Preparation method of blue luminous carbon dots for mitochondrial localization

Abstract

The invention relates to the crossing field of nano materials and biotechnology, and discloses preparation of mitochondrial positioning fluorescent carbon dots and application of the mitochondrial positioning fluorescent carbon dots in mitochondrial positioning. The mitochondrial positioning carbon dot is a blue fluorescent carbon dot prepared based on starch and by using ethylamine for N doping; the surface of the mitochondrial positioning carbon dot is slightly negatively charged, which indicates that the mitochondrial positioning mechanism is non-electrostatic adsorption; the fluorescence spectrum of the mitochondrial positioning carbon dot shows that the optimal excitation and the optimal emission are respectively 358nm and 448nm, and the absolute quantum yield is 84.84%; in a CCK-8 method cytotoxicity experiment, even if the concentration of the carbon dots is 320 mu g / mL, the survival rate of the cells can still reach 80% or above; the co-localization coefficient (Pearson coefficient) of the mitochondrial localization fluorescent carbon dot and a mitochondrial commercial probe is up to 0.91. The fluorescent carbon dots prepared by the method have the advantages of simple preparation process, good water solubility, high luminous efficiency, low cost and excellent cell imaging capability and biocompatibility, and are expected to replace traditional commercial mitochondrial fluorescent probes.

Description

[0001] manual Technical Field

[0002] The present invention relates to the intersection of nanomaterials and biotechnology, and in particular to a method for preparing carbon dots that can achieve precise positioning of mitochondria. Background Art

[0003] Energy is the basis of all life activities. Under normal physiological function, mitochondria convert ADP into ATP, which can be directly used by cells. They play a core role in energy conversion and production and are essential for cells to maintain a normal physiological state. Membrane potential (MMP) is an important indicator of the normal physiological function of mitochondria. At present, many types of fluorescent substances, such as inorganic / organic nanoparticles, fluorescent small molecules, precious metal nanoclusters, polymers, etc., are used for organelle imaging. These fluorescent dyes are widely used because of their high mitochondrial targeting and strong fluorescence signals. However, they also have many disadvantages, such as high price, poor water solubility, and complex synthesis. Moreover, traditional mitochondrial localization probes mainly rely on membrane potential-dependent targeting mechanisms for imaging. When the mitochondrial membrane potential (MMP) decreases or disappears, the localization effect of these probes is greatly reduced or the localization function is lost. In addition, due to toxicity, they may even cause mitochondrial autophagy. These shortcomings limit their application to a certain extent.

[0004] Patent application CN118064140A discloses a copper-doped mitochondrial targeted carbon dot, preparation method and application thereof, the method comprising: adding a carbon source, a copper source and a nitrogen source to a sulfuric acid solution in sequence, mixing to obtain a mixed solution; the mass ratio of the carbon source, the copper source and the nitrogen source is 1-15:1-23:1-20, then subjecting the mixed solution to a high temperature reaction, then centrifuging the solution obtained by the reaction, collecting the upper layer solution for filtration, dialysis, collecting a solution with a molecular weight of more than 800Da, and freeze-drying to obtain a copper-doped mitochondrial targeted carbon dot. The carbon dot has high cytotoxicity.

[0005] Patent application CN115975639A discloses a long-wavelength luminescent carbon dot targeting mitochondria and its preparation method. The present invention first uses p-phenylenediamine as a raw material and a solvent thermal method to prepare nitrogen-doped red-light carbon dots. Then, nitrogen-doped red-light carbon dots and carboxybutyltriphenylphosphine are used as reaction substrates, N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide are used as dehydrating agents and cross-linking agents, and amidation reactions are carried out at room temperature. After purification, long-wavelength luminescent carbon dots are obtained. The carbon dots use triphenylphosphine rich in positive charge as a targeting group and rely on the principle of electrostatic adsorption to target mitochondria. Summary of the invention

[0006] The technical scheme adopted by the present invention is: a mitochondrial-localized blue luminescent carbon dot, which is based on starch and prepared by a one-step hydrothermal method using ethylamine for N-doping and has blue fluorescence; the mitochondrial-localized carbon dot is slightly negatively charged and does not rely on the electrostatic attraction mechanism to image the mitochondria; the particle size of the mitochondrial-localized carbon dot is 2-3nm, the optimal excitation and optimal emission of the fluorescence spectrum are respectively 358nm and 448nm, the absolute quantum yield is 84.84%, the fluorescence lifetime is 9.652ns, and in the cytotoxicity experiment, even when the concentration of the carbon dot is 320μg / mL and the cytotoxicity is tested by the CCK-8 method, the survival rate can still reach more than 80%. The co-localization coefficient (Pearson coefficient) of the prepared mitochondrial-localized fluorescent carbon dot and the mitochondrial commercial probe (Mito Tracker Red CMXRos, MTR) is as high as 0.91.

[0007] Preferably, the particle size of the mitochondrial localized fluorescent carbon dots is 1.5-3.0 nm, and the average particle size is 2.24 nm.

[0008] The second aspect of the present invention provides a method for preparing mitochondrial localized fluorescent carbon dots, the method comprising the following steps:

[0009] 1. Disperse the soluble starch evenly in ultrapure water and place it in a water bath at 95°C for 30 minutes until the starch solution becomes a transparent gelatinous substance. During this process, shake the solution continuously to maintain its uniformity.

[0010] 2. Add different amounts of ethylamine to the gelatinized starch solution for N doping.

[0011] 3. After mixing, the mixed solution in step 2 is transferred to a stainless steel reactor with a polytetrafluoroethylene liner. The reactor is then placed in a forced air oven for reaction. After naturally cooling to room temperature, the product is a brown solution.

[0012] 4. The product obtained in step 3 was dialyzed using a dialysis bag with a molecular weight cutoff of 500 Da and freeze-dried for subsequent characterization.

[0013] Preferably, the mass of starch in step 1 is 0.75 g, and the volume of ultrapure water is 30 mL.

[0014] Preferably, the molar ratio of starch to ethylamine in step 2 is 1:1, 1:2, 1:3, 1:4, 1:5, 1:1:7, 1:8, 1:9, or 1:10.

[0015] Preferably, the reaction temperature in step 3 is 180° C. and the reaction time is 6 h.

[0016] Preferably, the product obtained in step 3 is centrifuged in a centrifuge at 10000 r / min for 10 min.

[0017] Preferably, the dialysis time in step 4 is 12 hours, and the ultrapure water is replaced every 2 hours.

[0018] The third aspect of the present invention provides a use of the mitochondrial localized fluorescent carbon dots described above in mitochondrial imaging.

[0019] Through the above technical solution, the outstanding features of the present invention are mainly:

[0020] 1. The preparation method of the present invention is simple to operate, the raw materials are cheap and easily available, and the yield of the prepared mitochondrial localized fluorescent carbon dots is high.

[0021] 2. The mitochondrial localization fluorescent carbon dots prepared by the present invention can accurately localize mitochondria and have good mitochondrial localization capabilities in different cell lines.

[0022] 3. The mitochondrial localized fluorescent carbon dots prepared in the present invention have strong luminescence efficiency, and their absolute fluorescence quantum yield reaches 84.84%.

[0023] 4. The mitochondrial localized fluorescent carbon dots prepared by the present invention have low biological toxicity. In the cytotoxicity experiment, even when the concentration of the carbon dots reaches 320 μg / mL, the cell survival rate still reaches more than 80%.

[0024] 5. The mitochondria-localized fluorescent carbon dots prepared by the present invention have good biocompatibility and have almost no effect on the normal physiological functions of cells after being localized in mitochondria.

[0025] 6. The preparation method of the present invention is simple, the raw materials are cheap and easily available, and large-scale preparation can be achieved.

[0026] 7. The carbon dots prepared by this method have greater potential for cell imaging due to their simple preparation, good water solubility and good safety, and are expected to replace traditional commercial mitochondrial fluorescent probes. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is a transmission electron microscope characterization image of the mitochondrial localized fluorescent carbon dots prepared in Example 1;

[0028] Figure 2 This is a high-resolution transmission electron microscope characterization image of the mitochondrial localized fluorescent carbon dots prepared in Example 1;

[0029] Figure 3 This is an atomic force microscope characterization image of the mitochondrial localized fluorescent carbon dots prepared in Example 1;

[0030] Figure 4 This is a Zeta potential characterization diagram of the mitochondrial localized fluorescent carbon dots prepared in Example 1;

[0031] Figure 5 is a Fourier transform infrared spectrum characterization diagram of the mitochondrial localized fluorescent carbon dots prepared in Example 1;

[0032] Figure 6 is an X-ray photoelectron spectrum characterization diagram of the mitochondrial localized fluorescent carbon dots prepared in Example 1;

[0033] Figure 7 is a fluorescence lifetime decay graph of mitochondrial localized fluorescent carbon dots prepared in Example 1;

[0034] Figure 8 It is the ultraviolet absorption spectrum and fluorescence spectrum characterization diagram of the mitochondrial localized fluorescent carbon dots prepared in Example 1;

[0035] Fig. 9 is an emission spectrum diagram of the mitochondria-localized fluorescent carbon dots prepared in Example 1 under different excitation light excitations;

[0036] Fig.10 This is the HeLa and HUVEC cytotoxicity graph of the mitochondrial localized fluorescent carbon dots prepared in Example 1;

[0037] Fig.11 The mitochondrial localization fluorescent carbon dots of Example 1 are co-localized fluorescence images with mitochondrial probes in HeLa and HUVEC cells, respectively. HeLa: a. Incubate blue fluorescent carbon dots for 4 hours; b. Mitochondria red commercial probe for 0.5 hours; c. Merge image; d. Co-localization analysis fluorescence intensity scatter plot, Pearson coefficient (PCC) and overlap coefficient (OLC); HUVEC: e. Incubate blue fluorescent carbon dots for 4 hours for 0.5 hours; f. Mitochondria red commercial probe; g. Merge image; h. Co-localization analysis fluorescence intensity scatter plot, Pearson coefficient (PCC) and overlap coefficient (OLC) (Channel 1: excitation wavelength 405, receiving wavelength 450±25nm, Channel 2: excitation wavelength 552nm, receiving range 600±25nm);

[0038] Fig.12 This is a fluorescence image of the mitochondria-localized fluorescent carbon dots prepared in Example 1 co-localized with mitochondria in HeLa cells when the membrane potential decreases or disappears;

[0039] Fig.13 This is a detection diagram of the effects of the mitochondrial localized fluorescent carbon dots and the mitochondrial cationic probe prepared in Example 1 on the cell membrane potential after imaging.

[0040] Fig.14 This is a comparison chart of cell imaging of the mitochondria-localized fluorescent carbon dots prepared in Example 1 and the blue mitochondrial commercial probe (MitoLiteBlue FX490) in a bacterial-contaminated environment.

[0041] Fig.15 This is a flow chart of the preparation of mitochondrial localized fluorescent carbon dots prepared in Example 1. DETAILED DESCRIPTION

[0042] Example 1

[0043] The preparation of fluorescent carbon dots in this embodiment includes the following steps:

[0044] Preparation of raw materials: Weigh starch, gelatinize in a 95°C water bath, add ethylamine so that the molar ratio of starch to ethylamine is 1:6.5, mix thoroughly and transfer to a hydrothermal reactor.

[0045] Reaction: In a hydrothermal reactor, the temperature is 180°C and the time is 6 hours to form a fluorescent carbon dot solution.

[0046] Purification: Centrifugation, filtration, and dialysis with a 500Da dialysis bag for 12 hours to obtain the target carbon dot solution.

[0047] The schematic diagram of this reaction is shown in Fig.15 The transmission electron microscopy results of the prepared fluorescent carbon dots are shown in Figure 1 The UV-visible absorption spectrum of the prepared fluorescent carbon dots is shown in Figure 8 The fluorescence emission spectra of the prepared fluorescent carbon dots under different excitation wavelengths are shown in Fig.10 .

[0048] Examples 2 to 11

[0049] The preparation steps of fluorescent carbon dots in Example 2 are the same as those in Example 1, except that the amounts of starch and ethylamine in step (1) are 1:1.

[0050] The preparation steps of fluorescent carbon dots in Example 3 are the same as those in Example 1, except that the ratio of starch to ethylamine in step (1) is 1:2.5.

[0051] The preparation steps of fluorescent carbon dots in Example 4 are the same as those in Example 1, except that the ratio of starch to ethylamine in step (1) is 1:5.

[0052] The preparation steps of fluorescent carbon dots in Example 5 are the same as those in Example 1, except that the ratio of starch to ethylamine in step (1) is 1:6.

[0053] The preparation steps of fluorescent carbon dots in Example 6 are the same as those in Example 1, except that the ratio of starch to ethylamine in step (1) is 1:6.5.

[0054] The preparation steps of fluorescent carbon dots in Example 7 are the same as those in Example 1, except that the ratio of starch to ethylamine in step (1) is 1:7.

[0055] The preparation steps of fluorescent carbon dots in Example 8 are the same as those in Example 1, except that the ratio of starch to ethylamine in step (1) is 1:8.

[0056] The preparation steps of fluorescent carbon dots in Example 9 are the same as those in Example 1, except that the ratio of starch to ethylamine in step (1) is 1:10.

[0057] The preparation steps of fluorescent carbon dots in Example 10 are the same as those in Example 1, except that the ratio of starch to ethylamine in step (1) is 1:14.

[0058] The preparation steps of fluorescent carbon dots in Example 11 are the same as those in Example 1, except that the ratio of starch to ethylamine in step (1) is 1:18.

[0059] Test Example 1

[0060] The particle size of the fluorescent carbon dots in Example 1 was measured using a transmission electron microscope (TEM, Hitachi HT-7800). Figure 1 As shown, the particle size distribution of carbon dots is between 1.5-3 nm, and the average particle size is 2.25 nm.

[0061] The thickness of the mitochondrial localized fluorescent carbon dots prepared in Example 1 was characterized using an atomic force microscope (AFM, Bruker Dimension Icon). Figure 2 As shown, the thickness of the fluorescent carbon dots is distributed between 1-1.5 nm, and the transmission image shows that the morphology of the fluorescent carbon dots is quasi-spherical.

[0062] (4) The Zeta potential of the mitochondria-localized fluorescent carbon dots prepared in Example 1 in an aqueous phase (pH 6.6-7.4) was measured using a dynamic light scattering instrument (Malvern ZS90 Zeta sizer), and the result was -6.09 mV.

[0063] (5) The absorption spectrum, excitation spectrum and emission spectrum of the mitochondrial localized fluorescent carbon dots prepared in Example 1 were characterized using an ultraviolet-visible spectrophotometer (Hitachi U-2910) and a fluorescence spectrometer (Hitachi F-4600). Figure 8 In addition, the absolute fluorescence quantum yield of the mitochondria-localized fluorescent carbon dots prepared in Example 1 was detected using a steady-state fluorescence spectrometer (Edinburgh FLS 1000), and the result was 84.84%.

[0064] Test Example 2

[0065] (1) Fourier transform infrared spectrometer (FTIR, Buker INVENIO S-type) was used to characterize the surface functional groups of the mitochondrial localized fluorescent carbon dots prepared in Example 1. As shown in the figure, at 3562 cm -1 The contraction of OH was observed at 3192 cm -1 The vibration contraction of NH was observed at the bottom of the X-ray diffraction pattern, and these hydrophilic groups make C dots have good dispersibility in water. There are three main peaks in the XPS spectrum: C 1s (284.5 eV), N 1s (398.7 eV) and O1s (532.2 eV).

[0066] Test Example 3

[0067] The imaging effect of the prepared fluorescent carbon dots on Hela cells is as follows:

[0068] A laser confocal microscope (CLSM, Leica TCS SP8) was used to capture the fluorescence image of the co-localization of the mitochondria-localized fluorescent carbon dots and the organelle probe obtained in Example 1. The method is as follows:

[0069] (1) HeLa cells (5×104 cells / mL) were seeded into a confocal microplate and cultured for 24 h.

[0070] (2) The cells were washed three times with phosphate-buffered saline (PBS), and 500 μL of 1640 medium containing 40 μg / mL mitochondrial-localized fluorescent carbon dots was added and incubated for 4 h.

[0071] (3) After washing the cells three times with PBS, add 500 μL of Mito Tracker Red CMXRos (MTR). The preparation method of Mito Tracker Red commercial probe is as follows: take 20 μL MTR (500×), dilute it with 1640 culture medium to 10 mL of 1640 culture medium, and incubate for another 30 min.

[0072] (4) Wash the cells three times with PBS and add phenol red-free 1640 medium for subsequent imaging. Fluorescence imaging of the cells was performed using a 63× oil objective lens of CLSM.

[0073] As can be seen from the figure, the fluorescence of mitochondrial localization fluorescent carbon dots and mitochondrial probes in HeLa cells almost completely overlapped. The Pearson correlation coefficient (PCC) and overlap coefficient (OLC) were 0.91 and 0.99 (HeLa), respectively. In addition, the repeatability of mitochondrial localization fluorescent carbon dots on mitochondrial imaging of different cells was also tested on normal cells (HUVEC). The results showed that the PCC and OLC of mitochondrial localization fluorescent carbon dots and mitochondrial probes were 0.90 and 0.99, respectively, indicating that mitochondrial localization fluorescent carbon dots have good mitochondrial localization ability in both tumor cells and normal cells.

[0074] Test Example 4

[0075] The effect of low or disappeared membrane potential on the mitochondria localization fluorescent carbon dots obtained in Example 1 was tested. The method is as follows: HeLa cells were treated with 10 μM carbonyl cyanide 3-chlorophenylhydrazone (CCCP) for 30 minutes in advance to reduce MMP. Then, they were incubated with mitochondria localization fluorescent carbon dots and Rhodamine 123 (a fluorescent probe that relies on electrostatic adsorption localization mechanism to localize mitochondria) for a period of time, and the changes in fluorescence intensity of cells treated with or without carbonyl cyanide 3-chlorophenylhydrazone (CCCP) were compared. As shown in the figure, compared with the control group, the fluorescence of mitochondria localized by Rho 123 was greatly weakened after CCCP treatment, while the fluorescence intensity of mitochondria localized by mitochondria localized by fluorescent carbon dots remained almost unchanged, showing an excellent mitochondrial localization ability that is independent of MMP. In addition, 4% paraformaldehyde (PFA) was used to treat cells for 20 minutes to fix the cells so that the mitochondrial membrane potential disappeared. After the membrane potential disappears, the fluorescence intensity of the mitochondria located by Rho 123 almost completely disappears. However, after the disappearance of MMP, the fluorescence of the mitochondria located by the mitochondria-localized fluorescent carbon dots can still be clearly observed. This indicates that the mitochondria-localized fluorescent carbon dots do not rely on electrostatic adsorption. (448nm laser intensity 10%, receiving range 470±10nm)

[0076] Test Example 5

[0077] The effects of the mitochondrial localization fluorescent carbon dots and mitochondrial probe imaging obtained in Example 1 on cell membrane potential were tested. The method is as follows: Hela cells were treated with mitochondrial localization fluorescent carbon dots (40 μg / mL) and mitochondrial cationic probe (MTR) respectively, and MMP was detected with Rho 123 after incubation for 4 hours. Fig.14It can be seen that the fluorescence intensity of Rho 123 did not decrease significantly after treatment with mitochondrial localized fluorescent carbon dots, which means that MMP remained almost unchanged. However, the fluorescence intensity decreased significantly after treatment with cationic probes, indicating that MMP was significantly reduced. This proves that mitochondrial localized fluorescent carbon dots have little effect on MMP and mitochondrial function. (448nm laser intensity 10%, receiving range 460±10nm)

[0078] Test Example 6

[0079] Comparison of the mitochondrial localization fluorescent carbon dots obtained in Test Example 1 with the mitochondrial probes Comparison of the cell imaging effects of the mitochondrial blue commercial dye (MiTo Lite Blue, MLB) in a bacterial environment Fig.15 As shown. The results show that even under the condition of cell contamination, it is still possible to accurately locate mitochondria and effectively distinguish cells from bacteria. However, traditional commercial fuels lose their original accurate positioning effect under the condition of cell contamination. (405 laser intensity 10%, receiving range 450±10nm)

[0080] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited thereto. For those skilled in the art, without departing from the principles of the present invention, several modifications and improvements can be made, all of which are included in the protection scope of the present invention.

Claims

1. A mitochondrial-localized blue luminescent carbon dot, characterized in that: The mitochondrial-localized carbon dots are based on starch and prepared by a one-step hydrothermal method using ethylamine for N-doping, and have blue fluorescence. The mitochondrial-localized carbon dots are slightly negatively charged and do not rely on electrostatic attraction mechanism to image mitochondria. The particle size of the mitochondrial-localized carbon dots is 2-3nm, and the optimal excitation and emission of the fluorescence spectrum are at 358nm and 448nm, respectively. The absolute quantum yield is 84.84%, and the fluorescence lifetime is 9.652ns. In the cytotoxicity experiment, even when the concentration of the carbon dots is 320μg / mL and the cytotoxicity is tested by CCK-8 method, the survival rate can still reach more than 80%. The co-localization coefficient (Pearson coefficient) of the prepared mitochondrial-localized fluorescent carbon dots and the commercial mitochondrial probe (Mito Tracker RedCMXRos, MTR) is as high as 0.

91.

2. The method for preparing starch carbon dots according to claim 1, characterized in that: Starch was added into distilled water and stirred sufficiently to obtain the starch solution, and then the starch solution was gelatinized at a water bath temperature of 95° C. for 0.5 h.

3. The mitochondrial localized carbon dots according to claim 1, characterized in that: The molar ratio of ethylamine for N-doping to starch monomer is 6.5:

1.

4. The method for synthesizing carbon dots as claimed in claim 3, characterized in that: The reaction temperature mentioned was 180°C and the reaction time was 6 h.

5. The mitochondrial-localized carbon dots according to claim 1, characterized in that: The separation and purification conditions after the synthesis of carbon dots were dialyzed using a 500Da dialysis bag and ultrapure water. The dialysis time was 12 h, and the ultrapure water was replaced every 2 h.

6. The mitochondrial-localized carbon dots according to claim 1, characterized in that: The particle size of mitochondrial localized carbon dots is concentrated in the range of 2-3 nm, with an average size of 2.24 nm.

7. The mitochondrial-localized carbon dots according to claim 1, characterized in that: The Zeta potential of the mitochondrial localized carbon dots is -6.09 eV.

8. The mitochondrial-localized carbon dots according to claim 1, characterized in that: The optimal excitation and emission of the fluorescence spectrum of mitochondrial-localized carbon dots are at 358 nm and 448 nm, respectively.

9. The mitochondrial-localized carbon dots according to claim 1, characterized in that: The absolute quantum yield of the mitochondria-localized carbon dots is 84.84%, and the fluorescence lifetime is 9.652ns.

10. The mitochondrial-localized carbon dots according to claim 1, characterized in that: Mitochondria-localized carbon dots have good biocompatibility and low cytotoxicity. When the concentration of carbon dots is 320 μg / mL and the cytotoxicity is tested by CCK-8 method, the survival rate can still reach more than 80%. While localizing, the healthy state of mitochondrial membrane potential is not damaged.

11. Use of the mitochondrial localized fluorescent carbon dots according to any one of claims 2 to 4 and 9 as mitochondrial blue fluorescent probes, wherein the co-localization degree with the commercial mitochondrial probe (Mito Tracker Red CMXRos, MTR) is as high as 0.91.

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