Preparation method of mitochondria-targeted blue luminescent carbon dots

Abstract

This invention relates to the interdisciplinary field of nanomaterials and biotechnology, and discloses the preparation of mitochondrial-targeting fluorescent carbon dots and their application in mitochondrial localization. These mitochondrial-targeting carbon dots are blue fluorescent carbon dots prepared based on starch and doped with ethylamine (N). The carbon dots have a slightly negative surface charge, indicating that the mitochondrial localization mechanism is non-electrostatic adsorption. The fluorescence spectrum of these carbon dots shows optimal excitation and emission at 358 nm and 448 nm, respectively, with an absolute quantum yield of 84.84%. In CCK-8 cytotoxicity assays, even at a carbon dot concentration of 320 μg / mL, cell viability remained above 80%. The colocalization coefficient (Pearson coefficient) between these mitochondrial-targeting fluorescent carbon dots and commercial mitochondrial probes is as high as 0.91. Due to its simple preparation process, good water solubility, high luminescence efficiency, low cost, superior cell imaging capabilities, and biocompatibility, the fluorescent carbon dots prepared by this method are expected to replace traditional commercial mitochondrial fluorescent probes.

Description

Technical Field

[0001] This invention relates to the interdisciplinary field of nanomaterials and biotechnology, specifically to a blue fluorescent carbon dot for precise mitochondrial localization, its preparation method, and its application. Background Technology

[0002] Energy is fundamental to all life activities. Mitochondria, as the cell's "energy factory," play a central role in energy metabolism by converting adenosine diphosphate (ADP) into adenosine triphosphate (ATP), which the cell can directly utilize, under normal physiological conditions. The mitochondrial membrane potential (MMP) is a crucial indicator of normal mitochondrial function. Currently, many fluorescent substances, such as inorganic / organic nanoparticles, fluorescent small molecules, noble metal nanoclusters, and polymers, are used for organelle imaging. Some commercially available dyes have been applied due to their high mitochondrial targeting ability and strong fluorescence signal; however, they also have many drawbacks, such as high cost, poor water solubility, and complex synthesis steps. More importantly, most traditional mitochondrial targeting probes rely on the negative mitochondrial membrane potential for targeted imaging. When the mitochondrial membrane potential (MMP) decreases or disappears, the targeting effect of these probes is greatly reduced or even lost, and their autotoxicity may induce mitochondrial autophagy. These shortcomings limit their widespread application to some extent.

[0003] Patent application CN118064140A discloses a copper-doped mitochondrial-targeting carbon dot, its preparation method, and its application. The method includes: sequentially adding a carbon source, a copper source, and a nitrogen source to a sulfuric acid solution and mixing them to obtain a mixed solution; the mass ratio of the carbon source, copper source, and nitrogen source is 1–15:1–23:1–20. The mixed solution is then placed in a high-temperature reaction environment, followed by centrifugation. The supernatant is collected, filtered, dialyzed, and the solution with a molecular weight greater than 800 Da is collected and lyophilized to obtain the copper-doped mitochondrial-targeting carbon dot. This carbon dot exhibits high cytotoxicity.

[0004] Patent application CN115975639A discloses a long-wavelength luminescent carbon dot targeting mitochondria and its preparation method. This invention first prepares nitrogen-doped red luminescent carbon dots using p-phenylenediamine as a raw material via a solvothermal method. Then, using the nitrogen-doped red luminescent carbon dots and carboxybutyltriphenylphosphine as reaction substrates, and N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide as dehydrating and crosslinking agents, an amidation reaction is carried out at room temperature. After purification, long-wavelength luminescent carbon dots are obtained. These carbon dots use positively charged triphenylphosphine as a targeting group, targeting mitochondria based on the principle of electrostatic adsorption. Summary of the Invention

[0005] The technical solution adopted in this invention is:

[0006] The carbon dots, prepared via a one-step hydrothermal method using starch as the carbon source and ethylamine as the nitrogen source, are nitrogen-doped blue fluorescent carbon dots. These mitochondrial-targeting carbon dots have a negatively charged surface, and their mitochondrial targeting mechanism is independent of electrostatic attraction. The particle size of these mitochondrial-targeting carbon dots ranges from 1.5 to 3.0 nm, with optimal excitation and emission at 358 nm and 448 nm, respectively. The absolute quantum yield is 84.84%, and the fluorescence lifetime is 9.652 ns. Cytotoxicity assays (CCK-8 assay) show that even at concentrations as high as 320 μg / mL, cell viability remains above 80%. The prepared mitochondrial-targeting fluorescent carbon dots exhibit a co-localization coefficient (Pearson correlation coefficient) of 0.91 with the commercial mitochondrial probe MitoTracker Red CMXRos (MTR), and this method enables large-scale preparation of mitochondrial-targeting fluorescent carbon dots.

[0007] Preferably, the mitochondrial-localized fluorescent carbon dots have a particle size of 1.5-3.0 nm and an average particle size of 2.25 nm.

[0008] (1) Disperse soluble starch evenly in ultrapure water and place it in a water bath at 95 ℃ for 30 min. The starch solution will be transparent and gelatinous. During this process, stir continuously to maintain the homogeneity of the solution.

[0009] (2) Add different amounts of ethylamine to the gelatinized starch solution to dope N.

[0010] (3) After mixing, the mixed solution from 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 brownish-brown solution.

[0011] (4) The product obtained in step (3) was dialyzed with a dialysis bag with a molecular weight cutoff of 500 Da and freeze-dried to obtain the mitochondrial-localized fluorescent carbon dots.

[0012] Preferably, the starch mass in step (1) is 0.75 g and the volume of ultrapure water is 30 mL.

[0013] Preferably, the molar ratio of starch to ethylamine in step (2) is 1:1 to 1:10, for example, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10. A more preferred molar ratio is 1:6.5.

[0014] Preferably, the reaction temperature in step (3) is 180 °C and the reaction time is 6 h.

[0015] Preferably, in step (4), the brownish-red solution obtained in step (3) is centrifuged before dialysis, and the supernatant is used for dialysis. More preferably, the centrifugation conditions are 10,000 r / min for 10 minutes.

[0016] The key features of this invention, based on the above technical solutions, are as follows: (1) Advantages of raw materials and process: The preparation method of the present invention is simple to operate, the raw materials starch and ethylamine are inexpensive and readily available, the biosafety is high, the cost is low, and the yield of mitochondrial localization fluorescent carbon dots is high.

[0017] (2) Performance advantages: The prepared carbon dots have excellent mitochondrial targeting ability, and still have a localization effect on mitochondria when the membrane potential decreases or disappears.

[0018] (3) Outstanding optical performance: The mitochondrial-localized fluorescent carbon dots have high brightness and an absolute fluorescence quantum yield of 84.84%, which is at the advanced level of similar materials.

[0019] (4) Excellent biocompatibility: The mitochondrial-localized fluorescent carbon dots have low cytotoxicity, have little effect on cell activity at concentrations up to 320 μg / mL, and have almost no effect on normal cell physiological functions.

[0020] (5) Great application potential: The mitochondrial-localized fluorescent carbon dots have good water solubility and high safety. Attached Figure Description

[0021] Figure 1 This is a transmission electron microscope (TEM) characterization image of the mitochondrial localized fluorescent carbon dots prepared in Example 1; Figure 2 This is a high-resolution transmission electron microscope (HRTEM) characterization image of the mitochondrial localized fluorescent carbon dots prepared in Example 1; Figure 3 This is an atomic force microscopy (AFM) characterization image of the mitochondrial localized fluorescent carbon dots prepared in Example 1; Figure 4 This is a Zeta potential characterization diagram of the mitochondrial localization fluorescent carbon dots prepared in Example 1; Figure 5 This is a Fourier transform infrared (FTIR) spectrum characterization of the mitochondrial localization fluorescent carbon dots prepared in Example 1; Figure 6 This is an X-ray photoelectron spectroscopy (XPS) characterization image of the mitochondrial-localized fluorescent carbon dots prepared in Example 1; Figure 7 This is a fluorescence lifetime decay curve of the mitochondrial-localized fluorescent carbon dots prepared in Example 1; Figure 8This is a characterization diagram of the UV-Vis absorption and fluorescence spectra of the mitochondrial-localized fluorescent carbon dots prepared in Example 1; Figure 9 These are the fluorescence emission spectra of the mitochondrial-localized fluorescent carbon dots prepared in Example 1 at different excitation wavelengths; Figure 10 This is a graph showing the results of the HeLa cytotoxicity assay (CCK-8) using mitochondrial-localized fluorescent carbon dots prepared in Example 1. Figure 11 The images show the co-localization fluorescence of mitochondrial fluorescent carbon dots and mitochondrial probes in HeLa cells, as described in Example 1. HeLa: a. Blue fluorescent carbon dots incubated for 4 h; b. Mitochondrial red commercial probe incubated for 0.5 h; c. Combined images; d. Co-localization analysis: fluorescence intensity scatter plot, Pearson correlation coefficient (PCC), and overlap coefficient (OLC) (Channel 1: excitation wavelength 405 nm, receiving range 450 ± 25 nm; Channel 2: excitation wavelength 552 nm, receiving range 600 ± 25 nm). Figure 12 This is a comparison of the imaging effects of mitochondrial localization fluorescent carbon dots and membrane potential-dependent probe Rhodamine 123 (Rho 123) prepared in Example 1 in HeLa cells when the mitochondrial membrane potential decreases or disappears. Figure 13 This is a graph showing the effect of the mitochondrial localization fluorescent carbon dots prepared in Example 1 on the mitochondrial membrane potential after imaging with the commercial cation probe MitoTrackerRed CMXRos (MTR).

[0022] Figure 14 This is a comparison image of cell imaging using the mitochondrial localization fluorescent carbon dots prepared in Example 1 and the commercial blue mitochondrial probe MitoLiteBlue FX490 (MLB) under a bacterial contamination environment.

[0023] Figure 15 This is a flowchart of the preparation process of the mitochondrial-localized fluorescent carbon dots prepared in Example 1. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of this invention.

[0025] Example 1: Preparation of mitochondrial-localized blue luminescent carbon dots This embodiment provides a method for preparing mitochondrial-localized blue luminescent carbon dots, including the following steps: (1) Preparation of raw materials: Weigh 0.75 g of soluble starch (potato source, purchased from Shanghai Maclean Biochemical Technology Co., Ltd.) and disperse it in 30 mL of ultrapure water. Place the mixture in a 95 ℃ water bath for gelatinization. After gelatinization, add ethylamine to the gelatinized starch solution to adjust the molar ratio of starch to ethylamine to 1:6.5, and stir thoroughly to mix well.

[0026] (2) Hydrothermal reaction: The mixed solution was transferred to a high-pressure reactor lined with polytetrafluoroethylene and reacted at 180 °C for 6 hours.

[0027] (3) Purification: After the reaction was completed, the solution was naturally cooled to room temperature to obtain a brown crude product solution. The solution was centrifuged, and the supernatant was taken and dialyzed in ultrapure water for 12 h using a dialysis bag with a molecular weight cutoff of 500 Da. Finally, the solution was freeze-dried to obtain solid blue luminescent carbon dots.

[0028] The schematic diagram of the preparation process is shown below. Figure 15 The transmission electron microscopy (TEM) characterization results of the product are shown in [the table below]. Figure 1 The ultraviolet-visible absorption spectrum is shown in [reference needed]. Figure 8 The fluorescence emission spectrum is shown in Figure 10 .

[0029] Examples 2 to 11: Effects of different material ratios on carbon dot properties The preparation steps of Examples 2 to 11 are exactly the same as those of Example 1, except that the molar ratio of starch to ethylamine in step 2 is changed. The specific ratios are as follows: The preparation steps of fluorescent carbon dots in Example 2 are the same as those in Example 1, except that the ratio of starch to ethylamine is 1:1.

[0030] 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 is 1:2.5.

[0031] 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 is 1:5.

[0032] 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 is 1:6.

[0033] 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 is 1:6.5.

[0034] 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 is 1:7.

[0035] 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 is 1:8.

[0036] 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 is 1:10.

[0037] 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 is 1:14.

[0038] 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 is 1:18.

[0039] (1) The particle size of the fluorescent carbon dots in Example 1 was detected using a transmission electron microscope (TEM, Hitachi HT-7800), and the results are as follows: Figure 1 As shown, the carbon dots have a particle size distribution between 1.5 and 3.0 nm, with an average particle size of 2.25 nm.

[0040] (2) The thickness of the mitochondrial-localized fluorescent carbon dots prepared in Example 1 was characterized using atomic force microscopy (AFM, Bruker Dimension Icon), and the results are as follows: Figure 2 As shown, the carbon dots are approximately 1-1.5 nm thick, which, together with the TEM results, indicates that they are quasi-spherical nanoparticles.

[0041] (3) The zeta potential of the mitochondrial localization fluorescent carbon dots prepared in Example 1 in aqueous phase (pH 6.6-7.4) was tested using a dynamic light scattering instrument (Malvern ZS90 Zeta sizer). The result was -6.09 mV. Figure 4 As shown.

[0042] (4) The absorption and fluorescence spectra of the carbon dots were characterized using a UV-Vis spectrophotometer (Hitachi U-2910) and a fluorescence spectrometer (Hitachi F-4600). The results are as follows: Figure 8 As shown, it has characteristic absorption at 358 nm and the strongest fluorescence emission at 448 nm.

[0043] (5) The absolute fluorescence quantum yield of carbon dots was determined by steady-state fluorescence spectrometry (Edinburgh FLS 1000), and the result was 84.84%.

[0044] (1) The surface functional groups of the mitochondrial-localized fluorescent carbon dots prepared in Example 1 were characterized using Fourier transform infrared spectroscopy (FTIR, Bruker INVENIO S-type). As shown in the figure, at 3562 cm⁻¹, the surface functional groups of the mitochondrial-localized fluorescent carbon dots prepared in Example 1 were characterized. -1 A stretching vibration peak of OH was observed at 3192 cm⁻¹. -1A stretching vibration peak of NH was observed at the point, and these hydrophilic groups enable the carbon dots to be well dispersed in water. There are three main peaks in the XPS full spectrum: C 1s (284.5 eV), N 1s (398.7 eV) and O 1s (532.2 eV).

[0045] (2) X-ray photoelectron spectroscopy (XPS) full spectrum ( Figure 6 The results show characteristic peaks for C 1s (284.5 eV), N 1s (398.7 eV) and O 1s (532.2 eV), confirming the presence of C, N and O elements and the successful doping of nitrogen.

[0046] The mitochondrial targeting ability of the fluorescent carbon dots prepared in Example 1 in HeLa and Hep G2 cells was tested using a laser confocal microscope (CLSM, Leica TCS SP8). The method is as follows: (1) HeLa cells (5×10 4 Inoculate (number per mL) into confocal dishes and incubate for 24 h.

[0047] (2) Wash the cells three times with phosphate-buffered saline (PBS) solution and incubate them for 4 h with 500 μL of 1640 complete medium containing 40 μg / mL mitochondrial-localized fluorescent carbon dots.

[0048] (3) After washing the cells three times with PBS, add 500 μL of the commercial mitochondrial red probe MitoTracker RedCMXRos (MTR). The preparation method of the commercial mitochondrial red probe is as follows: take 20 μL of MitoTracker Red CMXRos (MTR) 500× stock solution, dilute it to 10 mL with 1640 complete medium to obtain 1× working solution, and then take 500 μL of this working solution and add it to the cells for incubation for 30 min.

[0049] (4) Wash the cells three times with PBS and add phenol red-free 1640 complete medium for subsequent imaging. Perform fluorescence imaging of the cells using a CLSM 63× oil immersion lens.

[0050] The results are as follows Figure 11 As shown, the blue fluorescence of the carbon dots highly overlaps with the red fluorescence of MitoTracker Red CMXRos (MTR). Colocalization analysis revealed a Pearson correlation coefficient (PCC) of 0.91 and an overlap coefficient (OLC) of 0.99 in HeLa cells. This indicates that the carbon dots possess excellent mitochondrial targeting capabilities in cells.

[0051] To verify whether its targeting mechanism depends on mitochondrial membrane potential (MMP), the following method was used: HeLa cells were pretreated with 10 μM carbonyl cyanide 3-chlorophenylhydrazone (CCCP) for 30 min to reduce the mitochondrial membrane potential (MMP). Subsequently, cells were incubated with mitochondrial-localizing fluorescent carbon dots and Rhodamine 123 (a fluorescent probe for mitochondrial localization via electrostatic adsorption), respectively, for a period of time. The changes in fluorescence intensity were compared between cells treated with and without CCCP. Results are as follows: Figure 12 As shown, compared with the control group, the fluorescence of mitochondria localized by Rho 123 was significantly reduced after CCCP treatment, while the fluorescence intensity of mitochondria localized by mitochondrial localization fluorescent carbon dots remained almost unchanged, demonstrating excellent mitochondrial localization ability independent of mitochondrial membrane potential (MMP). Furthermore, cell fixation was achieved by treating cells with 4% paraformaldehyde (PFA) for 20 min, which simultaneously caused the mitochondrial membrane potential to disappear. After the membrane potential disappeared, the fluorescence intensity of mitochondria localized by Rho 123 almost completely disappeared. However, the fluorescence of mitochondria localized by mitochondrial localization fluorescent carbon dots could still be clearly observed after the disappearance of the mitochondrial membrane potential (MMP). This indicates that the localization of mitochondria by mitochondrial localization fluorescent carbon dots does not depend on electrostatic adsorption. (448 nm laser intensity 10%, receiving range 470±10 nm)

[0052] The effects of mitochondrial localization fluorescent carbon dots obtained in Example 1 and mitochondrial probe MitoTracker Red CMXRos (MTR) imaging on mitochondrial membrane potential were tested. The method is as follows: HeLa cells were treated with mitochondrial-localized fluorescent carbon dots (40 μg / mL) and the mitochondrial cation probe MitoTracker RedCMXRos (MTR), respectively. After incubation for 4 h, the mitochondrial membrane potential (MMP) was detected using Rho 123. Figure 13 It was found that the fluorescence intensity of Rho 123 did not decrease significantly after treatment with mitochondrial-targeted fluorescent carbon dots, indicating that the mitochondrial membrane potential (MMP) remained almost unchanged. However, treatment with a cation probe significantly reduced the fluorescence intensity, indicating a significant decrease in the mitochondrial membrane potential (MMP). This demonstrates that mitochondrial-targeted fluorescent carbon dots have minimal impact on the mitochondrial membrane potential (MMP) and mitochondrial function. (448 nm laser intensity 10%, receiving range 460±10 nm)

[0053] The mitochondrial localization fluorescent carbon dots obtained in Example 1 were compared with the mitochondrial probe. The results of cell imaging using the commercial mitochondrial blue dye MitoLite Blue FX490 (MLB) under bacterial contamination conditions were also compared. Figure 14 As shown in the figure. The results indicate that even under cell-contaminated conditions, precise mitochondrial localization can still be achieved, effectively distinguishing cells from bacteria. Traditional commercial dyes, however, lose their precise localization effect under cell-contaminated conditions. (405 laser intensity 10%, receiving range 450±10 nm) The preferred embodiments of the present invention have been described in detail above; however, 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 to the above embodiments. For those skilled in the art, several modifications and improvements can be made without departing from the principle of the present invention, and all such modifications and improvements are included within the protection scope of the present invention.

Claims

1. A mitochondria-targeted blue-emitting carbon dot, characterized in that, The mitochondrial-localized blue luminescent carbon dots were synthesized via a one-step hydrothermal method using starch as the carbon source and ethylamine as the nitrogen source, followed by N doping. These negatively charged carbon dots exhibit a mitochondrial imaging mechanism independent of electrostatic attraction. The particle size of these carbon dots ranges from 1.5 to 3.0 nm, with optimal excitation and emission at 358 nm and 448 nm, respectively. The absolute quantum yield is 84.84%, and the fluorescence lifetime is 9.652 ns. In the CCK-8 cytotoxicity assay, cell viability remained above 80% even at a concentration of 320 μg / mL. The Pearson correlation coefficient between these carbon dots and the commercial mitochondrial probe MitoTracker Red CMXRos (MTR) is 0.

91.

2. The method for preparing mitochondrial-localized blue luminescent carbon dots according to claim 1, characterized in that, The method includes The following steps are performed: starch is added to distilled water and stirred thoroughly to obtain the starch solution. Then, the starch solution is gelatinized in a water bath at 95°C for 0.5 hours. Subsequently, ethylamine is added to the gelatinized starch solution, wherein the molar ratio of ethylamine to the starch gelatinized and depolymerized to form glucose monomers is 6.5:1, the reaction temperature is 180°C, and the reaction time is 6 hours.

3. The method for preparing mitochondrial-localized blue luminescent carbon dots according to claim 2, characterized in that, The method also includes the following purification steps: the separation and purification conditions after the synthesis of blue luminescent carbon dots localized to mitochondria are dialysis using a 500 Da dialysis bag and ultrapure water for 12 hours, with the ultrapure water being replaced every 2 hours.

4. The mitochondrial-localized blue luminescent carbon dot according to claim 1, characterized in that, The mitochondrial-localized blue luminescent carbon dots are concentrated in the range of 1.5–3.0 nm in size, with an average size of 2.25 nm.

5. The mitochondrial-localized blue luminescent carbon dot according to claim 1, characterized in that, The zeta potential of the blue luminescent carbon dots located in mitochondria is -6.09 mV.

6. The mitochondrial-localized blue luminescent carbon dot according to claim 1, characterized in that, The blue luminescent carbon dots localized to mitochondria exhibit good biocompatibility and low cytotoxicity. At a concentration of 320 μg / mL, the cell viability can still reach over 80%, while having no significant effect on mitochondrial membrane potential.

7. The application of the mitochondrial-localized blue luminescent carbon dots as described in claim 1 as a mitochondrial blue fluorescent probe, characterized in that... In mitochondrial imaging of cells, the Pearson correlation coefficient between mitochondrial-localized blue luminescent carbon dots and the commercial mitochondrial probe MitoTracker Red CMXRos (MTR) was 0.91.

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