A fluorescent carbon dot targeting mitochondrial DNA, its preparation method and its bioimaging application

Abstract

This invention belongs to the field of fluorescent nanomaterial synthesis technology, and discloses a fluorescent carbon dot targeting mitochondrial DNA, its preparation method, and its bioimaging application. The reaction raw materials for this fluorescent carbon dot include 1,1,2,3-tetramethyl-1H-benzo[e]indolium iodide and urea. The 1,1,2,3-tetramethyl-1H-benzo[e]indolium iodide and urea are added to anhydrous ethanol and heated in a high-pressure reactor. The crude product obtained is purified by neutral alumina column chromatography using a dichloromethane-methanol mixture of different volume ratios as eluent. After rotary evaporation and concentration, the purified fluorescent carbon dots are finally obtained. The carbon dots of this invention can accurately locate mitochondrial DNA for real-time imaging, and simultaneously achieve dynamic visualization monitoring of mitochondrial DNA levels during apoptosis.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent nanomaterial synthesis technology, and particularly relates to a fluorescent carbon dot for mitochondrial DNA. Background Technology

[0002] Mitochondrial DNA (MDNA) is a unique genetic material composed of closed circular double-stranded structures located within mitochondria. It plays a crucial role not only in maintaining normal mitochondrial function but also in regulating various biological processes and the generation of several important bioactive molecules. Unlike nuclear DNA, mitochondrial DNA lacks the protection of introns and histones, making its structure relatively exposed. Furthermore, the electron transport chain in the mitochondrial matrix is ​​a major source of reactive oxygen species (ROS) within the cell, making mitochondrial DNA highly susceptible to oxidative attack and mutations. Studies have shown that its mutation rate is more than 10 times that of nuclear DNA, meaning that mitochondrial DNA is more sensitive to cellular damage and is an important indicator of cellular health. Mitochondrial DNA mutations have been proven to be a significant cause of various diseases, including genetic disorders, cancer, and immune disorders. Therefore, developing tools capable of ultrasensitive detection and clear imaging of mitochondrial DNA is of great importance for assessing cellular health. Such tools can be used not only for health assessment at the cellular and overall biological levels but also provide potential strategies for the early diagnosis of mitochondrial DNA-related diseases.

[0003] In recent years, carbon dots have attracted widespread attention due to their excellent properties such as simple synthesis, small size, tunable fluorescence emission, high photostability, and low biotoxicity. In fields such as bioimaging and biosensing, carbon dots exhibit high sensitivity and strong visualization capabilities, and are therefore increasingly used for the visualization of bioactive macromolecules in vivo, such as the mitochondrial-targeting fluorescent carbon dot disclosed in CN117106447A. However, regrettably, no fluorescent carbon dots specifically targeting mitochondrial DNA have yet been developed. Therefore, designing and synthesizing fluorescent carbon dots capable of specifically recognizing mitochondrial DNA is of significant research and application value for expanding the application areas of carbon dots and deepening our understanding of the biological functions of mitochondrial DNA. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes a fluorescent carbon dot targeting mitochondrial DNA, its preparation method, and its bioimaging applications.

[0005] To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0006] A method for preparing fluorescent carbon dots targeting mitochondrial DNA includes the following steps: 1,1,2,3-tetramethyl-1H-benzo[e]indolium iodide and urea are added to ethanol, followed by solvothermal treatment and purification. This invention uses urea containing electron-rich groups as the electron donor (D), indolium containing electron-withdrawing groups as the electron acceptor (A), and mitochondrial targeting groups to prepare carbon dots rich in amino groups and indolium. The unique core-shell structure of the carbon dots allows for the formation of a rotatable D-π-A structure between the π-conjugated carbon core and the surface electron-donating / accepting groups, endowing them with an active charge-transfer state. Under the synergistic effect of the electrostatic interaction of indolium and the hydrogen bonding of the amino groups, the carbon dots are confined within the grooves of mitochondrial DNA, forming a structure similar to a "rigid lock." This structure not only effectively inhibits π-π stacking between carbon dots but also greatly restricts the degree of freedom of intramolecular rotation and vibration, while enhancing the intramolecular charge-transfer effect, thereby significantly improving the fluorescence signal of the carbon dots.

[0007] Furthermore, in the above preparation method, the mass ratio of 1,1,2,3-tetramethyl-1H-benzo[e]indolium iodide to urea is 1:(0.6-1.82).

[0008] The temperature for the above-mentioned solvent heat treatment is 160-200℃, and the time is 8-12 h.

[0009] The purification was performed using neutral alumina column chromatography with dichloromethane and methanol as eluents.

[0010] The fluorescent carbon dots prepared by the above method have DNA recognition and mitochondrial targeting capabilities.

[0011] The above-mentioned fluorescent carbon dots targeting mitochondrial DNA are used in the preparation of mitochondrial DNA imaging agents.

[0012] The aforementioned mitochondrial DNA imaging agent is specifically used in the visualization and monitoring of the dynamic changes in mitochondrial DNA (mtDNA) content during apoptosis.

[0013] The beneficial effects of this invention are:

[0014] This invention utilizes 1,1,2,3-tetramethyl-1H-benzo[e]indolium iodide and urea as raw materials to prepare a fluorescent carbon dot targeting mitochondrial DNA via a solvothermal reaction. After interaction with DNA, the emission wavelength of the carbon dot redshifts from 610 nm to 623 nm, while the fluorescence intensity increases by approximately 6.4 times. Furthermore, the suitable amphiphilicity of this carbon dot promotes its rapid accumulation in mitochondria, enabling real-time monitoring and imaging of minute changes in mitochondrial DNA content. Therefore, the carbon dot of this invention provides a highly promising tool for future mitochondrial DNA-targeted therapy. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This is a transmission electron microscope image of the fluorescent carbon dots prepared in this invention.

[0017] Figure 2 This is the fluorescence emission spectrum of the fluorescent carbon dots selectively recognizing DNA according to the present invention.

[0018] Figure 3 This is a co-localization image of SMMC-7721 cells stained with carbon dots and a commercial mitochondrial probe. Scale bar: 20 μm.

[0019] Figure 4 This invention describes the toxic effects of fluorescent carbon dots on SMMC-7721 cells.

[0020] Figure 5 This is a nucleic acid digestion experiment. Scale bar: 20 μm.

[0021] Figure 6 This is a cellular confocal fluorescence image of real-time visualization of mitochondrial DNA level fluctuations using fluorescent carbon dots, as described in this invention. Scale bar: 20 μm.

[0022] Figure 7 This is a confocal fluorescence image of cells from which fluorescent carbon dots of the present invention are used to visualize and monitor fluctuations in mitochondrial DNA levels during the apoptosis process. Scale bar: 20 μm. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Example 1

[0025] The method for preparing fluorescent carbon dots targeting mitochondrial DNA in this embodiment includes the following steps:

[0026] (1) First, 15 mg of 1,1,2,3-tetramethyl-1H-benzo[e]indolium iodide and 9.1 mg of urea were dissolved in a glass sample vial containing 3 mL of anhydrous ethanol. Next, the sample vial containing the mixed solution was placed in a 25 mL high-pressure reactor lined with polytetrafluoroethylene. Subsequently, the reactor was placed in an oven at 180 °C and reacted for 10 h.

[0027] (2) After the reaction was completed, the crude product was purified by gradient elution using dichloromethane and methanol as eluents by neutral alumina column chromatography. Finally, the purified solution was further dried using a rotary evaporator to obtain solid product carbon dots, namely fluorescent carbon dots targeting mitochondrial DNA.

[0028] Example 2

[0029] The method for preparing fluorescent carbon dots targeting mitochondrial DNA in this embodiment includes the following steps:

[0030] (1) First, 15 mg of 1,1,2,3-tetramethyl-1H-benzo[e]indolium iodide and 18.2 mg of urea were dissolved in a glass sample vial containing 3 mL of anhydrous ethanol. Next, the sample vial containing the mixed solution was placed in a 25 mL high-pressure reactor lined with polytetrafluoroethylene. Subsequently, the reactor was placed in an oven at 180 °C and reacted for 10 h.

[0031] (2) After the reaction was completed, the crude product was purified by gradient elution using dichloromethane and methanol as eluents by neutral alumina column chromatography. Finally, the purified solution was further dried using a rotary evaporator to obtain solid product carbon dots, namely fluorescent carbon dots targeting mitochondrial DNA.

[0032] Example 3

[0033] The method for preparing fluorescent carbon dots targeting mitochondrial DNA in this embodiment includes the following steps:

[0034] (1) First, 15 mg of 1,1,2,3-tetramethyl-1H-benzo[e]indolium iodide and 27.3 mg of urea were dissolved in a glass sample vial containing 3 mL of anhydrous ethanol. Next, the sample vial containing the mixed solution was placed in a 25 mL high-pressure reactor lined with polytetrafluoroethylene. Subsequently, the reactor was placed in an oven at 180 °C and reacted for 10 h.

[0035] (2) After the reaction was completed, the crude product was purified by gradient elution using dichloromethane and methanol as eluents by neutral alumina column chromatography. Finally, the purified solution was further dried using a rotary evaporator to obtain solid product carbon dots, namely fluorescent carbon dots targeting mitochondrial DNA.

[0036] Example 4

[0037] The method for preparing fluorescent carbon dots targeting mitochondrial DNA in this embodiment includes the following steps:

[0038] (1) First, 15 mg of 1,1,2,3-tetramethyl-1H-benzo[e]indolium iodide and 18.2 mg of urea were dissolved in a glass sample vial containing 3 mL of anhydrous ethanol. Next, the sample vial containing the mixed solution was placed in a 25 mL high-pressure reactor lined with polytetrafluoroethylene. Subsequently, the reactor was placed in an oven at 200 °C for 8 h.

[0039] (2) After the reaction was completed, the crude product was purified by gradient elution using dichloromethane and methanol as eluents by neutral alumina column chromatography. Finally, the purified solution was further dried using a rotary evaporator to obtain solid product carbon dots, namely fluorescent carbon dots targeting mitochondrial DNA.

[0040] Example 5

[0041] The method for preparing fluorescent carbon dots targeting mitochondrial DNA in this embodiment includes the following steps:

[0042] (1) First, 15 mg of 1,1,2,3-tetramethyl-1H-benzo[e]indolium iodide and 18.2 mg of urea were dissolved in a glass sample vial containing 3 mL of anhydrous ethanol. Next, the sample vial containing the mixed solution was placed in a 25 mL high-pressure reactor lined with polytetrafluoroethylene. Subsequently, the reactor was placed in an oven at 160 °C and reacted for 12 h.

[0043] (2) After the reaction was completed, the crude product was purified by gradient elution using dichloromethane and methanol as eluents by neutral alumina column chromatography. Finally, the purified solution was further dried using a rotary evaporator to obtain solid product carbon dots, namely fluorescent carbon dots targeting mitochondrial DNA.

[0044] Example 6

[0045] The method for preparing fluorescent carbon dots targeting mitochondrial DNA in this embodiment includes the following steps:

[0046] (1) First, 15 mg of 1,1,2,3-tetramethyl-1H-benzo[e]indolium iodide and 9.1 mg of urea were dissolved in a glass sample vial containing 3 mL of anhydrous ethanol. Next, the sample vial containing the mixed solution was placed in a 25 mL high-pressure reactor lined with polytetrafluoroethylene. Subsequently, the reactor was placed in an oven at 170 °C and reacted for 11 h.

[0047] (2) After the reaction was completed, the crude product was purified by gradient elution using dichloromethane and methanol as eluents by neutral alumina column chromatography. Finally, the purified solution was further dried using a rotary evaporator to obtain solid product carbon dots, namely fluorescent carbon dots targeting mitochondrial DNA.

[0048] Example 7

[0049] The method for preparing fluorescent carbon dots targeting mitochondrial DNA in this embodiment includes the following steps:

[0050] (1) First, 15 mg of 1,1,2,3-tetramethyl-1H-benzo[e]indolium iodide and 27.3 mg of urea were dissolved in a glass sample vial containing 3 mL of anhydrous ethanol. Next, the sample vial containing the mixed solution was placed in a 25 mL high-pressure reactor lined with polytetrafluoroethylene. Subsequently, the reactor was placed in an oven at 190 °C for 9 h.

[0051] (2) After the reaction was completed, the crude product was purified by gradient elution using dichloromethane and methanol as eluents by neutral alumina column chromatography. Finally, the purified solution was further dried using a rotary evaporator to obtain solid product carbon dots, namely fluorescent carbon dots targeting mitochondrial DNA.

[0052] Example 8

[0053] The method for preparing fluorescent carbon dots targeting mitochondrial DNA in this embodiment includes the following steps:

[0054] (1) First, 15 mg of 1,1,2,3-tetramethyl-1H-benzo[e]indolium iodide and 9.1 mg of urea were dissolved in a glass sample vial containing 3 mL of anhydrous ethanol. Next, the sample vial containing the mixed solution was placed in a 25 mL high-pressure reactor lined with polytetrafluoroethylene. Subsequently, the reactor was placed in an oven at 200 °C for 8 h.

[0055] (2) After the reaction was completed, the crude product was purified by gradient elution using dichloromethane and methanol as eluents by neutral alumina column chromatography. Finally, the purified solution was further dried using a rotary evaporator to obtain solid product carbon dots, namely fluorescent carbon dots targeting mitochondrial DNA.

[0056] Example 9

[0057] The method for preparing fluorescent carbon dots targeting mitochondrial DNA in this embodiment includes the following steps:

[0058] (1) First, 15 mg of 1,1,2,3-tetramethyl-1H-benzo[e]indolium iodide and 27.3 mg of urea were dissolved in a glass sample vial containing 3 mL of anhydrous ethanol. Next, the sample vial containing the mixed solution was placed in a 25 mL high-pressure reactor lined with polytetrafluoroethylene. Subsequently, the reactor was placed in an oven at 160 °C and reacted for 12 h.

[0059] (2) After the reaction was completed, the crude product was purified by gradient elution using dichloromethane and methanol as eluents by neutral alumina column chromatography. Finally, the purified solution was further dried using a rotary evaporator to obtain solid product carbon dots, namely fluorescent carbon dots targeting mitochondrial DNA.

[0060] Implementation Results Example

[0061] The microstructure, DNA recognition ability, and mitochondrial targeting performance of the mitochondrial DNA fluorescent carbon dots prepared in this invention were studied and analyzed, and the results are as follows.

[0062] (1) Microscopic morphology

[0063] Figure 1 This is a transmission electron microscope (TEM) image of the fluorescent carbon dots prepared in Example 2. From... Figure 1 It can be seen that the carbon dots have a spherical or quasi-spherical structure. The average particle size of the carbon dots is 4.31 nm. High-resolution transmission electron microscopy images show that the carbon dots have a crystal plane spacing of 0.33 nm, corresponding to the (002) crystal plane of graphite, which means that the carbon dots have a graphitized structure.

[0064] (2) Optical properties

[0065] Figure 2 These are the fluorescence emission spectra of the fluorescent carbon dots prepared in Example 2 before and after reaction with DNA or RNA. The carbon dot stock solution (concentration 10 mg / mL, solvent: DMSO) was diluted in PBS buffer (pH 7.4) to a final concentration of 20 μg / mL. Subsequently, 250 μg / mL of RNA or DNA was added to the above solution, and the fluorescence emission spectra were collected separately. Figure 2 It can be seen that the addition of DNA significantly enhanced the fluorescence intensity of the carbon dots by about 6.4 times, while the addition of RNA only caused a slight increase of about 1.9 times.

[0066] (3) Mitochondrial targeting ability

[0067] Cells were co-incubated with carbon dots (10 μg / mL) solution and the commercial mitochondrial dye Mtio-Tracker (20 nM) for 15 min. After discarding the culture medium, SMMC-7721 cells were washed three times with phosphate-buffered saline (PBS, pH 7.4) to remove any untreated dye. Finally, imaging assays were performed, and the results are shown below. Figure 3 As shown. From Figure 3 As can be seen, the red fluorescence of carbon dots can overlap well with the green fluorescence of mitochondria, with a Pearson coefficient as high as 0.93, proving that carbon dots have excellent mitochondrial targeting ability.

[0068] (4) The toxic effect of carbon dots on SMMC-7721 cells

[0069] A series of carbon dots at different concentrations (0-30 μg / mL) were incubated in SMMC-7721 cells for 12 h, and tests were performed. The results are as follows: Figure 4 As shown, even at a carbon dot concentration as high as 30 μg / mL, cell viability still exceeded 90%. These results indicate that carbon dots have low cytotoxicity, which helps to further explore their application potential in biological experiments.

[0070] (5) Nucleic acid digestion experiment

[0071] SMMC-7721 cells were first pretreated with DNase I (5 U / mL) or RNase A (30 μg / mL) for 2 h. After pretreatment, the cells were washed three times with phosphate-buffered saline (PBS, pH 7.4), and then incubated with carbon dots (10 μg / mL). Finally, the cells were washed with PBS before imaging assays. Figure 5 The images shown are confocal fluorescence images of cells treated with DNase I or RNase A after carbon dot staining prepared in Example 1. It can be seen that, compared to untreated cells, the fluorescence of carbon dots showed almost no significant change after RNase A stimulation. In contrast, the intracellular DNA level in cells treated with DNase I decreased significantly, and the fluorescence of carbon dots illuminating mitochondria was weaker. These results indicate that carbon dots only produce a fluorescent signal when they bind to mitochondrial DNA.

[0072] (6) Exploring the ability of carbon dots to visualize fluctuations in mitochondrial DNA levels

[0073] First, SMMC-7721 cells were pretreated with ethidium bromide (50 ng / mL) for different durations (0, 6, 12, 24 h) to construct cell models with varying mitochondrial DNA content by inhibiting mitochondrial DNA transcription and replication. After pretreatment, SMMC-7721 cells were washed three times with phosphate-buffered saline (PBS, pH 7.4), followed by incubation with carbon dots (10 μg / mL). Finally, SMMC-7721 cells were washed with PBS before imaging assays. Figure 6 The image shows a confocal fluorescence image of cells treated with ethidium bromide after carbon dot staining, prepared in Example 1. It can be seen that the fluorescence intensity of the carbon dots in the cells gradually decreases with increasing ethidium bromide treatment time. These analytical results demonstrate that ethidium bromide can monitor the fluctuations in mitochondrial DNA levels in real time through changes in fluorescence signals.

[0074] Application examples

[0075] The fluorescent carbon dots prepared in Example 2 of this invention were applied to the visual monitoring of fluctuations in mitochondrial DNA levels during the apoptosis process. The specific steps are as follows:

[0076] First, SMMC-7721 cells were pretreated with different concentrations of catechol (0, 25, 50, 300 μM) for 12 h to induce apoptosis at different degrees. After treatment, SMMC-7721 cells were washed three times with phosphate-buffered saline (PBS, pH 7.4), and then incubated with carbon dots (10 μg / mL). Finally, SMMC-7721 cells were washed with PBS before imaging assays. Figure 7 These are confocal fluorescence images of cells treated with different concentrations of catechol after carbon dot staining, prepared in Example 1. It can be seen that, compared to normal cells that have not undergone apoptosis, the fluorescence intensity of the carbon dots gradually decreases with increasing apoptosis. This is because apoptosis leads to mitochondrial DNA damage, thereby reducing the amount of mitochondrial DNA bound to the carbon dots, thus causing a continuous decrease in the fluorescence intensity. The above analysis indicates that carbon dots can monitor subtle changes in mitochondrial DNA during the apoptosis process in real time and assess cell health.

[0077] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A fluorescent carbon dot targeting mitochondrial DNA, characterized in that, The reactants for the fluorescent carbon dots include 1,1,2,3-tetramethyl-1H-benzo[e]indolium iodide and urea.

2. The fluorescent carbon dots targeting mitochondrial DNA according to claim 1, characterized in that, The mass ratio of the 1,1,2,3-tetramethyl-1H-benzo[e]indolium iodide to urea is 1:(0.6-1.82).

3. The fluorescent carbon dots targeting mitochondrial DNA according to claim 1, characterized in that, The fluorescent carbon dots have DNA recognition and mitochondrial targeting capabilities.

4. The method for preparing fluorescent carbon dots targeting mitochondrial DNA according to claim 1, characterized in that, The steps are as follows: 1,1,2,3-Tetramethyl-1H-benzo[e]indolium iodide and urea are added to ethanol, and the product is obtained by solvothermal treatment and purification.

5. The method for preparing fluorescent carbon dots targeting mitochondrial DNA according to claim 4, characterized in that, The temperature of the solvothermal treatment is 160-200℃.

6. The method for preparing fluorescent carbon dots targeting mitochondrial DNA according to claim 5, characterized in that, The solvent heat treatment time is 8-12 h.

7. The method for preparing fluorescent carbon dots targeting mitochondrial DNA according to claim 6, characterized in that, The purification was performed using neutral alumina column chromatography.

8. The method for preparing fluorescent carbon dots targeting mitochondrial DNA according to claim 7, characterized in that, The neutral alumina column chromatography method uses dichloromethane and methanol as eluents.

9. The use of the fluorescent carbon dots targeting mitochondrial DNA as described in claim 1 in the preparation of mitochondrial DNA imaging agents.

10. The application according to claim 9, characterized in that, The mitochondrial DNA imaging agent is specifically used in the visualization and monitoring of dynamic changes in mitochondrial DNA content during apoptosis.

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