Fluorescent carbon quantum dots, their preparation methods, and their applications in organelle and organoid imaging and mitochondrial membrane potential monitoring

Abstract

The present invention discloses a kind of fluorescent carbon quantum dots, its preparation method and its application in organelle and organoid imaging, mitochondrial membrane potential monitoring, the method comprising the following steps: S1, 1,5-diaminoanthraquinone, vanillic acid and ethylenediamine are mixed in a solvent, hydrothermal reaction is carried out under heating, and the reaction is cooled to room temperature after completion; S2, the product obtained by step S1 is centrifuged, the centrifuge is taken, and rotary evaporation is carried out after adding silica gel, and then eluted with an eluent, the eluate is collected, and freeze-dried to obtain fluorescent carbon quantum dots; wherein the solvent is deionized water or ethanol, and the eluent is a mixture of dichloromethane and methanol. Both G-CDs and R-CDs provided by the present invention show excellent optical properties, as well as specific mitochondrial and lysosomal targeting properties, which enable them to be used for living organelle imaging and dynamic tracking. It is also verified that these carbon dots also have the ability to achieve efficient biological imaging in organoid systems.

Description

Technical Field

[0001] The present invention relates to the fields of biology and nanomaterials, and in particular to fluorescent carbon quantum dots, a preparation method thereof, and applications thereof in organelle and organoid imaging and mitochondrial membrane potential monitoring. Background Art

[0002] Mitochondria play a vital role in energy production and metabolism regulation within cells. They generate adenosine triphosphate (ATP) through oxidative phosphorylation, providing essential energy for cells. They also regulate intracellular calcium homeostasis and participate in apoptosis. Abnormal mitochondrial function can have serious consequences for cells and the human body, leading to impaired intracellular energy supply, oxidative stress, apoptosis, and metabolic disorders, severely impacting cellular structure and function. Lysosomes are primarily involved in intracellular degradation and digestion, including the degradation of endocytosed and extracellular substances. Lysosomal abnormalities affect intracellular metabolism and immune responses, leading to lysosome-related diseases, such as lysosomal storage diseases. These diseases are often characterized by the accumulation of specific substances in lysosomes, which can lead to multi-organ dysfunction and pose a serious risk to human health. Therefore, long-term, precise monitoring of mitochondria and lysosomes is crucial. This not only helps researchers gain a deeper understanding of the normal physiological functions of these organelles and their relationship to disease development, but also provides a crucial basis for early diagnosis, treatment, and prognostic assessment of diseases.

[0003] Organoids are simple cell-based in vitro models of tissue engineering and are miniaturized and simplified in vitro organ model systems. Studying the functions of cells and organs and their changes in disease states is also a common use of organoids. In order to further enhance the depth and breadth of organoid research, it is necessary to expand the library of probe tools suitable for organoid observation. Among them, fluorescent probes, as an important imaging tool, play an irreplaceable role in cell and tissue imaging. The present invention focuses on innovative research and development based on carbon dot fluorescent probes, aiming to develop new fluorescent probes with excellent optical properties, good biocompatibility and high targeting. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to address the deficiencies in the above-mentioned prior art and provide a fluorescent carbon quantum dot, a preparation method thereof and its application in organelle and organoid imaging and mitochondrial membrane potential monitoring.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows: In a first aspect of the present invention, a method for preparing fluorescent carbon quantum dots is provided, comprising the following steps:

[0006] S1. Mix 1,5-diaminoanthraquinone, vanillic acid, and ethylenediamine in a solvent, perform a hydrothermal reaction under heating, and cool to room temperature after the reaction is completed;

[0007] S2, centrifuging the product obtained in step S1, taking the centrifuge liquid, adding silica gel powder, rotary evaporation, eluting with an eluent, adding the silica gel powder to the upper end of the stationary phase of the chromatography column, and then eluting with an eluent, collecting the eluate, and freeze-drying to obtain fluorescent carbon quantum dots;

[0008] The solvent is deionized water or ethanol, and the eluent is a mixture of dichloromethane and methanol.

[0009] Preferably, the method for preparing fluorescent carbon quantum dots comprises the following steps:

[0010] S1. Mix 0.05 g of 1,5-diaminoanthraquinone, 0.05 g of vanillic acid, and 1 mL of ethylenediamine in 30 mL of deionized water, and perform hydrothermal reaction at 200°C for 8 hours. After the reaction is completed, cool to room temperature.

[0011] S2. Centrifuge the product obtained in step S1, take the centrifuge liquid, add 1.5 g of 300-400 mesh silica gel powder, and perform rotary evaporation. Add the silica gel powder to the upper end of the stationary phase of the chromatography column, then elute with an eluent, collect the eluate that emits bright green fluorescence, and freeze-dry to obtain green fluorescent carbon dots G-CDs;

[0012] The eluent is a mixture of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 10:1.

[0013] Preferably, the method for preparing fluorescent carbon quantum dots comprises the following steps:

[0014] S1. Mix 0.05 g of 1,5-diaminoanthraquinone, 0.05 g of vanillic acid, and 1 mL of ethylenediamine in 30 mL of ethanol, and perform hydrothermal reaction at 200°C for 8 hours. After the reaction is completed, cool to room temperature.

[0015] S2. Centrifuge the product obtained in step S1, take the centrifuge liquid, add 1.5 g of 300-400 mesh silica gel powder, and perform rotary evaporation. Add the silica gel powder to the upper end of the stationary phase of the chromatography column, then elute with an eluent, collect the eluate that emits bright red fluorescence, and freeze-dry to obtain red fluorescent carbon dots R-CDs;

[0016] The eluent is a mixture of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 20:1.

[0017] In a second aspect of the present invention, there is provided green fluorescent carbon dots (G-CDs), which are prepared by the method described above.

[0018] In a third aspect of the present invention, there is provided a red fluorescent carbon dot R-CDs, which is prepared by the method described above.

[0019] In a fourth aspect, the present invention provides a use of the green fluorescent carbon dots G-CDs described above in organelle imaging, wherein the green fluorescent carbon dots are used as fluorescent dyes for targeted imaging of mitochondria in living cells.

[0020] In a fifth aspect, the present invention provides a use of the green fluorescent carbon dots G-CDs described above in organoid imaging, wherein the green fluorescent carbon dots are used as fluorescent dyes for targeted imaging of mitochondria in organoids.

[0021] In a sixth aspect, the present invention provides a use of the green fluorescent carbon dots G-CDs as described above as a probe in monitoring mitochondrial membrane potential.

[0022] In a seventh aspect, the present invention provides an application of the red fluorescent carbon dots R-CDs described above in organelle imaging, wherein the green fluorescent carbon dots are used as fluorescent dyes for targeted imaging of lysosomes in living cells.

[0023] In an eighth aspect, the present invention provides an application of the red fluorescent carbon dots R-CDs described above in organelle imaging, wherein the green fluorescent carbon dots are used as fluorescent dyes for targeted imaging of lysosomes in organoids.

[0024] The beneficial effects of the present invention are:

[0025] The present invention uses the same precursors to synthesize two types of carbon dots through a bottom-up approach: G-CDs emitting at 515 nm and R-CDs emitting at 611 nm, with different fluorescence properties. Both G-CDs and R-CDs exhibit excellent optical properties and specific mitochondrial and lysosomal targeting, enabling their use in live organelle imaging and dynamic tracking. The G-CDs and R-CDs probes developed in this invention demonstrate significant potential for key cellular events, such as monitoring mitochondrial and lysosomal dynamics. Furthermore, these carbon dots have been demonstrated to be capable of efficient bioimaging in organoid systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The morphology and particle size distribution results of G-CDs and R-CDs;

[0027] Figure 2 FTIR spectra of G-CDs and R-CDs;

[0028] Figure 3 XPS spectra of G-CDs and R-CDs;

[0029] Figure 4 are the excitation and emission spectra of G-CDs and R-CDs;

[0030] Figure 5 Cell images of HeLa cells stained with G-CDs and R-CDs and commercial dyes for mitochondria and lysosomes;

[0031] Figure 6 Comparison of the results of using G-CDs and TMRE to monitor changes in mitochondrial membrane potential;

[0032] Figure 7 The results of using G-CDs as a probe to monitor the mitophagy process;

[0033] Figure 8 The results of R-CDs monitoring lysosome movement during lysosomal autophagy;

[0034] Figure 9 Experimental results of fine imaging of organoids using G-CDs and R-CDs. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below with reference to the embodiments so that those skilled in the art can implement the invention with reference to the description.

[0036] It should be understood that terms such as “having”, “including” and “comprising” used herein do not preclude the existence or addition of one or more other elements or combinations thereof.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Materials and reagents used in the following examples are commercially available unless otherwise specified. In the following examples, where specific conditions are not specified, the experiments were conducted under conventional conditions or those recommended by the manufacturer. Reagents and instruments used, where the manufacturer is not specified, are commercially available conventional products.

[0038] Example 1

[0039] This embodiment provides a green fluorescent carbon dot G-CDs, the preparation method of which includes the following steps:

[0040] S1. Mix 0.05 g of 1,5-diaminoanthraquinone, 0.05 g of vanillic acid, and 1 mL of ethylenediamine in 30 mL of deionized water, and perform hydrothermal reaction at 200°C for 8 hours. After the reaction is completed, cool to room temperature.

[0041] S2. Centrifuge the product obtained in step S1, discard the unreacted raw materials and by-products precipitated at the bottom, take the centrifuge liquid, add 1.5 g of 300-400 mesh silica gel powder, and fully dry it by rotary evaporation to allow the reaction product to adhere to the silica gel powder. Add the silica gel powder to the upper end of the stationary phase of the chromatography column, then elute with an eluent, collect the eluate that emits bright green fluorescence, and freeze-dry to obtain green fluorescent carbon dots G-CDs;

[0042] The eluent is a mixture of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 10:1.

[0043] This embodiment provides a red fluorescent carbon dot R-CDs, the preparation method of which is similar to that of green fluorescent carbon dots G-CDs, with only the solvent and eluent being different, and specifically includes the following steps:

[0044] S1. Mix 0.05 g of 1,5-diaminoanthraquinone, 0.05 g of vanillic acid, and 1 mL of ethylenediamine in 30 mL of ethanol, and perform hydrothermal reaction at 200°C for 8 hours. After the reaction is completed, cool to room temperature.

[0045] S2. Centrifuge the product obtained in step S1, take the centrifuge liquid, add 1.5 g of 300-400 mesh silica gel powder, and fully dry it by rotary evaporation to allow the reaction product to adhere to the silica gel powder. Add the silica gel powder to the upper end of the stationary phase of the chromatography column, and then elute with an eluent, and collect the eluate that emits bright red fluorescence to obtain red fluorescent carbon dots R-CDs;

[0046] The eluent is a mixture of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 20:1.

[0047] In order to facilitate storage and subsequent experimental use, the prepared G-CDs and R-CDs were dissolved in water and ethanol solutions with a concentration of 10 mg / mL, respectively.

[0048] Performance characterization and testing

[0049] Reference Figure 1 , are the morphology and particle size distribution of G-CDs and R-CDs, Figure 1 In the figure, G-CDs ( Figure 1 a) and R-CDs( Figure 1 c) TEM image. Statistical analysis of the sizes of 50 of the particles revealed that the average diameters of G-CDs and R-CDs were approximately 2.48 nm and 3.90 nm, respectively. To further explore the structural characteristics of the particles, high-resolution transmission electron microscopy was also performed. The above characterization revealed that the interplanar spacings of the two carbon dots were 0.210 nm and 0.209 nm, respectively, consistent with the (100) crystal plane of graphite.

[0050] Reference Figure 2 , which are the FTIR spectra of G-CDs and R-CDs. In the FTIR spectrum of G-CDs, the stretching vibration of CH is present at 2928 and 2863 cm -1 The stretching vibration range of C=O is 1620.9cm -1 to 1512.2cm -1The absorption band caused by the stretching vibration of CN is located at 1307.0 cm -1 , and 817.5cm -1 The peak at 3312.6 cm-1 corresponds to the bending vibration of CH. In the R-CD spectrum, the stretching vibrations of NH and CH are at 3312.6, 2972, and 2880 cm-1, respectively. -1 The peak values ​​are at 1087.6 and 1045.7 cm -1 The peak at 879 cm indicates the stretching of anhydride CO. -1 The sharp peak at is corresponding to the bending vibration of CH.The surface groups of G-CDs and R-CDs were identified by IR spectroscopy.

[0051] Reference Figure 3 , are the XPS spectra of G-CDs and R-CDs. In the XPS full spectrum analysis, G-CDs ( Figure 3 a) and R-CDs( Figure 3 The samples in b) all exhibit three significant peaks at C1s (284.8 eV and 285.1 eV, respectively), N 1s (399.1 eV), and O1s (532.0 eV and 532.1 eV, respectively), which reveal the presence of three main elements, C, O, and N, on the surface of these carbon dots.

[0052] Reference Figure 4 , are the excitation and emission spectra of G-CDs and R-CDs. It can be seen from the figure that the green carbon dots ( Figure 4 a) exhibits a strong emission peak at 515 nm, with the maximum excitation wavelength located at 460 nm. In addition, the red carbon dots ( Figure 4 b) It exhibits an optimal emission wavelength at 611 nm and an optimal excitation wavelength at 560 nm.

[0053] G-CDs and R-CDs application performance test:

[0054] 1. G-CDs tracking mitochondrial dynamics research

[0055] To use G-CDs to monitor changes in mitochondrial membrane potential during apoptosis and mitochondrial dynamics during mitophagy, HeLa cells were cultured in glass-bottomed culture dishes. Cells were pretreated with CCCP (10 mM) for 2 hours to induce apoptosis, a drug that alters the normal mitochondrial membrane potential. Subsequently, G-CDs (100 μg / mL) were used for staining at 37°C for 60 minutes, and fluorescence images of the cells were recorded.

[0056] Cells were initially incubated with G-CDs at 37°C for 60 min and then treated with 5 μg / mL rapamycin to induce mitophagy. Fluorescence images were captured at different time points, including without rapamycin and 5, 10, and 20 min after rapamycin addition.

[0057] 2. Study on Lysosome Dynamics Tracking by R-CDs

[0058] In order to accurately record and analyze the dynamic changes of lysosomes during apoptosis and cell necrosis, and thus better understand the role of lysosomes in these complex biological processes. A series of detailed experimental steps were designed in the experiment. HeLa cells were co-cultured with R-CDs at a concentration of 100 μg / mL for 60 minutes. Next, in order to simulate the cell stress environment, the treated cells were stimulated with 5 μM chloroquine. Immediately afterwards, a series of cell image sequences were captured using a confocal microscope at specific time points after chloroquine stimulation - 0, 4, 8, 12 and 16 minutes.

[0059] 3. Organoid Imaging of G-CDs and R-CDs

[0060] Fresh tumor tissue was finely minced and washed at least three times with ice-cold PBS mixed with 100 U / mL penicillin / streptomycin, 0.25 μg / mL amphotericin B (from Solarbio), and 10 μg / mL gentamicin (from Solarbio). The cells were then digested in DMEM containing 10% FBS, 1.5 mg / mL collagenase II (from Solarbio), 500 U / mL collagenase IV (from Solarbio), 20 μg / mL hyaluronidase (from Sigma), and 10 μM Y27632 (from Biogems), and incubated at 37°C for 30 minutes. After digestion, the dissociated tissue was filtered through a cell strainer. The cell suspension was then centrifuged for 5 minutes. The cell pellet was resuspended in a solution containing BME (reduced growth factor provided by Biomaterials Engineering) and distributed into a 48-well cell culture plate, with 30 μL of BME added to each well. After incubation at 37°C for 20 minutes, pre-prepared culture medium was added to each well. After 5 days of growth in the culture medium, 100 μg / mL of carbon dot solution was added to each well for fluorescence imaging of the organoid samples.

[0061] Reference Figure 5, which are cell images of HeLa cells stained with commercial dyes for mitochondria and lysosomes using G-CDs and R-CDs. The distribution of G-CDs in cells showed a high degree of consistency with that of mitochondria stained with Mito Tracker Red, with a Pearson correlation coefficient of 0.82. The merged image of R-CDs and lysosomes stained with Lyso Tracker Green showed even more significant consistency, with a Pearson correlation coefficient of 0.87. The above experimental results not only demonstrate the potential of G-CDs and R-CDs to localize to specific organelles in the cytoplasm, but also demonstrate their effectiveness as cell imaging and organelle labeling tools through quantitative analysis.

[0062] Reference Figure 6 Figure 2 shows a comparison of G-CDs and TMRE for monitoring changes in mitochondrial membrane potential. After CCCP treatment, the green fluorescence intensity of G-CDs in cells decreased, and its distribution was no longer confined to the cytoplasm. Furthermore, after CCCP treatment, the red fluorescence intensity of TMRE-stained cells also decreased significantly. Therefore, G-CDs exhibit similar fluorescence intensity decay trends compared to commercial probes for monitoring mitochondrial membrane potential.

[0063] Reference Figure 7 , as a result of using G-CDs as a probe to monitor mitophagy. After 10 minutes of treatment, a decrease in fluorescence intensity was observed, accompanied by the fragmentation of mitochondrial filaments, indicating the activation of mitophagy. After 20 minutes of treatment, the distinct structural shift in mitochondria caused by rapamycin became particularly pronounced: the previously clear mitochondrial structures became scattered within the cell, and this structural change was accompanied by a further decrease in fluorescence intensity compared to earlier observations. This phenomenon is likely the result of a series of changes in the cellular microenvironment triggered by rapamycin.

[0064] Reference Figure 8 , which is the result of R-CDs monitoring lysosome movement during lysosomal autophagy. Figure 8 ae are microscopic images acquired at 0, 4, 8, 12, and 16 minutes, respectively. To more clearly demonstrate the movement of lysosomes, images at different time points were combined, and the direction of lysosome movement is indicated by arrows in the insets. These experimental results demonstrate that R-CDs can effectively monitor and reveal the dynamic changes of lysosomes in cells as a probe.

[0065] Reference Figure 9The following is the result of a detailed imaging experiment using G-CDs and R-CDs on simulated miniature organs, organoids. By co-staining G-CDs with Mito Tracker Red, the mitochondria of intestinal organoids were labeled and observed. The imaging results are shown in the figure. At the same time, co-staining R-CDs with Lyso Tracker Green was used to accurately locate and image lysosomes in colon cancer organoids. The results of confocal microscopy are shown in the figure. Figure 9 fj in. Figure 9 ac are images of intestinal organoids stained with Dapi, G-CDs and Mito-Tracker Red, respectively, and the merged images are: Figure 9 e It can be observed that G-CDs have good overlap with the commercial mitochondrial staining probe; Figure 9 fh are intestinal organoids stained with Dapi, R-CDs and Lyso-Tracker Green, respectively, and the merged images: Figure 9 j It can be observed that R-CDs can mark lysosomes in organoid samples; further, G-CDs, R-CDs and Dapi were incubated with intestinal organoids, and detailed spatial structural images were obtained using three-dimensional fluorescence imaging technology, which are respectively Figure 9 k,l,m,, Figure 9 n is the merged image.

[0066] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A method for preparing fluorescent carbon quantum dots, characterized in that: The following steps are involved: S1. Mix 1,5-diaminoanthraquinone, vanillic acid, and ethylenediamine in a solvent, perform a hydrothermal reaction under heating, and cool to room temperature after the reaction is completed; S2, centrifuging the product obtained in step S1, taking the centrifuge liquid, adding silica gel powder, rotary evaporation, eluting with an eluent, adding the silica gel powder to the upper end of the stationary phase of the chromatography column, and then eluting with an eluent, collecting the eluate, and freeze-drying to obtain fluorescent carbon quantum dots; The solvent is deionized water or ethanol, and the eluent is a mixture of dichloromethane and methanol.

2. The method for preparing fluorescent carbon quantum dots according to claim 1, wherein The following steps are involved: S1. Mix 0.05 g of 1,5-diaminoanthraquinone, 0.05 g of vanillic acid, and 1 mL of ethylenediamine in 30 mL of deionized water. Hydrothermally react at 200°C for 8 hours. After the reaction is completed, cool to room temperature. S2. Centrifuge the product obtained in step S1, take the centrifuge liquid, add 1.5 g of 300-400 mesh silica gel powder, and perform rotary evaporation. Add the silica gel powder to the upper end of the stationary phase of the chromatography column, then elute with an eluent, collect the eluate that emits bright green fluorescence, and freeze-dry to obtain green fluorescent carbon dots G-CDs; The eluent is a mixture of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 10:

1.

3. The method for preparing fluorescent carbon quantum dots according to claim 1, wherein The following steps are involved: S1. Mix 0.05 g of 1,5-diaminoanthraquinone, 0.05 g of vanillic acid, and 1 mL of ethylenediamine in 30 mL of ethanol, and perform hydrothermal reaction at 200°C for 8 hours. After the reaction is completed, cool to room temperature. S2. Centrifuge the product obtained in step S1, take the centrifuge liquid, add 1.5 g of 300-400 mesh silica gel powder, and perform rotary evaporation. Add the silica gel powder to the upper end of the stationary phase of the chromatography column, then elute with an eluent, collect the eluate that emits bright red fluorescence, and freeze-dry to obtain red fluorescent carbon dots R-CDs; The eluent is a mixture of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 20:

1.

4. A green fluorescent carbon dot G-CDs, characterized in that It is prepared by the method according to claim 2.

5. A red fluorescent carbon dot R-CDs, characterized in that: It is prepared by the method according to claim 3.

6. A use of the green fluorescent carbon dots G-CDs according to claim 4 in organelle imaging, characterized in that: The green fluorescent carbon dots are used as fluorescent dyes for targeted imaging of mitochondria in living cells for non-disease diagnosis and treatment.

7. A use of the green fluorescent carbon dots G-CDs according to claim 4 in organoid imaging, characterized in that: The green fluorescent carbon dots are used as fluorescent dyes for targeted imaging of mitochondria in organoids for non-disease diagnosis and treatment.

8. Use of the green fluorescent carbon dots G-CDs as claimed in claim 4 as a probe for monitoring mitochondrial membrane potential for non-disease diagnosis and treatment.

9. A use of the red fluorescent carbon dots R-CDs according to claim 5 in organelle imaging, characterized in that: The red fluorescent carbon dots are used as fluorescent dyes for targeted imaging of lysosomes in living cells for non-disease diagnosis and treatment.

10. A use of the red fluorescent carbon dots R-CDs according to claim 5 in organelle imaging, characterized in that: The red fluorescent carbon dots are used as fluorescent dyes for targeted imaging of lysosomes in organoids for non-disease diagnosis and treatment.

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