Orange-emitting carbon dots with high photostability and preparation method and use thereof

Abstract

The present application provides a kind of carbon dot, it is prepared by solvothermal treatment with m-diethylamino phenol as raw material, and emits orange fluorescence.The carbon dot of the present application has high quantum yield, contains rich hydroxyl and ammonium ion on the surface, has high stability, and has strong mitochondria targeting ability.Moreover, the carbon dot of the present application has strong affinity with mitochondria, so that it can be aggregated in mitochondria in large quantities, the fluorescence is enhanced in low polarity environment, so that it is possible to observe the morphology of mitochondria without washing.Benefiting from the characteristics of washing-free and excellent light stability, the mitochondria dynamics in the process of apoptosis and mitochondrial autophagy of living cells can be monitored in real time, which provides a useful tool for the diagnosis and treatment strategy of mitochondrial related diseases.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent detection probes, specifically relating to an orange emitting carbon dot with high photostability, its preparation method, and its applications. Background Technology

[0002] Mitochondria are the site of oxidative phosphorylation and fatty acid β-oxidation, which provide energy for eukaryotic cells as ATP. In addition, mitochondria perform other functions in the cell, such as adipogenesis, signal transduction, and regulation of apoptosis. To accomplish these complex physiological functions, the integrity of the mitochondrial filamentary structure and the mitochondrial network is crucial, allowing all mitochondria to share the same metabolites and information. Furthermore, mitochondria interact with other intracellular organelles (such as lysosomes and lipid droplets) to respond to external stimuli, thereby maintaining cellular homeostasis. Therefore, long-term monitoring of mitochondrial dynamics is of great significance in pathological and physiological research.

[0003] Fluorescence imaging technology, with its advantages of high sensitivity, real-time tracking, and in-situ monitoring, is a powerful tool for studying cell dynamics. Developing suitable mitochondrial probes can enable long-term dynamic tracking of mitochondria in living cells, thus making the progression of mitochondrial biological events visible.

[0004] Carbon dots, as fluorescent nanomaterials with advantages such as good biocompatibility, strong cell penetration, bright fluorescence, high photostability, and simple synthesis, have become popular candidate materials in the field of bioimaging. Furthermore, some studies have shown that the surface functional groups of carbon dots (CDs) may determine their specific targeting abilities to different organelles. For example, cationic carbon dots modified with p-phenylenediamine and 4-carboxybutyltriphenylphosphine are used for nuclear targeting; morpholine-functionalized carbon dots are used for lysosome targeting; laurylamine-modified carbon dots are used for endoplasmic reticulum targeting; L-cysteine-rich chiral carbon dots are used for Golgi apparatus targeting; amphiphilic carbon dots based on the 2-dimethylamino-5-fluorobenzimidazole fluorophore are used for lipid droplet targeting; and lipophilic and surface group-mediated imaging of various organelles is also possible. Regarding reports on mitochondrial-targeting carbon dots, their targeting ability is usually attributed to the triphenylphosphine moiety, which is toxic to cells and requires additional modification steps for staining. In recent years, carbon dots with inherent mitochondrial targeting abilities dependent on positive charge and related surface functional groups (such as the lipophilic cation of rhodamine) have been reported, but studies on mitochondrial dynamic tracking are limited. Furthermore, short-wavelength excitation is considered phototoxic to cells; therefore, carbon dots emitted in blue or green by short-wavelength light excitation are generally unsuitable for cell labeling. Compared to organic molecular probes, carbon dots are less commonly used to study mitochondrial dynamics and their interactions with other organelles. Therefore, it is essential to develop carbon dots (CDs) with long-wavelength excitation, high stability, and inherent mitochondrial targeting capabilities for in-situ monitoring of mitochondrial dynamics.

[0005] The fluorescence of carbon dots (CDs) is related to the emission effects of the carbon nucleus, surface, molecular, and cross-linking states, and can be modulated by changing the reaction conditions. For cell imaging, materials with longer wavelengths of excitation and emission are needed to reduce phototoxicity and interference from autofluorescence. Some orange carbon dots with aggregation-induced emission and / or high quantum yield have been reported for use in the fabrication of light-emitting diodes or microcavity lasers, pH encryption, and zebrafish embryo imaging. For orange-emitting CDs used for organelle targeting, researchers prepared Golgi-targeting carbon dots (L-CQDs) with an emission wavelength of 601 nm and a quantum yield (QY) of 10.76% through a one-step solvothermal treatment of L-cysteine ​​and neutral red. Further research on carbon dots with excellent biocompatibility, longer emission wavelengths, high photostability, and good mitochondrial targeting remains important to provide a wider range of options and tools for the diagnosis and treatment strategies of mitochondrial-related diseases. Summary of the Invention

[0006] The purpose of this invention is to provide an orange emitting carbon dot with high photostability, its preparation method, and its uses.

[0007] The present invention provides a carbon dot, which is prepared by solvothermal treatment of m-diethylaminophenol as raw material, and the carbon dot emits orange fluorescence.

[0008] Furthermore, the above-mentioned solvothermal treatment is performed in a mixed solvent of ethanol, water, and acid.

[0009] Furthermore, the molar volume ratio of the above-mentioned m-diethylaminophenol to the solvent is (2-6) mmol: (20-60) mL.

[0010] Furthermore, the molar volume ratio of the above-mentioned m-diethylaminophenol to the solvent is 4 mmol: 40 mL.

[0011] Furthermore, the above heat treatment conditions are: 150-200℃ for 4-8 hours.

[0012] Furthermore, the above heat treatment conditions are 180℃ for 6 hours.

[0013] Furthermore, the aforementioned carbon dots are spherical with an average diameter of 3–4 nm.

[0014] The present invention also provides a method for preparing the above-mentioned carbon dots, comprising the following steps:

[0015] (1) Dissolve m-diethylaminophenol in a solvent and heat treat it at 150-200°C for 4-8 hours; the solvent is a mixture of ethanol, water and acid.

[0016] (2) Filtration, concentration, and column chromatography were used to purify and collect the components that emitted orange fluorescence.

[0017] Further, the molar volume ratio of m-diethylaminophenol to solvent in step (1) above is (2-6) mmol: (20-60) mL; preferably 4 mmol: 40 mL.

[0018] Furthermore, the heat treatment described in step (1) above is a treatment at 180°C for 6 hours.

[0019] Furthermore, the filtration described in step (2) above is a filtration using several 0.22μm microporous membranes.

[0020] Furthermore, the column chromatography purification in step (2) above uses dichloromethane and methanol as eluents.

[0021] This invention also provides the application of the above-mentioned carbon dots as fluorescent probes.

[0022] Furthermore, the aforementioned fluorescent probe is a mitochondrial-targeting fluorescent probe.

[0023] The beneficial effects of this invention are as follows: The orange-emitting carbon dots of this invention have high quantum yield, abundant hydroxyl and ammonium ions on their surface, high stability, and strong mitochondrial targeting ability. Furthermore, the strong affinity of the carbon dots for mitochondria causes them to aggregate extensively within mitochondria, resulting in enhanced fluorescence in low-polarity environments, making washless observation of mitochondrial morphology possible. Thanks to the washless nature and excellent photostability, mitochondrial dynamics during live cell apoptosis and mitophagy can be monitored in real time. Therefore, O-CDs provide a useful tool for studying diagnostic and therapeutic strategies for mitochondrial-related diseases.

[0024] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0025] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0026] Figure 1 Images of carbon dots (CDs) synthesized in different solvents under 365-nm UV light irradiation.

[0027] Figure 2(A) TEM image of O-CDs. Scale bar: 20 nm. (B) Size distribution of O-CDs. (C) Zeta potential of O-CDs in water. (D) FTIR spectrum of O-CDs. (E) 1H HMR spectrum of O-CDs. (F) XPS spectrum of O-CDs. High-resolution (G)C 1s, (H)N 1s, and (I)O 1s spectra of O-CDs.

[0028] Figure 3 (A) Absorption spectra of O-CDs. (B) Excitation and emission spectra of O-CDs. (C) Excitation-independent fluorescence spectra of CDs. (D) Normalized emission spectra of O-CDs in different solvents. (E) Relative fluorescence intensity at 565 nm in various solvents. (F) Emission spectrum of O-CDs in a methanol-glycerol mixture. (G) Emission spectra of O-CDs in solutions with different pH values. (H) Fluorescence intensity in solutions containing metal ions and amino acids. (I) Kinetics of O-CDs, Mito-Tracker Green (MTG), Mito-Tracker Deep Red (MTDR), Rhodamine 123 (Rho 123), Hoechst, and BODIPY.

[0029] Figure 4 The absolute PLQY of O-CD in (A) water and (B) ethanol.

[0030] Figure 5 The fluorescence decay spectrum of O-CDs in water.

[0031] Figure 6 The fluorescence intensity of O-CDs in water stored at room temperature for different time periods.

[0032] Figure 7 To evaluate the cytotoxicity of O-CDs using CCK-8.

[0033] Figure 8 CLSM images of live HeLa cells stained with different concentrations of O-CDs for 20 minutes. Scale bar: 10 micrometers.

[0034] Figure 9 CLSM images of HeLa live cells stained with 5 μg / mL O-CD at different time points. Scale bar: 10 μm.

[0035] Figure 10 CLSM images of HeLa cells treated with O-CD alone (control) or with O-CD and 4℃ / one endocytosis inhibitor (amiloride, CPZ, genistein, NaN3, and MβCD), and corresponding normalized FL intensities. Scale bar: 10 μm.

[0036] Figure 11 Confocal fluorescence and colocalization images of HeLa cells stained with O-CDs and commercial organelle probes: (A) MTDR, (B) Hoechst 33342, (C) LTG, and (D) BODIPY 493 / 503. 3D images (vertical, front view, and left view) of HeLa cells stained with O-CDs and cultured with Hoechst 33342 (E) or BODIPY (F). Scale bar, 10 μm.

[0037] Figure 12 (A) Schematic diagram and CLSM image of O-CDs stained cells before and after PBS washing. Scale bar: 10 μm. (B) Schematic diagram and CLSM image of normal HeLa cells and cells treated with cccp or 4% PFA. Scale bar: 10 μm.

[0038] Figure 13 CLSM images of different cell lines co-cultured with O-CD and MTDR. Scale bar: 10 μm.

[0039] Figure 14 The relative fluorescence intensity of O-CDs at different time points after continuous irradiation with a 543nm laser for 20 min in CLSM images of HeLa cells.

[0040] Figure 15 Mitochondrial dynamics in living cells. (A) O-CDs stained confocal image of cells. Schematic diagram of mitochondrial fusion and fission. (B) Magnified confocal fluorescence image of mitochondrial fission in orange and iridescent colors within the green box in (A), where iridescent colors represent signal intensity. (C) Magnified confocal fluorescence image of mitochondrial fusion and the orange and iridescent colors within the white box in (A), where iridescent colors represent signal intensity. (D) Schematic diagram of mitochondrial fusion and fission. (E) Two-color confocal image of mitochondrial-ld contact in HeLa cells. The enlargement of the boxed area indicates a dynamic interaction between mitochondria and lipid droplets. (F) Schematic diagram of mitochondria encapsulating lipid droplets, scale bar: 10 μm.

[0041] Figure 16 (A) Dynamic schematic of mitochondria and LDs after treatment with H2O2 (left) and rapamycin (right). (B) O-CDs and bodipy-stained CLSM of HeLa cells after incubation with H2O2 (B) and rapamycin (C) for 0, 5, 10, and 20 minutes, as well as ImageJ-processed mitochondrial threshold and scaffold images. Scale bar: 10 μm.

[0042] Figure 17The relative fluorescence intensity of O-CDs in DMEM medium containing H2O2 or rapamycin under continuous 543 nm laser irradiation. Detailed Implementation

[0043] The chemical reagents and equipment involved in this invention are as follows:

[0044] Amiloride, m-diethylaminophenol, chlorpromazine, methyl-β-cyclodextrin, and genistein were purchased from Shanghai Aladdin Reagent Co., Ltd. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was purchased from Maclean Biochemical Co., Ltd. Other chemicals were purchased from Chengdu Kelon Chemical Reagent Factory. BODIPY493 / 503 was from Thermo Fisher. Hoechst 33342, MitoTracker Green (MTG), Rhodamine 123 (Rho 123), and Lyso-Tracker Green (LTG) were all purchased from Beyotime Biotechnology Research Institute. MitoTracker Deep Red FM (MTDR) was purchased from Yisheng Biotechnology Co., Ltd. Dulbecco's Modified Eagle Medium (DMEM) and Hank's balanced salt solution (HBSS) were both from HyClone. Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. All chemicals were analytical grade and were not further purified before use.

[0045] Transmission electron microscopy (TEM) was performed using a FEI Tecnai G2 F20 TEM. Fourier transform infrared (FTIR) spectroscopy was acquired using a Nicolet 6700 infrared spectrophotometer. X-ray photoelectron spectroscopy (XPS) analysis was performed using a Kratos Axis Ultra DLD spectrometer. Nuclear magnetic resonance (NMR) spectroscopy was recorded using a Bruker BioSpin AV11-600MHz spectrophotometer. UV-Vis spectra were measured using a U-2910 UV-Vis spectrophotometer. Fluorescence spectra, absolute quantum yields, fluorescence lifetimes, and kinetic spectra of O-CDs and commercial probes were recorded using a Horiba fluorolog-3 microscope. All confocal images were acquired using a ZEISS LSM 880 confocal microscope, and the colocalization coefficients of O-CDs were determined using the ZEISS LSM 880 confocal microscope software.

[0046] Unless otherwise stated, all other raw materials and equipment used in this invention are known products, obtained by purchasing commercially available products.

[0047] The experimental characterization method involved in this invention is as follows:

[0048] Cell culture and cytotoxicity studies:

[0049] HeLa cells and NIH 3T3 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin at 37°C and 5% CO2 humidity. L929 cells were cultured in MEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin. Cytotoxicity was assessed using the Cell Counting Kit-8 (CCK-8).

[0050] Cellular uptake pathway:

[0051] To investigate the internalization pathway of O-CDs, HeLa cells were pretreated at 37°C for 1 hour with 4°C, NaN3, chlorpromazine (CPZ), genistein (GEN), amiloride (AMI), and methyl-β-cyclodextrin (MβCD), followed by the addition of O-CDs. After culturing HeLa cells with O-CDs for 10 minutes, fluorescence imaging was performed using a Zeiss LSM 880.

[0052] Cell imaging and colocalization:

[0053] The potential of the obtained O-CDs for cell imaging was tested using HeLa cells as a model cell. HeLa cells (1×10⁻⁶) were used... 5 Cells were seeded at 10000 mcg / mL in glass-bottomed cell culture dishes and incubated for 24 h. For colocalization experiments, MitoTracker Deep Red (MTDR, 50 nM) was injected into the culture dishes and incubated at 37 °C for 30 min. Cells were then washed three times with phosphate-buffered saline (PBS) to remove unbound dye molecules. Imaging was performed directly after staining with O-CDs (5 μg / mL) for 10 min. Images were obtained using a Zeiss LSM 880 confocal laser scanning microscope. Cosizing experiments were performed using commercial organelle probes containing Lyso-Tracker Green, BODIPY 493 / 503, and Hoechst, following similar methods. O-CDs: λex = 543 nm; MitoTracker Deep Red: λex = 633 nm; Lyso-Tracker Green: λex = 488 nm; BODIPY 493 / 503: λex = 488 nm; Hoechst: λex = 405 nm. Colocalization coefficients were calculated using ZEN blue software.

[0054] To verify the mitochondrial targeting mechanism, 20 μM CCCP or 4% paraformaldehyde was added to the culture dish and incubated for 30 min before imaging.

[0055] Long-term cell tracking:

[0056] HeLa cells were seeded in glass-bottomed cell culture dishes and incubated at 37°C for 24 h. After incubation with 5 μg / mL O-CDs for 10 min, fluorescence imaging was performed.

[0057] To monitor mitochondrial dynamics during apoptosis and mitophagy, HeLa cells were first cultured in glass-bottomed dishes for 24 h, then stained with BODIPY 493 / 503 (2 μM) for 30 min, followed by staining with O-CDs (5 μg / mL) at 37 °C for 10 min. Before imaging, apoptosis or mitosis was induced with H2O2 (10 mM) or rapamycin (5 μg / mL), respectively. Fluorescence images were recorded at different time points (0, 5, 10, and 20 min).

[0058] Example 1: Synthesis of carbon dot O-CDs of the present invention

[0059] 4 mmol of m-diethylaminophenol was dissolved in a mixture of 40 mL of water / ethanol and hydrochloric acid (ratio: 19.1 mL water, 19.1 mL ethanol, 1.8 mL 37 wt.% hydrochloric acid). The solution was then transferred to a 100 mL polytetrafluoroethylene autoclave and heated at 180 °C for 6 h. After cooling to room temperature, large particles were removed by filtration through a 0.22 μm microporous membrane. The resulting solution was then concentrated and purified by column chromatography using dichloromethane and methanol as eluents. Finally, the orange emission component was collected and lyophilized to obtain a brown powder.

[0060] Comparative Example 1

[0061] 4 mmol of m-diethylaminophenol was dissolved in a mixture of 40 mL of water and ethanol. The solution was then transferred to a 100 mL high-pressure reactor and heated at 180 °C for 6 h. After cooling to room temperature, large particles were removed by filtration through a 0.22 μm microporous membrane. The resulting solution was then concentrated and purified by column chromatography using dichloromethane and methanol as eluents. Finally, the orange emission component was collected and lyophilized to obtain a brown powder.

[0062] The following experimental examples demonstrate the beneficial effects of the present invention.

[0063] Experimental Example 1: Structural Characterization of O-CDs

[0064] like Figure 1As shown, the crude products obtained in different solvents (water / ethanol and water / ethanol / HCl) exhibited different colors under 365 nm UV light. With ethanol and water as solvents, the product mainly exhibited blue fluorescence, which is unsuitable for biological applications. Inspired by previous research, acidic conditions can promote longer wavelength emission of CDs, so we introduced HCl into the reaction solvent to improve the emission performance of CDs. As expected, when using a mixed solvent of ethanol, water, and HCl, the prepared O-CDs exhibited brighter and longer emission, indicating greater potential as cell imaging probes, which was confirmed in subsequent experiments.

[0065] TEM images show that O-CDs are spherical with an average diameter of approximately 3.70 nm. Figure 2 AB). Furthermore, the zeta potential of O-CDs ( Figure 2 C) is approximately +23.97 mV, indicating a positive surface charge, essential for mitochondrial targeting. Infrared spectroscopy reveals the surface functional groups of hydroxyl and ammonium groups in O-CDs. (Example: ...) Figure 2 As shown in D, 3000~3500cm -1 The stretching absorption bands at this point are composed of -OH and -NH2 groups, ranging from 2975 to 2465 cm⁻¹. -1 The strong tensile absorption band at that point is -NH3. + Tensile vibrations confirmed that the diethylamino group can undergo N-deethylation to generate -NH2, and further protonation to generate -NH3. + This triggers orange fluorescence emission. Furthermore, at 1614 and 1451 cm⁻¹... -1 C=C / C=O bonds were observed at 1231cm. -1 The absorption peak at 1350–1270 cm⁻¹ corresponds to the CO tensile vibration. -1 The characteristic absorption peaks between them indicate the presence of CN. This is confirmed by NMR spectroscopy (…). Figure 2 E) was used to elucidate the chemical shifts of CDs, and several domain peaks representing aromatic structures were observed in the range of 6.5–7.5 ppm. Furthermore, 1 The chemical shifts of 3.2–3.6 ppm and 2.2–2.6 ppm in ¹H NMR correspond to OH and NH, respectively. Further analysis of the chemical composition of O-CDs was performed using XPS data. (Full XPS spectrum) Figure 2 F) shows that O-CDs contain C, N, and O elements, with contents of 85.11%, 6.01%, and 8.89%, respectively. From Figure 2The high-resolution spectra of GI show that the three fitted peaks at 284.6, 285.9, and 289.6 eV in the C 1s spectrum represent the chemical structures of CC / C=C, CN, and CO; the two fitted peaks at 399.2 eV and 400.3 eV in the N 1s spectrum represent pyridine N and pyrrole N; and the two fitted peaks at 532.5 and 530 eV in the O 1s spectrum represent the presence of the C=O and CO bands.

[0066] The above results collectively confirm that the elemental composition, surface functional groups, hydroxyl groups, amino groups, and ammonium ions on the surface of O-CDs give them excellent hydrophilicity and mitochondrial targeting ability.

[0067] Experimental Example 2: Optical Properties of O-CDs

[0068] The optical properties of O-CDs were then investigated. UV-vis spectra ( Figure 3 A) A significant peak is observed at 532 nm, followed by optimal emission at 565 nm under 535 nm excitation, as shown below. Figure 3 As shown in Figure B, the inset shows the fluorescence under ultraviolet (365 nm) illumination. Figure 3 C exhibits excitation-independent properties in the excitation range of 460 nm to 540 nm, indicating that the luminescence originates solely from surface defects. Furthermore, the absolute quantum yields in water and ethanol are 5.79% and 12.61%, respectively. Figure 4 The lifetime of O-CDs was calculated to be 2.48 ns. Figure 5 The polar responsiveness of O-CDs was investigated using organic solvents of different polarities. The results showed that the maximum emission of O-CDs remained almost unchanged with increasing polarity. Figure 3 D). Furthermore, the effect of polarity on FL intensity was investigated using the fluorescence intensity at 565 nm, such as... Figure 3 As shown in Figure E, the emission of O-CDs in water is weaker than in most organic solvents. This difference between water and organic solvents paves the way for wash-free imaging, as intracellular organelles have less polarity than water. It has been reported that mitochondrial viscosity can increase under physiological and pathological conditions; therefore, we measured the fluorescence response of O-CDs to viscosity in mixtures of glycerol and methanol in different proportions. Figure 3 As shown in Figure F, the maximum fluorescence intensity only increased by about two times when the viscosity increased from 0 to 100% glycerol, indicating very low sensitivity to viscosity. The sensitivity of fluorescence properties to metal ions or certain interfering substances may lead to unreliable imaging; therefore, we further investigated the stability of O-CDs in different pH, ionic, and amino acid environments. Figure 3As shown in GH, the fluorescence intensity of O-CDs remained almost unchanged under physiological pH or interference from various metal ions, indicating that O-CDs have high chemical stability. Further comparison with the photostability of commercial MTG, MTDR, Rho 123, Hoechst, and BODIPY showed fluorescence reductions of 24.78%, 13.5%, 7.6%, 21.2%, and 36.3%, respectively, while the fluorescence of the synthesized O-CDs remained almost unchanged after 30 min of testing. Figure 3 I). Furthermore, the fluorescence stability of O-CDs dispersed in water during room temperature storage was investigated, such as… Figure 6 As shown, the results indicate that O-CDs have good photostability and still exhibit high fluorescence intensity even after 21 days.

[0069] These results indicate that O-CDs have good anti-interference ability and high photostability under various environments, which is beneficial for cell imaging.

[0070] Experiment 3: Cytotoxicity and Mitochondrial Imaging

[0071] Before using fluorescent O-CDs for bioimaging, HeLa cells were used as a model cell to assess cytotoxicity. CCCK-8 assay ( Figure 7 The results showed that after culturing for 24 hours at a dose of 40 μg / mL, more than 80% of HeLa cells survived, indicating that O-CDs have good biocompatibility.

[0072] Based on the excellent optical properties and biocompatibility of O-CDs, we further investigated their imaging capabilities in live cells. HeLa cells were incubated for 24 h in glass dishes with different concentrations (0.5, 1, 5, 10 μg / mL) of O-CDs. Figure 8 The CLSM images showed bright fluorescence signals in the cytoplasm under 543 nm laser excitation, with fluorescence intensity varying with O-CD concentration. We then investigated the absorption efficiency of O-CDs by calculating the fluorescence intensity at different time points within the same view. Figure 9 As shown, FL intensity increased with increasing incubation time, and the FL intensity at 10 min was sufficient for imaging. Subsequent cell imaging was performed at a concentration of 5 μg / mL and an incubation time of 10 min.

[0073] Because HeLa cells have good cell permeability, we investigated the O-CD uptake pathway using four endocytosis inhibitors (CPZ, a clathrin-mediated endocytosis inhibitor; AMI, a macrocytosis inhibitor; GEN, a vesicle-mediated endocytosis inhibitor; and MβCD, a lipid raft-mediated endocytosis inhibitor). We also verified whether endocytosis is energy-dependent through 4°C and NaN3 treatment, as 4°C treatment reduced enzyme activity and ATP production, while NaN3 blocked electron transport chain transfer, thereby inhibiting energy metabolism. Figure 10 CLSM images showed that the FL intensity of cells treated with GEN, 4℃, and NaN3 was significantly reduced, indicating fewer O-CDs entering the cells. These results suggest that O-CD uptake is energy-dependent and depends on the endocytic pathway mediated by pore proteins.

[0074] To verify the mitochondrial-specific targeting ability of O-CDs, a co-localization experiment was performed. HeLa cells were stained with O-CDs and commercially available MTDR. Figure 11 As shown in Figure A, the orange fluorescence of O-CDs is consistent with the red fluorescence of MTDR, with a superposition factor of 0.90, confirming that O-CDs have excellent mitochondrial targeting ability in living cells. Furthermore, commercial organelle targeting fluorescent probes BODIPY493 / 503 were used for LDs, LTG for lysosomes, and Hoechst 33342 for the nucleus for co-localization experiments. Figure 11 The fluorescence signals of these probes showed much lower colocalization coefficients than MTDR (Hoechst 0.21, LTG 0.50, BODIPY 0.48), confirming the high mitochondrial specificity of O-CDs. Mitochondrial-specific imaging capabilities were then detected in L929 and 3T3 cells, respectively. Figure 13 The results showed that O-CDs stained the filamentous structures of different cells and had a high co-localization coefficient, indicating that O-CDs have excellent mitochondrial targeting ability. The distribution of mitochondria was observed using three-dimensional images of HeLa cells. Figure 11 As shown in Figure E, the blue channels representing the cell nucleus are only present in the cell center, while the orange channels representing filamentous mitochondria are distributed throughout the cytoplasm. Similarly, the relative positions of mitochondria and LDs are shown in Figure E. Figure 11 As shown in F, some LDs are attached to or wrapped by mitochondria, which facilitates the transport of fatty acids from LDs to mitochondria.

[0075] Interestingly, during the imaging process, we found that the image intensity of cells washed with PBS three times and those not washed three times was the same. Figure 12A) This is likely due to the lower polarity of the mitochondrial environment compared to aqueous solutions, resulting in a higher concentration of O-CDs in the mitochondria compared to the culture medium. Therefore, synthesized O-CDs can be used for wash-free imaging, aiming to save operation time and provide uninterrupted visualization of mitochondrial dynamics. Typically, traditional mitochondrial probes rely on negative MMPs, accumulating in the inner membrane through electrostatic attraction. To investigate the targeting mechanism of our O-CDs, we treated cells with CCCP, a typical MMP-lowering drug, and 4% paraformaldehyde (PFA), a drug used for cell fixation, from... Figure 12 In the results of B, we found that the filamentous structures representing mitochondria were no longer clear after CCCP treatment, and the fluorescence emitted by mitochondria decreased but did not disappear completely, indicating that the binding of O-CDs to mitochondria mainly occurs through electrostatic attraction. Furthermore, a weak fluorescence signal could still be observed after 4% PFA fixation, suggesting that other interactions, such as hydrogen bonding interactions between hydroxyl and amino groups and thiol groups of mitochondrial proteins, may also play an important role in the interaction between mitochondria and O-CDs.

[0076] The above results indicate that the O-CDs of the present invention have good biocompatibility, can specifically target mitochondria, and have good wash-free imaging performance.

[0077] Experiment Example 4: Dynamic Tracking of Mitochondria

[0078] Mitochondria are highly dynamic organelles in cells. The morphological changes, fusion, and fission of mitochondria are a rapid and dynamic process, considered closely related to physiological and pathological states. For dynamic monitoring, fluorescent dyes should possess good resistance to photobleaching. This ability of O-CDs was verified by recording fluorescence signals after 20 minutes of continuous laser irradiation. Figure 14 As shown, the fluorescence of HeLa cells stained with O-CDs showed almost no reduction. Due to their good photostability and bright fluorescence, we further used mitochondrial-targeted O-CDs to achieve long-term mitochondrial tracking (…). Figure 15 AC). For example Figure 15 As shown in Figure B, mitochondrial fission can be clearly observed. At this stage, the mitochondria indicated by the white circles gradually divide, and the fluorescence intensity at the two ends of the division increases. Furthermore, Figure 15 C also recorded mitochondrial fusion. Initially, the mitochondrial branching was linear, separating from other mitochondria, then became curved, connecting with another mitochondrial (marked by white arrows). Subsequently, the branch returned and fused with another branch; the fusion site was slightly brighter than before and could be observed through relative fluorescence intensity images. A schematic diagram of mitochondrial fusion and fission observed by O-CD staining is shown below. Figure 15 As shown in D.

[0079] Intracellular homeostasis is the result of the coordinated action of multiple organelles within the cell. It has been reported that mitochondria may interact with other organelles, such as lipid droplets (LDs), lysosomes, and the endoplasmic reticulum, through contact. Visualization of the dynamic interactions between mitochondria and other organelles will facilitate the study of molecular mechanisms. LDs are the main storage sites for neutral lipids within the cell, thus providing mitochondria with the fatty acids required for β-oxidation. Therefore, close contact between mitochondria and LDs is essential for substance exchange. Next, mitochondria and lipid droplets were co-stained with O-CDs and BODIPY staining, respectively, to image the mitochondrial-lipid droplet interaction. Figure 15 As shown in Figure EF, the orange mitochondria are arc-shaped, with green lipid droplets in contact with the outer edge of the arc. After a few seconds, the mitochondria change shape and envelop the lipid droplets. This morphological change may increase the contact surface area, thereby improving the efficiency of substance transport, which facilitates mitochondrial-lipid droplet interactions.

[0080] Under pathological conditions, the morphology and function of mitochondria change, differing from their normal state. To investigate the dynamic interaction between mitochondria and LDs under different biological processes, HeLa cells were treated with H2O2 to induce apoptosis, and the dynamic changes in mitochondria and LDs over the next 20 minutes were recorded. The changes in mitochondria and LDs after H2O2 treatment are shown below. Figure 16 As shown in A (left), its relative signal strength is recorded. Figure 16 As shown in Figure B, the mitochondrial filamentary structure in the CLSM image changed from clear to blurry, eventually almost completely disappearing. ImageJ-processed images of the mitochondrial skeleton showed a fragmented mitochondrial network, indicating that oxidative damage disrupted the mitochondrial structure. However, it had little effect on the morphology of LDs, consistent with previous reports that LDs are less sensitive to H2O2-induced apoptosis than mitochondria. The rapid collapse of mitochondrial structure resulted in minimal exchange of substances and information between LDs and mitochondria. Therefore, the number and volume of LDs did not change significantly during apoptosis. Mitophagy is a fundamental biological phenomenon in cells, playing a crucial role in maintaining the dynamic homeostasis of cell numbers. Therefore, we will next investigate the dynamic changes of mitochondria and LDs during mitophagy, with changes before and after rapamycin treatment as shown in Figure B. Figure 16 As shown in A (right). HeLa cells were stained with O-CDs and BODIPY and then treated with 50 μg / mL rapamycin to induce autophagy. Figure 16As shown in Figure C, mitochondrial fluorescence weakened and filamentous structures shortened after 10 minutes, indicating that mitophagy had occurred. Twenty minutes after the addition of rapamycin, the mitochondrial filamentous structures became punctate, and the fluorescence was weaker than before, possibly due to changes in the surrounding environment caused by rapamycin. Simultaneously, the number and volume of lipid droplets around the mitochondria significantly increased, possibly due to the delayed consumption of liposomes by the mitochondria during mitophagy. In summary, we clearly observed the different behaviors of mitochondria during apoptosis and mitophagy. The near disappearance of fluorescence intensity can be attributed to reagent treatment, as it caused changes in cell state and the subtle influence of the reagent itself on fluorescence. Figure 17 During H2O2-induced apoptosis, mitochondria are severely damaged, as evidenced by the diffusion of CDs into the cytoplasm and nucleolus. Mitochondria also break down into smaller fragments during mitophagy; therefore, this difference can be used to distinguish between apoptosis and mitosis. Furthermore, abnormal mitochondrial morphology leads to functional impairment and LD homeostasis imbalance.

[0081] Mitochondrial morphology and the interaction between mitochondria and LDs are associated with cancer, diabetes, and neurodegenerative diseases. Therefore, the O-CDs provided by this invention offer a powerful tool for the study of these diseases.

[0082] In summary, this invention provides orange-emitting carbon dots (O-CDs) with a quantum yield of 12.61%, abundant hydroxyl and ammonium ions on their surface, high stability, and strong mitochondrial targeting ability. Furthermore, the strong affinity for mitochondria leads to the large-scale aggregation of O-CDs within mitochondria, and their fluorescence is enhanced in low-polarity environments, enabling wash-free observation of mitochondrial morphology. Thanks to their wash-free properties and excellent photostability, real-time monitoring of mitochondrial dynamics during live-cell apoptosis and mitophagy is possible. Therefore, O-CDs provide a useful tool for studying diagnostic and therapeutic strategies for mitochondrial-related diseases.

Claims

1. A carbon dot, characterized in that, It is prepared by solvent heat treatment using m-diethylaminophenol as a raw material, and the carbon dots emit orange fluorescence; The solvothermal treatment is performed in a mixed solvent of ethanol, water, and acid. The heat treatment conditions are: 180~200℃ for 6~8 hours.

2. The carbon dot as described in claim 1, characterized in that, The heat treatment conditions are 180℃ for 6 hours.

3. The method for preparing carbon dots according to claim 1 or 2, characterized in that, Includes the following steps: (1) Dissolve m-diethylaminophenol in a solvent and heat treat it at 180~200℃ for 6~8h; the solvent is a mixture of ethanol, water and acid. (2) Filtration, concentration, and column chromatography were used to purify and collect the components that emitted orange fluorescence.

4. The preparation method according to claim 3, characterized in that, In step (1), the molar volume ratio of m-diethylaminophenol to solvent is (2~6) mmol: (20~60) mL.

5. The preparation method according to claim 4, characterized in that, In step (1), the molar volume ratio of m-diethylaminophenol to solvent is 4 mmol: 40 mL.

6. The preparation method according to claim 3, characterized in that, The heat treatment in step (1) is 180℃ for 6 hours.

7. The preparation method according to claim 3, characterized in that, The column chromatography purification in step (2) uses dichloromethane and methanol as eluents.

8. The use of the carbon dots as described in claim 1 or 2 in the preparation of fluorescent probes.

9. The application as described in claim 8, characterized in that, The fluorescent probe is a mitochondrial-targeting fluorescent probe.

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