Carbon dot, preparation method and application thereof, and antitumor drug

Abstract

The invention discloses a carbon dot, a preparation method and application thereof, and an antitumor drug, and relates to the technical field of carbon materials. The carbon dot comprises a pyridine-mediated electron donor-acceptor structure formed by a nitrogen-containing heterocyclic aromatic compound, an acrylamide derivative and ethidene diamine, the catalytic performance of the carbon dot is improved through structure-enhanced charge transfer, and the blank of rational design guidance of the current carbon-based nano-enzyme is filled up; the carbon dot has dual catalytic activity of peroxidase and oxidase, can efficiently generate active oxygen, can down-regulate the biological regulation function of a PI3K / AKT signal channel, realizes direct intervention on a tumor cell anti-oxidation defense system, amplifies the active oxygen mediated killing effect, and makes up the blank of conversion between the chemical structure and the biological function of the carbon dot at present. The preparation method of the carbon dots is simple and convenient, does not need complex reaction process and operation, and is low in production cost, simple in production process and suitable for large-scale production.

Description

Technical Field

[0001] This invention relates to the field of carbon materials technology, and more specifically, to a carbon dot, its preparation method and application, and anti-tumor drugs. Background Technology

[0002] Carbon dots, as one of the most widely studied carbon-based nanomaterials, have become a promising class of nanozymes due to their low toxicity, excellent biocompatibility, ultra-small size, tunable band structure, and low preparation cost. Many studies have shown that the surface modification and core structure of carbon dots are related to their enzyme-like activity. However, the catalytic performance of carbon-based nanozymes is generally considered to be lower than that of metal-based nanozymes, which greatly limits their application.

[0003] The synthesis of pyridine-rich carbon dot structures is challenging due to the unclear structure-activity relationship of carbon dots and the lack of rational design guidance for their acquisition. Although many ingenious carbon dots have been developed through empirical synthesis, these carbon dots generally lack biological-level molecular regulatory functions, which greatly limits their application in the biological field, especially in tumor therapy. Therefore, constructing a bridge between chemical structure and biological function to endow carbon dots with the desired biomolecular regulatory functions remains another challenge.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a carbon dot, its preparation method and application, and an anti-tumor drug, so as to solve or improve the above-mentioned technical problems.

[0006] This invention is implemented as follows: In a first aspect, embodiments of the present invention provide a carbon dot comprising a pyridine-mediated electron donor-acceptor structure; The electron donor-acceptor structure is formed by nitrogen-containing heterocyclic aromatic compounds, acrylamide derivatives, and ethylenediamine.

[0007] Secondly, embodiments of the present invention provide a method for preparing carbon dots as described above, comprising the following steps: A nitrogen-containing heterocyclic aromatic compound, an acrylamide derivative, ethylenediamine, and water are mixed in a certain proportion and subjected to a solvothermal reaction under a power of 200W-2000W to obtain carbon dots.

[0008] Thirdly, embodiments of the present invention provide the application of carbon dots as described above or carbon dots prepared by the aforementioned method in the preparation of antitumor drugs, wherein the tumor includes at least one of solid tumors and hematologic malignancies.

[0009] Fourthly, embodiments of the present invention provide an antitumor drug comprising carbon dots as described above or carbon dots prepared by the methods described above and pharmaceutically acceptable excipients.

[0010] The present invention has the following beneficial effects: The carbon dots provided in this invention are prepared using nitrogen-containing heterocyclic aromatic compounds, acrylamide derivatives, and ethylenediamine as raw materials. The prepared carbon dots contain an electron donor-acceptor structure, which enhances the catalytic performance of the carbon dots through enhanced charge transfer, thus filling the gap in the current rational design guidance of carbon-based nanoenzymes.

[0011] Carbon dots possess dual catalytic activity, similar to both peroxidases and oxidases, enabling them to efficiently generate reactive oxygen species. They also have a bioregulatory function that downregulates the PI3K / AKT signaling pathway, thereby achieving direct intervention on the antioxidant defense system of tumor cells, amplifying the killing effect mediated by reactive oxygen species, and filling the current gap in the transformation between the chemical structure and biological function of carbon dots.

[0012] Carbon dots can be prepared directly by reacting nitrogen-containing heterocyclic aromatic compounds, acrylamide derivatives and ethylenediamine in an aqueous solution under solvothermal conditions. This method requires no complicated reaction process or operation, has low production cost and simple production process, and is suitable for large-scale production. Attached Figure Description

[0013] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0014] Figure 1 This is a schematic diagram illustrating the mechanism of transformation between the chemical structure and biological function of the carbon dot N-CDs obtained in Example 1. Figure 2 This is a schematic diagram illustrating the preparation process of carbon dot N-CDs obtained in Example 1; Figure 3 Morphology and particle size analysis of carbon dot N-CDs obtained in Example 1: (a) TEM morphology image; (b) dynamic light scattering; Figure 4 The ultraviolet absorption spectrum and two-position fluorescence spectrum are shown: (a) CDs1 prepared in Comparative Example 1; (b) CDs2 prepared in Comparative Example 2; (c) Carbon point N-CDs prepared in Example 1; Figure 5 For the 1H NMR spectra: (a) CDs1 prepared in Comparative Example 1; (b) CDs2 prepared in Comparative Example 2; (c) Carbon point N-CDs prepared in Example 1; Figure 6 Structural characterization of the carbon dot N-CDs prepared in Example 1: (a) Raman spectrum; (b) FT-IR spectrum; (c) XPS full spectrum; (d) N1s high-resolution spectrum of N-CDs; (e) C1s high-resolution spectrum of N-CDs; (f) O1s high-resolution spectrum of N-CDs. Figure 7 Paramagnetic resonance spectra of CDs1 prepared in Comparative Example 1, CDs2 prepared in Comparative Example 2, and carbon dot N-CDs prepared in Example 1: (a) aqueous solution of hydrogen peroxide; (b) DMSO solution; Figure 8 The DCFH probe was used to detect the level of ROS generated by the carbon N-CDs prepared in Example 1 in water and hydrogen peroxide aqueous solution; Figure 9 The results detected by DCFH after incubating CT26 cells with the carbon dot N-CDs prepared in Example 1 are as follows: (a) ROS fluorescence intensity; (b) CLSM imaging and mitochondrial membrane potential detection. Figure 10 Transcriptomic studies of CT26 cells treated with carbon dot N-CDs prepared in Example 1: (a) GO functional enrichment analysis of significantly altered gene sets; (b) GSEA enrichment analysis of reactive oxygen species pathway after N-CDs treatment; (c) Volcano plot comparing CT26 cells in the N-CDs-treated and PBS-treated groups; (d) Csf1 / Csf1r / Pik3r1 / Akt1 / Nfe2l2 pathway network; (e) GSEA enrichment analysis of the PI3K / AKT pathway after N-CDs treatment; (f) Heatmap of changes in Csf1 / Csf1r / Pik3r1 / Akt1 / Nfe2l2 gene expression in CT26 cells in the N-CDs-treated and PBS-treated groups. Figure 11 Results of CT26 cells treated with carbon N-CDs prepared in Example 1: (a) Cell viability assay results; (b) Dead / live double staining image; Figure 12 The results of tumor testing after 15 days of treatment with the carbon dot N-CDs prepared in Example 1 in a mouse subcutaneous tumor-bearing model are as follows: (a) tumor volume; (b) tumor weight. Detailed Implementation

[0015] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0016] In a first aspect, embodiments of the present invention provide a carbon dot comprising a pyridine-mediated electron donor-acceptor structure; The electron donor-acceptor structure is formed by nitrogen-containing heterocyclic aromatic compounds, acrylamide derivatives, and ethylenediamine.

[0017] The pyridine-mediated electron donor-acceptor structure-enhanced charge transfer improves the catalytic performance of carbon dots, filling the gap in the rational design guidance of carbon-based nanozymes. Carbon dots possess dual catalytic activities of peroxidase and oxidase-like substances, can efficiently generate reactive oxygen species, and can downregulate the biological regulatory function of the PI3K / AKT signaling pathway, achieving direct intervention on the antioxidant defense system of tumor cells, amplifying the killing effect mediated by reactive oxygen species, and filling the gap in the transformation between the chemical structure and biological function of carbon dots.

[0018] Nitrogen-containing heterocyclic aromatic compounds, due to their strong electron adsorption capacity, can serve as electron acceptor structures, creating the adsorption and active sites required for catalysis. Acrylamide derivatives can act as electron donors, and their electron-donating effect is further enhanced after cross-linking due to the elongated skeleton. Ethylenediamine serves as a linker for electron donors and acceptors.

[0019] In an optional embodiment, the ratio of nitrogen-containing heterocyclic aromatic compound, acrylamide derivative and ethylenediamine is (0.1-1) g: (0.1-1) g: (2-8) mL.

[0020] In an optional embodiment, the ratio of nitrogen-containing heterocyclic aromatic compound, acrylamide derivative and ethylenediamine is (0.3-0.7) g : (0.3-0.7) g : (3-5) mL.

[0021] For example, the ratio of the nitrogen-containing heterocyclic aromatic compound, the acrylamide derivative, and ethylenediamine can be selected from any one of 0.3g:0.3g:3mL, 0.4g:0.4g:3mL, 0.3g:0.4g:4mL, 0.5g:0.3g:3mL, 0.5g:0.5g:3mL, 0.5g:0.5g:4mL, 0.5g:0.5g:5mL, 0.6g:0.7g:3mL, and 0.7g:0.7g:5mL, or other values ​​within the range of (0.3-0.7)g:(0.3-0.7)g:(3-5)mL.

[0022] In an optional embodiment, the nitrogen-containing heterocyclic aromatic compound is selected from at least one of citric acid, melamine, cyanuric acid diamide, trichloroisocyanuric acid, and nicotinic acid.

[0023] It should be noted that nitrogen-containing heterocyclic aromatic compounds, especially pyrrole compounds, will form cross-linked pyridine rings during microwave reactions. As an electron-withdrawing group, the pyridine ring can act as an electron acceptor, creating the adsorption and active centers necessary for catalysis by modulating the charge distribution of adjacent carbon skeletons.

[0024] Furthermore, nitrogen-containing heterocyclic aromatic compounds include citric acid.

[0025] In an optional embodiment, the acrylamide derivative is selected from at least one of N,N-methylenebisacrylamide, N,N-(1,2-dihydroxyethylene)bisacrylamide, N,N-methylenebismethylacrylamide, N-isopropylacrylamide, and N-hydroxymethylacrylamide.

[0026] It should be noted that acrylamide derivatives are a class of strong crosslinking agents, which can generate a large number of amino groups during the synthesis of carbon dots. Amino groups, as electron-rich groups, can act as electron donors to form donor-acceptor structures with pyridine structures. Under microwave conditions, through crosslinking, carbonization, and other steps, carbon dots with electron-acceptor structures uniformly dispersed in an electron-donating conjugated system can be prepared. These carbon dots exhibit significantly enhanced nanozyme catalytic performance due to enhanced charge transfer, further generating reactive oxygen species at the cellular level.

[0027] Furthermore, acrylamide derivatives include N,N-methylenebisacrylamide.

[0028] For example, when the nitrogen-containing heterocyclic aromatic compound is citric acid and the acrylamide derivative is N,N-methylenebisacrylamide, the carbon dots have uniformly dispersed sp atoms rich in amino groups. 2 In the conjugated structure, pyridine and amino groups form an electron donor-acceptor structure.

[0029] And / or, the particle size of the carbon dots is 6nm-10nm.

[0030] Secondly, embodiments of the present invention provide a method for preparing carbon dots as described above, comprising the following steps: A nitrogen-containing heterocyclic aromatic compound, an acrylamide derivative, ethylenediamine, and water are mixed in a certain proportion and subjected to a solvothermal reaction under a power of 200W-2000W to obtain carbon dots.

[0031] In an optional embodiment, the power of the solvothermal reaction is 850W-1200W, and the time is 3min-10min; In an optional embodiment, the amount of water used is 25 mL to 50 mL, and the water is selected from at least one of ultrapure water, double-distilled water, deionized water, and purified water.

[0032] In an optional embodiment, the process includes post-processing after the solvothermal reaction, which includes purification and lyophilization.

[0033] In an optional implementation, the purification process takes 24-72 hours and uses a dialysis bag with a molecular weight cutoff of 500-1000 Da.

[0034] It should be noted that the purification process of the present invention utilizes the principle of dialysis. In other embodiments of the present invention, a dialysis bag with a suitable molecular weight cutoff can be selected as needed.

[0035] Freeze-drying involves placing the dialyzed solution in relevant equipment. This invention does not impose any particular limitations on the equipment used for freeze-drying; it can be selected appropriately as needed.

[0036] Thirdly, embodiments of the present invention provide the application of carbon dots as described above or carbon dots prepared by the aforementioned method in the preparation of antitumor drugs, wherein the tumor includes at least one of solid tumors and hematologic malignancies.

[0037] It should be noted that the antitumor drug prepared by carbon dots provided by this invention has biological molecular regulatory functions.

[0038] For nanozyme-based tumor catalytic therapy, the oxidative stress of tumor cells themselves is a significant factor hindering the effectiveness of nanozymes. The PI3K / AKT signaling pathway in tumor cells is one of the core mechanisms for resisting oxidative stress, maintaining survival, and promoting proliferation. Abnormal and persistent activation of this pathway is crucial for tumor cell survival in high-pressure, hypoxic, and highly reactive oxygen species (ROS) microenvironments. The PI3K / AKT pathway systematically enhances the tumor cell's ability to scavenge, repair, and survive oxidative damage through a multi-target, network-like approach. Therefore, inhibiting this pathway has become an important strategy for improving the efficacy of ROS-dependent antitumor therapies (including nanozyme therapy). When developing novel carbon-based nanozymes, considering whether they can interfere with or inhibit survival-promoting pathways such as PI3K / AKT, or in combination with pathway inhibitors, is one of the key approaches to overcoming tumor oxidative defenses and achieving more effective tumor killing.

[0039] In optional implementations, at least one of the following application methods is included: (1) Carbon dots downregulate the expression of the Csf1 gene in tumor cells; (2) Carbon dots downregulate the activity of the PI3K / AKT pathway in tumor cells; further reduce the antioxidant capacity of cells and further enhance the cell killing effect of ROS (in vitro reactive oxygen species) generated by nanozymes.

[0040] Carbon dots can further inhibit the PI3K / AKT pathway by downregulating the Csf1 gene, leading to downregulation of Nfe2l2 expression, thereby weakening the antioxidant capacity of tumor cells and enhancing the tumor-killing effect of nanozymes mediated by ROS. Therefore, the carbon dot N-CDs of this invention possess the dual functions of enzyme-like catalytic activity and bioregulatory capabilities.

[0041] (3) Carbon dots generate reactive oxygen species at the cellular level; the carbon dots of the present invention can be used as nanozymes similar to peroxidase (POD) and oxidase (OXD) to generate reactive oxygen species at the cellular level.

[0042] In optional embodiments, the solid tumor is selected from liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, pancreatic cancer, nasopharyngeal carcinoma, lung cancer, gastric cancer, adrenocortical carcinoma, adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, atypical teratoma, rhabdomyosarcoma, basal cell carcinoma, bile duct carcinoma, bladder cancer, bone cancer, brain tumor, bronchial tumor, Burkitt lymphoma, carcinoid tumor, cardiac tumor, bile duct epithelial carcinoma, chordoma, colorectal cancer, craniopharyngioma, ductal carcinoma in situ, germinal tumor, etc. Endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, intracranial germ cell tumors, gonadal germ cell tumors, eye cancer, fallopian tube cancer, gallbladder cancer, head and neck cancer, hypopharyngeal cancer, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, lip cancer, oral cancer, Merkel cell carcinoma, malignant mesothelioma, multiple endocrine syndrome, mycosis fungoides, nasal and sinus cancer, neuroblastoma, non-small cell lung cancer, ovarian cancer, pancreatic neuroendocrine tumors, islet cell tumors, papilloma, paraganglioma. At least one of the following: sinus and nasal cavity carcinoma, parathyroid carcinoma, penile cancer, pharyngeal cancer, pituitary adenoma, pleural blastoma, primary peritoneal carcinoma, retinoblastoma, salivary gland tumor, sarcoma, Cézare syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, testicular cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, endometrial and uterine sarcoma, vaginal cancer, vascular tumor, vulvar cancer, and single myeloma.

[0043] In an optional embodiment, the hematologic malignancy is selected from at least one of B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), B-cell prolymphoblastic leukemia, blastic plasmacytoid dendritic cell tumor, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell-follicular lymphoma, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell tumor, Waldenstrom's macroglobulinemia, and preleukemia.

[0044] Fourthly, embodiments of the present invention provide an antitumor drug comprising carbon dots as described above or carbon dots prepared by the methods described above and pharmaceutically acceptable excipients.

[0045] In an alternative embodiment, the pharmaceutically acceptable excipient is selected from at least one of desiccants, antioxidants, stabilizers, binders, dispersants, fillers, buffers, and coating materials.

[0046] In optional embodiments, the dosage form of the antitumor drug is selected from at least one of capsules, tablets, microcapsules, injections, suppositories, sprays, powders, soft capsules, drop pills, honey pills, pills, granules, honey-infused pastes, sustained-release preparations, oral liquid preparations, chewable tablets, oral tablets, transdermal patches, and effervescent tablets.

[0047] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0048] Example 1 This embodiment provides a method for preparing carbon dots, mainly including two processes: crosslinking and carbonization. The specific implementation steps are as follows, and a schematic diagram of the preparation process can be found in [the following diagram]. Figure 2 : A nitrogen-containing heterocyclic aromatic compound (citric acid, 0.5 g), an acrylamide derivative (N,N-methylenebisacrylamide, 0.5 g), and ethylenediamine (3 mL) were dissolved in 30 mL of double-distilled water. The solution was then subjected to a solvothermal reaction in a microwave oven and heated at 1000 W for 5 min. After cooling to room temperature, the resulting solid product was dissolved in 30 mL of double-distilled water to prepare a suspension.

[0049] The suspension was purified in a dialysis bag (molecular weight cutoff of 500 Da) for 72 h. Finally, the aqueous solution in the dialysis bag was collected, lyophilized, and carbon dots were obtained, denoted as N-CDs.

[0050] Example 2 This embodiment provides a method for preparing carbon dots, the implementation steps of which are the same as those in Embodiment 1, the only difference being: The volume of ethylenediamine is 4 mL.

[0051] Example 3 This embodiment provides a method for preparing carbon dots, the implementation steps of which are the same as those in Embodiment 1, the only difference being: The volume of ethylenediamine is 5 mL.

[0052] Example 4 This embodiment provides a method for preparing carbon dots, the implementation steps of which are the same as those in Embodiment 1, the only difference being: The power of the solvothermal reaction is 500W.

[0053] Example 5 This embodiment provides a method for preparing carbon dots, the implementation steps of which are the same as those in Embodiment 1, the only difference being: The power of the solvothermal reaction is 1500W.

[0054] Example 6 This embodiment provides a method for preparing carbon dots, the implementation steps of which are the same as those in Embodiment 1, the only difference being: The solvothermal reaction time is 2 minutes.

[0055] Example 7 This embodiment provides a method for preparing carbon dots, the implementation steps of which are the same as those in Embodiment 1, the only difference being: The solvothermal reaction time is 10 min.

[0056] Comparative Example 1 This comparative example provides a method for preparing carbon dots, the implementation steps of which are the same as those in Example 1, the only difference being: The acrylamide derivative is missing. The resulting carbon dots are denoted as CDs1.

[0057] Comparative Example 2 This comparative example provides a method for preparing carbon dots, the implementation steps of which are the same as those in Example 1, the only difference being: A nitrogen-containing heterocyclic aromatic compound is missing. The resulting carbon dots are denoted as CDs2.

[0058] Test Example 1 This test example describes the morphology of the N-CDs prepared in Example 1 using transmission electron microscopy (TEM) and dynamic light scattering (DLS) measurements. The results are as follows: Figure 3 As shown: (a) TEM topography image; (b) dynamic light scattering.

[0059] from Figure 3 It can be seen that N-CDs are uniformly distributed in aqueous solution. High-resolution transmission electron microscopy (HRTEM) images show that N-CDs have lattice fringes of 0.21 nm, attributed to the (100) crystal plane of graphene. TEM and DLS results show that the particle size of N-CDs is approximately 8.3 nm.

[0060] Test Example 2 This test example performs UV absorption and fluorescence tests on the N-CDs prepared in Example 1, CDs1 obtained in Comparative Example 1, and CDs2 obtained in Comparative Example 2, respectively. The test results are shown in the figure. Figure 4 (a) CDs1 prepared in Comparative Example 1; (b) CDs2 prepared in Comparative Example 2; (c) N-CDs prepared in Example 1.

[0061] from Figure 4 It can be seen that the absorption of CDs1 in Comparative Example 1 and CDs2 in Comparative Example 2 is concentrated in the ultraviolet region, and the two-dimensional fluorescence spectra show that the emission centers of both are below 500 nm. In contrast, the N-CDs of Example 1 have a wide absorption range between 600 nm and 800 nm, and have two emission centers at 538 nm and 618 nm, indicating that the N-CDs prepared in Example 1 have a different structure from CDs1 in Comparative Example 1 and CDs2 in Comparative Example 2.

[0062] Test Example 3 In this test case, N-CDs prepared in Example 1, CDs1 prepared in Comparative Example 1, and CDs2 prepared in Comparative Example 2 were subjected to nuclear magnetic resonance (NMR) analysis. 1 H NMR) test analysis, relevant results are shown in […]. Figure 5 (a) CDs1 prepared in Comparative Example 1; (b) CDs2 prepared in Comparative Example 2; (c) N-CDs prepared in Example 1.

[0063] from Figure 5 As can be seen, the 1H NMR spectrum of CDs1 in Comparative Example 1 shows a chemical shift of a hydrogen atom on the pyridine ring, which can act as an electron acceptor. The 1H NMR spectrum of CDs2 in Comparative Example 2 shows a strong chemical shift of an amino hydrogen atom. Moreover, the chemical shift of 6 ppm-7 ppm is attributed to a hydrogen atom on the benzene ring, indicating the presence of a conjugated system in CDs2 of Comparative Example 2 after microwave reaction.

[0064] The 1H NMR spectrum of the N-CDs in Example 1 showed the presence of an amino shift signal (6 ppm-9 ppm), a pyridine peak, and an aromatic hydrogen shift (6 ppm-7 ppm). These results indicate that only the N-CDs in Example 1 contain a pyridine ring as an electron acceptor and an amino group connected to the conjugated system as an electron donor.

[0065] Test Example 4 This test case characterizes the structure of the N-CDs prepared in Example 1. The relevant results are shown in [link to relevant documentation]. Figure 6 (a) Raman spectrum; (b) FT-IR spectrum; (c) XPS full spectrum; (d) N1s high-resolution spectrum of N-CDs; (e) C1s high-resolution spectrum of N-CDs; (f) O1s high-resolution spectrum of N-CDs.

[0066] from Figure 6 It can be seen that the Raman spectrum (a) of N-CDs is at 1570 cm⁻¹ -1 (G-band) and 1370 cm -1 Two are shown near the (D band), corresponding to sp respectively. 2 E2g vibration modes and sp domain 2 Structural defects or partially disordered structures within the domain. Higher G-band intensity indicates spc in N-CDs. 2 It has a rich domain content.

[0067] The chemical structure was characterized using Fourier transform infrared spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS). In the FT-IR spectrum (b), N-CDs are visible at 1555 cm⁻¹. -1 and 1441 cm -1 An absorption band is observed at 3270 cm⁻¹, corresponding to the stretching vibration of the pyridine ring, confirming the presence of an electron-withdrawing pyridine group in N-CDs. -1 1358 cm -1 and 1298 cm -1 The absorption bands at these locations correspond to NH, OH, and the aromatic amine CN, respectively, which can serve as evidence of an amino-rich electron donor.

[0068] Comprehensive XPS (c) spectroscopy revealed a nitrogen-rich N-CD composition of 14.17 at.%. Characteristic peaks for pyridine nitrogen (398.9 eV), amino nitrogen (399.6 eV), and pyrrole nitrogen (400.1 eV) were observed in the N1s spectrum (d), with pyridine nitrogen being dominant, accounting for approximately 50% of all nitrogen species. Characteristic peaks for C═C, C─C (284.4 eV), C─N, C─O (285.5 eV), and C═O (287.5 eV) were observed in the C1s spectrum (e). Peaks for C═O (530.9 eV) and C─O, O─H (531.8 eV) were observed in the O1s spectrum (f). These results strongly confirm the pyridine-activated donor-acceptor structure of the N-CDs.

[0069] Test Example 5 The superoxide radical generation capacity of N-CDs prepared in Example 1, CDs1 prepared in Comparative Example 1, and CDs2 prepared in Comparative Example 2 was tested using electron paramagnetic resonance (EPR) technology.

[0070] It should be noted that 5,5-dimethyl-1-pyrrolino-N-oxide (DMPO) is a free radical scavenger (purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., item number 222-011-1). In aqueous solution, it can capture hydroxyl radicals and exhibits four characteristic signals with an intensity ratio of 1:2:2:1; in dimethyl sulfoxide (DMSO) solution, it can capture superoxide anions and exhibit four characteristic signals with an intensity ratio of 1:1:1:1. Electron paramagnetic resonance (EPR) spectroscopy was used to detect the EPR spectra of DMPO (1%) + CDs1 (10 mg / mL), DMPO (1%) + CDs2 (10 mg / mL), and DMPO (1%) + N-CDs (10 mg / mL) in hydrogen peroxide aqueous solution and DMSO, respectively. The relevant results are shown in […]. Figure 7 (a) Hydrogen peroxide aqueous solution; (b) DMSO solution.

[0071] from Figure 7 It can be seen that CDs1 in Comparative Example 1 and CDs2 in Comparative Example 2 did not produce any hydroxyl radicals or superoxide anion signals in hydrogen peroxide aqueous solution and DMSO, but N-CDs in Example 1 showed strong response signals in both solutions, indicating that N-CDs can catalyze the generation of reactive oxygen species by hydrogen peroxide or oxygen.

[0072] Test Example 6 This test example examines the reactive oxygen species (ROS) generation capacity of the N-CDs prepared in Example 1.

[0073] It should be noted that 2,7-dichlorofluorescein (DCFH) is a reactive oxygen species (ROS) probe (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number H131224). It is non-fluorescent itself, but emits green fluorescence after being oxidized by ROS. Therefore, its fluorescence signal can be detected to determine its ROS generation capacity. Fluorescence spectra of DCFH (4 μM) and N-CDs (5 ppm) in different solvents were detected using a fluorescence spectrometer in the range of 490 nm to 650 nm. Quantitative analysis was performed using the fluorescence intensity at 525 nm. The relevant results are shown in […]. Figure 8 .

[0074] from Figure 8It can be seen that the fluorescence intensity of the aqueous solution containing both N-CDs and DCFH is stronger than that of the aqueous solution containing DCFH and carbon dots themselves, indicating that N-CDs may catalyze the generation of reactive oxygen species in the aqueous solution. Replacing the aqueous solution with an aqueous solution of hydrogen peroxide further enhances the fluorescence intensity of DCFH, which may be due to the simultaneous catalysis of hydrogen peroxide by N-CDs to generate hydroxyl radicals.

[0075] Test Example 7 This test used mouse colorectal cancer cells (CT26) as a tumor cell model to test the ability of N-CDs prepared in Example 1 to generate reactive oxygen species (ROS) in cells. In vitro ROS detection was performed using confocal laser scanning microscopy (CLSM). Specifically, CT26 cells were seeded in confocal culture dishes and cultured overnight. Afterward, the culture medium was replaced with 1 mL of fresh medium containing PBS solution and 200 μg / mL N-CDs. After 2 hours of incubation, the culture medium was replaced with 1 mL of fresh serum-free medium containing 10 μM DCFH, and incubation continued at 37 °C for 30 minutes. Cells were then washed with fresh serum-free medium, and the fluorescence intensity at 525 nm was measured using CLSM imaging or a microplate reader. The results are shown in [link to relevant results]. Figure 9 (a) ROS fluorescence intensity; (b) CLSM imaging and mitochondrial membrane potential detection.

[0076] from Figure 9 It can be seen that after treatment with N-CDs under dark conditions, the intracellular DCFH fluorescence intensity increased with the increase of N-CDs concentration, indicating that N-CDs have nanozyme activity and can generate ROS at the cellular level. Specifically, the increase in fluorescence intensity of the hydroxyl radical-specific probe hydroxyphenyl fluorescein (HPF) and the commercial probe superoxide anion clearly confirms that N-CDs can act as nanozymes similar to peroxidase (POD) and oxidase (OXD) to generate reactive oxygen species at the cellular level.

[0077] Furthermore, the use of JC-1 to detect mitochondrial membrane potential confirmed that the cytotoxicity of N-CDs was mediated through a ROS-induced mitochondrial pathway. This was confirmed by a significant increase in the green fluorescence of JC-1 monomers, indicating mitochondrial depolarization following N-CD treatment.

[0078] Test Example 8 In this test case, cells were treated with N-CDs prepared in Example 1 for 24 h. All RNA from the cells was collected, and RNA expression and pathway enrichment were analyzed. The relevant results are shown below. Figure 10(a) GO functional enrichment analysis of significantly altered gene sets; (b) GSEA enrichment analysis of reactive oxygen species pathways after N-CDs treatment; (c) Volcano plot comparing CT26 cells in N-CDs-treated and PBS-treated groups; (d) Csf1 / Csf1r / Pik3r1 / Akt1 / Nfe2l2 pathway network; (e) GSEA enrichment analysis of PI3K / AKT pathway after N-CDs treatment; (f) Heatmap of changes in Csf1 / Csf1r / Pik3r1 / Akt1 / Nfe2l2 gene expression in CT26 cells in N-CDs-treated and PBS-treated groups.

[0079] In gene set enrichment analysis (GSEA)(b), the reactive oxygen species (ROS) pathway was significantly upregulated, with a normalized enrichment score (NES) of 2.011 (p = 0.002), which is considered further strong evidence that N-CDs regulate cellular ROS. Then, statistical data on all altered genes were collected, among which colony-stimulating factor 1 (Csf1), one of the most significantly downregulated genes, attracted attention. Csf1 is a cytokine that is autocrine in certain cell lines (e.g., colorectal cancer cell lines). It binds to its receptor Csf1r and regulates the cellular antioxidant molecule Nfe2l2 through the PI3K / AKT pathway. Therefore, the enrichment of the PI3K / AKT pathway was examined. The results showed an NES value of -1.840 (p = 0.000), indicating that N-CDs can downregulate the PI3K / AKT pathway, thereby reducing cellular antioxidant capacity and further enhancing the cytotoxic effect of ROS generated by nanozymes.

[0080] Furthermore, we analyzed the specific expression of genes related to the PI3K / AKT pathway, and the results showed that all genes were significantly downregulated, ultimately leading to the downregulation of the key cellular antioxidant gene Nfe2l2. Based on these results, we elucidated the mechanism by which N-CDs exert ROS-induced cell killing at the cellular level. On the one hand, pyridine-activated donor-acceptor N-CDs can act as POD and OXD nanozymes to generate reactive oxygen species. In addition, the chemical structure of N-CDs endows them with biological activity. At the cellular level, N-CDs can further inhibit the PI3K / AKT pathway by downregulating the Csf1 gene, leading to downregulation of Nfe2l2 expression, thereby weakening the antioxidant capacity of tumor cells and enhancing the nanozyme-mediated ROS-induced tumor killing effect. These results indicate that N-CDs possess a dual function of enzyme-like catalytic activity and bioregulatory capabilities.

[0081] Test Example 9 This test case used the Cell Count Kit-8 (CCK-8) method to assess the in vitro cytotoxicity of N-CDs. Briefly, 200 μL of culture medium containing CT26 cells was diluted with 1 × 10⁻⁶ cells per well. 5Cells were seeded at a density of 100 μL in 96-well plates and cultured overnight. The medium was then replaced with 100 μL of fresh medium containing different concentrations (0-1000 μg / mL) of N-CD. After 24 hours of culture, the medium was replaced with 100 μL of fresh medium containing 10% CCK-8. After 2 hours of incubation, absorbance was measured at 450 nm using a microplate reader. Calcein-AM and propidium iodide (PI) were used for double staining of live and dead cells to distinguish between them. Calcein-AM is a fluorescently labeled reagent for live cells; it can penetrate the cell membrane and emits strong green fluorescence after hydrolysis within the cell. Propidium iodide (PI) cannot penetrate the cell membrane but can enter dead cells and intercalate into their DNA, thus emitting red fluorescence. By using these two reagents simultaneously, live and dead cells can be distinguished under a fluorescence microscope: live cells show green fluorescence, and dead cells show red fluorescence. The relevant results are shown in [link to results]. Figure 11 (a) Cell viability assay results; (b) Dead / live double staining image.

[0082] from Figure 11 As can be seen from the results, (a) cell viability assays showed a direct dose-dependent relationship between N-CDs exposure and cancer cell death, clearly linking the observed cytotoxicity to ROS generated by N-CDs. (b) Double staining of live and dead cells also showed similar results.

[0083] Test Case 10 This test case established a subcutaneous tumor model in mice. Mice were randomly divided into 4 groups (n=5) and treated for 15 days. To establish the CT26 tumor-bearing mouse model, 1×10⁻⁶ tumor cells were used... 5 One CT26 cell (100 μL PBS solution) was subcutaneously injected into each mouse. Animals were housed in an SPF-grade animal facility at 22°C with a 12-hour light / 12-hour dark cycle and free access to rodent food and water. After 7 days, when the tumor volume reached approximately 50 mm... 3 The medication was administered intravenously every other day for a total of four injections. The concentration of N-CDs was 1 mg / mL, and the injection volume was 200 μL. Relevant results are shown in [link to relevant results]. Figure 12 (a) Tumor volume; (b) Tumor weight.

[0084] from Figure 12 It can be seen that after 15 days of treatment, N-CDs showed a significant tumor-suppressing effect, indicating that N-CDs can also exert catalytic performance in vivo by generating reactive oxygen species and downregulating the PI3K / AKT pathway to achieve tumor killing.

[0085] In summary, the carbon dots provided in this invention comprise a pyridine-mediated electron donor-acceptor structure formed from a nitrogen-containing heterocyclic aromatic compound, an acrylamide derivative, and ethylenediamine. This structure enhances charge transfer, improving the catalytic performance of antitumor nanozymes prepared from these carbon dots, filling a gap in the current rational design guidance for carbon-based nanozymes. The carbon dots possess dual catalytic activity, resembling both peroxidases and oxidases, and can efficiently generate reactive oxygen species (ROS). This can downregulate the bioregulatory function of the PI3K / AKT signaling pathway, enabling direct intervention on the antioxidant defense system of tumor cells and amplifying the ROS-mediated killing effect, thus filling the current gap in the transformation between the chemical structure and biological function of carbon dots. The carbon dot preparation method is simple, requiring no complex reaction processes or operations, resulting in low production costs and a simple production process, making it suitable for large-scale production.

[0086] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A carbon dot, characterized by, The carbon dots include pyridine-mediated electron donor-acceptor structures; The electron donor-acceptor structure is formed from a nitrogen-containing heterocyclic aromatic compound, an acrylamide derivative, and ethylenediamine.

2. The carbon dot of claim 1, wherein, The ratio of the nitrogen-containing heterocyclic aromatic compound, the acrylamide derivative, and the ethylenediamine is (0.1-1) g : (0.1-1) g : (2-8) mL; Preferably, the ratio of the nitrogen-containing heterocyclic aromatic compound, the acrylamide derivative, and the ethylenediamine is (0.3-0.7) g: (0.3-0.7) g: (3-5) mL.

3. The carbon dot of claim 1, wherein, The nitrogen-containing heterocyclic aromatic compound is selected from at least one of citric acid, melamine, cyanuric acid diamide, trichloroisocyanuric acid, and nicotinic acid; And / or, the acrylamide derivative is selected from at least one of N,N-methylenebisacrylamide, N,N-(1,2-dihydroxyethylene)bisacrylamide, N,N-methylenebismethylacrylamide, N-isopropylacrylamide and N-hydroxymethylacrylamide; And / or, the particle size of the carbon dots is 6nm-10nm.

4. A method for preparing carbon dots according to any one of claims 1 to 3, characterized in that, Includes the following steps: A nitrogen-containing heterocyclic aromatic compound, an acrylamide derivative, ethylenediamine, and water are mixed in a certain proportion and subjected to a solvothermal reaction under a power of 200W-2000W to obtain carbon dots.

5. The production method according to claim 4, characterized by, The power of the solvothermal reaction is 850W-1200W, and the time is 3min-10min; And / or, the amount of water used is 25mL-50mL, and the water is selected from at least one of ultrapure water, double-distilled water, deionized water and purified water; And / or, the process further includes post-processing after the solvothermal reaction is completed, including purification and lyophilization. And / or, the purification process takes 24-72 hours and uses a dialysis bag with a molecular weight cutoff of 500-1000 Da.

6. Use of the carbon dots according to any one of claims 1-3 or the carbon dots prepared by the method according to any one of claims 4-5 in the preparation of an antitumor drug, characterized in that, The tumor includes at least one of solid tumors and hematologic malignancies.

7. Use according to claim 6, characterized in that, Including at least one of the following application methods: (1) Carbon dots downregulate the expression of the Csf1 gene in tumor cells; (2) Carbon dots downregulate the activity of the PI3K / AKT pathway in tumor cells; (3) Carbon dots generate reactive oxygen species at the cellular level.

8. Use according to claim 6, characterized in that, The solid tumors mentioned are selected from liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, pancreatic cancer, nasopharyngeal carcinoma, lung cancer, gastric cancer, adrenocortical carcinoma, adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, atypical teratoma, rhabdomyosarcoma, basal cell carcinoma, bile duct carcinoma, bladder cancer, bone cancer, brain tumor, bronchial tumor, Burkitt lymphoma, carcinoid tumor, cardiac tumor, bile duct epithelial carcinoma, chordoma, colorectal cancer, craniopharyngioma, ductal carcinoma in situ, germinal tumor, and endometrial cancer. Ependymoma, esophageal cancer, olfactory neuroblastoma, intracranial endoderm tumors, gonadal germ cell tumors, eye cancer, fallopian tube cancer, gallbladder cancer, head and neck cancer, hypopharyngeal cancer, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, lip cancer, oral cancer, Merkel cell carcinoma, malignant mesothelioma, multiple endocrine syndrome, mycosis fungoides, nasal and sinus cancer, neuroblastoma, non-small cell lung cancer, ovarian cancer, pancreatic neuroendocrine tumors, islet cell tumors, papilloma, paraganglioma. At least one of the following: sinus and nasal cavity carcinoma, parathyroid carcinoma, penile cancer, pharyngeal cancer, pituitary adenoma, pleural pulmonary blastoma, primary peritoneal carcinoma, retinoblastoma, salivary gland tumor, sarcoma, Cézare syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, testicular cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, endometrial and uterine sarcoma, vaginal cancer, vascular tumor, vulvar cancer, and single myeloma; And / or, the hematologic malignancy is selected from at least one of B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), B-cell prolymphoblastic leukemia, blastic plasmacytoid dendritic cell tumor, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell-follicular lymphoma, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell tumor, Waldenstrom macroglobulinemia, and preleukemia.

9. An antitumor drug, characterized in that, It includes carbon dots as described in any one of claims 1-3 or carbon dots prepared by the preparation method as described in any one of claims 4-5, and pharmaceutically acceptable excipients.

10. The antitumor drug according to claim 9, characterized in that, The pharmaceutically acceptable excipients are selected from at least one of the following: desiccants, antioxidants, stabilizers, binders, dispersants, fillers, buffers, and coating materials; And / or, the dosage form of the antitumor drug is selected from at least one of capsules, tablets, microcapsules, injections, suppositories, sprays, powders, soft capsules, drop pills, honey pills, pills, granules, honey-infused pastes, sustained-release preparations, oral liquid preparations, chewable tablets, oral tablets, transdermal patches, and effervescent tablets.

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