Quercetin carbon dot nano material, preparation method and application

Abstract

The invention relates to the technical field of pharmacy, in particular to a quercetin carbon dot nanometer material and a preparation method and application thereof.According to the quercetin carbon dot nanometer material, quercetin serves as a carbon source, quercetin molecules are made into the quercetin carbon dot nanometer material with the nanometer size through a hydrothermal reaction, and the quercetin carbon dot nanometer material is prepared into the quercetin carbon dot nanometer material with the nanometer size. The quercetin carbon dot nano material is in a spherical shape, and the diameter of the quercetin carbon dot nano material is 3.92 nm to 5.7 nm; the temperature of the hydrothermal reaction is 180-200 DEG C, and the time is 8-9 hours. The quercetin carbon dots are synthesized by using quercetin drug molecules as precursors, the toxicity is low, the safety is higher, and the serious toxic and side effects and sequelae accompanied by traditional radiotherapy drugs in laryngeal cancer treatment are reduced. And the carbon dot nano material has the characteristics of good water solubility and strong biocompatibility, so that the drug-derived carbon dots not only maintain some original pharmacological activities of quercetin drug molecules, but also show a plurality of enhanced treatment effects.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and more specifically to a quercetin carbon dot nanomaterial, a preparation method and an application thereof. Background Art

[0002] Laryngeal cancer is a malignant tumor that occurs in the larynx. Currently, surgical resection and radiotherapy are the main treatments for laryngeal cancer. Early laryngeal cancer is treated with a single treatment method of primary lesion resection or radiotherapy alone, with a cure rate of 80% to 90%. However, most patients are diagnosed with locally advanced T3, T4 or regional lymph node metastasis, and the 5-year survival rate is usually less than 50%. Due to the importance of the larynx in society and its key role in voice, swallowing, and the quality of life of patients, the clinical research focus of laryngeal cancer is to preserve and reconstruct laryngeal function while effectively controlling the tumor. In most advanced patients, laryngeal preservation surgery + radiotherapy or synchronous radiotherapy CRT is used as an alternative to total laryngectomy to preserve laryngeal function. However, for the larynx, a "small organ" with delicate structure and complex function, a pathologically negative surgical margin of only 3mm to 5mm outside the junction of normal tissue is not a truly safe margin at the molecular level. The special anatomical structure of the head and neck and the abundant cervical lymph nodes increase the possibility of recurrence and metastasis. At the same time, radiotherapy lacks specificity, laryngeal cancer is resistant to traditional radiotherapy drugs, and is often accompanied by serious toxic side effects and sequelae, making radiotherapy for laryngeal cancer ineffective.

[0003] With the development of nanoscience and nanotechnology, more and more nanomaterials are used in anti-tumor therapy, such as gold nanoparticles Au NPs and zinc oxide nanoparticles ZnO NPs. However, these nanomaterials contain metal elements and have poor safety. Therefore, it is necessary to develop a new nanomaterial that can be used for anti-tumor therapy. Summary of the invention

[0004] To solve the above problems, the present invention provides a quercetin carbon dot nanomaterial, a preparation method and an application thereof, for improving the radiotherapy effect.

[0005] The present invention is achieved through the following technical solutions: A quercetin carbon dot nanomaterial is disclosed. Quercetin is used as a carbon source. Quercetin molecules are prepared into nano-sized quercetin carbon dot nanomaterials through hydrothermal reaction. The quercetin carbon dot nanomaterial is spherical and has a diameter of 3.92nm-5.7nm. The temperature of the hydrothermal reaction is 180°C-200°C and the time is 8h-9h.

[0006] Preferably, the method for preparing the quercetin carbon dot nanomaterial specifically comprises the following steps: The quercetin is dissolved in water, and the pH is adjusted to 8.7-9.3 with a saturated NaOH aqueous solution to increase the solubility of the quercetin molecule to obtain a mixture.

[0007] The mixture was subjected to a hydrothermal reaction, cooled to room temperature, centrifuged, and the supernatant was collected, filtered, and dialyzed to obtain quercetin carbon dot nanomaterials, wherein the molecular weight cutoff of the dialysis was 1000Da.

[0008] Preferably, the room temperature is 25°C.

[0009] Preferably, the centrifugal speed is 3000 rpm to 3500 rpm, and the centrifugal time is 10 min to 15 min.

[0010] Preferably, filtering is performed using a device with a filter pore diameter of 0.20 µm to 0.24 µm.

[0011] Preferably, the dialysis time is 24h~26h.

[0012] Preferably, the water is deionized water.

[0013] The application of the quercetin carbon dot nanomaterial in preparing a drug for treating laryngeal cancer.

[0014] A chemotherapy drug for laryngeal cancer, wherein the chemotherapy drug uses the quercetin carbon dot nanomaterial according to claim 1 as the only active ingredient.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention discloses a quercetin carbon dot nanomaterial. Quercetin is used as a carbon source, and quercetin molecules are made into quercetin carbon dot nanomaterials with nanometer size through hydrothermal reaction. The quercetin carbon dot nanomaterial is spherical with a diameter of 3.92nm~5.7nm; the temperature of the hydrothermal reaction is 180℃~200℃, and the time is 8h~9h. The present invention uses quercetin drug molecules as precursors to synthesize quercetin carbon dots, which have low toxicity and higher safety, and reduce the serious toxic side effects and sequelae associated with traditional radiotherapy drugs in the treatment of laryngeal cancer. And the carbon dot nanomaterial has the characteristics of good water solubility and strong biocompatibility, so that the drug-derived carbon dots not only retain some original pharmacological activities of the quercetin drug molecules, but also show a variety of enhanced therapeutic effects. In addition, the quercetin carbon dots have good biocompatibility, low toxicity and stable physical and chemical properties. Therefore, quercetin carbon dot nanomaterials prepared using quercetin molecules as carbon source provide a new strategy for inhibiting the growth and metastasis of laryngeal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 This is a characteristic diagram of the Que-CDs of the present invention.

[0018] Figure 1 In the figure, A is a schematic diagram of the synthesis route of Que-CDs; B is a transmission electron microscope, or TEM image, of quercetin carbon dots. Scale bar: 0.2 μm; C is a particle size distribution histogram based on TEM images; D is the fluorescence emission spectrum of Que-CDs, 10 μg / mL; E is the Fourier transform infrared, or FT-IR spectrum, of Que-CDs; F is the X-ray diffraction, or XRD pattern, of Que-CDs; G is the full scan X-ray photoelectron spectroscopy, or XPS pattern, of quercetin carbon dots; H: high-resolution C1s XPS spectrum.

[0019] Figure 2 This is a graph showing the effects of Que-CDs of the present invention on the growth, migration and invasion of TU686 cells.

[0020] Figure 2 In the figure, A is the morphological changes of TU686 cells after treatment with different groups; B is the clone formation test; TU686 cells were cultured for 14 days after being treated with quercetin and Que-CDs; C is the CCK-8 test result; D is the corresponding statistical chart of the clone formation test.

[0021] Figure 3 This is a graph showing the effect of Que-CDs of the present invention on the migration of TU686 cells.

[0022] Figure 3 In the figure, A is the effect of Que-CDs on TU686 cell migration; B is the corresponding statistical graph of the effect of Que-CDs on TU686 cell migration, scale bar = 500 μm.

[0023] Figure 4 This is a graph showing the effect of Que-CDs of the present invention on the invasion of TU686 cells.

[0024] Figure 4 In the figure, A is the Transwell assay display; B is the corresponding statistical graph of the Transwell assay, scale bar = 200 μm; C is the Western blot analysis result of migration-related proteins; D is the corresponding statistical graph of Western blot analysis of migration-related proteins, *p <0.05,** p <0.01,*** p <0.001.

[0025] Figure 5 This is a graph showing the experimental results of the effect of Que-CDs on the cell cycle of TU686 cells.

[0026] Figure 5 In the figure, A is the Control group, i.e., the cell cycle detection result diagram of the control group; B is the Cit-CDs cell cycle detection result diagram; C is the Que cell cycle detection result diagram; D is the Que-CDs cell cycle detection result diagram; and E is a statistical diagram of the cell cycle detection results.

[0027] Figure 6 This is a graph showing the experimental results of the effect of Que-CDs of the present invention on apoptosis of TU686 cells.

[0028] Figure 6 In the figure, A is the Control group, i.e., the cell apoptosis detection result diagram of the control group; B is the cell apoptosis detection result diagram of Cit-CDs; C is the cell apoptosis detection result diagram of Que; D is the cell apoptosis detection result diagram of Que-CDs; and E is the statistical diagram of the cell apoptosis detection results.

[0029] Figure 7 This is a diagram showing the live and dead cell staining results of TU686 cells using Que-CDs of the present invention.

[0030] Figure 7 In the figure, A is the live and dead cell staining result diagram; B is the corresponding statistical diagram of live and dead cell staining.

[0031] Figure 8 Western blot analysis of apoptosis-related proteins in TU686 cells by Que-CDs of the present invention.

[0032] Figure 8 In the figure, A is a Western blot analysis of CDK1 and CCNB1 proteins, with β actin as the internal reference protein; B is a statistical graph corresponding to the Western blot analysis of CDK1 and CCNB1 proteins; C is a Western blot analysis of Bax, Bcl2 and p53 proteins, with β actin as the internal reference protein; D is a statistical graph corresponding to the Western blot analysis of Bax, Bcl2 and p53 proteins. DETAILED DESCRIPTION

[0033] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively below, and preferred embodiments of the present invention are provided. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0035] The inventive concept of the present invention is as follows: Laryngeal cancer is a malignant tumor that occurs in the larynx. However, for the larynx, a "small organ" with delicate structure and complex functions, a pathologically negative surgical margin of only 3mm to 5mm outside the junction of normal tissue is not a truly safe margin at the molecular level. The special anatomical structure of the head and neck and the abundant cervical lymph nodes increase the possibility of recurrence and metastasis. At the same time, radiotherapy lacks specificity, and laryngeal cancer is resistant to traditional radiotherapy drugs, and is often accompanied by serious toxic side effects and sequelae, making radiotherapy for laryngeal cancer ineffective.

[0036] With the development of nanoscience and nanotechnology, more and more nanomaterials are used in anti-tumor therapy, such as gold nanoparticles Au NPs and zinc oxide nanoparticles ZnO NPs. However, these nanomaterials contain metal elements and have poor safety. Therefore, it is necessary to develop a new nanomaterial that can be used for anti-tumor therapy.

[0037] Based on this, the present invention provides a quercetin carbon dot nanomaterial, which uses quercetin as a carbon source, and makes quercetin molecules into quercetin carbon dot nanomaterials with nanometer size through hydrothermal reaction, wherein the quercetin carbon dot nanomaterial is spherical with a diameter of 3.92nm~5.7nm; the temperature of the hydrothermal reaction is 180℃~200℃, and the time is 8h~9h. The present invention uses quercetin drug molecules as precursors to synthesize quercetin carbon dots, which has low toxicity and higher safety, and reduces the serious toxic side effects and sequelae associated with traditional radiotherapy drugs in the treatment of laryngeal cancer. In addition, the carbon dot nanomaterial has the characteristics of good water solubility and strong biocompatibility, so that the drug-derived carbon dots not only retain some of the original pharmacological activities of the quercetin drug molecules, but also show a variety of enhanced therapeutic effects. During the formation of carbon dots, the active groups in the quercetin molecules are partially retained on the surface of the carbon dots, and these groups can maintain the original pharmacological activities of quercetin, such as antioxidant, anti-inflammatory, and anti-cancer. Through the nano-size effect and surface effect of carbon dots, the water solubility, stability, and bioavailability of quercetin carbon dot nanomaterials are significantly improved; because quercetin carbon dot nanomaterials have good water solubility, they are better dispersed in the aqueous phase, thereby improving their solubility and distribution in the body; due to their small size and improved solubility, they can be more effectively taken up by cells, improving bioavailability, thereby enhancing their pharmacological activity. Experiments have shown that the quercetin carbon dot nanomaterial of the present invention not only retains the original pharmacological activity of quercetin, but also exhibits an enhanced therapeutic effect due to the nano effect, thereby more effectively inhibiting the growth and metastasis of laryngeal cancer.

[0038] In addition, the nanostructure and surface characteristics of carbon dots make the physical and chemical properties of quercetin carbon dot nanomaterials more stable than those of original quercetin molecules, making them less likely to degrade during storage and use, and they have good biocompatibility.

[0039] The beneficial effects of the present invention are described below by means of specific embodiments: Example 1 A method for preparing quercetin carbon dot nanomaterial.

[0040] Dissolve 0.33mmol 0.10g quercetin in 10mL deionized water. Subsequently, gradually add saturated NaOH aqueous solution to adjust the pH to 8.7 to obtain a mixture; transfer the obtained mixture to a 25ml Teflon-lined autoclave and perform a hydrothermal reaction at 180°C for 8h. After the reaction, the solution was naturally cooled to room temperature, centrifuged at 3000rpm for 10min, and then the supernatant was collected and filtered with a 0.20µm filter. Subsequently, dialyze with deionized water for 24h using a dialysis bag with a molecular weight cutoff of 1000Da. During the dialysis process, the deionized water was replaced every 6h to obtain quercetin carbon dot nanomaterials, namely Que-CDs, which were freeze-dried to obtain Que-CDs powder. The schematic diagram of the synthesis route of quercetin carbon dots is shown in the figure. Figure 1 As shown in A.

[0041] Example 2 A method for preparing quercetin carbon dot nanomaterial.

[0042] 0.33mmol 0.10g quercetin was dissolved in 10mL deionized water. Subsequently, a saturated NaOH aqueous solution was gradually added to adjust the pH to 9.0 to obtain a mixture; the obtained mixture was transferred to a 25ml Teflon-lined autoclave and subjected to a hydrothermal reaction at 190°C for 8.5h. After the reaction, the solution was naturally cooled to room temperature, centrifuged at 3200rpm for 12min, and then the supernatant was collected and filtered with a 0.22µm filter. Subsequently, the quercetin carbon dot nanomaterial, i.e., Que-CDs, was obtained by dialyzing with deionized water for 25h using a dialysis bag with a molecular weight cutoff of 1000Da, and the deionized water was replaced every 6h during the dialysis process, and then freeze-dried to obtain Que-CDs powder.

[0043] Example 3 A method for preparing quercetin carbon dot nanomaterial.

[0044] 0.33mmol 0.10g quercetin was dissolved in 10mL deionized water. Subsequently, a saturated NaOH aqueous solution was gradually added to adjust the pH to 9.3 to obtain a mixture; the obtained mixture was transferred to a 25ml Teflon-lined autoclave and subjected to a hydrothermal reaction at 200°C for 9h. After the reaction, the solution was naturally cooled to room temperature, centrifuged at 3500rpm for 15min, and then the supernatant was collected and filtered with a 0.24µm filter. Subsequently, the quercetin carbon dot nanomaterial, i.e., Que-CDs, was obtained by dialyzing with deionized water for 26h using a dialysis bag with a molecular weight cutoff of 1000Da, and the deionized water was replaced every 6h during the dialysis process, and freeze-dried to finally obtain Que-CDs powder.

[0045] Comparative Example 1: A method for preparing citric acid carbon dot nanomaterials 1.0mM 0.19g of citric acid was dissolved in 10mL of deionized water. The resulting mixture was transferred to a 25mL polytetrafluoroethylene-lined autoclave and hydrothermally reacted at 200°C for 12h. After the reaction was completed, the solution was cooled naturally to room temperature. The large amount of precipitate formed was separated by centrifugation at 3000 rpm for 10 minutes. The supernatant was then collected and filtered through a 0.22 micron filter membrane to remove any remaining small precipitates. Subsequently, a dialysis bag with a molecular weight cutoff of 1000Da was used for 24h dialysis with deionized water, and the deionized water was replaced every 6h during the dialysis process to separate the citric acid carbon dot nanomaterials, namely Cit-CDs. The resulting Cit-CDs were collected, freeze-dried, and stored for subsequent use.

[0046] Experimental Example 1 The size and surface morphology of Que-CDs in Example 1 were elucidated by transmission electron microscopy (TEM).

[0047] like Figure 1 As shown in Figures B and C, TEM images show that Que-CDs are spherical, have a uniform size distribution and are well dispersed, with an average diameter of 4.81 nm. Figure 1 The fluorescence spectrum of Que-CDs shown in D in Figure 5 exhibits significant fluorescence emission at 562 nm, with excitation wavelengths ranging from 515 to 540 nm, which is consistent with their uniform size distribution. Figure 1 The Fourier transform infrared spectrum (FT-IR spectrum) of Que-CDs shown in Figure E shows five obvious absorption bands, located at 3425 cm -1 、2967cm -1 、1638cm -1 、1512cm -1 and 1323cm -1 , corresponding to OH, CH, C=O, C=C, and CO functional groups. These data indicate that the surface of Que-CDs is rich in -OH and -COOH functional groups and confirm that Que-CDs retain some functional groups of the parent Que molecule. Figure 1 As shown in Figure F, X-ray diffraction, or XRD, tests indicate the presence of amorphous carbon components in Que-CDs, due to the presence of a broad diffraction peak at 26°. Figure 1As shown in G in Figure 1, the elemental composition and functional groups of Que-CDs were further characterized using X-ray photoelectron spectroscopy, or XPS. The full scan spectrum of Que-CDs can identify the characteristic peaks of C 1s and O 1s, which are located at 281eV and 529eV, respectively. Elemental quantitative analysis shows that Que-CDs are composed of 65.78% by mass of C and 25.60% by mass of O. Figure 1 As shown in Figure 1, the high-resolution XPS spectrum can be decomposed into three peaks. The peak at 284.8 eV is attributed to C=C / CC, while the peaks at 286.0 eV and 287.9 ​​eV are assigned to CO and C=O in the carboxyl functional group, respectively. The above characterization results verify the successful synthesis of carbon dots.

[0048] Experimental Example 2 The ability of Que-CDs to inhibit laryngeal cancer proliferation was verified.

[0049] The proliferation rate of cells was compared by CCK8 method. 4 100 μL of cells were inoculated in a 96-well plate and cultured overnight. Then different concentrations of quercetin, i.e. Que, and the quercetin carbon dot nanomaterials prepared in Example 1, i.e. Que-CDs, were added respectively, and the different concentrations were set to 0 μg / ml, 2 μg / ml, 4 μg / ml, 8 μg / ml, 16 μg / ml, 32 μg / ml and 64 μg / ml, respectively. After 24 hours of treatment, 90 μL Cell Counting kit-8 was added, that is, 10 μL of culture medium was added to the CCK-8 solution. CCK-8 was purchased from Beijing Coolbo Technology Co., Ltd., China, and incubated at 37°C for 4 hours. The absorbance at 450 nm was detected with an enzyme reader, and the enzyme reader was purchased from TECAN, Switzerland. The results are as follows Figure 2 As shown in Figure C, Que and Que-CDs showed dose-dependent anticancer activity, and after treatment with 16 μg / mL Que-CDs, cell viability was significantly decreased compared with the Que group at the same dose.

[0050] The effects of different groups on laryngeal cancer cell TU686. The experiment was divided into four groups: Control group, Cit-CDs group, Que group and Que-CDs group. The control group was cultured in serum-free medium for 24 hours; the Cit-CDs group was a citric acid carbon dot group, which was exposed to 16 μg / mL of citric acid carbon dot nanomaterials obtained in Comparative Example 1 for 24 hours; the Que group was a quercetin administration group, which was exposed to 16 μg / mL of quercetin for 24 hours; the Que-CDs group was a quercetin carbon dot administration group, which was exposed to 16 μg / mL of quercetin carbon dot nanomaterials obtained in Example 1 for 24 hours. The results showed that compared with the other three groups, the cells in the Que-CDs group were significantly stretched and shrunk, as shown in the figure. Figure 2As shown in A.

[0051] The inhibitory effect of Que-CDs on TU686 cell proliferation was further verified by clone formation assay. 3 The cells were seeded in a 6-well plate and cultured with serum-free medium for 14 days as the control group; the citric acid carbon dot nanomaterials obtained in Comparative Example 1 were exposed to 16 μg / mL for 14 days as the Cit-CDs group; 16 μg / mL quercetin was used to expose for 14 days as the Que group; and 16 μg / mL quercetin carbon dot nanomaterials obtained in Example 1 were exposed to 14 days as the Que-CDs group and treated for 14 days. The cells were then washed with phosphate buffered solution, i.e. PBS, and fixed with 4% paraformaldehyde for 15 minutes. After removing the 4% paraformaldehyde, the cells were washed with PBS, stained with crystal violet solution for 15 minutes, and then washed with PBS. Finally, the 6-well plates were dried and the number of cell clusters was calculated. The results showed that compared with the cell colonies in the control group, Que group and Cit-CDs group, the Que-CDs group had a significant reduction, such as Figure 2 As shown in B and D. The decrease in cell viability and cell colonies indicated that Que-CDs inhibited the proliferation of TU686 cells.

[0052] Experimental Example 3 The ability of Que-CDs to inhibit laryngeal cancer migration and invasion was verified.

[0053] The inhibition of laryngeal cancer migration by quercetin carbon dots was verified by wound healing assay. 5 The cells were cultured in a 6-well plate and scratched with a 10 μL pipette tip. The cells were then gently rinsed with PBS and cultured in serum-free medium for 48 hours as the control group; the citric acid carbon dot nanomaterials obtained in Comparative Example 1 were exposed to 16 μg / mL for 48 hours as the Cit-CDs group; 16 μg / mL quercetin was used for exposure for 48 hours as the Que group; and the quercetin carbon dot nanomaterials obtained in Example 1 were exposed to 16 μg / mL for 48 hours as the Que-CDs group and treated for 14 days. Images were taken with a microscope purchased from OLYMPUS, Tokyo, Japan, and analyzed with Image J software purchased from NIH, USA. Compared with the control group, Que group and Cit-CDs group, the mobility of the Que-CDs group was lower, as shown in Figure 2. Figure 3 As shown in A and B in Figure 3. Western blotting further demonstrated the effect of quercetin carbon dots on the expression levels of migration-related proteins. After Que-CDs treatment, the expression of migration-related proteins MMP2 and MMP9 was significantly reduced, as shown in Figure 3. Figure 4 As shown in C and D.

[0054] The ability of quercetin carbon dots to inhibit laryngeal cancer invasion was verified by Transwell invasion assay. First, cells were cultured in 6-well plates, and serum-free medium was used for 48 hours as the control group; 16 μg / mL of citric acid carbon dot nanomaterials obtained in Comparative Example 1 were exposed for 48 hours as the Cit-CDs group; 16 μg / mL of quercetin was used for 48 hours as the Que group; and 16 μg / mL of quercetin carbon dot nanomaterials obtained in Example 1 were exposed for 48 hours as the Que-CDs group for 14 days. Then, the diluted matrix gel was added to the upper layer and stored at 4°C. Then, 4×10 4 100 μL of cells were transferred to the upper layer and cultured in a medium without FBS. At the same time, the lower layer was treated with a medium containing 20% ​​FBS by volume. After being kept at 37°C for 48 hours, the chamber was removed and washed with PBS. The cells were fixed with 4% paraformaldehyde and stained with crystal violet. Finally, the cells were washed with PBS and observed under a microscope. The results showed that compared with the control group, Que group and Cit-CDs group, Que-CDs treatment had the most significant reduction in cell invasion, as shown in Figure 2. Figure 4 As shown in A and B.

[0055] Experimental Example 4 The ability of Que-CDs to promote laryngeal cancer apoptosis was verified.

[0056] The study detected the cell cycle by flow cytometry and found that Que-CDs treatment led to an increase in the proportion of TU686 cells in the G1 phase, such as Figure 5 The percentage of apoptotic cells was detected by Annexin V-FITC / PI apoptosis detection kit, which was purchased from Hangzhou Lianke Biotechnology, China. 5 Cells were seeded in 6-well plates and cultured with serum-free medium for 24 hours as the control group; the citric acid carbon dot nanomaterials obtained in Comparative Example 1 were exposed to 16 μg / mL for 24 hours as the Cit-CDs group; 16 μg / mL quercetin was used for exposure to 24 hours as the Que group; and 16 μg / mL quercetin carbon dot nanomaterials obtained in Example 1 were exposed to 16 μg / mL for 24 hours as the Que-CDs group and treated for 14 days. The cells were harvested with EDTA-free trypsin and washed with PBS. The cells were resuspended in 500 μL binding buffer and incubated with 5 μL Annexin V-FITC and 10 μL PI for 15 minutes. The cells were analyzed by flow cytometry. The results showed that the apoptosis rate of cells treated with Que-CDs was the highest, increasing from 26.2% to 46.1%. Figure 6 As shown in A~E.

[0057] The ratio of live cells to dead cells was detected using an animal live-dead cell viability / cytotoxicity assay kit purchased from Proteintech, Wuhan, China. 5 The cells were seeded in a 6-well plate and cultured in serum-free medium for 12 hours as the control group; the citric acid carbon dot nanomaterials obtained in Comparative Example 1 were exposed to 16 μg / mL for 12 hours as the Cit-CDs group; 16 μg / mL quercetin was used for exposure for 12 hours as the Que group; and 16 μg / mL quercetin carbon dot nanomaterials obtained in Example 1 were exposed for 12 hours as the Que-CDs group and treated for 14 days. The cells were then harvested with EDTA-free trypsin, washed with PBS, and 2 μL of calcein AM and 4.5 μL of PI were added to 500 μL of serum-free medium and incubated for 20 minutes. The stained cells were observed under a microscope. The live cell staining results showed that the proportion of dead cells in the Que-CDs group was higher than that in the control group, Que group and Cit-CDs group, as shown in Figure 2. Figure 7 As shown in A and B in Figure 2. In addition, the protein expression of pro-apoptotic factors Bax and p53 in the Que-CDs group was significantly higher than that in the Que group, and the protein expression of anti-apoptotic factor Bcl2 was significantly lower than that in the Que group. At the same time, the results showed that the protein expression levels of CCNB1 and CDK1 in the Que-CDs group were significantly lower than those in the Que group, as shown in Figure 2. Figure 8 As shown in A to D in FIG. 1 , the results show that the quercetin carbon dot nanomaterial of the present invention significantly increases the apoptosis level of laryngeal cancer cells and induces G1 phase arrest in TU686 cells.

[0058] It should be noted that the present invention also verifies the size and surface morphology, the ability to inhibit laryngeal cancer proliferation, the ability to inhibit laryngeal cancer migration and invasion, and the ability to promote laryngeal cancer apoptosis of the Que-CDs obtained in Example 2 and Example 3, and the results are the same as the verification results of Example 1.

[0059] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0060] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A quercetin carbon dot nanomaterial, characterized in that: Using quercetin as a carbon source, the quercetin molecules are made into quercetin carbon dot nanomaterials with nanometer size through hydrothermal reaction, wherein the quercetin carbon dot nanomaterials are spherical with a diameter of 3.92nm-5.7nm; The temperature of the hydrothermal reaction is 180° C. to 200° C., and the time is 8 h to 9 h.

2. The method for preparing quercetin carbon dot nanomaterials according to claim 1, characterized in that: The specific steps include: Dissolving quercetin in water, adjusting the pH to 8.7-9.3 with a saturated aqueous NaOH solution, to obtain a mixture; The mixture was subjected to a hydrothermal reaction, cooled to room temperature, centrifuged, and the supernatant was collected, filtered, and dialyzed to obtain quercetin carbon dot nanomaterials, wherein the molecular weight cutoff of the dialysis was 1000Da.

3. The method for preparing quercetin carbon dot nanomaterials according to claim 2, characterized in that: The centrifugal speed is 3000 rpm to 3500 rpm, and the centrifugal time is 10 min to 15 min.

4. The method for preparing quercetin carbon dot nanomaterials according to claim 2, characterized in that: Filter using a device with a filter pore diameter of 0.20µm~0.24µm.

5. The method for preparing quercetin carbon dot nanomaterials according to claim 2, characterized in that: The dialysis time is 24h~26h.

6. The method for preparing quercetin carbon dot nanomaterials according to claim 2, characterized in that: The water is deionized water.

7. Use of the quercetin carbon dot nanomaterial as claimed in claim 1 in the preparation of a drug for treating laryngeal cancer.

8. A chemotherapy drug for laryngeal cancer, characterized in that: The chemotherapy drug has the quercetin carbon dot nanomaterial described in claim 1 as the only active ingredient.

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