Synthesis method of red luminescent carbon dots with potential of targeting diagnosis and treatment of hepatocellular carcinoma

Abstract

The application relates to a synthesis method of red luminescent carbon dots with a targeting diagnosis and treatment potential of hepatocellular carcinoma, which comprises the following steps: 1) adding 5-fluorouracil and indocyanine green into deionized water, uniformly mixing, and forming a uniform mixed solution; 2) heating the mixed solution obtained in the step 1) at 160-200 DEG C for 5-9 h, and cooling to room temperature; 3) filtering and dialyzing the solution obtained in the step 2), and obtaining a pure 5-FICD solution; and 4) freeze-drying the solution obtained in the step 3), and obtaining the product. The method takes anticancer drug 5-fluorouracil and photosensitizer indocyanine green as precursors, synthesizes a low-toxicity red light carbon dot through a simple hydrothermal method, improves the shortcoming that a chemotherapy drug has a large side effect, and realizes the targeted killing of hepatocellular carcinoma cells.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of carbon dot synthesis, and particularly relates to a synthesis method of red luminescent carbon dots with potential for targeted diagnosis and treatment of hepatocellular carcinoma. BACKGROUND

[0002] Chemotherapy is one of the most effective means of cancer treatment, and most chemotherapy drugs affect normal cells while acting on tumors, so how to enhance the effect of chemotherapy drugs and reduce their side effects is one of the main challenges of cancer treatment. Carbon dots are a kind of fluorescent nanomaterials, which are widely used in the biomedical field due to their simple preparation process, good biocompatibility, excellent photoluminescence performance and low toxicity.

[0003] Carbon dots can retain part of the active functional groups of the precursor during carbonization, thereby retaining the characteristics of the precursor. 5-fluorouracil is a commonly used basic chemotherapy drug in cancer treatment, which inhibits the synthesis of DNA by inhibiting thymidylate synthetase, but it is difficult to precisely target cancer cells, and it has a great impact on normal cells when killing cancer cells. Indocyanine green is a near-infrared cyanine fluorescent agent widely used in the biomedical field. It is selectively absorbed by liver cells after entering the human body, and is excreted by metabolic action in normal liver cells, but it is slowly metabolized by hepatocellular carcinoma cells, which causes retention. In addition, indocyanine green is a widely used phototherapeutic agent that produces reactive oxygen species and causes local heating under laser irradiation, and has excellent phototherapeutic effects. Therefore, carbon dots synthesized from 5-fluorouracil and indocyanine green as precursors are expected to have both targeted diagnostic and apoptosis-inducing abilities for hepatocellular carcinoma cells. Based on this, the present application is developed. SUMMARY

[0004] The present application aims to overcome the defects of the prior art and provide red luminescent carbon dots with potential for targeted diagnosis and treatment of hepatocellular carcinoma, which are synthesized from anticancer drug 5-fluorouracil and photosensitizer indocyanine green as precursors, and a low-toxicity red light carbon dot is obtained by a simple hydrothermal method, which improves the disadvantage of large side effects of chemotherapy drugs and realizes targeted killing of hepatocellular carcinoma cells.

[0005] The present application also provides a synthesis method and application of the above-mentioned red luminescent carbon dots with potential for targeted diagnosis and treatment of hepatocellular carcinoma.

[0006] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0007] A synthesis method of red luminescent carbon dots with potential for targeted diagnosis and treatment of hepatocellular carcinoma, which specifically comprises the following steps:

[0008] 1) 5-Fluorouracil (5-FU) and indocyanine green (ICG) are added to deionized water, stirred or ultrasonically treated to mix evenly, to form a uniform mixed solution;

[0009] 2) The mixed solution obtained in step 1) is transferred to a polytetrafluoroethylene high-pressure reaction kettle and heated at 160-200°C for 5-9h, and cooled to room temperature;

[0010] 3) The solution obtained in step 2) is filtered and dialyzed to obtain a pure 5-FICD solution;

[0011] 4) The solution obtained in step 3) is poured into a glass dish, and then freeze-dried in a freeze-drier at -40--60°C to obtain black solid 5-FICD.

[0012] Specifically, in step 1), the mass ratio of 5-fluorouracil and indocyanine green can be 14-20:1. In the mixed solution, the concentration of 5-fluorouracil can be 10-12 mg / mL, and the concentration of indocyanine green can be 0.5-0.7 mg / mL.

[0013] Specifically, in step 3), a filter membrane with a pore size of 0.2-0.4 μm can be used for filtration, and preferably a filter membrane with a pore size of 0.22 μm is used for filtration.

[0014] Further, in step 3), the dialysis can be performed in a dialysis bag with a molecular weight cut-off of 1000-1200 Da using deionized water for 45-50 h. Preferably, the dialysis is performed in a dialysis bag with a molecular weight cut-off of 1000 Da using deionized water for 48 h.

[0015] The present application provides a red luminescent carbon dot (5-FICD) with potential for targeted diagnosis and treatment of hepatocellular carcinoma synthesized by the above method.

[0016] The present application also provides the use of the above red luminescent carbon dot (5-FICD) with potential for targeted diagnosis and treatment of hepatocellular carcinoma as an infrared fluorescence imaging mediated photothermal tumor treatment drug.

[0017] The present application also provides a method for targeting and treating liver cancer, the specific steps of which are as follows:

[0018] 1) Prepare a PBS buffer solution with a pH of 7.4 and a concentration of 10 mM, and dissolve 5-FICD in the PBS buffer solution to prepare a solution with a concentration of 200 μg / mL;

[0019] 2) The solution obtained in step 1) is added to different fluorescence cuvettes, and detected on a fluorescence spectrophotometer;

[0020] 3) For photothermal effect detection experiments, the solution obtained in step 1) is irradiated with a laser, and the temperature change of the solution is recorded by an infrared camera.

[0021] 4) For photodynamic effect detection experiment, the solution obtained in step 1) is irradiated by using a laser, and the solution is added into different fluorescence cuvettes for detection on a fluorescence spectrophotometer;

[0022] 5) For cell fluorescence imaging experiment, 5-FICD is first added for incubation, and then the fluorescence change in the red channel is observed;

[0023] 6) For cell flow experiment, 5-FICD is first added for incubation, and then the apoptosis of cells in different channels is observed;

[0024] 7) For 3D tumor sphere experiment, 5-FICD is added for incubation, and the change of tumor sphere morphology over time is observed.

[0025] Most of the chemotherapy drugs will have great side effects on other normal cells when exerting drug efficacy, thereby causing certain influence on the health of patients. Therefore, it is of great significance to develop a nano drug with low toxicity and capable of targeting liver cancer cells. For this purpose, a red luminescent carbon dot (5-FICD) is designed and synthesized in the application, which not only retains the properties of the precursors 5-fluorouracil and indocyanine green, can be enriched in liver cancer cells for imaging and killing, but also has the low toxicity and good biocompatibility of the carbon dot itself, so as to reduce the toxic side effects on normal liver cells. The cell experiment proves that 5-FICD can effectively target liver cancer cells and has low toxicity to normal liver cells. The low toxicity and targeting ability of 5-FICD can provide a new reference for the early diagnosis and treatment of liver cancer patients.

[0026] The application takes the anticancer drug 5-fluorouracil and the photosensitizer indocyanine green as precursors, and synthesizes a low-toxicity red luminescent carbon dot (5-FICD) through a simple hydrothermal method, which improves the disadvantage of large side effects of chemotherapy drugs and realizes the targeted killing of liver cancer cells. Compared with the prior art, the application has the following advantages and beneficial effects:

[0027] 1) The red luminescent carbon dot (5-FICD) synthesized in the application can be enriched in liver cancer cells;

[0028] 2) The red luminescent carbon dot (5-FICD) synthesized in the application retains the characteristics of indocyanine green and 5-fluorouracil, and realizes the targeted treatment of liver cancer cells;

[0029] 3) The application can monitor the diagnosis and treatment process through fluorescence imaging. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 The transmission electron microscope image (a) and the particle size distribution diagram (b) of 5-FICD;

[0031] Figure 2 Fluorescence emission spectrum of 5-FICD;

[0032] Figure 3 Maps of photothermal (a) and photodynamic (b) effects of 5-FICD under laser irradiation;

[0033] Figure 4 Dark field (F.L.) and merge (Merge) images (a) and fluorescence intensity contrast image (b) of 5-FICD imaging of liver cancer cells over time;

[0034] Figure 5 Imaging images (a) and fluorescence intensity contrast images (b) of 5-FICD in different cells;

[0035] Figure 6 Flow cytometry image of 5-FICD;

[0036] Figure 7 3D tumor sphere model of 5-FICD. DETAILED DESCRIPTION

[0037] The technical solutions of the present application are further described in detail below in conjunction with examples, but the scope of protection of the present application is not limited thereto.

[0038] In the following examples, the raw materials used are ordinary commercially available products that can be purchased directly or prepared using conventional techniques in the art.

[0039] Room temperature refers to 25±5℃.

[0040] Example 1

[0041] A synthesis method of a red luminescent carbon dot (5-FICD) with potential for targeted diagnosis and treatment of hepatocellular carcinoma, specifically comprising the following steps:

[0042] 1) Mix 170 mg of 5-FU and 10 mg of ICG, grind and mix uniformly, then dissolve in 15 mL of deionized water, and ultrasonically treat the mixed solution for 10 min to mix uniformly,

[0043] 2) Transfer the mixed solution obtained in step 1) to a 30 mL stainless steel high-pressure reaction kettle lined with polytetrafluoroethylene, heat at 180℃ for 7 h. After the reaction is completed, cool to room temperature;

[0044] 3) Filter the solution obtained in step 2) with a 0.22 μm filter membrane, then dialyze the solution in a dialysis bag with a molecular weight cutoff of 1000 Da for 48 h with deionized water, finally pour into a glass dish, and then freeze-dry in a freeze-dryer at -40--60℃ for 6-8 h to obtain black powder 5-FICD.

[0045] Figure 1 Transmission electron microscope image and particle size distribution of 5-FICD prepared in this example are given. It can be seen that the product is uniformly distributed without aggregation. The particle size is 3.5 ± 1 nm, indicating the successful synthesis of 5-FICD. Figure 1

[0046] Example 2

[0047] A PBS buffer solution with pH = 7.4 and a concentration of 10 mM was prepared, and 5-FICD was dissolved in the PBS buffer solution with pH = 7.4 to prepare a carbon dot solution with a concentration of 200 μg / mL. 2.5 mL of the carbon dot solution was taken and added to a fluorescence cuvette, and the fluorescence spectrum was detected on a fluorescence spectrometer.

[0048] Figure 2 The fluorescence emission spectrum of 5-FICD is given. Figure 2 It can be seen that the emission peak of 5-FICD at 607 nm is almost unchanged as the excitation wavelength increases from 330 nm to 610 nm. Figure 2

[0049] Example 3

[0050] Photothermal effect detection experiment: Different concentrations of 5-FICD solution (50, 100, 200, and 400 μg / mL) were placed in test tubes, and the test tubes were irradiated with a fixed laser of 1.0 W / cm 3 , 660 nm for 350 s. The temperature change of the solution was observed and recorded using an infrared camera.

[0051] Photodynamic effect detection experiment: 10 μL of active oxygen indicator 2,7-dichlorofluorescein diacetate (DCFH-DA) was added to test tubes containing 2 mL of 5-FICD solution (200 μg / mL) and water, respectively, and the test tubes were irradiated with a fixed laser of 1.0 W / cm 3 , 660 nm for 5 min. The fluorescence intensity was recorded every 30 s using a fluorescence spectrometer.

[0052] Figure 3 The graphs of the photothermal (a) and photodynamic effects (b) of 5-FICD under laser irradiation are given. Figure 3 It can be seen in a that after laser irradiation, the 5-FICD solution releases heat to cause temperature rise, indicating that 5-FICD can produce photothermal effect and has concentration dependence. Figure 3 It can be seen in b that 5-FICD releases active oxygen after being irradiated with a laser, oxidizing DCFH to highly fluorescent DCF. This indicates that 5-FICD can produce active oxygen. ​​

[0053] Example 4

[0054] A PBS buffer solution with pH = 7.4 and a concentration of 10 mM was prepared, and a 5-FICD PBS solution with a concentration of 1 mg / mL, i.e. a carbon dot solution, was prepared. A cell culture solution was prepared, specifically: fetal bovine serum (FBS) : cell culture medium (DMEM) = 1:9 by volume. The carbon dot solution was added to the cell culture solution to make the concentration 200 μg / mL. Then, incubation was performed with liver cancer cells HepG-2 in a 37°C incubator with a CO2 concentration of 5% for different time periods (0.5h, 1h, 3h, 5h, 8h). After incubation, washing was performed three times with a PBS buffer solution (pH = 7.4), and then imaging of the liver cancer cells was observed under a confocal microscope, and the fluorescence intensity shown in the image was read and compared.

[0055] Figure 4 An imaging diagram of liver cancer cells with 5-FICD changing over time is given. It can be seen from the diagram that the fluorescence intensity of 5-FICD in HepG-2 cells increases over time ( Figure 4 ), and 5-FICD can be observed to enter liver cancer cells within 30 min.

[0056] Example 5

[0057] A PBS buffer solution with pH = 7.4 and a concentration of 10 mM was prepared, and a 5-FICD PBS solution with a concentration of 1 mg / mL, i.e. a carbon dot solution, was prepared. The carbon dot solution was added to a cell culture solution to make the concentration 200 μg / mL. Then, incubation was performed with human normal liver cells HL-7702, human liver cancer cells HepG-2, human normal breast cells MCF-10a, human breast cancer cells MCF-7, and human lung cancer cells A549 in a 37°C incubator with a CO2 concentration of 5% for 12 h. After incubation, washing was performed three times with a PBS buffer solution (pH = 7.4), and then imaging of the liver cancer cells was observed under a confocal microscope, and the fluorescence intensity shown in the image was read and compared.

[0058] Figure 5 An imaging diagram of 5-FICD in different cells is given. It can be seen from the diagram that 5-FICD has obvious fluorescence in HepG-2 cells, while weak fluorescence appears in the other cells ( Figure 5 ). It can be seen that 5-FICD has the ability to target liver cancer cell imaging.

[0059] Example 6

[0060] Prepare a 10 mM PBS buffer solution with pH 7.4, and a 1 mg / mL 5-FICD PBS solution (carbon dot solution). Add the carbon dot solution to the cell culture medium to achieve a concentration of 200 μg / mL. Then, co-incubate with HepG-2 liver cancer cells at 37°C and 5% CO2 for 24 h, using a 1.0 W / cm² solution. 3 Cells were irradiated with a 660 nm laser for 5 min, then incubated for 12 h. After incubation, 1 ml of trypsin (EDTA) was added, and the cells were allowed to stand for 1 min before centrifugation to collect the cells. The cells were then resuspended in 100 μL of PBS buffer, and 5 μL of Annexin V-APC and 10 μL of 7-AAD (both commercially available kits were used; instructions were followed) were added for staining. The cells were incubated at room temperature for 5 min. Finally, flow cytometry was used to analyze the apoptosis of cell samples from different groups. A blank group supplemented with culture medium and a 5-FU group supplemented with 5-FU solution were used as controls.

[0061] Figure 6 Flow cytometry images of 5-FICD are shown. The figures show that 5-FICD can induce apoptosis in liver cancer cells, with the apoptosis rate in the 5-FICD+Laser group reaching 85.93%. Figure 6 This indicates that 5-FICD has potential therapeutic effects on liver cancer cells.

[0062] Example 7

[0063] Prepare a 1% agarose gel (add 200 mg agarose to 20 mL of deionized water and heat in a metal bath at 120 °C). While still hot, add 150 μL of agarose gel to each well of a 96-well plate. Allow the plate to cool naturally at room temperature. Then, seed approximately 1000 HepG-2 cells into each well of the 96-well plate and add FBS:DMEM medium at a ratio of 1:9 to a final volume of 500 μL. Incubate the plate at 37 °C with 5% CO2 for 3 days, observing cell morphology. On day 4, replace the medium by aspirating 250 μL of medium and replenishing with 250 μL of fresh medium. On day 7, observe cell aggregation and growth into tumor spheroids, ready for further experiments.

[0064] Select appropriate wells as the blank group, blank + laser group, 5-FICD group, and 5-FICD + laser control group, respectively. Add the prepared 5-FICD solution to the 96-well plate (add culture medium to the blank group). Changes in the morphology of tumor spheres can be observed over time. Figure 7 ).

[0065] Figure 7The tumor spheroid morphology over time is given. It can be seen in the figure: in the blank group with / without laser irradiation, the tumor spheroid morphology is stable after 7 days, which shows that the laser itself has no obvious effect on the tumor spheroid. The 5-FICD group without laser irradiation, the tumor spheroid morphology changes after 7 days of culture, which shows that 5-FICD itself can induce tumor cell apoptosis. In the 5-FICD+Laser group, the tumor spheroid morphology changes after 3 days of culture, and by the 7th day, the tumor spheroid morphology has completely collapsed, and the complete tumor spheroid morphology cannot be observed. These results show that laser-induced 5-FICD has a stronger therapeutic effect and has a significant killing effect on hepatocellular carcinoma cells.

Claims

1. A method for synthesizing red luminescent carbon dots, characterized in that, Specifically, the steps include the following: 1) Add 5-fluorouracil and indocyanine green to deionized water, mix well, and form a homogeneous mixed solution; 2) Heat the mixed solution obtained in step 1) at 160-200℃ for 5-9 h, and then cool it to room temperature; 3) Filter and dialyze the solution obtained in step 2) to obtain a pure 5-FICD solution; 4) Pour the solution obtained in step 3) into a glass dish, and then freeze-dry it in a freeze dryer at -40~-60℃ to obtain 5-FICD solid; In step 1), the mass ratio of 5-fluorouracil to indocyanine green is 14-20:

1.

2. The method for synthesizing red luminescent carbon dots as described in claim 1, characterized in that, In step 3), filtration is performed using a 0.2-0.4 μm filter membrane.

3. The method for synthesizing red luminescent carbon dots as described in claim 1, characterized in that, In step 3), dialyze with deionized water in a 1000-1200 Da dialysis bag for 45-50 h.

4. The red luminescent carbon dots synthesized using any one of the methods described in claims 1 to 3.

5. The use of the red luminescent carbon dots as described in claim 4 as a tumor photothermal therapy drug mediated by infrared fluorescence imaging.

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