence quantum yields (Φ) of LT-M, LT-S and LT-N in glycerol were measured to be 0.251, 0.259 and 0.243, respectively, which are much higher than those in water ( Figure 3 B). In addition, the fluorescence of LT-M, LT-S, and LT-N in glycerol increases with decreasing temperature and exhibits good reversibility ( Figure 4 In addition, all probes have pH-stable fluorescence ( Figure 5 ), good viscosity selectivity ( Figure 6 ), and excellent physiological viscosity anti-interference ability ( Figure 7 ). These results indicate that LT-M, LT-S, and LT-N are excellent viscosity probes.

[0099] Table 1 Fluorescence quantum yields of three probes in solvents water and glycerol

[0100]

[0101] Cell membrane imaging

[0102] Before applying LT-M, LT-S, and LT-N to cell imaging, their cytotoxicity was evaluated using the MTT assay. Figure 8 As shown, after culturing cells with different concentrations (0-20 μM) of LT-M, LT-S and LT-N for 24 h, the cell viability was good (>90%), indicating that they all had low cytotoxicity.

[0103] Then, the performance of the three probes in cell membrane imaging was investigated, e.g. Fig. 9As shown in A, after the three probes were incubated with cells for 0.5 h, the cell border showed bright red fluorescence, while there was no fluorescence in the cytoplasm, indicating that they were all cell membrane probes. It is worth noting that with the extension of the incubation time, even after 12 h, only the cell membrane of the cells stained with LT-M showed bright red fluorescence. However, the cells stained with LT-S and LS-N showed obvious fluorescence in the cytoplasm after 4 and 6 h, respectively, indicating that they were internalized. In addition, we observed that the fluorescence of LT-S and LS-N was completely internalized into the cell interior at 6 h and 12 h, respectively. Compared with LT-S, some LT-N remained on the plasma membrane after 6 h of cell staining. These results show that all three probes can be used for cell membrane imaging, but their residence time on the cell membrane varies greatly (LT-M>LT-N>LT-S). Only LT-M can be imaged for a long time for at least 12 h. The reason may be that both LT-M and LT-N molecules contain two positive charges. They can better bind to the cell membrane (phospholipid bilayer structure) with negative charges on the surface through electrostatic attraction, thereby increasing their ability to anchor to the cell membrane. In addition, LT-M contains a rigid and stereogenic quaternary ammonium group, which further prevents LT-M from penetrating the cell membrane, making it anchored on the cell membrane for long-term imaging among the three probes. Therefore, we next focused on LT-M.

[0104] The co-localization imaging experiment of LT-M and DiO (a commercial cell membrane probe) showed that the fluorescence overlap between LT-M and DiO was very good, and the Pearson correlation coefficient was very high (P = 0.88), proving that LT-M is a cell membrane probe ( Fig. 9 B). However, after 2 hours, DiO was almost completely internalized, indicating that LT-M has a significant advantage over DiO in long-term imaging of cell membranes ( Fig. 9 C). We further prolonged the incubation time of LT-M with cells and found that at 16 h, LT-M tended to be internalized. However, even after 24 h of incubation with cells, bright fluorescence still appeared on the cell membrane ( Fig.10 ), indicating that there are still a lot of LT-M residues on the cell membrane, further proving the ability of LT-M to image the cell membrane for a long time.

[0105] We also demonstrated the universality of LT-M for long-term imaging of cell membranes in different cell types ( Fig.11 ). In addition, LT-M can rapidly image cell membranes within 10 seconds ( Fig.12 ), the concentration used for imaging cell membranes can be as low as 200 nM ( Fig.13 In addition, since the background fluorescence of LT-M in water is almost zero, we also found that the washing process before imaging is unnecessary. In other words, it can be used for wash-free imaging ( Fig.14). To test the photostability of LT-M in cells, we used a 561nm strong laser to continuously irradiate LT-M-stained cells for 15 minutes. The results showed that the fluorescence of LT-M was very stable ( Fig.15 ), indicating that LT-M has good photostability. All the results show that LT-M is an excellent cell membrane probe that can not only rapidly image the cell membrane but also provide long-term imaging.

[0106] Imaging of CM viscosity changes. Next, we investigated whether LT-M could be used to detect changes in cell membrane viscosity. Monensin and nystatin can stimulate an increase in cell viscosity, so we compared imaging of cells stimulated with and without monensin or nystatin. Fig.16 As shown in A, the fluorescence on the cell membrane of HeLa cells treated with monensin and nystatin was significantly enhanced by LT-M imaging. It is well known that lowering the temperature will also increase cell viscosity. Fig.16 As shown in B, using LT-M staining, the fluorescence on the cell membrane does show a clear increase when the temperature is lowered. These data indicate that LT-M can image changes in cell membrane viscosity.

[0107] The viscosity of cancer cell membranes is higher than that of normal cell membranes. Based on the viscosity sensitivity of LT-M, it is expected to show brighter fluorescence on cancer cell membranes. Fig.16 As shown in Figure C, when cancer cells (HeLa and HepG2 cells) and normal cells (HEK 293T and RAW 264.7) were stained with 10 μM LT-M and imaged under the same conditions, the cell membrane of cancer cells indeed showed brighter fluorescence, indicating that LT-M can distinguish cancer cells from normal cells based on the different viscosities on the cell membrane. Fig.17 As shown, when HEK 293T cells were stimulated with monensin and nystatin, the fluorescence on the cell membrane became brighter, which was consistent with the results of HeLa cells ( Fig.16 A) These data further demonstrate that LT-M can image changes in viscosity of cell membranes.

[0108] Monitoring changes in CM viscosity during ferroptosis. Ferroptosis is a cell death process discovered in recent years that is caused by iron imbalance and is characterized by lipid peroxidation and high iron dependence. Lipid peroxidation causes changes in cell membrane structure and composition, affecting the viscosity of the intracellular environment. However, changes in CM viscosity during ferroptosis are still unclear. This prompted us to use LT-M to study changes in CM viscosity during ferroptosis. Erastin-induced ferroptosis, such as Fig.18As shown, after pre-treatment of cells with different concentrations of Erastin, cell shrinkage was observed, indicating successful induction of ferroptosis. In addition, LT-M showed that the fluorescence in the CM of cells treated with Erastin increased compared with the control group, and gradually increased with the increase of Erastin concentration, indicating that the viscosity of CM increased during ferroptosis. Ferrostatin 1 (Fer-1) is an effective inhibitor of ferroptosis that can prevent the massive accumulation of intracellular cytoplasmic and lipid reactive oxygen species (ROS).

[0109] It is noteworthy that when cells were pretreated with Erastin and Fer-1 and then stained with LT-M, the fluorescence intensity of CM was comparable to that of the control group, but significantly lower than that of cells treated with Erastin ( Fig.18 (e in B). These results suggest that CM viscosity increases during ferroptosis and that this process can be detected by LT-M.

[0110] Monitoring changes in CM viscosity caused by hyperglycemia. It is well known that diabetes is a chronic disease characterized by hyperglycemia. Under hyperglycemic conditions, membrane proteins on the cell membrane become abnormal, resulting in impaired intracellular glucose uptake. This can lead to abnormal intracellular energy metabolism, oxidative stress, and inflammation, causing damage to the cell membrane structure and function, and leading to changes in the membrane microenvironment. However, to date, there are no fluorescent probes for detecting changes in cell membrane viscosity caused by hyperglycemia. Therefore, we established a hyperglycemic cell injury model by stimulating cells with high concentrations of glucose and performed imaging studies using LT-M. Fig.19 As shown in AC, after incubating HeLa cells with increasing glucose concentration (50-150 mM) for 12 h, the cell membrane fluorescence gradually increased after staining with LT-M. Flow cytometry obtained the same results ( Fig.19 ). This indicates that the viscosity of the cell membrane increases after hyperglycemia stimulation. Similar results were also observed in hyperglycemic HEK 293T cells ( Fig. 20 ). In addition, when cells were incubated with high concentrations of glucose, the fluorescence intensity on the cell membrane gradually increased with the extension of the stimulation time ( Fig.21 ). These results indicate that LT-M can detect changes in cell membrane viscosity caused by hyperglycemia, providing a possibility for the study of diabetes or diabetic complications.

[0111] LT-M in vivo imaging of diabetic nephropathy. Secondly, since most water-soluble molecules are metabolized and excreted in the urine within a few minutes after intravenous injection into mice, it is not conducive to in vivo tracking imaging.

[0112] Considering that diabetes is a very common metabolic disease in the world, it is often accompanied by serious complications and damage to major internal organs. Based on the viscosity sensitivity of the LT-M probe and its excellent cell membrane imaging ability, LT-M was further used for imaging of diabetic mice. The diabetic mouse model was established by intraperitoneal injection of STZ (streptozotocin), and normal mice were intraperitoneally injected with normal saline as the control group. After injection of STZ, the blood glucose of the mice increased significantly, indicating that the diabetic model was successfully established. Fig. 22 As shown in AB, after the injection of LT-M, the fluorescence signal in the kidney area on both sides of the diabetic mice gradually increased over time, and the fluorescence signal was brightest at about 15 minutes, which was significantly higher than that of the control group (about 2.2 times), indicating that the viscosity of the kidneys of diabetic mice was upregulated. This shows that LT-M is a probe that can image diabetic nephropathy in vivo. Subsequently, the above mice were killed and the main organs were separated for imaging examination. Fig. 22 As shown in Figure C, among the organs of normal mice and diabetic mice, only the kidneys have fluorescent signals, indicating that LT-M is mainly located in the kidneys. In contrast, the fluorescence of the kidneys of diabetic mice is significantly brighter, indicating that the viscosity of the kidneys of diabetic mice is significantly increased. Secondly, LT-M and H&E frozen section staining were used to observe kidney tissue. Fig.23 As shown in the figure, after LT-M staining under the same conditions, the fluorescence of the kidney tissue sections of diabetic mice was significantly higher than that of normal mice, which is consistent with the in vivo imaging results, further indicating that the viscosity of the kidneys of diabetic mice is increased. In addition, the tissue images also showed that LT-M is located on the cell membrane. H&E staining results ( Fig. 22 E) shows that there are significant differences in kidney tissue between the control group and the diabetic group. The kidney tissue of the diabetic group clearly showed glomerular nodular sclerosis and renal capsule expansion, indicating kidney damage in diabetic mice. Therefore, LT-M provides a unique molecular probe tool for the diagnosis of diabetes and diabetic nephropathy from the perspective of cell membrane microenvironment.

[0113] Other beneficial effects:

[0114] This application synthesized and studied three new cell membrane targetable viscosity-responsive probes, LT-M, LT-S, and LT-N. Among them, LT-M introduced a 1,4-diazoheterocyclic [2.2.2] octane quaternary ammonium salt group to achieve long-term (>12h) specific imaging of the cell membrane. LT-M has excellent cell membrane imaging performance, can sensitively sense changes in cell membrane viscosity, and can distinguish between tumor cells and normal cells. LT-M successfully detected changes in cell membrane viscosity during ferroptosis and high glucose stimulation, and found that cell membrane viscosity increased during these processes. In addition, LT-M can target the kidneys and has been successfully used to detect abnormal changes in viscosity of renal cells and tissues in diabetic mice. In summary, this work provides valuable probes for long-term cell membrane imaging, cell membrane viscosity detection, and the diagnosis and evaluation of diabetic nephropathy.

[0115] The specific implementation of the present invention described above does not constitute a limitation on the protection scope of the present invention. Any other corresponding changes and modifications made based on the technical concept of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A fluorescent probe, characterized in that Its structural formula is one or more of the following structural formulas: or Wherein, R1 represents one of C1-C6 alkyl groups; R2 represents or R3 represents one of C1-C6 alkyl groups, and R4 represents one of C1-C6 alkyl groups.

2. The fluorescent probe according to claim 1, characterized in that Its structural formula is one or more of the following structural formulas:

3. A method for preparing a fluorescent probe, characterized in that: The following steps are involved: The compound The solvent and piperidine are mixed to obtain a mixture, and the mixture is stirred at 75-85° C. to react to obtain a fluorescent probe; the compound is or 4. The method for preparing a fluorescent probe according to claim 3, characterized in that: The reaction time is 5-8 hours under stirring at 75-85°C.

5. Use of the fluorescent probe according to any one of claims 1 to 2 or the fluorescent probe prepared by the preparation method according to any one of claims 3 to 4 in cell membrane imaging.

6. Use of the fluorescent probe according to any one of claims 1 to 2 or the fluorescent probe prepared by the preparation method according to any one of claims 3 to 4 in a fluorescent probe for distinguishing cancer cells from normal cells.

7. Use of the fluorescent probe according to any one of claims 1 to 2 or the fluorescent probe prepared by the preparation method according to any one of claims 3 to 4 as a viscosity probe.

8. Use of the fluorescent probe according to any one of claims 1 to 2 or the fluorescent probe prepared by the preparation method according to any one of claims 3 to 4 in detecting cell membrane viscosity.

9. Use of the fluorescent probe according to any one of claims 1 to 2 or the fluorescent probe prepared by the preparation method according to any one of claims 3 to 4 in detecting changes in cell membrane viscosity caused by hyperglycemia.

10. The use according to claim 9, characterized in that: The cell membrane viscosity change is the cell membrane viscosity change in the kidney.

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