A kind of nucleolus-targeted red fluorescent carbon dots and its preparation method and application

Abstract

The present invention provides a nucleolus-targeted red fluorescent carbon dot and its preparation method and application, belonging to the technical field of nucleolus-targeted red fluorescent carbon dots; the technical problem to be solved is: to provide a nucleolus-targeted red fluorescent carbon dot and its preparation and application methods; the technical solution adopted to solve this technical problem is: the carbon dots are prepared by using p-phenylenediamine and L-tryptophan as precursors, and GdCl3·6H2O and FeCl3·6H2O as gadolinium source and iron source respectively, and Gd-doped carbon dots p-CDs-Gd and Fe-doped carbon dots p-CDs-Fe are respectively prepared by a one-step hydrothermal method, and then the obtained dark red liquid is freeze-dried to obtain the solid powders of p-CDs-Gd and p-CDs-Fe; the present invention is applied to the preparation and application of nucleolus-targeted red fluorescent carbon dots.

Description

Technical Field

[0001] The present invention provides a nucleolus-targeted red fluorescent carbon dot and its preparation method and application, belonging to the technical field of nucleolus-targeted red fluorescent carbon dots. Background Art

[0002] The nucleolus is the most obvious structure in the interphase nucleus of eukaryotic cells. Its functions are closely related to cell growth, proliferation, cycle regulation, aging and stress response. As a diagnostic biomarker and a potential target for treating various human diseases, through the visualization study of the nucleolus, the effect during cancer treatment can be evaluated. And existing studies have shown that directly delivering chemotherapeutic drugs into the cell nucleus can kill cancer cells more effectively than delivering them into the cytoplasm or other organelles. Nucleolus-targeted carriers help deliver drugs into the cell nucleus and improve the treatment effect. It can be seen that nucleolus-targeted imaging is of great significance for the early disease diagnosis and treatment in biomedicine.

[0003] Carbon dots (CDs) are a class of emerging carbon nanomaterials with a size <10 nm. Due to their extremely low cytotoxicity, excellent water solubility and unique photoluminescence properties, they have good application prospects. In addition, CDs have the advantages of organic molecules (small size and easy to modify) and carbon materials (good biocompatibility and excellent stability). These combined properties can also make CDs ideal candidates for FL bioimaging agents.

[0004] Currently, most of the nuclear-targeted CDs for anti-tumor treatment focus on the short-wavelength (blue and green) emission region, which cannot avoid the interference of autofluorescence in organisms and may cause damage to tissues and cells. Although long-wavelength emission CDs have advantages such as large tissue penetration depth, low light scattering / absorption level, and negligible interference from autofluorescence in biological tissue background, however, there is a blank in the current research on applying long-wavelength emission CDs to target the cell nucleus in anti-tumor treatment, which will all affect the effectiveness of nuclear-targeted CDs in cell imaging and drug delivery. Therefore, developing long-wavelength emission nucleolus-targeted carbon dots is of great significance for the fluorescence tracer of drugs and the feedback of treatment effect for the integrated diagnosis and treatment. Summary of the Invention

[0005] In order to overcome the deficiencies existing in the prior art, the technical problem to be solved by the present invention is to provide a nucleolus-targeted red fluorescent carbon dot and its preparation and application methods.

[0006] To solve the above technical problems, the technical solution adopted by the present invention is as follows: A kind of nucleolus-targeted red fluorescent carbon dots, the carbon dots are prepared by using p-phenylenediamine and L-tryptophan as precursors, and GdCl3⋅6H2O and FeCl3⋅6H2O as gadolinium source and iron source, respectively preparing Gd-doped carbon dots p-CDs-Gd and Fe-doped carbon dots p-CDs-Fe by a one-step hydrothermal method, and then freeze-drying the obtained dark red liquid to obtain the solid powders of p-CDs-Gd and p-CDs-Fe.

[0007] A preparation method of nucleolus-targeted red fluorescent carbon dots, comprising the following preparation steps:

[0008] Weigh 2-8 mg of p-phenylenediamine p-PD and 2-8 mg of L-tryptophan, completely dissolve them in 20-80 mL of deionized water, add 10-40 μL of GdCl3⋅6H2O and FeCl3⋅6H2O with a concentration of 50-200 nM respectively, transfer the mixture to a polytetrafluoroethylene-lined autoclave, and react at 80 °C for 8-12 h;

[0009] After the autoclave is naturally cooled, centrifuge the completely reacted solution at 5000-10000 rpm for 5-10 min, and dry the obtained solution in a freeze dryer to form a solid powder, which is p-CDs-Gd and p-CDs-Fe.

[0010] An application of nucleolus-targeted red fluorescent carbon dots in cytotoxicity testing, including a method for performing cytotoxicity testing on SMMC-7721 cells using p-CDs-Gd or p-CDs-Fe, and the specific steps are as follows:

[0011] Grow SMMC-7721 cells in DMEM culture medium containing 10-20% fetal bovine serum and 1-5% penicillin-streptomycin, and culture them in an environment of 30-40 °C and 1-5% CO2;

[0012] Seed the cells in the logarithmic growth phase into a 96-well plate at a density of 1×10 3 to 3×10 3 , and culture them in an incubator for 24-48 h;

[0013] After the cells are completely adhered, divide the cells into a control group and an experimental group, with 6 replicate wells in each group, and add p-CDs-Gd or p-CDs-Fe with concentrations of 0, 25, 50, 100, 200, and 400 μg / mL respectively, and continuously treat for 24 h and 48 h;

[0014] Then add 10-20 μL of freshly prepared MTT solution with a concentration of 1-5 mg / mL to the cells and incubate for 4-10 hours;

[0015] After removing the original culture medium, add 100 - 200 μL of DMSO solution to each well. Shake the well plate and let it stand for 10 - 15 min until the DMSO completely dissolves the blue-violet crystals;

[0016] Place the 96-well plate into the microplate reader, measure the absorbance OD value of each well at 450 nm, and record it;

[0017] Finally, calculate the cell survival rate. The calculation formula is:

[0018] Cell survival rate (%) = (Average OD value of the experimental group / Average OD value of the control group) × 100%.

[0019] An application of a nucleolus-targeted red fluorescent carbon dot in cell imaging, including a method for imaging in SMMC-7721 cells using p-CDs-Gd or p-CDs-Fe. The specific steps are as follows:

[0020] Seed SMMC-7721 cells in the logarithmic growth phase at a density of 1×10 4 to 3×10 4 per well in a 24-well plate and incubate for 24 - 48 h;

[0021] After the cells adhere to the wall, replace the culture medium with 1 - 5 mL of culture medium containing 100 - 200 μL of p-CDs-Gd solution with a concentration of 1 - 5 mg / mL, or replace the culture medium with 1 - 5 mL of culture medium containing 100 - 250 μL of p-CDs-Fe solution with a concentration of 1 - 5 mg / mL. After co-incubating with the cells for 6 - 12 h, take out the 24-well plate, wash the cells in each well 3 times with PBS, and then add 100 - 500 µL of 1 - 4% paraformaldehyde solution to each well to fix for 5 - 10 min;

[0022] After fixation, wash 3 times with PBS again, and perform fluorescence imaging on the cells using a Cytation5 imaging reader.

[0023] An application of a nucleolus-targeted red fluorescent carbon dot in the detection of nuclear targeting mechanism, including a method for detecting the nuclear targeting mechanism in cells using p-CDs-Gd or p-CDs-Fe. The specific steps are as follows:

[0024] Seed SMMC-7721 cells in the logarithmic growth phase at a density of 1×10 4 to 3×10 4 per well in a 24-well plate and incubate for 24 - 48 h;

[0025] After the cells adhere completely, fix the cells with methanol at 1 - 4 °C for 1 - 10 min, and then permeabilize the cell membrane with 1 - 5% TritonX-100 for 2 - 10 min;

[0026] After that, the cells were divided into three groups, and PBS solution for control experiment, DNase solution with a concentration of 10 - 25 µg / mL, and RNase solution with a concentration of 10 - 25 µg / mL were added respectively, and incubation continued for 2 - 10 h;

[0027] After that, 1 - 5 mL of culture medium containing 100 - 200 μL of p-CDs-Gd solution with a concentration of 1 - 5 mg / mL was added to each well for incubation for 3 - 12 h, or 1 - 5 mL of culture medium containing 100 - 250 μL of p-CDs-Fe solution with a concentration of 1 - 5 mg / mL was added to each well for incubation for 3 - 12 h;

[0028] After staining, fluorescence imaging was performed using a Cytation5 imaging reader.

[0029] The beneficial effects of the present invention compared with the prior art are as follows: In order to solve the problems that most of the currently used nuclear-targeted CDs for anti-tumor treatment are concentrated in the short-wavelength (blue and green) emission regions, which cannot avoid the interference of biological autofluorescence and may cause damage to tissues and cells, etc., the present invention provides a preparation method of red fluorescent carbon quantum dots and their application in nucleolus imaging; The m-phenylenediamine and L-tryptophan used for preparing the carbon dot precursors are relatively common, cheap and easily available, and the one-step hydrothermal method used for preparation is very simple and easy to operate. The carbon dots prepared based on this method have a long emission wavelength, can enhance tissue penetration ability, avoid the interference of biological autofluorescence and the damage to tissues and cells, and can be used as good nucleolus-targeted probes. Brief Description of the Drawings

[0030] The following further describes the present invention with reference to the drawings:

[0031] Figure 1 It is the ultraviolet-visible absorption spectrum of p-CDs-Gd prepared in Example 1 of the present invention;

[0032] Figure 2 It is the infrared spectrum of p-CDs-Gd prepared in Example 1 of the present invention;

[0033] Figure 3 It is the excitation wavelength-dependent spectrum of p-CDs-Gd prepared in Example 1 of the present invention;

[0034] Figure 4 It is the X-ray photoelectron spectrum of p-CDs-Gd prepared in Example 1 of the present invention;

[0035] Figure 5 It is the EDS spectrum of p-CDs-Gd prepared in Example 1 of the present invention;

[0036] Figure 6 TEM image of p-CDs-Gd prepared in Example 1 of the present invention;

[0037] Figure 7 Schematic diagram of the cytotoxicity test results of p-CDs-Gd on SMMC-7721 cells in Example 4 of the present invention;

[0038] Figure 8 Schematic diagram of the imaging results of p-CDs-Gd in SMMC-7721 cells in Example 6 of the present invention;

[0039] Figure 9 Schematic diagram of the imaging results of p-CDs-Gd in SMMC-7721 cells (with and without DNase and RNase treatment) in Example 8 of the present invention;

[0040] Figure 10 UV-Vis absorption spectrum of p-CDs-Fe prepared in Example 2 of the present invention;

[0041] Figure 11 Infrared spectrum of p-CDs-Fe prepared in Example 2 of the present invention;

[0042] Figure 12 Excitation wavelength-dependent spectrum of p-CDs-Fe prepared in Example 2 of the present invention;

[0043] Figure 13 X-ray photoelectron spectrum of p-CDs-Fe prepared in Example 2 of the present invention;

[0044] Figure 14 EDS spectrum of p-CDs-Fe prepared in Example 2 of the present invention;

[0045] Figure 15 TEM image of p-CDs-Fe prepared in Example 2 of the present invention;

[0046] Figure 16 Schematic diagram of the cytotoxicity test results of p-CDs-Fe on SMMC-7721 cells in Example 5 of the present invention;

[0047] Figure 17 Schematic diagram of the imaging results of p-CDs-Fe in SMMC-7721 cells in Example 7 of the present invention;

[0048] Figure 18 Schematic diagram of the imaging results of p-CDs-Fe in SMMC-7721 cells (with and without DNase and RNase treatment) in Example 9 of the present invention. Detailed implementation manners

[0049] A nucleolus-targeted red fluorescent carbon dot provided by the present invention, its preparation method and application, specifically in the field of fluorescence imaging, providing a preparation method of red nucleolus-targeted carbon dots and their application in cell imaging; the carbon dots use p-phenylenediamine and L-tryptophan as precursors, and GdCl3·6H2O and FeCl3·6H2O as gadolinium source and iron source, and Gd-doped carbon dots (p-CDs-Gd) and Fe-doped carbon dots (p-CDs-Fe) are respectively prepared by a one-step hydrothermal method; the imaging diagrams of the carbon dots in SMMC-7721 cells in the present invention show that both the carbon dots p-CDs-Gd and p-CDs-Fe can be used to image the targeted nucleolus, and the preparation method of the carbon dots is simple, can target the nucleolus without modification, and the long-wavelength emission can also avoid the interference of biological autofluorescence and damage to tissues and cells.

[0050] The present invention is realized by the following technical solutions:

[0051] The nucleolus-targeted red fluorescent carbon dots provided by the present invention, the carbon quantum dots are specifically: using p-phenylenediamine and L-tryptophan as precursors, and GdCl3·6H2O and FeCl3·6H2O as gadolinium source and iron source, Gd-doped carbon dots (p-CDs-Gd) and Fe-doped carbon dots (p-CDs-Fe) are respectively prepared by a one-step hydrothermal method, and then the obtained dark red liquid is freeze-dried to obtain p-CDs-Gd and p-CDs-Fe solid powders, which are the nucleolus-targeted carbon quantum dots.

[0052] The specific preparation method of the carbon dots is as follows:

[0053] (1) Accurately weigh 4 mg of p-phenylenediamine p-PD and 4 mg of L-tryptophan, completely dissolve them in 40 mL of deionized water, respectively add 20 μL of GdCl3·6H2O and FeCl3·6H2O with a concentration of 100 nM, transfer the mixture to a polytetrafluoroethylene-lined autoclave, and react at 80 °C for 12 hours;

[0054] (2) After the reaction kettle cools naturally, centrifuge the completely reacted solution (10000 rpm, 10 min). Dry the obtained solution into a solid powder in a freeze dryer, which is p-CDs-Gd and p-CDs-Fe.

[0055] The following further describes the embodiments of the present invention in detail with reference to the accompanying drawings:

[0056] Example 1: The preparation method of the carbon dots p-CDs-Gd is as follows:

[0057] Weigh 4 mg of p-phenylenediamine (p-PD) and 4 mg of L-tryptophan, completely dissolve them in 40 mL of deionized water, then add 20 μL of GdCl3·6H2O with a concentration of 100 nM. Transfer the mixture to a polytetrafluoroethylene-lined autoclave and react at 80 °C for 12 hours. After the autoclave cools naturally, centrifuge the completely reacted solution (10000 rpm, 10 min). Dry the obtained solution in a freeze dryer to form a solid powder for subsequent use.

[0058] Example 2: The preparation method of carbon dots p-CDs-Fe is as follows:

[0059] Weigh 4 mg of p-phenylenediamine (p-PD) and 4 mg of L-tryptophan, completely dissolve them in 40 mL of deionized water, then add 20 μL of FeCl3·6H2O with a concentration of 100 nM. Transfer the mixture to a polytetrafluoroethylene-lined autoclave and react at 80 °C for 12 hours. After the autoclave cools naturally, centrifuge the completely reacted solution (10000 rpm, 10 min). Dry the obtained solution in a freeze dryer to form a solid powder for subsequent use.

[0060] Example 3: For the characterization of p-CDs-Gd in Example 1, its property characterization is as Figures 1-6 shown, specifically:

[0061] Figure 1 As shown in the UV absorption spectrum of p-CDs-Gd, two different absorption peaks are shown at about 270 nm and 500 nm, which come from the π-π* energy level transition of the C=C bond and the n-π* transition of the C-N or C-O structure respectively;

[0062] Figure 2 This is the infrared spectrum of p-CDs-Gd, indicating that p-CDs-Gd is rich in functional groups such as O-H / N-H, C-H, C=O, C=C, and C-O;

[0063] Figure 3 This is the emission spectrum of p-CDs-Gd at different excitation wavelengths, showing that p-CDs exhibits a maximum emission wavelength at 630 nm, and under the excitation wavelength of 480 nm, the maximum fluorescence emission is generated;

[0064] Figure 4 This is the X-ray photoelectron spectroscopy of p-CDs-Gd, indicating that p-CDs-Gd is mainly composed of three elements: C, N, and O;

[0065] Figure 5 This is the EDS spectrum of p-CDs-Gd, indicating the presence of C, N, O, and Gd elements in the structure of p-CDs-Gd;

[0066] Figure 6 It is the transmission electron microscopy image of p-CDs-Gd, indicating that the particle size of p-CDs-Gd is approximately 8.3 nm.

[0067] Example 3: Regarding the characterization of p-CDs-Fe in Example 2, its property characterization is as Figures 10-15 shown, specifically:

[0068] Figure 10 As shown in the ultraviolet absorption spectrum of p-CDs-Fe in, two different absorption peaks are shown at approximately 270 nm and 500 nm, originating from the π-π* energy level transition of the C=C bond and the n-π* transition of the C-N or C-O structure, respectively;

[0069] Figure 11 It is the infrared spectrum of p-CDs-Fe, indicating that p-CDs-Fe is rich in functional groups such as O-H / N-H, C=O, C=C, and C-O;

[0070] Figure 12 It is the emission spectrum of p-CDs-Fe at different excitation wavelengths, showing that p-CDs-Fe exhibits a maximum emission wavelength at 630 nm, and under the excitation wavelength of 480 nm, the maximum fluorescence emission is generated;

[0071] Figure 13 It is the X-ray photoelectron spectroscopy of p-CDs-Fe, indicating that p-CDs-Fe is mainly composed of three elements: C, N, and O;

[0072] Figure 14 It is the EDS spectrum of p-CDs-Fe, indicating the presence of C, N, O, and Fe elements in the Gd-CDs structure;

[0073] Figure 15 It is the transmission electron microscopy image of p-CDs-Fe, indicating that the particle size of p-CDs-Fe is approximately 2.7 nm.

[0074] Example 4: The toxicity test of p-CDs-Gd on SMMC-7721 cells was carried out. The specific method is as follows:

[0075] SMMC-7721 cells were grown in DMEM culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and cultured in an environment of 37°C and 5% CO2. The cells in the logarithmic growth phase were seeded at 3×10 per well 3The cells were inoculated into 96-well plates at a density of 100 μg / mL and cultured in an incubator for 24 h. After the cells were completely attached to the wall, they were divided into a control group and an experimental group, with 6 replicate wells in each group. Different concentrations of p-CDs-Gd (0, 25, 50, 100, 200, 400 μg / mL) were added for continuous treatment for 24 h and 48 h. Then, freshly prepared MTT solution (10 μL, 5 mg / mL) was added to the cells and incubated for 4 h. After the original culture medium was drawn out, 100 μL of DMSO solution was added to each well, and the well plate was gently shaken and placed for 10-15 min until DMSO completely dissolved the blue-purple crystals. The 96-well plate was placed in an ELISA reader, and the absorbance value (OD) of each well at 450 nm was tested and recorded. The cell survival rate was calculated using the formula: Cell survival rate (%) = (average OD value of the experimental group / average OD value of the control group) × 100%.

[0076] like Figure 7 The figure shows the toxicity test results of p-CDs-Gd on SMMC-7721 cells, which shows that when the concentration of p-CDs-Gd is 400 µg / mL, the survival rate of SMMC-7721 cells is still higher than 85%, proving that p-CDs-Gd has very low toxicity to SMMC-7721 cells and can be ignored.

[0077] Example 5: Toxicity test of SMMC-7721 cells using p-CDs-Fe, the specific method is as follows:

[0078] SMMC-7721 cells were grown in DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2. Cells in the logarithmic growth phase were plated at 3×10 3 The cells were inoculated into 96-well plates at a density of 100 μL and cultured in an incubator for 24 h. After the cells were completely attached to the wall, they were divided into a control group and an experimental group, with 6 replicate wells in each group. Different concentrations of p-CDs-Fe (0, 25, 50, 100, 200, 400 μg / mL) were added to the cells for continuous treatment for 24 h and 48 h. Then, freshly prepared MTT solution (10 μL, 5 mg / mL) was added to the cells and incubated for 4 h. After the original culture medium was drawn out, 100 μL of DMSO solution was added to each well, and the well plate was gently shaken and placed for 10-15 min until DMSO completely dissolved the blue-purple crystals. The 96-well plate was placed in a microplate reader, and the absorbance value (OD) of each well at 450 nm was tested and recorded. The cell survival rate was calculated using the formula: Cell survival rate (%) = (average OD value of the experimental group / average OD value of the control group) × 100%.

[0079] like Figure 16Figure showing the cytotoxicity test results of p-CDs-Fe against SMMC-7721 cells, indicating that when the concentration of p-CDs-Fe is 400 µg / mL, the survival rate of SMMC-7721 cells is still about 85%, proving that the toxicity of p-CDs-Fe to SMMC-7721 cells is very low and negligible.

[0080] Example 6: Imaging was performed in SMMC-7721 cells using p-CDs-Gd. The specific method was as follows:

[0081] SMMC-7721 cells in the logarithmic growth phase were seeded in 24-well plates at a density of 1×10 4 and incubated for 24 h. After the cells adhered, the culture medium was replaced with 1 mL of culture medium containing p-CDs-Gd solution (200 μL, 1 mg / mL). After co-incubating with the cells for 6 h, the 24-well plates were taken out. The cells in each well were washed 3 times with PBS, and then 500 µL of 4% paraformaldehyde solution was added to each well to fix for 10 min. After fixation, the cells were washed 3 times with PBS again, and fluorescence imaging of the cells was performed using a Cytation5 imaging reader.

[0082] As Figure 8 Figure showing the imaging of p-CDs-Gd in SMMC-7721 cells. It can be seen that in SMMC-7721, the nucleoli showed bright red fluorescence, and the state of the nucleoli could be clearly seen, indicating that p-CDs-Gd is a good nucleolar imaging agent.

[0083] Example 7: Imaging was performed in SMMC-7721 cells using p-CDs-Fe. The specific method was as follows:

[0084] SMMC-7721 cells in the logarithmic growth phase were seeded in 24-well plates at a density of 1×10 4 and incubated for 24 h. After the cells adhered, the culture medium was replaced with 1 mL of culture medium containing p-CDs-Fe solution (250 μL, 1 mg / mL). After co-incubating with the cells for 6 h, the 24-well plates were taken out. The cells in each well were washed 3 times with PBS, and then 500 µL of 4% paraformaldehyde solution was added to each well to fix for 10 min. After fixation, the cells were washed 3 times with PBS again, and fluorescence imaging of the cells was performed using a Cytation5 imaging reader.

[0085] As Figure 17 Figure showing the imaging of p-CDs-Fe in SMMC-7721 cells. It can be seen that in SMMC-7721, the nucleoli showed bright red fluorescence, and the state of the nucleoli could be clearly seen, indicating that p-CDs-Fe is a good nucleolar imaging agent.

[0086] Example 8: Study on the nuclear targeting mechanism of p-CDs-Gd in cells:

[0087] SMMC-7721 cells in the logarithmic growth phase were seeded in 24-well plates at a density of 1×10 4 and incubated for 24 h. After the cells were completely adherent, the cells were fixed with methanol at 4 °C for 1 min, and then the cell membrane was permeabilized with 1% Triton X-100 for 2 min. Then the cells were divided into three groups and incubated with PBS solution (control experiment), DNase (25 μg / mL), and RNase (25 μg / mL) for another 2 h. Then 1 mL of culture medium containing p-CDs-Gd solution (200 μL, 1 mg / mL) was added to each well and incubated for 3 h. After staining, fluorescence imaging was performed using a Cytation5 imaging reader.

[0088] As Figure 9 shown in the imaging results of SMMC-7721 cells, it can be seen that bright red fluorescence can be emitted from the nucleoli of the control group and the experimental group treated with RNase, while almost no fluorescence can be observed in the nucleoli of the experimental group treated with DNase. This indicates that p-CDs-Gd binds to DNA and emits fluorescence. After treatment with DNase, the binding ability of the nucleoli to p-CDs-Gd is weakened or even lost, so the nucleoli do not emit fluorescence.

[0089] Example 9: Study on the nuclear targeting mechanism of p-CDs-Fe in cells:

[0090] SMMC-7721 cells in the logarithmic growth phase were seeded in 24-well plates at a density of 1×10 4 and incubated for 24 h. After the cells were completely adherent, the cells were fixed with methanol at 4 °C for 1 min, and then the cell membrane was permeabilized with 1% Triton X-100 for 2 min. Then the cells were divided into three groups and incubated with PBS solution (control experiment), DNase (25 μg / mL), and RNase (25 μg / mL) for another 2 h. Then 1 mL of culture medium containing p-CDs-Fe solution (250 μL, 1 mg / mL) was added to each well and incubated for 3 h. After staining, fluorescence imaging was performed using a Cytation5 imaging reader.

[0091] As Figure 18It is the imaging result diagram of SMMC-7721 cells. It can be seen that the nucleoli of the control group and the experimental group treated with RNase can emit bright red fluorescence, while the nucleoli of the experimental group treated with DNase hardly emit fluorescence. This indicates that p-CDs-Fe emits fluorescence by binding to DNA. After being treated with DNase, the binding ability of the nucleoli to p-CDs-Fe weakens or even disappears, so the nucleoli do not emit fluorescence.

[0092] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A kind of nucleolus-targeted red fluorescent carbon dots, characterized in that: Carbon dots were prepared by using p-phenylenediamine and L-tryptophan as precursors, and GdCl3⋅6H2O and FeCl3⋅6H2O as gadolinium source and iron source respectively. Through a one-step hydrothermal method, Gd-doped carbon dots p-CDs-Gd and Fe-doped carbon dots p-CDs-Fe were prepared, and then the obtained dark red liquid was freeze-dried to obtain the solid powders of p-CDs-Gd and p-CDs-Fe.

2. The preparation method of the nucleolus-targeted red fluorescent carbon dots according to claim 1, wherein: It includes the following preparation steps: (1) Weigh 2-8 mg of p-phenylenediamine p-PD and 2-8 mg of L-tryptophan, completely dissolve them in 20-80 mL of deionized water, add 10-40 μL of GdCl3⋅6H2O and FeCl3⋅6H2O with a concentration of 50-200 nM respectively, transfer the mixture to a polytetrafluoroethylene-lined autoclave, and react at 80 °C for 8-12 h; (2) After the reaction kettle cools naturally, centrifuge the completely reacted solution at 5000-10000 rpm for 5-10 min, and dry the obtained solution in a freeze dryer to form a solid powder, which is p-CDs-Gd and p-CDs-Fe.

3. Use of the nucleolus-targeting red fluorescent carbon dots according to claim 1 in cytotoxicity testing, characterized in that: It includes a method for performing toxicity tests on SMMC-7721 cells using p-CDs-Gd or p-CDs-Fe. The specific steps are as follows: Grow SMMC-7721 cells in DMEM culture medium containing 10-20% fetal bovine serum and 1-5% penicillin-streptomycin, and culture them in an environment of 30-40 °C and 1-5% CO2; Cells in the logarithmic growth phase were seeded into a 96-well plate at a density of 1×10 3 to 3×10 3 , and cultured in an incubator for 24 - 48 h; After the cells are completely adherent, divide the cells into a control group and an experimental group, with 6 replicate wells in each group, and add p-CDs-Gd or p-CDs-Fe with concentrations of 0, 25, 50, 100, 200, and 400 μg / mL respectively and continuously treat for 24 h and 48 h; Then add 10-20 μL of freshly prepared MTT solution with a concentration of 1-5 mg / mL to the cells and incubate for 4-10 hours; After extracting the original culture medium, add 100-200 μL of DMSO solution to each well, shake the well plate and place it for 10-15 min until the DMSO completely dissolves the blue-violet crystals; Put the 96-well plate into an enzyme-linked immunosorbent assay (ELISA) reader, measure the absorbance value OD of each well at 450 nm, and record it; Finally, calculate the cell survival rate. The calculation formula is: Cell survival rate (%) = (average OD value of the experimental group / average OD value of the control group) × 100%.

4. Use of the nucleolus-targeted red fluorescent carbon dots according to claim 1 in cell imaging, characterized in that: It includes a method for imaging using p-CDs-Gd or p-CDs-Fe in SMMC-7721 cells. The specific steps are as follows: Seed SMMC-7721 cells in the logarithmic growth phase at a density of 1×10 4 to 3×10 4 per well in a 24-well plate and incubate for 24 - 48 h; After the cells are adherent, replace the culture medium with 1-5 mL of culture medium containing 100-200 μL of p-CDs-Gd solution with a concentration of 1-5 mg / mL, or replace the culture medium with 1-5 mL of culture medium containing 100-250 μL of p-CDs-Fe solution with a concentration of 1-5 mg / mL. After co-incubating with the cells for 6-12 h, take out the 24-well plate, wash the cells in each well 3 times with PBS, and then add 100-500 µL of 1-4% paraformaldehyde solution to each well and fix for 5-10 min; After fixation, wash the cells three times with PBS, and perform fluorescence imaging of the cells using a Cytation5 imaging reader.

5. Use of the nucleolus-targeted red fluorescent carbon dots according to claim 1 in the detection of nuclear targeting mechanism, characterized in that: It includes a method for detecting the nuclear targeting mechanism of p-CDs-Gd or p-CDs-Fe in cells. The specific steps are as follows: Seed SMMC-7721 cells in logarithmic growth phase at a density of 1×10 4 to 3×10 4 per well in a 24-well plate and incubate for 24 - 48 h; After the cells are completely adherent, fix the cells with methanol at 1-4 °C for 1-10 min, and then permeabilize the cell membrane with 1-5% TritonX-100 for 2-10 min; Then divide the cells into three groups, and continue to incubate them with PBS solution for control experiment, DNase solution with a concentration of 10-25 μg / mL, and RNase solution with a concentration of 10-25 μg / mL for 2-10 h; Then add 1-5 mL of culture medium containing 100-200 μL of p-CDs-Gd solution with a concentration of 1-5 mg / mL to each well and incubate for 3-12 h, or add 1-5 mL of culture medium containing 100-250 μL of p-CDs-Fe solution with a concentration of 1-5 mg / mL to each well and incubate for 3-12 h; After staining, perform fluorescence imaging using a Cytation5 imaging reader.

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