Temperature-sensitive fluorescent probe and preparation method and application thereof
阅读说明：本技术 一种温敏荧光探针及其制备方法和应用 (Temperature-sensitive fluorescent probe and preparation method and application thereof ) 是由 白钢 侯媛媛 申福葵 杨雯 于 2021-06-29 设计创作，主要内容包括：本发明公开了一种温敏荧光探针及其制备方法和应用,属于化学生物学领域。本发明温敏荧光探针的分子式为C-(33)H-(42)ClN-3O,分子量为531.3016；所述温敏荧光探针的制备方法包括以下步骤：制备酯基保护罗丹明B；制备羟基修饰罗丹明B；制备氯代罗丹明B；制备温敏荧光探针。本发明所制备的温敏荧光探针RhBⅣ,具有良好的温度敏感性,且靶向细胞线粒体,可以在活细胞和活体小动物上实时观测线粒体温度变化,可用于调控能量代谢的药物筛选,以及活体动物水平实时监测脏器温度的变化,为相关药效学评价提供新的检测方法,具有良好的应用前景。(The invention discloses a temperature-sensitive fluorescent probe and a preparation method and application thereof, belonging to the field of chemistry and biology. The molecular formula of the temperature-sensitive fluorescent probe is C 33 H 42 ClN 3 O, molecular weight 531.3016; the preparation method of the temperature-sensitive fluorescent probe comprises the following steps: preparing ester group protected rhodamine B; preparing hydroxyl modified rhodamine B; preparing chlororhodamine B; and preparing the temperature-sensitive fluorescent probe. The temperature-sensitive fluorescent probe RhB IV prepared by the invention has good temperature sensitivity, targets cell mitochondria, can observe the temperature change of the mitochondria in real time on living cells and living small animals, can be used for screening medicines for regulating and controlling energy metabolism and monitoring the level of the living animals in real timeThe change of the organ temperature provides a new detection method for the related pharmacodynamics evaluation, and has good application prospect.)
1. The temperature-sensitive fluorescent probe is characterized in that the molecular formula of the probe is C33H42ClN3O, molecular weight of 531.3016, and structural formula as follows:
2. the method for preparing a temperature-sensitive fluorescent probe according to claim 1, comprising the steps of:
step 1, preparing ester group protected rhodamine B: rhodamine B and K2CO3Reacting with iodomethane to obtain ester group protected rhodamine B;
the structural formula of ester group protected rhodamine B is shown in the specification
Step 2, preparing hydroxyl modified rhodamine B: the ester group protects rhodamine B to react with lithium aluminum hydride to obtain hydroxyl modified rhodamine B;
the structural formula of the hydroxyl modified rhodamine B is shown as
Step 3, preparing chlororhodamine B: reacting the hydroxyl modified rhodamine B with thionyl chloride to obtain chloro rhodamine B;
the structural formula of the chloro rhodamine B is shown as
Step 4, preparing a temperature-sensitive fluorescent probe: the chlororhodamine B, piperidine and Na2CO3And reacting to obtain the temperature-sensitive fluorescent probe.
3. The method for preparing a temperature-sensitive fluorescent probe according to claim 2, wherein the reaction solvent in the step 1 is anhydrous acetone, the reaction temperature is 65 ℃, and the reaction time is 12 hours.
4. The method for preparing a temperature-sensitive fluorescent probe according to claim 2, wherein the reaction solvent in the step 2 is anhydrous tetrahydrofuran, the reaction temperature is room temperature, and the reaction time is 12 hours.
5. The method for preparing a temperature-sensitive fluorescent probe according to claim 2, wherein the reaction solvent in the step 3 is anhydrous dichloromethane, the reaction temperature is room temperature, and the reaction time is 12 hours.
6. The preparation method of the temperature-sensitive fluorescent probe according to claim 2, wherein the reaction solvent in the step 4 is an anhydrous DMF solution, and the reaction conditions are as follows: the reaction was carried out in an ice bath for 30 minutes and then at room temperature for 12 hours.
7. The method for preparing a temperature-sensitive fluorescent probe according to claim 2, wherein the step of quenching the reaction with hydrochloric acid is further included after the target compound is generated in the steps 1 to 4.
8. The use of a temperature-sensitive fluorescent probe according to claim 1 in an ex vivo live cell biomarker.
Technical Field
The invention relates to the field of chemistry and biology, in particular to a temperature-sensitive fluorescent probe and a preparation method and application thereof.
Background
The abundance of the aromatic fluorescent compound is expressed to some extent by the magnitude of its fluorescence intensity. The generation of fluorescence and the magnitude of the fluorescence response intensity are not only influenced by the conjugated structure of the compound, but also related to the external environment. The environmental factors involved include temperature, polarity of the solvent, pH of the solvent, and concentration of the fluorescent molecules. The temperature can affect to some extent the fluorescence intensity of the compounds in solution. In general, the higher the temperature, the lower the quantum yield of fluorescence, and the lower the fluorescence intensity, because the higher the temperature, the faster the molecular motion, the higher the number of collisions between molecules, and the greater the energy loss, so that the fluorescence quantum yield decreases, and vice versa, the fluorescence quantum yield increases.
Mitochondria are energy factories of cells and play a crucial role in cell life. Mitochondrial temperature is an important indicator of mitochondrial function, and maintenance of mitochondrial temperature is the basis for cell growth, differentiation and survival, and has important significance. Therefore, the observation of the change in the temperature of cells using the change in the signal of a molecular probe (i.e., a fluorescence thermometer) has attracted considerable attention in the biological field. Mitochondrial dysfunction is closely associated with many diseases, such as metabolic diseases like diabetes, cardiovascular diseases, neurodegenerative and neuromuscular diseases, cancer, gastrointestinal diseases and liver diseases. The research on the temperature change of mitochondria is very important for understanding and controlling the molecular mechanism of cell function, so the development of the fluorescence probe related to mitochondria has important significance and has important promotion effect on the development of drugs and the mechanism research.
Various types of fluorescence thermometers have been reported, for example, using inorganic nanoparticles [ Fischer L H et al, Angew Chem Int Ed Engl,2011,50(20): 4546-. The chinese patent of the invention discloses two methods for preparing temperature sensitive probes, namely fluorescent temperature sensitive probes based on complex visible light excitation [ application number: CN201610802438.0 and a quantum dot temperature-sensitive probe [ CN201510906169.8] based on interface defects. The fluorescent probe which can be used for detecting the temperature change of organisms at present usually consists of a positioning group and a fluorescence detection group, and the whole is fat-biased molecule. This results in their inaccurate localization to intracellular mitochondria. In view of the rapid diffusion of heat into the extracellular environment, the prior art generally places a temperature probe in close proximity to the organelles to target the heat source of the organelles to better sense the change in the heat generated by the organelles. However, the distance between the fluorescent probe and the heat source is also considered to be a critical factor in terms of accuracy of temperature measurement, i.e., the temperature probe is placed in a position much closer to the organelle rather than exactly as the temperature measured on the mitochondria.
Rhodamine B (rhodamine B, RhB) is a fluorescent dye of the catechol type, with tautomerism of spiro ring opening and closing. Two aromatic rings in the rhodamine B are connected by oxygen atoms opposite to carbon atoms to form a six-membered ring, so that the rhodamine B has a rigid planar structure to form a large conjugated system. The spirolactam in the rhodamine probe can induce ring opening in the presence of a specific object, so that the color and fluorescence of the probe are changed. Meanwhile, because many modifiable binding sites exist in the rhodamine framework, the generation of fluorescence of the rhodamine framework is influenced by chemically modifying specific sites on the rhodamine framework. Arai et al synthesized a temperature sensitive fluorescent probe MitoThermoyellow based on rhodamine design, which has mitochondrial targeting ability, although it exhibited sensitivity to temperature changes at the cellular level, but since MitoThermoyellow had an excitation wavelength close to the emission wavelength (excitation wavelength 540nm, emission wavelength 570nm), strong mutual interference, and insufficient red shift of its emission wavelength, high endogenous background, it could not be used for in vivo imaging analysis, and is currently limited to cell-level analysis [ Arai S et al. Chem Commun (Camb),2015,51(38): 8044-. The molecular structure of the mitothermyolow probe is as follows:
although rhodamine B-based fluorescence-derived molecular probes have been widely used in the fields of biology and medicine, temperature-sensitive probes currently available for detection of living animals have not been reported.
Disclosure of Invention
The invention aims to provide a temperature-sensitive fluorescent probe, and a preparation method and application thereof, so as to solve the problems in the prior art, and the temperature-sensitive fluorescent probe can target mitochondria in cells and can be used for level detection of living cells and living animals.
In order to achieve the purpose, the invention provides the following scheme:
one of the purposes of the invention is to provide a temperature-sensitive fluorescent probe, and the molecular formula of the probe is C33H42ClN3O, molecular weight of 531.3016, and structural formula as follows:
the invention also aims to provide a preparation method of the temperature-sensitive fluorescent probe, which comprises the following steps:
step 1, preparing ester group protected rhodamine B: rhodamine B and K2CO3Reacting with iodomethane to obtain ester group protected rhodamine B;
the structural formula of ester group protected rhodamine B is shown in the specification
Step 2, preparing hydroxyl modified rhodamine B: the ester group protects rhodamine B to react with lithium aluminum hydride to obtain hydroxyl modified rhodamine B;
the structural formula of the hydroxyl modified rhodamine B is shown as
Step 3, preparing chlororhodamine B: reacting the hydroxyl modified rhodamine B with thionyl chloride to obtain chloro rhodamine B;
the structural formula of the chloro rhodamine B is shown as
Step 4, preparing a temperature-sensitive fluorescent probe: the chlororhodamine B, piperidine and Na2CO3And reacting to obtain the temperature-sensitive fluorescent probe.
Further, the reaction solvent of step 1 is anhydrous acetone, the reaction temperature is 65 ℃, and the reaction time is 12 hours.
Further, the reaction solvent of step 2 is anhydrous tetrahydrofuran, the reaction temperature is room temperature, and the reaction time is 12 hours.
Further, the reaction solvent in step 3 is anhydrous dichloromethane, the reaction temperature is room temperature, and the reaction time is 12 hours.
Further, the reaction solvent of step 4 is anhydrous DMF solution, and the reaction conditions are: the reaction was carried out in an ice bath for 30 minutes and then at room temperature for 12 hours.
Further, the step 1-4 comprises a step of quenching the reaction with hydrochloric acid after the target compound is produced.
The invention also aims to provide the application of the temperature-sensitive fluorescent probe in-vitro living cells.
The technical conception of the invention is as follows:
mitochondria have a bilayer membrane structure. The outer membrane is a limiting membrane and the inner membrane is folded inwardly to form ridges. The inner and outer membranes are not connected to form a membrane potential of about-180 mV. Currently, most mitochondrial localization fluorescent probes are based on the property of mitochondrial negative membrane potential. Staining was performed using a positively charged fluorophore or introducing a positively charged group into the fluorophore targeting mitochondria. The temperature-sensitive probe RhB IV in the invention contains positive charges of quaternary ammonium structure, so that mitochondrial targeting can be realized. Because the optimal excitation wavelength of the temperature-sensitive fluorescent probe RhBIV is 520nm and the optimal emission wavelength is 592nm, the interference of endogenous fluorescent substances can be effectively avoided, and the endogenous background is low.
The invention discloses the following technical effects:
(1) the optimal excitation wavelength of the temperature-sensitive fluorescent probe (RhBIV) prepared by the invention is 520nm, the optimal emission wavelength is 592nm, the difference between the optimal excitation wavelength and the optimal emission wavelength is large, and mutual interference is avoided;
(2) the fluorescence intensity of the temperature-sensitive probe RhB IV has good temperature-sensitive responsiveness, the fluorescence value response of the temperature-sensitive probe RhB IV is reduced along with the temperature rise in the range of 27-41 ℃, the fluorescence intensity of each change of high and low temperatures is not obviously different, and good linearity and stability are presented;
(3) cell viability detection and mouse acute toxicity tests prove that the temperature-sensitive probe RhB IV prepared by the invention has lower cytotoxicity which is 10 DEG-4The concentration below mol/L has no influence on the cell activity, and the single injection administration of less than 100mg/kg has no obvious toxicity on mice;
(4) the temperature-sensitive probe RhB IV prepared by the invention can be positioned on mitochondria in cells, can effectively indicate the temperature change of the mitochondria of the cells, and can be used for activity evaluation of the mitochondria of the cells. In vivo imaging analysis of a small animal injected with a temperature-sensitive probe RhB IV from a tail vein of a mouse shows that the RhB IV probe is mainly distributed in the liver after administration, reaches a peak value after about 1.5 hours, can be effectively maintained for 6 hours, and can be used for monitoring the body temperature change of the mouse;
(5) the temperature-sensitive fluorescent probe RhB IV prepared by the invention has good temperature sensitivity, targets cell mitochondria, can observe the temperature change of mitochondria in real time on living cells and living small animals, can be used for screening medicines for regulating and controlling energy metabolism and monitoring the change of organ temperature in real time at the level of the living animals, provides a new detection method for related pharmacodynamic evaluation, and has good application prospect.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a synthetic route of a temperature-sensitive fluorescent probe RhB IV of the present invention;
FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of a temperature-sensitive probe RhB IV;
FIG. 3 is the nuclear magnetic resonance carbon spectrum of the temperature sensitive probe RhB IV;
FIG. 4 is an HPLC liquid chromatogram of a temperature sensitive probe RhB IV;
FIG. 5 is a high resolution mass spectrogram of a temperature sensitive probe RhB IV;
FIG. 6 is a graph of the fluorescence intensity of RhB, MitoThermoyellow probes and temperature sensitive fluorescent probe RhB IV at the optimal excitation wavelength and the fluorescence spectrum as a function of temperature;
FIG. 7 is a cytotoxicity test of a temperature-sensitive fluorescent probe RhB IV on Hepg2 cells;
FIG. 8 is a co-localization study of a temperature-sensitive fluorescent probe RhB IV and a mitochondrial dye MitoTracker;
FIG. 9 is a temperature sensitivity test of a temperature sensitive fluorescent probe RhB IV on Hepg2 cells; wherein, the graph A is a fluorescence intensity-temperature change curve of a temperature-sensitive probe RhB IV based on the cell level; the graph B is the stability investigation of the temperature-sensitive probe RhB IV;
FIG. 10 is a graph showing the effect of a temperature sensitive fluorescent probe RhB IV on the change of cell temperature when detecting a drug;
FIG. 11 shows the distribution and application of temperature sensitive fluorescent probe RhB IV in living animals; wherein, the graph A is the distribution condition in the body of the mouse after 30 minutes of administration; panel B is a study of metabolism in mice following dosing; and the graph C is the change of the organ temperature of the mouse before and after the detection of the drug intervention by using a temperature-sensitive probe RhB IV.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
Example 1: synthesis of temperature-sensitive probe RhB IV
1) 500mg (1.04mmoL,1.0eq) of rhodamine B and 290mg (2.08mmoL,2.0eq) of K2CO3Placing the mixture into a 50mL round-bottom flask, adding 10mL of anhydrous acetone solution under the protection of nitrogen, slowly dropwise adding 240mg of methyl iodide, heating to 65 ℃, and reacting for 12 hours. After the reaction of the raw material rhodamine B is detected by TLC, the reaction is quenched by 1mol/L hydrochloric acid. The organic phase was dried over anhydrous sodium sulfate, concentrated in vacuo, and purified by silica gel column to give 420mg of ester-protected rhodamine B (RhBI) at 82.1% yield.
2) 400mg (0.81mmol,1.0eq) of ester group-protected rhodamine B (RhBI) is placed in a 50mL round-bottomed flask, 10mL of anhydrous tetrahydrofuran solution is added under the protection of nitrogen, 63mg (1.62mmol,2.0eq) of lithium aluminum hydride is slowly added under ice bath, and the reaction is carried out at room temperature for 12 hours. After TLC detection of the disappearance of the raw materials, 1mol/L hydrochloric acid was added to quench the reaction, the reaction was extracted with ethyl acetate and washed with saturated saline three times, the organic phase was dried over anhydrous sodium sulfate, concentrated in vacuo and purified with a silica gel column to give 278mg of hydroxyl-modified rhodamine B (RhB II) with a yield of 74%.
3) 250mg (0.54mmol,1.0eq) of hydroxyl-modified rhodamine B (RhBII) is placed in a 50mL round-bottom flask, 5mL of anhydrous dichloromethane solution is added under the protection of nitrogen, 193mg (1.62mmol,3.0eq) of thionyl chloride is slowly added under ice bath, and the reaction is carried out at room temperature for 12 hours. After TLC detection of the disappearance of the starting material, the reaction was quenched with 1mol/L sodium bicarbonate, extracted with ethyl acetate and washed three times with saturated brine, and the organic phase was dried over anhydrous sodium sulfate, concentrated in vacuo and purified by silica gel column to give 229mg of chlororhodamine B (RhBIII) in 88% yield.
4) A mixture of 200mg (0.42mmol,1.0eq) of chlororhodamine B (RhBIII) and 71mg (0.84mmol,2.0eq) of piperidine is placed in a 25mL round-bottomed flask, 8mL of anhydrous DMF solution is added under argon protection, and 174mg (1.26mmol,3.0eq) of Na is added under ice bath2CO3And 70mg (0.42mmol,1.0eq) of KI were reacted in ice bath for 30 minutes, followed by reaction at room temperature for 12 hours. After TLC detection, the raw material is quenched by 1mol/L hydrochloric acid, extracted by ethyl acetate and washed by saturated saline solution for three times, the organic phase is dried by anhydrous sodium sulfate and then concentrated in vacuum to obtain a crude product of a final product, and the crude product is further purified by a silica gel column to obtain 206mg of temperature sensitive probe RhB IV, wherein the yield is 67%. The synthetic route of the temperature-sensitive probe RhB IV is shown in figure 1.
The Nuclear Magnetic Resonance (NMR) detection result of the temperature-sensitive probe RhB IV is as follows:1H NMR(400MHz,CDCl3)δ7.13–7.02(m,3H),6.79(d,J＝8.6Hz,2H),6.37(d,J＝2.6Hz,2H),6.26(dd,J＝8.6,2.6Hz,2H),5.68(s,1H),3.61(s,2H),3.31(q,J＝7.1Hz,8H),2.41(s,4H),1.55(t,J＝5.6Hz,4H),1.46–1.41(m,2H),1.14(t,J＝7.0Hz,12H).13C NMR(100MHz,CDCl3) δ 152.0,147.4,131.3,130.6,130.3,128.0,125.2,113.0,107.4,98.7,62.5,54.6,44.4,26.1,24.6,12.7 as shown in fig. 2 and 3.
And (3) detecting the purity of the temperature-sensitive probe RhB IV by adopting a High Performance Liquid Chromatography (HPLC), wherein the used instruments and chromatographic conditions are as follows:
the instrument, Shimadzu high performance liquid chromatograph LC-15C, is equipped with binary pump, SPD-15C ultraviolet detector, CTO-15C column incubator, LC-Solution 15C workstation, Agilent Eclipse Plus C18 chromatographic column (4.6X 100mm, 3.5 μm).
The chromatographic conditions comprise a mobile phase A of 0.05 percent phosphoric acid water and a mobile phase B of acetonitrile. Setting a program, wherein the phase B is set as follows: 0.01-6 minutes, 4-45%; 45-65% of the total amount of the catalyst for 6-8 minutes; 65-85% for 8-12 minutes; 85-100% of the solution for 12-22 minutes; 22-25 minutes, 100%. Flow rate: 1.00mL/min, column temperature: 28 ℃, detection wavelength: 550 nm. The HPLC purity of the temperature sensitive probe RhB IV is 99.2% by integration calculation, as shown in FIG. 4.
Carrying out High Resolution Mass Spectrometry (HRMS) detection on the temperature-sensitive probe RhBIV, [ M-Cl ]]+The calculated high resolution mass spectrum HRMS is: 496.3322, found: 496.3326, as shown in fig. 5.
According to the results of NMR and HRMS identification, the finally prepared compound is a target product, the temperature-sensitive probe RhB IV is mauve powder, the melting point is 187 ℃, and the molecular formula is C33H42ClN3O, molecular weight 531.3016, is easily soluble in water and ethanol, and slightly soluble in acetone, chloroform, hydrochloric acid, etc.
Example 2: thermo-sensitive probe RhB IV fluorescence spectrum detection and thermo-sensitive investigation
Using a multifunctional microplate reader (Spark, Austria) pair 10-5And dissolving the mol/L temperature-sensitive probe RhBIV in physiological saline for fluorescence scanning, and determining that the optimal excitation wavelength is 520nm and the optimal emission wavelength is 592 nm. Then, under the same concentration conditions, fluorescence spectrum scanning was performed on rhodamine B (RhB), a MitoThermoyellow probe (synthesized by Shizu Seienseopropyco Co., Ltd., entrusted) and a temperature sensitive probe RhB IV at an optimum excitation wavelength of 520nm, and the effect of temperature response was examined.
The experimental results show that: the fluorescence intensity of rhodamine B (RhB) is hardly influenced by temperature; the fluorescence intensity attenuation of RhB IV shows better temperature dependence along with the temperature rise; whereas the change in fluorescence amplitude of the MitoThermoYellow probe was small (as shown in FIG. 6). The optimal emission wavelength 592nm of the temperature-sensitive probe RhB IV is obviously higher than the emission wavelength 570nm of MitoThermoyellow, which shows that the temperature-sensitive probe RhB IV has better fluorescence responsiveness and temperature sensitivity and can be used for subsequent function evaluation.
Example 3: cytotoxicity and single-oral acute toxicity investigation of temperature-sensitive probe RhB IV
The Cell Counting Kit-8(CCK-8) Kit is used for detecting the influence of different doses of temperature-sensitive probes RhB IV on the activity of HepG2 cells, and the experimental method is as follows:
1) the recovered HepG2 cells were placed in DMEM complete medium (containing 10% FBS and 1% double antibody) and placed in 5% CO2Culturing at 37 deg.C in incubator. When the cells grow until the fusion degree is 50-60%, adding 100 mu L of serum-free culture medium containing temperature-sensitive probes RhBIV with different concentrations into each hole, and incubating overnight; discarding the culture medium, and adding a mixture of 10 μ L of CCK-8 solution and 90 μ L of PBS solution; placing the culture plate in an incubator for incubation for 30 minutes; the absorbance was measured by a microplate reader (Spark, Austria) according to the kit instructions, and the detection wavelength was 450 nm.
2) The experimental setup was calibrated with a control group without drug and a blank group without cells and drug. The experiment was repeated 8 times, the mean was taken and the effect of the probe on cell viability was counted by analysis of variance. Calculating the formula: cell survival rate is [ (As-Ab)/(Ac-Ab) ] × 100%. Wherein, As represents the absorbance of the experimental group; ac represents the absorbance of the control group; ab represents blank absorbance.
3) The experimental result is shown in FIG. 7, when the RhB IV concentration of the temperature-sensitive probe is less than 10-4At mol/L, RhB IV has less toxicity to HepG2 liver cancer cells, and when the concentration exceeds 5x 10-3At mol/L, the survival rate of HepG2 liver cancer cells is reduced. Thus, option 10-4The concentration below mol/L is the safe dose for cell level detection.
4) The rhodamine B is used as a food additive, and the safe dosage of the rhodamine B is larger. By referring to the intravenous administration dosage, 20 g of Kunming mice are selected to be subjected to intravenous injection of RhB IV, and when the single injection administration is less than 100mg/kg, the mice do not find obvious toxicity.
Example 4: cell localization of temperature sensitive Probe RhB IV
Culturing HepG2 cells according to the conditions of example 3, digesting with trypsin when the cells grow to 70-80%, transferring the cells to a confocal dish (2 mL each), and placing in CO2The incubator continued overnight. When the cells are fused to 50%, the culture medium is discarded, the cells are washed three times by PBS, and then 10 diluted by serum-free culture medium is added-6Putting the mol/L temperature-sensitive probe RhB IV in CO2The incubator was allowed to incubate for 6 hours. After 3 PBS washes, mitochondrial dye was added(10-7moL/L) for 15 minutes, washed 3 times with PBS and immediately subjected to confocal imaging analysis under a confocal microscope on HepG2 cells (LSM 800with Airyscan, germany). Mitochondrial dyesThe detection is carried out by adopting 644nm wavelength excitation and 665nm emission wavelength detection. The temperature-sensitive probe RhBIV is excited at the wavelength of 540nm and detected at the emission wavelength of 600 nm. The experimental result shows that the green fluorescence generated by the temperature-sensitive probe can be mixed with mitochondrial dyeThe red fluorescence of (a) can be displayed as orange by partial coincidence, and the Mannesmann co-localization coefficient is 0.96, as shown in FIG. 8. Proves that the temperature-sensitive probe can interact with mitochondria in cells and can be used for detecting the temperature change of the mitochondria.
Example 5: thermo-sensitivity examination of RhB IV probe based on HepG2 cells
1) HepG2 cells were cultured under the conditions described in example 3, and when the cells grew to 70-80%, they were digested and inoculated into a black light-shielding 96-well plate at a cell density of 104/well and 200. mu.L/well, and then added to a final concentration of 10-5And (3) placing the mol/L temperature-sensitive probe RhB IV in an incubator for further culturing for 20 minutes. Adding 4% of polyAfter the cells are fixed by the formaldehyde solution for 15 minutes, the cells are washed by PBS (phosphate buffer solution) which is pre-treated at the constant temperature of 27 ℃ for three times, and then are quickly placed in a temperature-controlled microplate reader, a temperature rise program is set (the temperature rises by 2 ℃ per minute), the temperature range of 27-41 ℃ is taken, the fluorescence intensity change is respectively detected, the excitation wavelength is set to 520nm, and the emission wavelength is set to 592 nm. The experiment was repeated 6 times, and the change of fluorescence intensity was expressed as an average value, and as a result, as shown in fig. 9A, the fluorescence value response gradually decreased with the increase of temperature, and a good linear relationship was exhibited in the range of 27 ℃ to 41 ℃. The linear fit satisfies the equation: Y-780.5X +45871 (where X is temperature and Y is fluorescence intensity) was calculated to decrease fluorescence intensity by 55% from 27 ℃ to 41 ℃ with an average decrease in fluorescence response of about 3.92% per 1 ℃ increase.
2) In order to examine the stability of the fluorescence intensity of the temperature-sensitive probe RhB IV corresponding to the temperature, the temperature of the enzyme label plate is increased from 27 ℃ to 41 ℃, then the temperature is decreased from 41 ℃ to 27 ℃, the detection is repeated for 3 times under the same conditions that the excitation wavelength is 520nm and the emission wavelength is 592nm, the stability of the probe to the temperature change is evaluated by counting the change of the fluorescence value, the result is shown in figure 9B, along with the repeated change of the temperature, the fluorescence intensity of the temperature-sensitive probe can be found to be along with the repeated change of the temperature, the fluorescence intensity of each change of the high temperature and the low temperature has no obvious difference, and the temperature-sensitive probe RhB IV has good temperature stability and reproducibility.
Example 6: method for detecting influence of drug on cell temperature change based on temperature-sensitive probe RhB IV
Genipin is a natural inhibitor of mitochondrial uncoupling protein (UCP), and can effectively reduce mitochondrial temperature. In the embodiment, oxidative phosphorylation inhibitor carbonyl cyanide metachlorophenylhydrazone (CCCP) is adopted to cause mitochondria to generate high fever, and then a temperature-sensitive probe RhB IV is used for investigating the antipyretic effect of genipin on a cellular level.
Specifically, HepG2 cells were cultured according to the method of example 3, and the cultured HepG2 cells were seeded in a black light-shielding 96-well plate at a cell density of 104Per well, each at a concentration of 10-4mol/L、10-5mol/L、10-6mol/L and 10-7mol/L genipin, incubation at 37 ℃After 6 hours, wash three times with PBS, add 5X 10-5mol/L carbonyl cyanide metachlorophenylhydrazone (CCCP) incubation for 30 min, washing three times with PBS, and addition of 10-5And (3) continuously incubating the temperature-sensitive probe RhBIV of mol/L for 20 minutes, washing by PBS, and measuring the change of fluorescence intensity under an enzyme-labeling instrument. Excitation wavelength 520nm and emission wavelength 592 nm. And simultaneously setting a blank group and a model group, wherein the blank group is not added with CCCP and genipin, and the model group is not added with genipin.
The experimental results are shown in fig. 10, compared with the blank group, the fluorescence value of the model group is reduced obviously after the CCCP is added, which indicates that the CCCP can induce mitochondrial fever. Genipin can increase the fluorescence intensity of rhbiv probes in a concentration-dependent manner compared to the model group, and genipin exhibits a concentration-dependent antipyretic activity (P < 0.01;. P <0.001) compared to the model group.
Example 7: temperature-sensitive probe RhB IV-based in-vivo detection of body temperature change of small animals
Taking 20 g Kunming mice, injecting a temperature-sensitive probe RhBIV of 10mg/Kg into the tail vein, detecting the distribution condition of the probe in the mice by using a small animal living body imager (NightOWL II LB983, Germany) after administration for 30 minutes, and obtaining the result as shown in figure 11A, wherein the temperature-sensitive probe is mainly enriched in the liver, the kidney and the lymph tissue of the mice; dynamic observations revealed that the fluorescence intensity of rhboiv probe molecules remained essentially stable in the liver for approximately 60 to 120 minutes, peaked for 90 minutes, and lasted for 6 hours (4 animals per group). Thus, the optimal image acquisition time was determined to be 90 minutes as shown in fig. 11B.
In order to investigate the influence of the drug on the body temperature change of the mouse, the body temperature change of the small animal is detected by a 2, 4-dinitrophenol induced mouse fever model and a temperature sensitive probe RhBIV. A blank group, a 2, 4-dinitrophenol fever group, an antadine group and a genipin administration group (4 in each group) are set in the experiment, and the relative change of the body temperature of the mice before and after administration is respectively examined through the change of the fluorescence intensity of a temperature-sensitive probe RhB IV of the temperature-sensitive probe. As shown in FIG. 11C, 2, 4-dinitrophenol significantly increased the body temperature of mice and the fluorescence intensity of the temperature sensitive probe RhBIV was attenuated (compared to the blank, # # P < 0.01). Both the antadine (20mg/Kg) and genipin (28mg/Kg) showed good antipyretic effects (P <0.001, P <0.05 compared to the model group). The experimental result shows that the temperature-sensitive probe RhB IV can detect the change of the body temperature of a mouse on the living body level, and has good application prospect.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.