Preparation method and application of fluorescent probe for dynamic imaging of cell microvesicles and mitochondria

Abstract

The invention discloses a preparation method and application of a fluorescent probe for dynamic imaging of cell microvesicles and mitochondria, the fluorescent probe is a cationic indole-coumarin fluorescent probe (IC-Cn, n is 1-5) with different aliphatic side chains, IC-C1 and IC-C2 can specifically mark MVs, and the production, movement, fusion and division processes of the MVs can be clearly observed; iC-C3, IC-C4 and IC-C5 can locate MVs and Mito, and the invention relates to the technical field of biological imaging analysis. According to the preparation method and application of the fluorescent probe for dynamic imaging of the cell microvesicles and the mitochondria, the interaction is researched through a fluorescence lifetime imaging microscope (FLIM), and the dynamic process of MVs derived from the mitochondria is disclosed. The fluorescent probe disclosed by the invention has good light stability and low phototoxicity, and the probe has excellent biocompatibility, selectivity and light stability by selecting indole and coumarin as parent bodies, so that the fluorescent probe can specifically mark cell microvesicles, cell microvesicles and mitochondria and emit bright red fluorescence.

Description

Technical Field

[0001] The present invention relates to the technical field of biological imaging analysis, and in particular to the preparation of a fluorescent probe IC-Cn for in situ imaging of cell microvesicle dynamics and the interaction between cell microvesicles and mitochondria, and its application in biomedical research. Background Art

[0002] Cellular microvesicles (MVs) play an important role in intercellular communication, tissue homeostasis, and the occurrence and development of diseases. However, due to the lack of specific labeling methods, in-depth research on MVs is greatly limited. Traditional methods for studying MVs, such as isolating MVs from biological samples and analyzing their components, can determine some of the sources of MVs, but cannot provide information on their real-time dynamic changes in living cells. Existing MV labeling technologies, such as fluorescent protein transfection, immunofluorescence labeling, and lipid dye staining, all have certain defects. For example, the biomarkers used in gene-encoded protein reporter genes and immunofluorescence labeling are also widely present in the cell body, resulting in poor selectivity and high background signals during observation; the method of extracting MVs from biological fluids and labeling them with fluorescent dyes before injecting them into the organism can study their biological distribution and function, but cannot track their origin and secretion process, and the extraction process may change the structure and composition integrity of MVs. Therefore, the development of new fluorescent probes that can specifically label MVs is of great significance for a deep understanding of the physiological and pathological functions of MVs. Summary of the invention

[0003] 1. Technical issues to be resolved

[0004] In view of the shortcomings of the prior art, the present invention provides a method for preparing and using a fluorescent probe for dynamic imaging of cell microvesicles and mitochondria, which has the advantages of simple structure, easy synthesis, good optical stability and low cytotoxicity. Among them, IC-C1 and IC-C2 can specifically label cell microvesicles and can be used to monitor the dynamics of cell microvesicles in real time; while IC-C3, IC-C4 and IC-C5 can specifically label cell microvesicles and mitochondria and can be used to monitor the interaction between cell microvesicles and mitochondria in real time.

[0005] (II) Technical solution

[0006] To achieve the above objectives, the present invention is implemented by the following technical scheme: a fluorescent probe for dynamic imaging of cell microvesicles and mitochondria, wherein the fluorescent probe is a fluorescent probe having different aliphatic side chains (C n H 2n+1 , n is 1 to 5) of the cationic indole-coumarin fluorescent probe, denoted as IC-Cn, and different aliphatic side chains are C n H 2n+1, wherein n is 1 to 5, and the structural formula of the fluorescent probe is as follows:

[0007]

[0008] The present invention also provides a method for preparing a fluorescent probe for dynamic imaging of cell microvesicles and mitochondria, specifically comprising: firstly reacting 2,3,3-trimethylindole with iodinated alkane to synthesize an intermediate, then reacting the intermediate with 7-diethylaminocoumarin-3-aldehyde to synthesize the microvesicle, and the microvesicle and mitochondria-specifically labeled fluorescent probe;

[0009] The specific reaction equation is as follows:

[0010]

[0011] Preferably, the molar ratio of the 2,3,3-trimethylindole to the iodinated alkane is 1:1.

[0012] Preferably, the 2,3,3-trimethylindole and iodinated alkane are reacted at 80°C.

[0013] Preferably, the 2,3,3-trimethylindole and iodinated alkane are reacted under ethanol reflux conditions for 24 hours.

[0014] The present invention also provides cell microvesicles and mitochondria-specific labeled fluorescent probes for imaging the dynamics of microvesicles in living cells and real-time tracking the interaction between microvesicles and mitochondria in living cells.

[0015] The cell microvesicles, cell microvesicles and mitochondria-specific fluorescent probes of the present invention have an excitation wavelength of 570 nm and a maximum emission wavelength of 650 nm. The molecule has a rotatable single bond, which makes its fluorescence intensity weak in aqueous solution. When combined with the target analyte, it emits bright red fluorescence and the fluorescence intensity increases. This series of probes does not respond to other analytes and exhibits excellent selectivity. This series of probes has little effect on the activity of SH-SY5Y cells and shows good biocompatibility. This series of probes has a wash-free function and exhibits excellent resistance to photobleaching, which is suitable for long-term tracking and dynamic imaging. This series of probes can be used to monitor the dynamics of microvesicles in cells in real time; specifically label mitochondria and microvesicles in cells for real-time monitoring of the interaction between microvesicles and mitochondria in cells.

[0016] (III) Beneficial effects

[0017] The present invention provides a method for preparing and using a fluorescent probe for dynamic imaging of cell microvesicles and mitochondria. Compared with the prior art, it has the following beneficial effects:

[0018] (1) The preparation method and application of the fluorescent probe for dynamic imaging of cell microvesicles and mitochondria, by selecting indole and coumarin as the parent, the probe has excellent biocompatibility, selectivity and photostability, so that it can specifically mark cell microvesicles, cell microvesicles and mitochondria and emit bright red fluorescence.

[0019] (2) The preparation method and application of the fluorescent probe for dynamic imaging of cell microvesicles and mitochondria solve the problems of time-consuming and labor-intensive, expensive, invasive cell damage, and inability to monitor cell microvesicles in real time in the prior art. The present invention provides a near-infrared emitting small molecule fluorescent probe that can monitor the dynamics of cell microvesicles in real time by fluorescence imaging. The synthetic method is simple, the cytotoxicity is low, and long-term real-time imaging can be performed. At the same time, the influence of the molecule's own concentration and the biologically active molecules in the cell can be avoided. Fluorescence imaging can accurately and real-timely observe the dynamic changes of microvesicles and mitochondria in cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The fluorescent probe IC-C1 of the present invention 1 H NMR spectrum;

[0021] Figure 2 The fluorescent probe IC-C1 of the present invention 13 H NMR spectrum;

[0022] Figure 3 is the HRMS spectrum of the fluorescent probe IC-C1 of the present invention;

[0023] Figure 4 The fluorescent probe IC-C2 of the present invention 1 H NMR spectrum;

[0024] Figure 5 The fluorescent probe IC-C2 of the present invention 13 H NMR spectrum;

[0025] Figure 6 is the HRMS spectrum of the fluorescent probe IC-C2 of the present invention;

[0026] Figure 7 The fluorescent probe IC-C3 of the present invention 1 H NMR spectrum;

[0027] Figure 8 The fluorescent probe IC-C3 of the present invention 13 H NMR spectrum;

[0028] Fig. 9 is the HRMS spectrum of the fluorescent probe IC-C3 of the present invention;

[0029] Fig.10The fluorescent probe IC-C4 of the present invention 1 H NMR spectrum;

[0030] Fig.11 The fluorescent probe IC-C4 of the present invention 13 H NMR spectrum;

[0031] Fig.12 is the HRMS spectrum of the fluorescent probe IC-C4 of the present invention;

[0032] Fig.13 The fluorescent probe IC-C5 of the present invention 1 H NMR spectrum;

[0033] Fig.14 The fluorescent probe IC-C5 of the present invention 13 H NMR spectrum;

[0034] Fig.15 is the HRMS spectrum of the fluorescent probe IC-C5 of the present invention;

[0035] Fig.16 This is the localization image of the fluorescent probe IC-Cn of the present invention in SH-SY5Y cells;

[0036] Fig.17 It is the cytotoxicity spectrum of the fluorescent probes IC-C1 and IC-C2 of the present invention to SH-SY5Y cells;

[0037] Fig.18 It is the phototoxicity and photobleaching resistance spectrum of the fluorescent probes IC-C1 and IC-C2 of the present invention;

[0038] Fig.19 It is the fluorescence intensity selectivity spectrum of the fluorescent probe IC-C1 of the present invention in the presence of different analytes;

[0039] Fig. 20 It is the selectivity spectrum of the fluorescent probe IC-C1 of the present invention for monitoring cell microvesicles;

[0040] Fig.21 It is the dynamic spectrum of the fluorescent probe IC-C1 of the present invention monitoring cell microvesicles;

[0041] Fig. 22 The cytotoxicity, phototoxicity and photobleaching resistance of the fluorescent probes IC-C3, IC-C4 and IC-C5 of the present invention to SH-SY5Y cells are shown in the graphs;

[0042] Fig.23 It is the fluorescence intensity selectivity spectrum of the fluorescent probe IC-C4 of the present invention in the presence of different analytes;

[0043] Fig.24 It is the selective spectrum of the fluorescent probe IC-C4 of the present invention to monitor cell microvesicles and mitochondria;

[0044] Fig.25 It is a graph of the interaction between cell microvesicles and mitochondria monitored by the fluorescent probe IC-C4 of the present invention. DETAILED DESCRIPTION

[0045] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0046] See also Figure 1-Figure 25 The embodiments of the present invention provide three technical solutions: cell microvesicles, cell microvesicles and mitochondria-specific fluorescent probes; a method for preparing the probes; and the application of the probes to monitor cell microvesicles, the interactions between cell microvesicles and mitochondria, specifically including the following embodiments:

[0047] Embodiment 1

[0048] The present invention is a series of cell microvesicles, cell microvesicles and mitochondria selective fluorescent probes IC-Cn (n is 1 to 5), which can selectively mark cell microvesicles, microvesicles and mitochondria, and the absorption wavelength of the series of probes is 570nm, and the emission wavelength is 650nm, and the dynamics of cell microvesicles and the interaction between cell microvesicles and mitochondria can be tracked in real time by fluorescence or fluorescence lifetime imaging;

[0049] The structural formula of the series of fluorescent probes is as follows:

[0050]

[0051] Embodiment 2

[0052] This example describes in detail the synthesis steps of cell microvesicles, cell microvesicles and mitochondria-specific fluorescent probe IC-Cn (n is 1 to 5).

[0053] Step 1: Add alkane methane (12.6 mmol) and 2,3,3-trimethylindole (12.6 mmol) to 20 mL of acetonitrile respectively, reflux for 24 h, filter the precipitate, and wash with acetonitrile to obtain an intermediate, which is directly used in the next reaction without purification.

[0054] Step 2: The intermediate (2.5 mmol) and 7-diethylaminocoumarin-3-aldehyde (2.5 mmol) were added to 20 mL of ethanol and refluxed for 24 h, the solvent was removed by rotary evaporation, and purified by silica gel chromatography, eluted with CH2Cl2:CH3OH=10:1 (V / V), to obtain a purple-black solid IC-Cn (n is 1 to 5), i.e., the cell microvesicles, cell microvesicles and mitochondria-specific fluorescent probes. Its characterization data are as follows Figure 1-15 shown.

[0055] Embodiment 3

[0056] With respect to the cell microvesicles, cell microvesicles and mitochondria-specific fluorescent probes prepared in Example 2, their properties are specifically characterized in this example.

[0057] 1. Please refer to Fig.16 , the probes IC-C1 and IC-C2 specifically label microvesicles (MVs) in cells, and IC-C3, IC-C4, and IC-C5 specifically label mitochondria and microvesicles in cells.

[0058] 2. Please refer to Fig.17 , the probes IC-C1 and IC-C2 showed low toxicity in SH-SY5Y cells, indicating that IC-C1 and IC-C2 had good biocompatibility.

[0059] 3. Please refer to Fig.18 Compared with probe IC-C2, probe IC-C1 exhibits lower phototoxicity and better resistance to photobleaching. Both IC-C1 and IC-C2 can label cell vesicles, but IC-C1 is more suitable for long-term, real-time monitoring of the dynamics of cell microvesicles.

[0060] 4. Please refer to Fig.19 , probe IC-C1 only showed fluorescence intensity selectivity for DOPC liposomes in vitro.

[0061] 5. Please refer to Fig. 20 The probe IC-C1 can specifically label microvesicles without staining other extracellular vesicles (such as exosomes and apoptotic bodies), and its specificity can be verified by immunofluorescence and other fluorescent dyes.

[0062] VI. Please refer to Fig.21 , the probe IC-C1 was used to monitor the dynamics of cell vesicles, including the movement of microvesicles along cell pseudopodia and the fusion and fission of microvesicles inside and outside the cell.

[0063] VII. Please refer to Fig. 22Among probes IC-C3, IC-C4 and IC-C5, probe IC-C4 has better resistance to photobleaching, lower cytotoxicity and weaker phototoxicity. IC-C3, IC-C4 and IC-C5 can all label cell vesicles and mitochondria, and IC-C4 is more suitable for long-term and real-time monitoring of the interaction between the two.

[0064] 8. Please refer to Fig.23 , probe IC-C4 only showed fluorescence intensity and fluorescence lifetime selectivity for DOPC liposomes in vitro.

[0065] IX. Please refer to Fig.24 The probe IC-C4 can specifically label cell microvesicles and mitochondria without staining other extracellular vesicles (such as exosomes and apoptotic bodies), and its specificity can be verified by immunofluorescence and other fluorescent dyes.

[0066] 10. Please refer to Fig.25 The probe IC-C4 is used to monitor the interaction between cellular microvesicles and mitochondria, including the generation process of mitochondria-derived microvesicles, the fusion process with lysosomes, and the process of excretion from the cell.

[0067] In summary, the near-infrared cell microvesicles, cell microvesicles and mitochondria selective fluorescent probes of the present invention have good biocompatibility, resistance to photobleaching and phototoxicity. This series of probes shows excellent localization when performing cell imaging and will not stain non-target cell components. The application of this series of probes can monitor the cell microvesicles themselves and the interactions between cell microvesicles and mitochondria in real time and over a long period of time.

[0068] In summary, the present invention selects indole and coumarin as the parent, so that the probe has excellent biocompatibility, selectivity and photostability, so that it can specifically mark cell microvesicles, cell microvesicles and mitochondria and emit bright red fluorescence. The present invention solves the problems of time-consuming and labor-intensive, expensive, cell invasive damage, and inability to monitor cell microvesicles in real time in the prior art. The present invention provides a near-infrared emitting small molecule fluorescent probe that can monitor the dynamics of cell microvesicles in real time by fluorescence imaging. The synthesis method is simple, the cytotoxicity is small, and long-term real-time imaging can be performed, while avoiding the influence of the concentration of the molecule itself and the biologically active molecules in the cell. The dynamic changes of microvesicles and mitochondria in cells can be accurately and real-time observed by fluorescence imaging.

[0069] Meanwhile, the contents not described in detail in this specification belong to the prior art known to those skilled in the art.

[0070] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0071] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A fluorescent probe for dynamic imaging of cell microvesicles and mitochondria, characterized in that: The fluorescent probes are chromatographically distinct from each other with different aliphatic side chains (C n H 2n+1 , n is 1 to 5) of the cationic indole-coumarin fluorescent probe, denoted as IC-Cn, and different aliphatic side chains are C n H 2n+1 , wherein n is 1 to 5, and the structural formula of the fluorescent probe is as follows:

2. The method for preparing a fluorescent probe for dynamic imaging of cell microvesicles and mitochondria according to claim 1, characterized in that: Specifically, 2,3,3-trimethylindole and iodinated alkane are first reacted to synthesize an intermediate, and then the intermediate is reacted with 7-diethylaminocoumarin-3-aldehyde to synthesize the microvesicles, and the microvesicles and mitochondria-specific labeled fluorescent probes are synthesized; The specific reaction equation is as follows:

3. The method for preparing a fluorescent probe for dynamic imaging of cell microvesicles and mitochondria according to claim 2, characterized in that: The molar ratio of the 2,3,3-trimethylindole to the iodinated alkane is 1:

1.

4. The method for preparing a fluorescent probe for dynamic imaging of cell microvesicles and mitochondria according to claim 2, characterized in that: The 2,3,3-trimethylindole and iodinated alkane react at 80°C.

5. The method for preparing a fluorescent probe for dynamic imaging of cell microvesicles and mitochondria according to claim 2, characterized in that: The 2,3,3-trimethylindole and iodinated alkane react under ethanol reflux conditions for 24 hours.

6. Use of the fluorescent probe for dynamic imaging of cell microvesicles and mitochondria according to claim 1 to observe the dynamics of cell microvesicles and track the interaction between cell microvesicles and mitochondria in real time.

Images