CN115611798A - pH triggered charge reversible fluorescent probe and application thereof in cell imaging - Google Patents
pH triggered charge reversible fluorescent probe and application thereof in cell imaging Download PDFInfo
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Abstract
The invention belongs to the technical field of advanced materials and biomedicine, relates to clinical diagnosis materials, and particularly relates to a pH triggered charge reversible fluorescent probe and application thereof in cell imaging. The chemical structure of the pH triggering charge reversible fluorescent probe is
The fluorescent probe provided by the invention can stain LDs and nucleolus with two different colors at the same time, and the physical contact between LDs and nucleolus can be visualized for the first time.
Description
Technical Field
The invention belongs to the technical field of advanced materials and biomedicine, relates to clinical diagnosis materials, and particularly relates to a pH triggered charge reversible fluorescent probe and application thereof in cell imaging.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
To the inventors' study, it is understood that although various kinetics of Lipid Droplets (LDs) and their cytoplasmic functions have been intensively explored, the functions and changes of LDs in the nucleus are still unclear. The only prior art for non-destructive detection of changes in LDs in the nucleus of cells using fluorescent probes is immunofluorescence microscopy using BODIPY 558 (a commercial LDs probe) and Hoechst 33342 (a commercial nuclear probe). However, nucleoli, an important component of the nucleus, has not been considered in exploring the function of LDs in the nucleus. Furthermore, although changes in nuclear LDs can be explored by co-staining experiments, the uptake rates of the two different types of probes are different and may interfere with simultaneous imaging. Therefore, the development of a two-color multi-target fluorescent probe, such as simultaneous detection of LDs and nucleoli, can provide an effective means for further exploring the functional significance of LDs in the nucleus. Unfortunately, the design of nucleolar probes has been a challenge due to the lack of specific RNA targeting groups, while labeling nucleoli and LDs have become more valuable.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide a pH-triggered charge reversible fluorescent probe and application thereof in cell imaging.
In order to achieve the purpose, the technical scheme of the invention is as follows:
in one aspect, a pH triggered charge reversible fluorescent probe has a chemical structure shown below:
its chemical name is 2- ((1E, 3E) -4- (4- (dimethylamino) phenyl) but-1, 3-dien-1-yl) -3- (2-hydroxyethyl) -1, 1-dimethyl-1H-benzo [ e ] indol-3-ium iodide, noted LD-Nu.
On the other hand, the preparation method of the pH triggered charge reversible fluorescent probe comprises the steps of obtaining the fluorescent probe LD-Nu by using the compound 1 according to the following reaction route;
in a third aspect, the preparation composition comprises an active ingredient and an auxiliary material, wherein the active ingredient is the pH triggered charge reversible fluorescent probe.
In a fourth aspect, the application of the pH triggering charge reversible fluorescent probe in pH detection is provided. Researches show that the fluorescence of the pH triggered charge reversible fluorescent probe provided by the invention can generate blue shift in an acidic environment relative to an alkaline environment, so that the pH change can be sensitively detected.
In a fifth aspect, the application of the above-mentioned pH triggered charge reversible fluorescent probe in cell imaging is to stain lipid droplets, nucleolus or stain lipid droplets and nucleolus simultaneously in cells by using the pH triggered charge reversible fluorescent probe.
And in a sixth aspect, the cell imaging detection kit comprises an active component and a buffer solution, wherein the active component is the pH triggered charge reversible fluorescent probe or the preparation composition.
The invention constructs a pH triggered charge reversible fluorescent probe (LD-Nu) based on the pH value and charge difference between LDs and nucleolus and based on a cyclization and ring-opening mechanism. The test results confirmed that the LD-Nu probe stains LDs and nucleolus simultaneously in two different colors, visualizing for the first time the physical contact between LDs and nucleolus. The fluorescence of the probe LD-Nu can generate blue shift in an acid environment relative to an alkaline environment, so that the sensitive detection of pH change is realized. In the case of simultaneous presence of RNA and DNA, LD-Nu can preferentially bind RNA, which provides a precondition for specific staining of nucleoli in the nucleus by the probe. Meanwhile, the relationship between LDs and nucleolus is further researched, and the result shows that the interaction between the LDs and the nucleolus is more easily influenced by the abnormality of LDs than that of the nucleolus. In addition, cell imaging results showed that LDs in cytoplasm and nucleus could be observed using LD-Nu probe, and interestingly, LDs in cytoplasm were more susceptible to external stimuli than LDs in nucleus.
In conclusion, the pH triggered charge reversible fluorescent probe provided by the invention can specifically dye LDs and nucleolus in a soft acidic or weakly alkaline environment and can further explore the interaction relationship between the LDs and the nucleolus.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are included to illustrate an exemplary embodiment of the invention and not to limit the invention.
FIG. 1 variation of the absorbance and fluorescence spectra with pH (a, b, c) for the probe LD-Nu (10. Mu.M), E x (b)＝380nm，E x (c) =603nm. (d) In I 727nm /I 470nm Fluorescence ratio versus pH. (e) pH and log [ (I) max -I)/(I-I min )]A graph of the relationship (c). The y-intercept is the pKa value of the probe (6.24. + -. 0.79). (f) Photostability of LD-Nu (10. Mu.M) probe at pH 4.0 and 9.0.
FIG. 2 pH sensing mechanism of LD-Nu: (a) Probe LD-Nu in DMSO-d before and after NaOH addition 6 In (1) 1 H NMR spectra, (b) optimal geometry of LD-Nu in ring-opened and cyclized form.
FIG. 3 shows unimolecular configuration of the positively charged form of LD-Nu in single crystal cells.
FIG. 4 MTT results of cell viability of HepG2, heLa, A549 and RAW264.7 after 24h culture of LD-Nu at various concentrations.
FIG. 5 fluorescence imaging of LD-Nu stained HepG2 cells (2. Mu.M, 10 min). (a): green channel, (b): red channel, (c): DIC, (d): merged images of channels a and b, (e) merged images of channels a and c, (f): merged images of channels b and c. (g-i): channels a, c, and e are enlarged images marked with solid line boxes, respectively. (j-l): the magnified images of channels b, d, f are marked with dashed boxes. Green channel: e x ＝405nm，E m =420-550nm; red channel: e x ＝633nm，E m 650-850nm. Sign boardRuler (a-f) =20 μm, ruler (g-l) =5 μm.
FIG. 6 fluorescence imaging of LD-Nu stained HeLa cells (2. Mu.M, 10 min). (a): green channel, (b): red channel, (c): DIC, (d): merged images of channels a and b, (e) merged images of channels a and c, (f): merged images of channels b and c. (g-i): channels a, c and e are enlarged images marked with solid line boxes, respectively. (j-l): the magnified images of channels b, d, f are marked with dashed boxes. Green channel: e x ＝405nm，E m =420-550nm; red channel: e x ＝633nm，E m =650-850nm. Scale (a-f) =20 μm, scale (g-l) =5 μm.
FIG. 7 fluorescence imaging of LD-Nu stained RAW cells (2. Mu.M, 10 min). (a): green channel, (b): red channel, (c): DIC, (d): merged images of channels a and b, (e) merged images of channels a and c, (f): merged images of channels b and c. (g-i): channels a, c, and e are enlarged images marked with solid line boxes, respectively. (j-l): the magnified images of channels b, d, f are marked with dashed boxes. Green channel: e x ＝405nm，E m =420-550nm; red channel: e x ＝633nm，E m =650-850nm. Scale (a-f) =20 μm, scale (g-l) =5 μm.
FIG. 8 fluorescent image of HepG2 cells under LD-Nu (2. Mu.M, 10 min) with green and red fluorescence channels. The method comprises the following steps of (a) two-dimensional fluorescence image, (b) solid line frame marking panel (a) enlarged image, (c) panel (b) three-dimensional image, and (d) panel (c) three-dimensional perspective correction plane image. Green channel: e x ＝405nm，E m =420-550nm; red channel: e x ＝633nm，E m =650-850nm. Scale (a) =40 μm, scale (b, c) =10 μm, and scale (d) =5 μm.
FIG. 9 fluorescence titration spectra of LD-Nu (10. Mu.M) versus RNA response, E x =603nm. b) The graphs were fitted to the Scatchard equation with an RNA binding constant of 2.0 x 10 6 M -1 Fitting constant R 2 =0.947. c) The probe LD-Nu is butted with an RNA molecule, and d) the two-dimensional interaction map is detailed.
FIG. 10 a) fluorescence titration spectra of LD-Nu (10. Mu.M) on DNA (0-3750. Mu.g/mL), E x =603nm; b) Root of herbaceous plantAccording to the curve fitted by Scatchard equation, the binding constant of DNA is 2.8 × 10 5 M -1 Fitting constant R 2 =0.925; c) LD-Nu (10. Mu.M) fluorescence emission spectra at the same concentration 2500. Mu.g/mL for RNA and DNA.
FIG. 11 fluorescence (green, red channel), DIC and their pooled images of HepG2 cells stained with LD-Nu (2. Mu.M, 10 min) after 4h incubation with DNase and RNase. Arrows indicate changes in nucleoli. Green channel: e x ＝405nm，E m =420-550nm; red channel: e x ＝633nm，E m =650-850nm. Scale =20 μm.
FIG. 12 images of fluorescence (green, red channel), DIC and their incorporation in HepG2 cells stained with LD-Nu (2. Mu.M, 10 min) after 4h incubation with DNase and RNase digestion. Arrows indicate changes in nucleoli. Green channel: e x ＝405nm，E m =420-550nm; red channel: e x ＝633nm，E m 650-850nm. Scale =20 μm.
FIG. 13 blue channel, DIC and pooled images after DAPI staining at different times after 4h incubation of HepG2 cells with DNase digestion. Blue channel: e x ＝405nm，E m And =420-550nm. Scale =20 μm.
FIG. 14 intensity profiles of fluorescence (green channel, red channel), DIC and their pooled images and white arrows of LD-Nu (2. Mu.M, 10 min) stained HepG2 cells after incubation with PFOA for various times under normal conditions. Overlay of FIG. 1: combining the green and red channels with DIC; overlay of FIG. 2: merging the red and green channel images; green channel: e x ＝405nm，E m =420-550nm; red channel: e x ＝633nm，E m 650-850nm. Scale =10 μm.
FIG. 15 Normal State and H 2 O 2 Fluorescence (green channel, red channel), DIC, pool, magnification and LD-Nu (2. Mu.M, 10 min) staining patterns at different ratios of HepG2 cells after culture. Amplifying an image: regions marked with solid boxes in the images are merged. Green channel: e x ＝405nm，E m =420-550nm; red channel: e x ＝633nm，E m 650-850nm. Scale with a measuring device=20 μm, scale (enlargement) =5 μm.
FIG. 16 Normal State and H 2 O 2 Fluorescence (green channel, red channel), DIC, pooled images, magnified images and LD-Nu (2. Mu.M, 10 min) staining patterns at different ratios of HeLa cells after culture. Amplifying an image: regions marked with solid boxes in the images are merged. Green channel: e x ＝405nm，E m =420-550nm; red channel: e x ＝633nm，E m =650-850nm. Scale =20 μm, scale (zoom) =5 μm.
FIG. 17 fluorescence (green channel, red channel), DIC, combined magnified images, and different ratios of LD-Nu stained RAW cells (2. Mu.M, 10 min), after incubation with LDL for 12h under normal conditions. Amplifying an image: regions marked with solid boxes in the images are merged. Green channel: e x ＝405nm，E m =420-550nm, red channel: e x ＝633nm，E m =650-850nm. Scale =20 μm, scale (magnification) =5 μm.
FIG. 18 fluorescence (green channel, red channel), DIC, pooled images, magnified images and different ratios of LD-Nu (2. Mu.M, 10 min) stained cells of HepG2 cells after incubation with LPS under normal conditions. Amplifying the image: regions marked with solid boxes in the images are merged. Green channel: e x ＝405nm，E m =420-550nm; red channel: e x ＝633nm，E m =650-850nm. Scale =20 μm, scale (zoom) =5 μm.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. 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.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
In view of the fact that the existing same type of fluorescent probe cannot stain lipid droplets and nucleoli at the same time, the invention provides a pH triggered charge reversible fluorescent probe and application thereof in cell imaging.
In an exemplary embodiment of the present invention, a pH triggered charge reversible fluorescent probe is provided, which has a chemical structure as follows:
its chemical name is 2- ((1E, 3E) -4- (4- (dimethylamino) phenyl) but-1, 3-dien-1-yl) -3- (2-hydroxyethyl) -1, 1-dimethyl-1H-benzo [ e ] indol-3-ium iodide, noted LD-Nu.
In another embodiment of the invention, the preparation method of the pH triggered charge reversible fluorescent probe is provided, and comprises the steps of obtaining the fluorescent probe LD-Nu by using the compound 1 according to the following reaction route;
in some embodiments, compound 1 is quaternized with iodoethanol to obtain compound 2, and compound 2 is condensed with compound 3 to obtain fluorescent probe LD-Nu.
In some embodiments, compound 1 is heated to reflux with iodoethanol in chloroform to obtain compound 2. The molar ratio of the compound 1 to the iodoethanol is 1.
In some embodiments, the compound 2 is added into ethanol, heated and dissolved, then the compound 3 is added, heated and refluxed, and reacted for at least 48 hours, so as to obtain the fluorescent probe LD-Nu.
According to a third embodiment of the invention, a preparation composition is provided, which comprises an active ingredient and an auxiliary material, wherein the active ingredient is the pH triggered charge reversible fluorescent probe.
The auxiliary materials of the invention are pharmaceutic auxiliary materials, such as solubilizer, emulsifier, stabilizer, preservative and the like.
In a fourth embodiment of the invention, the application of the pH triggered charge reversible fluorescent probe in pH detection is provided. Researches show that the fluorescence of the pH triggered charge reversible fluorescent probe provided by the invention can generate blue shift in an acidic environment relative to an alkaline environment, so that the pH change can be sensitively detected.
In a fifth embodiment of the present invention, there is provided a use of the above-mentioned pH-triggered charge-reversible fluorescent probe in cell imaging, wherein the cell imaging is lipid drop staining, nucleolus staining or simultaneous staining of lipid drop and nucleolus in cells by using the pH-triggered charge-reversible fluorescent probe.
The cell is a living cell, and in some embodiments, the living cell is an immortalized cell or a normal cell. Specifically, the immortalized cell is a HeLa, hepG2 or A549 cell; the normal cells are RAW264.7 cells.
The application in cell imaging can be used for the purpose of diagnosis and treatment of diseases, and can also be used for the purpose of diagnosis and treatment of non-diseases. The purpose of the diagnosis and treatment of non-diseases may be scientific research, such as research on cytotoxicity and cell staining, research on specific RNA responses, and research on further exploring the interaction between LDs and nucleoli; it may also refer to the use for the manufacture of a medicament, for example for the manufacture of a cell imaging agent.
In a sixth embodiment of the invention, a cell imaging detection kit is provided, which comprises an active component and a buffer solution, wherein the active component is the above pH triggered charge reversible fluorescent probe or the above preparation composition.
In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.
Example 1
Synthetic route of LD-Nu
4mL (25.2 mmol) of the compound 2, 3-trimethylbenzindole and 30mL of chloroform were added to a 100mL round-bottom flask and stirred well. Then 2mL (25.2 mmol) of iodoethanol is added into the solution, the mixture is heated and refluxed for reaction for more than 48 hours, and the reaction is monitored by column chromatography. After the reaction is finished, the system is cooled to room temperature and filtered to obtain 3- (2-hydroxyethyl) -1, 2-trimethyl-1 h-benzo [ e)]Indole-3-ammonium iodide. 1 HNMR(DMSO-d 6 ,400MHz)δ(ppm)：8.39(d,J＝8.4Hz,1H),8.29(d,J＝8.9Hz,1H),8.22(d,J＝8.2Hz,1H),8.14(d,J＝8.9Hz，1H),7.76(dt,J＝24.2,7.4Hz,2H),5.32(s，1H),4.72(t,J＝5.1Hz,2H),3.94(t,J＝5.1Hz,2H),2.92(s,3H),1.78(,6H)。
Then 3- (2-hydroxyethyl) -1, 2-trimethyl-1 h-benzo [ e ]]Indole-3-ammonium iodide (0.2g, 0.525mmol) and ethanol (30 mL) were added to a three-necked flask and heated at 55 ℃ until dissolved. Then, 4-dimethylaminocinnamaldehyde (0.11g, 0.606mmol) was added to be completely dissolved, and the temperature was raised to reflux for 48 hours or more. The reaction was monitored by column chromatography. After the reaction was completed, the system was cooled to room temperature and filtered to obtain 2- ((1E, 3E) -4- (4- (dimethylamino) phenyl) but-1, 3-dien-1-yl) -3- (2-hydroxyethyl) -1, 1-dimethyl-1 h-benzo [ e ]]Indole-3-ammonium iodide. 1 H NMR(400MHz,DMSO-d 6 )δ(ppm)：8.63(d,J＝16.1Hz,1H),8.54(d,J＝1.9Hz,1H),8.45(d,J＝8.5Hz,1H),8.31-8.15(m,3H),8.09(d,J＝9.0Hz,1H),7.92-7.76(m,3H),7.75-7.66(m,2H),7.32(dd,J＝9.1,2.6Hz,1H),7.04(d,J＝2.6Hz,1H),5.27(s,1H),4.91(t,J＝5.0Hz,2H),3.98(t,J＝5.0Hz,2H),3.14(s,6H),2.08(s,6H). 13 C NMR(101MHz,DMSO-d 6 )δ(ppm)：181.87,155.47,153.18,151.72,139.71,137.22,133.05,131.80,131.00,130.46,128.69,127.42,126.99,124.41,123.57,123.39,113.78,112.85,112.07,59.20,53.28,49.00,26.65.HRMS(m/z)：calcd for C 28 H 31 IN 2 O：538.15；found：411.25(M-I) + 。
Example 2
Cell culture and imaging
HeLa and HepG2 cells were cultured in HDMEM medium containing 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin. RAW264.7 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum/1% penicillin/streptomycin. A549 cells were cultured in f12k medium containing 10% fetal bovine serum and 1% penicillin/streptomycin. Toxicity of the probe to mammalian cells was determined by methylthiazoloyldiphenyltetrazolium bromide (MTT) method. HeLa, hepG2 and RAW264.7 cells were incubated with 2. Mu.M LD-Nu in medium for 20min and observed under a fluorescent microscope from the green and red channels. And imaging the fluorescence of the red channel and the green channel by using a 3D imaging technology, and observing the position relation between LDs and nucleolus. HepG2 cells were incubated with 5. Mu.M DAPI and observed under a fluorescent microscope.
Example 3
LD-Nu pH sensing behavior and mechanism experiment thereof
In order to verify whether the probe responds to the pH change, a pH titration experiment is carried out on the probe LD-Nu, and the obtained absorption spectrum and fluorescence spectrum are shown in figures 1 a-c. As shown in FIG. 1a, the maximum absorption peak of the probe LD-Nu gradually changes from 380nm to 600nm as the pH value decreases. Furthermore, when the probe was excited at 380nm, the fluorescence intensity of LD-Nu gradually increased with increasing pH, and a maximum emission peak appeared around 470nm (FIG. 1 b). When the probe was excited at 600nm, an opposite change in fluorescence intensity was observed, with a strong emission peak around 727nm (FIG. 1 c). Furthermore, the fluorescence intensity ratio (I) between 727 and 470nm 727nm /I 470nm ) The ratiometric fluorescent response of probe LD-Nu to pH is shown (FIG. 1 d). Meanwhile, the pKa of LD-Nu was calculated to be 6.24 according to FIG. 1 e. In addition, the photostability of LD-Nu was also investigated, as shown in FIG. 1f, the fluorescence intensity ratio (I) 727nm /I 470nm ) The LD-Nu of (2) is stable at pH 4.0 and 9.0 even after 20 minutes of continuous xenon lamp irradiation, which is advantageous for the LD-Nu to image cells for a long time. These experimental results preliminarily confirm that the fluorescence of the probe LD-Nu can generate blue shift under an acidic environment relative to an alkaline environment, thereby realizing sensitive detection of pH change.
To further confirm that the blue shift of the probe LD-Nu in alkaline environment is due to cyclization reaction induced by LD-Nu in DMSO-d 6 In 1 The H NMR spectrum gave a spectrum before and after addition of NaOH. As shown in FIG. 2a, hydroxyl hydrogen (red dotted line) is evidentThe "s" peak of (a) disappears in the spectrum after addition of NaOH. More importantly, the two methyl protons on the aniline ring (1 and 2) are in DMSO-d 6 Is represented by an "s" peak, but in DMSO-d 6 After changing to two "s" peaks (3 and 4), sodium hydroxide was added. To explain this phenomenon, the geometry of the probe LD-Nu in different states was optimized using Gauss software. As can be seen from fig. 2b, in the ring-opened form, the benzene ring, the two double bonds and the benzazole indole are coplanar, so that the two methyl protons (1 and 2) can be symmetrically distributed on both sides of the plane, resulting in the same shielding effect. In the cyclized form, the benzindole group is not coplanar with the other two groups, so that the shielding effect of the two methyl protons (3 and 4) is different, thus giving rise to two "s" peaks. Thus, the probe LD-Nu can gradually transition from a positively charged form to cyclization with increasing pH. Further, the structure of LD-Nu in the ring-opened form was confirmed by single crystal X-ray diffraction analysis (CCDC No. 2192276, FIG. 3).
Example 4
Experiment on cytotoxicity and staining Capacity of Living cell of LD-Nu
Cytotoxicity is a key parameter for evaluating fluorescent probes, and therefore MTT assay of probe LD-Nu was performed. As shown in FIG. 4, hepG2, heLa, A549 and RAW264.7 cells were stained with LD-Nu (2, 4, 6, 8 and 10. Mu.M) for 24h, and all cell viability remained higher than 87%. Therefore, LD-Nu probes show negligible cytotoxicity for live cell imaging in a short time (e.g., 30 min) and can be used to analyze biological behavior inside the cell. According to the designed mechanism, the LD-Nu probe can stain LDs and nucleolus simultaneously, and the LD-Nu is used for staining HepG2 cells for the first time to verify the cell imaging capability. As shown in fig. 5a-d, both the green (a) and red (b) channels fluoresce, but they show different distributions, shapes and sizes. Green fluorescence appears randomly within the cell as "droplets", while red fluorescence is concentrated in the nucleus as larger entities. To further determine the staining location of LD-Nu, the fluorescence of the red and green channels were merged with DIC images, respectively (FIGS. 5 e-f). First, the average diameter of the green "droplets" is almost 1-2 μm, which is consistent with the size of LDs in the relevant report. In addition, since the refractive index of LDs is higher than other structures in the cell, it appears as a black dot in the DIC image. Thus, the black map in the DIC image of FIG. 5e should be LDs, while the green fluorescence in FIG. 5g can be merged well with the black map according to FIG. 5 i. In addition, as shown in the magnified images of FIGS. 5j-l, the red fluorescence in FIG. 5j fused very well with the nucleolus in the DIC image of FIG. 5 k. In combination, the probe LD-Nu can stain LDs and nucleolus simultaneously, showing green and red fluorescence, respectively. Furthermore, similar phenomena were observed in HeLa (fig. 6) and RAW (fig. 7) cells. More fortunately, in FIGS. 5i and 5l, an LDs was imaged near the nucleolus, as indicated by the white arrows in FIGS. 5i and 5l, suggesting that there may be close or even physical association between the LDs and the nucleolus.
To more accurately observe the relationship between LDs and nucleoli, cells were visualized in multiple dimensions using 3D imaging techniques. As shown in FIG. 8, a cell in panel a was selected for magnification, with a green LDs just above the red nucleolus in the XY plane (FIG. 8 b). Then, a 3D image of the cell was obtained (fig. 8 c), and the physical connection between LDs and nucleolus was observed in three dimensions XY, XZ, YZ (fig. 8D). The experimental fact proves that physical contact does exist between LDs and nucleolus, provides solid evidence for the existence of nucleus LDs, and lays a foundation for exploring the interaction between LDs and nucleolus.
Example 5
Experiment of response of specific RNA of LD-Nu
The development of RNA probes has been a challenge due to the lack of specific targeting groups. In order to investigate in detail the RNA targeting mechanism of probe LD-Nu, this example performed a series of experiments. First, in vitro titration fluorometric assays of the response of LD-Nu to RNA and DNA, respectively, were performed. As shown in FIG. 9a, the fluorescence intensity of LD-Nu gradually increased with the addition of RNA. The RNA binding constant of the probe LD-Nu is 2.0 x 10 6 M -1 (FIG. 9 b), indicating that it has a higher affinity for RNA. Meanwhile, although the fluorescence intensity of LD-Nu increases with the addition of DNA, its binding constant is only 2.8X 10 5 M -1 Is much smaller than the binding constant of LD-Nu to RNA (FIG. 10)). Therefore, in the case where RNA and DNA are present simultaneously, LD-Nu can bind preferentially to RNA, which provides a precondition for the probe to specifically stain nucleoli in the nucleus.
To further confirm the binding mechanism of the probe LD-Nu to RNA, this example performed open-loop molecular docking analysis of LD-Nu. As shown in FIG. 9c, in the open loop format, there are three major binding sites between RNA and the probe LD-Nu. First, the positively charged N atom in the ring forms a salt bridge with the phosphate group of A14, and the protonated xylidine group in the weakly acidic environment of the core also forms a salt bridge with G18. In addition, the hydroxyl groups form two typical hydrogen bonds with a15 and G8, respectively. In addition, several weak hydrogen bonds are also observed between LD-Nu and U6 and G17. Thus, the open-loop form of LD-Nu can specifically stain RNA due to electrostatic interactions and hydrogen bonding. The two-dimensional interaction pattern of RNA with LD-Nu is shown in FIG. 9 d.
Finally, to demonstrate that probe LD-Nu stains nucleoli intracellularly by targeted RNA, RNA and DNA digestion experiments. As shown in FIG. 11, bright fluorescence was still observed in the red channel in the nucleolus of HepG2 cells after digestion with deoxyribonuclease (DNase) for 4 h. However, no red fluorescence was observed in the nucleoli after the same time of digestion with ribonuclease (RNase). The same phenomenon was observed in HeLa cells (fig. 12). In addition, in order to confirm that DNA in the nucleus was sufficiently completely eliminated after 4h of DNase digestion, DAPI (a commercial DNA probe) was used as a control in this example. As can be seen in FIG. 13, blue fluorescence only appears in the cell membrane and not in the nucleus after DNase digestion at 4h, 2min DAPI staining. When the staining time of DAPI was extended to 20min, only weak blue fluorescence appeared in the cytoplasm, but not in the nucleus. Thus, digestion with DNase for 4h was sufficient to remove all DNA in the nucleus. As can be seen from the above experiments, the LD-Nu probe can specifically stain nucleoli because it is directed against RNA and because there are strong hydrogen bonds and electrostatic interactions between them.
Example 6
Tracking relationships between LDs and nucleoli
The LD-Nu probe allows two-color imaging of the LDs and nucleolus and visualization of their physical contact according to the above experiment. Therefore, it is speculated that LD-Nu may be used to further explore the interaction relationship between two subcellular structures. As mentioned above, LDs store large amounts of fatty acids, which may be released into the cytoplasm as substrates for lipid synthesis and protein modification, if necessary. However, the role of fatty acids in the nucleus is not clear.
According to recent reports, PFOA can interfere with the normal activity of fatty acids because it has fatty acid-like properties, a highly hydrophobic, rigid perfluorocarbon carbon tail, and a strongly polar carboxyl head. Thus, first, cells were induced with PFOA and fatty acids were studied in LDs
Interaction with nucleolus. As shown in figure 14, LDs (yellow arrows) that were originally in physical contact with the nucleolus gradually move away from the nucleolus with the addition of PFOA. After 10min of PFOA treatment, the distance between LDs and nucleolus was gradually increased to 4 μm. Meanwhile, with the addition of PFOA, 3 originally dispersed LDs with a diameter of about 2.5 μm gradually accumulate into large LDs with a diameter of 5 μm, which is consistent with the relevant report. This is probably because PFOA addition interferes with the normal functioning of fatty acids, resulting in restricted activity of LDs, blocking the interaction of LDs with nucleoli, triggering the accumulation of LDs. Thus, fatty acids play a crucial role in their interactions, even affecting the physical contact of LDs with the kernel.
In addition, apoptosis, which is an important physiological process for eliminating unnecessary or abnormal cells, plays an important role in the stabilization of the internal environment and thus, this example investigated the changes of LDs and nucleoli in the apoptotic process. As can be seen from the DIC image of FIG. 15, H 2 O 2 After treatment of HepG2 cells, several blebs appeared around the plasma membrane, indicating that the cells were already in the middle and late apoptotic stages. The nucleolus size in the apoptotic cells increased significantly, with an average diameter from 3.41 μm to 5.70 μm, indicating that apoptosis has a large effect on nucleolus morphology. The magnified images show that there is also physical contact between LDs and nucleoli in apoptotic cells. The same phenomenon was observed in HeLa cells (fig. 16). This may be due to interactions between LDs and nucleoliIn use, movement of LDs is dominant, while the nucleolus is in passive contact. Thus, the association between LDs and nucleolus is maintained after mild injury to the nucleolus, but is blocked after the fatty acid activity in LDs is disturbed.
Finally, it is also worth noting that the probe can penetrate the nuclear membrane, thus allowing simultaneous visualization of cytosolic and nuclear LDs. To date, the function of LDs in the cytoplasm has been well studied, while the role in the nucleus has not been clear. Therefore, the research on the relationship of LDs in two environments has great significance for the deep understanding of the intracellular lipid network. Therefore, this example first investigated the changes in LDs during cell foaming with LD-Nu probe. As shown in fig. 17, the red-green channel fluorescence intensity ratio decreased from 0.91 to 0.66 after 12h incubation of RAW cells with LDL, indicating an increase in the overall level of LDs in the cells. However, after LDL treatment, the ratio of the green fluorescence intensity of the nuclei to that of the whole cells decreased from 0.07 to 0.03, and physical contact between LDL and nucleoli (white arrows) remained. Thus, although the addition of LDL almost doubled the number of LDs in the cells, there was no significant effect on the number and function of nuclear LDs. In addition, similar phenomena are observed during inflammatory stimuli. As can be seen in FIG. 18, the amount of LDs in the nucleus and their contact with the nucleolus is not significantly disturbed, although the overall green fluorescence intensity is reduced after LPS treatment of the cells. These experimental results indicate that cellular foaming and inflammatory stimuli can increase or decrease LDs in the cytoplasm, respectively, without significant effects on LDs in the nucleus. This is probably because LDs near the nucleolus are produced in the endoplasmic reticulum near the nuclear membrane and can be transported directly to the nucleus, thereby largely avoiding interference from stimulus within the cytoplasm.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A pH triggered charge reversible fluorescent probe is characterized in that the chemical structure is as follows:
2. a preparation method of a pH triggered charge reversible fluorescent probe is characterized by comprising the steps of obtaining a fluorescent probe LD-Nu by using a compound 1 according to the following reaction route;
3. the method for preparing the pH triggered charge reversible fluorescent probe as claimed in claim 2, wherein the compound 1 is quaternized with iodoethanol to obtain the compound 2, and the compound 2 is condensed with the compound 3 to obtain the fluorescent probe LD-Nu.
4. The method for preparing a pH triggered charge reversible fluorescent probe as claimed in claim 2, wherein the compound 2 is obtained by heating and refluxing the compound 1 and iodoethanol in chloroform.
5. The method for preparing the pH triggered charge reversible fluorescent probe as claimed in claim 2, wherein the compound 2 is added into ethanol, heated and dissolved, then the compound 3 is added, heated and refluxed, and reacted for at least 48 hours, so as to obtain the fluorescent probe LD-Nu.
6. A preparation composition, which comprises an active ingredient and an auxiliary material, and is characterized in that the active ingredient is the pH triggered charge reversible fluorescent probe as claimed in claim 1.
7. Use of the pH triggered charge reversible fluorescent probe of claim 1 in detecting pH.
8. Use of the pH triggered charge reversible fluorescent probe of claim 1 in cellular imaging of lipid droplets, nucleoli or both lipid droplets and nucleoli in cells using the pH triggered charge reversible fluorescent probe.
9. The use of the pH triggered charge reversible fluorescent probe for imaging cells as claimed in claim 8, wherein the cells are immortalized or normal cells.
10. A cell imaging detection kit, which comprises an active component and a buffer solution, and is characterized in that the active component is the pH triggered charge reversible fluorescent probe as claimed in claim 1 or the preparation composition as claimed in claim 6.
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| US3384487A (en) * | 1964-09-01 | 1968-05-21 | Eastman Kodak Co | Butadienyl dyes for photography |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US3384487A (en) * | 1964-09-01 | 1968-05-21 | Eastman Kodak Co | Butadienyl dyes for photography |
| US20190375941A1 (en) * | 2018-06-11 | 2019-12-12 | Institute For Stem Cell Biology And Regenerative Medicine (Instem) | Compounds as fluorescent probes, synthesis and applications thereof |
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| Title |
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| HUAMIAO ZHANG，等: "A general strategy to increase emission shift of two-photon ratiometric pH probes using a reversible intramolecular reaction of spiro-oxazolidine", 《SPECTROCHIMICA ACTA PART A: MOLECULAR AND BIOMOLECULAR SPECTROSCOPY》, vol. 246, pages 119035 * |
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