Carbon dot assembly, preparation method therefor, and use thereof

Abstract

The application belongs to the technical field of carbon nanomaterials, and particularly relates to a carbon dot assembly, a preparation method and application thereof. The application provides a carbon dot assembly. Compared with traditional carbon dots, a donor-acceptor structure promotes the charge separation process in the carbon dot assembly, so that electrons can be transferred from the core as an electron donor to the interface as an electron acceptor, and then interact with the surrounding oxygen to promote the generation of superoxide free radicals and photo-generated holes. The carbon dot assembly provided by the application has excellent properties, can specifically generate type I reactive oxygen species, oxidize important biological substrates through photo-generated holes, and thus realize efficient anti-tumor photodynamic therapy. In addition, the carbon dot assembly has a larger particle size, and is more conducive to being targeted to tumor tissues through the enhanced permeability and retention effect of tumor tissues, and thus realizes efficient tumor killing.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of carbon nanomaterials, and particularly relates to a carbon dot assembly and a preparation method and application thereof. BACKGROUND

[0002] Photodynamic therapy is an effective tumor treatment method, which has the advantages of low side effects, controllable treatment time and space, good tissue selectivity, and small trauma. Photodynamic therapy relies on the active oxygen generated by the reaction of photosensitizers with surrounding substrates or oxygen under light irradiation. According to the types of active oxygen generated, photodynamic therapy can be divided into type I photodynamic therapy and type II photodynamic therapy. Type I photodynamic therapy mainly generates type I active oxygen, such as hydroxyl radicals and superoxide radicals, through electron or proton transfer; and type II photodynamic therapy mainly generates type II active oxygen, such as singlet oxygen, through charge transfer. Compared with type II photodynamic therapy, type I photodynamic therapy has less dependence on oxygen. Considering that the tumor tissue microenvironment has the characteristic of hypoxia, type I photodynamic therapy is more advantageous for tumor treatment. In addition, the active oxygen generated by photodynamic therapy can also effectively kill bacteria, and compared with traditional antibacterial therapy, photodynamic sterilization will not produce drug resistance.

[0003] Carbon dots (CDs) are a kind of zero-dimensional photoluminescent carbon nanomaterials with a size of less than 10 nm, which were first discovered in 2004. Because of its simple preparation, easy availability of raw materials, low cost, good water solubility and biocompatibility, it is widely used in various fields such as drug delivery, photothermal therapy and photodynamic therapy of tumors. However, in the aspect of photodynamic therapy of tumors, most of the carbon dots reported at present generate singlet oxygen, play a role in type II photodynamic therapy, and have poor treatment effect on tumors with hypoxic microenvironment. Effective charge transfer is the key to the generation of active oxygen, and the ability of molecules or particles to generate type I active oxygen can be significantly improved by enhancing charge transfer. However, there is still a lack of effective strategies to enhance the charge transfer inside carbon dots.

[0004] Therefore, it is urgent to develop a strategy to promote the charge transfer inside carbon dots, so as to make carbon dots specifically generate type I active oxygen, and further apply them to the photodynamic therapy of tumors with hypoxic microenvironment and microbial infection.

[0005] In view of this, the present application is proposed. SUMMARY

[0006] The present application aims at at least solving one of the above technical problems existing in the prior art.

[0007] To this end, the principle of the present application is:

[0008] The application provides a donor-acceptor type carbon dot assembly, the structure of the carbon dot assembly has carbon dots gathered in a core as an electron donor and an interface formed by fused molecules containing electron-withdrawing groups as an electron acceptor, and the interface between different carbon dots and the outer layer of the carbon dots are both composed of fused molecules containing electron-withdrawing groups. Charge separation can occur efficiently inside the carbon dot assembly with this structure, promoting light-induced electron transfer. Under light conditions, the excited electrons are transferred to the interface of the carbon dot assembly and further transferred to the surrounding oxygen, generating a large amount of superoxide free radicals. At the same time, the photo-generated holes are stabilized in the core of the carbon dot assembly and have a certain oxidation ability, which can oxidize important biological substrates. The above carbon dot assembly is obtained through the donor-acceptor strategy, which can specifically generate type I reactive oxygen species - superoxide free radicals, and promotes the application of carbon dots in tumor treatment.

[0009] It can be seen that the core of the application is the electron transfer between the interface between the carbon dot core and the electron acceptor material, rather than the material composition or particle size of the carbon dot, or the specific type of electron acceptor material. Those skilled in the art should know that carbon dots prepared by other methods can also be prepared into carbon dot assemblies by the method of the application. Other types of solvents can also be used to prepare the carbon dots, such as the formic acid carbon nanodots, acetic acid carbon nanodots and acetone carbon nanodots prepared by different solvents in CN116173209A, which can also be used to prepare the carbon dot assemblies of the application. Further, carbon dots prepared from other raw materials other than citric acid and urea can also be used to prepare the carbon dot assemblies of the application. Similarly, electron acceptor materials with other electron-withdrawing groups can form carbon dot assemblies with similar electron acceptor material interfaces by the preparation method of the application, and also have similar technical effects of the application.

[0010] The first aspect of the application aims to provide a carbon dot assembly.

[0011] The second aspect of the application aims to provide a preparation method of a carbon dot assembly.

[0012] The third aspect of the application aims to provide an application of a carbon dot assembly.

[0013] The fourth aspect of the application aims to provide an antiseptic.

[0014] The fifth aspect of the application aims to provide a medicine.

[0015] In order to achieve the above-mentioned purposes of the application, the technical scheme adopted by the application is:

[0016] In a first aspect, the present invention provides a carbon dot assembly comprising an electron donor core and electron acceptor interfaces between and on the surface of the electron donor core; the electron donor core comprising carbon dots.

[0017] Preferably, the electron acceptor material is bonded to carbon dots via chemical bonds.

[0018] Preferably, the chemical bond includes at least one of covalent bond, ionic bond, and metallic bond.

[0019] In a first aspect of the invention, a carbon dot assembly refers to an aggregate of carbon dots formed by an electron acceptor material bonded to multiple carbon dots via covalent or non-covalent bonds. In this carbon dot assembly, the electron acceptor material encapsulates multiple carbon dots, and the mononoid-absorbing groups of the electron acceptor material form electron-accepting interfaces with the surfaces of different carbon dots. In this carbon dot assembly, the carbon dots and the electron acceptor material are tightly bonded, rather than being a simple physical mixture or dispersion.

[0020] Preferably, the electron acceptor material comprises a material containing electron-withdrawing groups.

[0021] Preferably, the electron-withdrawing group includes one or more of nitro, cyano, halogen, alkynyl, carboxyl, sulfonic acid, formyl, and acyl groups.

[0022] Preferably, the electron-withdrawing group includes a cyano group.

[0023] In one embodiment of the invention, the carbon dot assembly is formed by the combination of carboxyl groups on aggregated carbon dots and hydroxyl groups on a material containing electron-withdrawing groups via ester bonds.

[0024] In one embodiment of the present invention, the electron acceptor material comprises 2,3-dicyanohydroquinone.

[0025] Preferably, the particle size of the carbon dots is 1 to 10 nm.

[0026] Preferably, the particle size of the carbon dots is 1.5 to 3.5 nm.

[0027] The carbon dots in this invention have a large number of sp... 2 Carbon dots with conjugated domains and electron-donating properties.

[0028] In one embodiment of the present invention, the carbon dots are prepared by a solvothermal reaction of citric acid and urea.

[0029] Preferably, the mass ratio of citric acid to urea is 1:(1-5); more preferably, it is 1:(2-4).

[0030] Preferably, the solvent is at least one selected from N,N-dimethylformamide, dimethyl sulfoxide, formic acid, acetic acid, acetone, and N-methylpyrrolidone; more preferably, the solvent is N,N-dimethylformamide.

[0031] Preferably, the reaction temperature is 120–200°C and the reaction time is 0.5–24 h.

[0032] Preferably, the particle size of the carbon dot assembly is 15–30 nm; more preferably, it is 15–20 nm.

[0033] A second aspect of the present invention provides a method for preparing a carbon dot assembly according to the first aspect of the present invention, comprising the following steps:

[0034] The carbon dots and electron acceptor materials are mixed with the solution and reacted to obtain the desired result.

[0035] Preferably, the reaction includes at least one of solvothermal reaction, microwave reaction, deposition reaction, and calcination reaction.

[0036] Preferably, the reaction is a solvothermal reaction.

[0037] Preferably, the temperature of the solvothermal reaction is 40–200°C; and the time of the solvothermal reaction is 0.5–24 h.

[0038] Preferably, the solvent includes at least one of N,N-dimethylformamide, dimethyl sulfoxide, formic acid, acetic acid, acetone, and N-methylpyrrolidone; a mixture of two or more of these may also be selected, with no limit on the mixing ratio.

[0039] In one embodiment of the present invention, the carbon dot assembly is prepared by carbon dot solution and electron acceptor material under solvothermal conditions.

[0040] Preferably, the carbon dots are prepared using citric acid and urea as raw materials, and the mass ratio of citric acid to urea is 1:(1-5); more preferably, it is 1:(2-4).

[0041] Preferably, the mass ratio of the carbon dots to the electron acceptor material is 1:(0.1-3).

[0042] Preferably, the reaction temperature is 80–200°C and the reaction time is 0.5–24 h.

[0043] In some embodiments of the present invention, the reaction temperature of the solvothermal reaction is 80–200°C, for example, 80°C, 90°C, 100°C, 110°C, 120°C, 130°C, 140°C, 145°C, 150°C, 155°C, 160°C, 165°C, 170°C, 175°C, 180°C, 185°C, 190°C, 195°C, or 200°C; the reaction time is 0.5–24 h, for example, 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, or 24 h.

[0044] Furthermore, in some embodiments, the product obtained from the solvothermal reaction is purified by centrifugation at a speed of 8000 rpm for 10 minutes each time, for a total of three centrifugations.

[0045] A third aspect of the invention provides the application of the carbon dot assembly of the first aspect of the invention in 1) to 3):

[0046] 1) Preparation of reactive oxygen species;

[0047] 2) Preparation of bactericides;

[0048] 3) Preparation of anti-tumor drugs.

[0049] Preferably, the bactericide is a photocatalytic bactericide.

[0050] Preferably, the antitumor drug is a photocatalytic drug.

[0051] Preferably, the tumor comprises at least one of a solid tumor and a hematoma; more preferably, it comprises a solid tumor.

[0052] Preferably, the solid tumors include 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, appendiceal 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, endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, intracranial germinal tumor, gonadal germ cell tumor, eye cancer, fallopian tube cancer, gallbladder cancer, head and neck cancer, hypopharyngeal cancer, Kaposi's sarcoma, kidney cancer, and Langerhans cell histiocytoma. Cellular hyperplasia, laryngeal cancer, lip cancer, oral cancer, Merkel cell carcinoma, malignant mesothelioma, multiple endocrine neoplasia syndrome, mycosis fungoides, nasal cavity and sinus cancer, neuroblastoma, non-small cell lung cancer, ovarian cancer, pancreatic neuroendocrine tumor, islet cell tumor, papilloma, paraganglioma, sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pituitary adenoma, pleural pulmonary blastoma, primary peritoneal cancer, 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.

[0053] Preferably, 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.

[0054] In one embodiment of the present invention, the tumor is breast cancer.

[0055] Those skilled in the art will anticipate that, since oxidative stress is a common mechanism for treating cancer, reactive oxygen species can exert their therapeutic effects on tumors through multiple means, such as promoting tumor apoptosis, directly causing tumor cell necrosis, and promoting autophagic cell death in tumor cells. Therefore, since the prior art does not disclose specific tumor types that resist reactive oxygen species, the specific type of tumor will not affect the application of this invention.

[0056] A fourth aspect of the present invention provides a bactericide comprising the carbon dot assembly of the first aspect of the present invention.

[0057] Preferably, the bactericide includes other bactericidal components.

[0058] Preferably, the other bactericidal components include, but are not limited to, at least one of antibiotics and antimicrobial peptides.

[0059] Preferably, the bactericide further includes a pharmaceutically acceptable carrier or excipient.

[0060] A fifth aspect of the present invention provides an antitumor drug comprising a carbon dot assembly of the first aspect of the present invention.

[0061] Preferably, the drug also includes other antitumor drugs.

[0062] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0063] Preferably, the pharmaceutically acceptable excipients include, but are not limited to, at least one of desiccants, antioxidants, stabilizers, binders, dispersants, fillers, buffers, and coating materials.

[0064] Preferably, the dosage form of the drug includes at least one of the following: 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.

[0065] The beneficial effects of this invention are:

[0066] This invention provides a carbon dot assembly, consisting of a core formed by aggregated carbon dots with electron-donating properties and an interface formed by molecules containing electron-withdrawing groups. Compared to traditional carbon dots, the carbon dot assembly has a larger particle size, which is more conducive to its targeting of tumor tissue through enhanced permeability and retention effects characteristic of tumor tissue, thereby achieving highly efficient tumor killing. The carbon dot assembly provided by this invention has excellent properties, capable of specifically generating type I reactive oxygen species while oxidizing important biological substrates through photogenerated holes, thereby achieving highly efficient anti-tumor photodynamic therapy. Compared with traditional small molecule photosensitizers, the carbon dot assembly has better water solubility, which is more conducive to its application in organisms. The carbon dot assembly provided by this invention kills pathogenic microorganisms by generating reactive oxygen species through a photodynamic process. Its killing effect is not affected by the current drug resistance status of the microorganisms and does not induce drug resistance in the microorganisms. The donor-acceptor type carbon dot assembly provided by this invention is simple to prepare, directly by reacting carbon dots with electron-donating properties and electron acceptor materials containing electron-withdrawing groups under solvothermal conditions, without the need for complex reaction processes and operations. Attached Figure Description

[0067] 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.

[0068] Figure 1 This is a schematic diagram illustrating the preparation process of a donor-acceptor type carbon dot assembly according to one embodiment of the present invention.

[0069] Figure 2 The images show transmission electron microscopy (TEM) images (a, b) of supra-CDs from Example 1 of the present invention, TEM image (c) of CDs from Comparative Example 1, and their particle size distribution diagram (d).

[0070] Figure 3 The images show the ultraviolet-visible (UV-Vis) absorption spectra of supra-CDs from Example 1 of this invention, CDs from Comparative Example 1, the molecule DCHQ containing an electron-withdrawing group, and a simple mixture of CDs and DCHQ.

[0071] Figure 4 Fluorescence spectra of DCFH solutions containing reactive oxygen species probes after different illumination times (a), DCFH solutions containing CDs of Comparative Example 1 (b), and DCFH solutions containing supra-CDs of Example 1 (c), and their fluorescence intensity at 525 nm is quantitatively plotted (d).

[0072] Figure 5Fluorescence spectra of DHR123 solutions containing superoxide radical probes (a), DHR123 solutions containing CDs of Comparative Example 1 (b), and DHR123 solutions containing supra-CDs of Example 1 (c) after different illumination times, and quantitative fluorescence intensity at 528 nm (d).

[0073] Figure 6 Electron paramagnetic resonance spectra of DMSO solutions containing CDs and radical scavenger DMPO (comparative Example 1) and DMSO solutions containing supra-CDs and radical scavenger DMPO (Example 1) after different illumination times (a), and quantitative spectrum of their signal intensity at 335.6 mT (c).

[0074] Figure 7 Electron paramagnetic resonance (EPR) spectra of DMSO solutions of CDs in Comparative Example 1 (a) and supra-CDs in Example 1 (b) after different illumination times, and quantitative signal intensity diagrams at 337.5 mT (c); EPR spectra of DMSO solutions of supra-CDs in Example 1 after 5 min of illumination followed by cessation of illumination at different times (d) and quantitative signal intensity diagrams at 337.5 mT (e).

[0075] Figure 8 The UV-Vis absorption spectra of BH4 solution (a) and NADH solution (b) after different illumination times with the addition of supra-CDs from Example 1 are shown.

[0076] Figure 9 Cytotoxicity tests were conducted on mouse breast cancer cells (4T1) under dark and light conditions using CDs of different concentrations from Comparative Example 1 (a) and supra-CDs of Example 1 (b), as well as tests were conducted on the ability of CDs of Comparative Example 1 and supra-CDs of Example 1 to generate reactive oxygen species (c) and superoxide radicals (d) in 4T1 cells.

[0077] Figure 10 The growth curves of mouse tumors in different treatment groups are shown in (a), the tumor weight of mice 16 days after treatment is shown in (b), the photographs of mouse tumors 16 days after treatment are shown in (c), and typical comparison images of mouse tumors before and after treatment are shown in (d).

[0078] Figure 11 The antibacterial rate of supra-CDs of Example 1 at different concentrations was tested against Staphylococcus aureus under light conditions. Detailed Implementation

[0079] The following will describe the concept and technical effects of the present invention clearly and completely with reference to the embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0080] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0081] Example 1: A method for preparing a donor-acceptor type carbon dot assembly

[0082] This embodiment provides a method for preparing a donor-acceptor type carbon dot assembly, including the following steps:

[0083] 1 g of citric acid and 2 g of urea were dispersed in 10 mL of N,N-dimethylformamide and sonicated for 30 min to aid dissolution. The mixture was then placed in a high-pressure reactor with a polytetrafluoroethylene liner and subjected to a solvothermal reaction in a high-temperature oven at 180 °C for 4 h. After the reaction cooled to room temperature, 1 g of 2,3-dicyanohydroquinone (DCHQ) was added to the solution, and the solvothermal reaction was continued in a high-temperature oven at 180 °C for 1 h. After the reaction cooled to room temperature, the reaction solution was removed, and two volumes of anhydrous ethanol were added. The mixture was centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the precipitate was collected. The mixture was washed three times with anhydrous ethanol and the precipitate was collected. The precipitate was freeze-dried to obtain a black solid, which was the donor-acceptor type carbon dot assembly (supra-CDs).

[0084] Example 2

[0085] This embodiment provides a method for preparing a donor-acceptor type carbon dot assembly, which differs from Example 1 only in that the second solvothermal reaction time is 0.5 h.

[0086] Example 3

[0087] This embodiment provides a method for preparing a donor-acceptor type carbon dot assembly, which differs from Example 1 only in that the second solvothermal reaction time is 2 hours.

[0088] Example 4

[0089] This embodiment provides a method for preparing a donor-acceptor type carbon dot assembly, which differs from Example 1 only in that the temperature of the second solvothermal reaction is 160°C.

[0090] Example 5

[0091] This embodiment provides a method for preparing a donor-acceptor type carbon dot assembly, which differs from Example 1 only in that the temperature of the second solvothermal reaction is 140°C.

[0092] Example 6

[0093] This embodiment provides a method for preparing a donor-acceptor type carbon dot assembly, which differs from Example 1 only in that the temperature of the second solvothermal reaction is 120°C.

[0094] Example 7

[0095] This embodiment provides a method for preparing a donor-acceptor type carbon dot assembly, which differs from Example 1 only in that the temperature of the second solvothermal reaction is 100°C.

[0096] Example 8

[0097] This embodiment provides a method for preparing a donor-acceptor type carbon dot assembly, which differs from Example 1 only in that the temperature of the second solvothermal reaction is 80°C.

[0098] Example 9

[0099] This embodiment provides a method for preparing a donor-acceptor type carbon dot assembly, which differs from Example 1 only in that the mass of DCHQ added is 500 mg.

[0100] Example 10

[0101] This embodiment provides a method for preparing a donor-acceptor type carbon dot assembly, which differs from Example 1 only in that the mass of DCHQ added is 200 mg.

[0102] Comparative Example 1

[0103] This comparative example provides a method for preparing carbon dots, including the following steps:

[0104] 1 g of citric acid and 2 g of urea were dispersed in 10 mL of N,N-dimethylformamide and sonicated for 30 min to aid dissolution. The mixture was then placed in a high-pressure reactor with a polytetrafluoroethylene liner and subjected to a solvothermal reaction in a high-temperature oven at 180 °C for 4 h. After the reaction cooled to room temperature, the reaction solution was removed, and twice the volume of anhydrous ethanol was added. The mixture was centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the precipitate was collected. Anhydrous ethanol was added again, and the mixture was washed three times. The precipitate was then collected and freeze-dried to obtain a black solid, which was the carbon dots (CDs).

[0105] Example 1

[0106] The morphology of supra-CDs obtained in Example 1 and CDs obtained in Comparative Example 1 was measured by transmission electron microscopy (TEM) and dynamic light scattering (DLS). The results are as follows: Figure 2 As shown. Figure 2 These are TEM and DLS images of supra-CDs in Embodiment 1 and CDs in Comparative Example 1 of the present invention.

[0107] from Figure 2 It can be seen that CDs and supra-CDs are uniformly distributed in aqueous solution. High-resolution transmission electron microscopy (HRTEM) images show that CDs have lattice fringes of 0.21 nm, attributed to the (100) crystal plane of graphene. supra-CDs are composed of multiple aggregated CDs fused together and also have lattice fringes of 0.21 nm. DLS results show that the particle size of CDs is approximately 2.5 nm and that of supra-CDs is approximately 20 nm.

[0108] Example 2

[0109] UV absorption tests were performed on supra-CDs obtained in Example 1, CDs obtained in Comparative Example 1, raw material DCHQ, and mixtures of CDs and DCHQ.

[0110] Figure 3 The results showed that CDs had two characteristic absorption peaks at 416 nm and 550 nm, while DCHQ had a characteristic absorption peak at 348 nm. The mixture of CDs and DCHQ had characteristic absorption peaks of both CDs and DCHQ, while supra-CDs showed a new characteristic absorption peak at 389 nm, proving the successful synthesis of supra-CDs and that supra-CDs is not a simple physical mixture of CDs and DCHQ.

[0111] Example 3

[0112] The reactive oxygen species (ROS) generation capacity of supra-CDs obtained in Example 1 and CDs obtained in Comparative Example 1 was tested. 2,7-Dichlorofluorescein (DCFH) is an ROS probe; it is non-fluorescent itself but emits green fluorescence after being oxidized by ROS. Therefore, its ROS generation capacity can be determined by detecting its fluorescence signal. The DCFH used was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number H131224. The fluorescence spectra of DCFH (4 μM), DCFH (4 μM) + CDs (5 ppm), and DCFH (4 μM) + supra-CDs (5 ppm) in the range of 490 nm to 650 nm were detected by a fluorescence spectrometer after different times of white light irradiation. The fluorescence intensity at 525 nm was selected for quantitative analysis.

[0113] Figure 4 The results showed that the fluorescence of the DCFH solution itself did not increase significantly under light irradiation; however, in the presence of CDs, the fluorescence of the DCFH solution gradually increased after light irradiation, indicating that CDs can generate a small amount of reactive oxygen species; compared with CDs, the fluorescence enhancement of the DCFH solution after light irradiation was more obvious in the presence of supra-CDs, indicating that supra-CDs can generate more reactive oxygen species than CDs.

[0114] Example 4

[0115] The superoxide radical generating capacity of supra-CDs obtained in Example 1 and CDs obtained in Comparative Example 1 was tested. Dihydrorhodamine 123 (DHR23) is a superoxide radical probe. It is non-fluorescent itself, but emits green fluorescence after being oxidized by superoxide radicals. Therefore, its superoxide radical generating capacity can be determined by detecting its fluorescence signal. The DHR123 used was purchased from Shanghai Maokang Biotechnology Co., Ltd., catalog number MX4455. The fluorescence spectra of DHR123 (4μM), DHR23 (4μM)+CDs (5ppm), and DHR123 (4μM)+supra-CDs (5ppm) in the range of 510nm to 650nm were detected by a fluorescence spectrometer after white light irradiation for different times. The fluorescence intensity at 528nm was selected for quantitative analysis.

[0116] Figure 5 The results showed that the fluorescence of DHR123 solution itself did not increase significantly under light irradiation; however, in the presence of CDs, the fluorescence of DHR123 solution gradually increased after light irradiation, indicating that CDs can generate a small amount of superoxide radicals; compared with CDs, the fluorescence enhancement of DHR123 solution after light irradiation was more obvious in the presence of supra-CDs, indicating that supra-CDs can generate more superoxide radicals than CDs.

[0117] Example 5

[0118] The superoxide radical generating capacity of supra-CDs obtained in Example 1 and CDs obtained in Comparative Example 1 was tested using electron paramagnetic resonance (EPR) spectroscopy. 5,5-Dimethyl-1-pyrrolline-N-oxide (DMPO) is a radical scavenger that can capture superoxide radicals in dimethyl sulfoxide (DMSO) solution, exhibiting four characteristic signals with an intensity ratio of 1:1:1:1. The DMPO used was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., product number 222-011-1. The EPR spectra of DMPO (1%) + CDs (100 ppm) and DMPO (1%) + supra-CDs (100 ppm) after different times of white light irradiation were detected by EPR spectroscopy.

[0119] Figure 6 The results showed that, in the presence of CDs, the DMPO solution exhibited four characteristic signals of 1:1:1:1 after light irradiation, indicating that CDs could generate a small amount of superoxide radicals. Compared with CDs, in the presence of supra-CDs, the DMPO solution also exhibited four characteristic signals of 1:1:1:1 after light irradiation, but the signal intensity was significantly stronger, indicating that supra-CDs could generate more superoxide radicals than CDs.

[0120] Example 6

[0121] The photogenerated hole generation capability of supra-CDs obtained in Example 1 and CDs obtained in Comparative Example 1 was tested using electron paramagnetic resonance (EPR) technology.

[0122] Figure 7 The results showed that the DMSO solution of CDs had almost no EPR signal under illumination, while the DMSO solution of supra-CDs showed a gradually increasing EPR signal under illumination, indicating that supra-CDs can generate photogenerated holes under illumination. After 5 minutes of illumination, the EPR signal of the DMSO solution of supra-CDs was monitored at different time intervals. The results showed that the EPR signal of the DMSO solution of supra-CDs persisted even after the illumination was stopped. After 40 minutes, the EPR signal remained above 80% of the highest value. These results indicate that photogenerated holes can persist for a long time, proving that there is effective long-term charge separation within supra-CDs.

[0123] Example 7

[0124] Using tetrahydrobiopterin (BH4) and reduced coenzyme I (NADH) as representatives, the oxidation capacity of photogenerated holes produced by supra-CDs obtained in Example 1 was tested. BH4 was purchased from MCE (MedChemExpress), catalog number HY-107383; NADH was purchased from Beijing Solarbio Science & Technology Co., Ltd., catalog number IN0020. The visible-ultraviolet absorption spectra of supra-CDs (50ppm) + BH4 (100μM) and supra-CDs (50ppm) + NADH (100μM) after different times of white light irradiation were measured using a UV-Vis spectrophotometer.

[0125] Figure 8 The results showed that with prolonged illumination, the absorption peak of BH4 at 300 nm decreased, while the absorption peak of its oxidation product, dihydrobiopterin (BH2), at 280 nm increased, indicating that photogenerated holes generated by supra-CDs under illumination can oxidize BH4. The oxidation test results for NADH showed that with prolonged illumination, the absorption peak of NADH at 340 nm decreased, while the absorption peak of its oxidation product at 260 nm increased, indicating that photogenerated holes generated by supra-CDs under illumination can oxidize NADH. These results demonstrate that photogenerated holes generated by supra-CDs under illumination can oxidize some important biological substrates, thereby affecting cell growth.

[0126] Example 8

[0127] Using mouse breast cancer cells (4T1) as a tumor cell model, the killing ability of supra-CDs obtained in Example 1 and CDs obtained in Comparative Example 1 against tumor cells and their ability to generate reactive oxygen species in cells were tested. 4T1 cells were seeded at a density of 8000 cells per well in 96-well plates and incubated overnight in a cell culture incubator. The culture medium in the wells was then replaced with 100 μL of cell culture medium containing different concentrations (500, 250, 100, 50, 25, 10, 5, 0 ppm) of CDs or supra-CDs, respectively. After 12 h of culture, the light-treated group was illuminated with a white light flashlight for 10 min, and cultured for another 12 h. CCK-8 reagent was then added, and the OD value of each well at 450 nm was measured using a microplate reader, and cell viability was calculated.

[0128] Figure 9The results showed that both CDs and supra-CDs exhibited only weak cytotoxicity under dark conditions; under light conditions, both CDs and supra-CDs showed increasing cytotoxicity with concentration. At a concentration of 500 ppm, the cell viability after CDs treatment was approximately 36%, while the cell viability after supra-CDs treatment was only 4%, indicating that supra-CDs had a stronger tumor cell killing ability under light conditions. Subsequently, the ability of supra-CDs and CDs to generate reactive oxygen species (ROS) in tumor cells was measured. The ROS assay was performed in the following groups: supra-CDs, L (white light irradiation for 10 min), CDs+L, and supra-CDs+L. 4T1 cells were cultured at 3 × 10⁶ cells per well. 4 Cells were seeded at a density of 100 μL in 8-well chambers and incubated overnight in a cell culture incubator. The culture medium in each chamber was then replaced with 200 μL of cell culture medium containing 500 ppm CDs or supra-CDs, and cultured for another 12 h. After replacing the culture medium in the chambers with medium containing DCFH or DHR123, the light-illuminated groups were exposed to white light for 10 min, incubated in cell culture medium for 30 min, and then the green fluorescence of each group was observed using a confocal microscope. The results showed that under light conditions, CDs produced only a small amount of reactive oxygen species (ROS) in tumor cells, while supra-CDs produced a large amount of ROS, primarily superoxide radicals.

[0129] Example 9

[0130] BALB / c mice subcutaneously loaded with breast cancer cells (4T1) were used as an animal model to test the inhibitory effects of supra-CDs obtained in Example 1 and CDs obtained in Comparative Example 1 on tumor growth at the animal level. This was achieved by subcutaneously injecting mice with 100 μL of a solution containing 1×10⁻⁶ breast cancer cells (4T1). 6One week after treatment, mice were randomly divided into five groups: PBS, L (10 min white light irradiation), supra-CDs, CDs+L, and supra-CDs+L, with five mice in each group. Each group received a tail vein injection of 100 μL of PBS, CDs (10 mg / kg), or supra-CDs (10 mg / kg). Twelve hours later, the mice in the L, CDs+L, and supra-CDs groups received 10 min of white light irradiation. Tumor volume was measured for the next two days. On day 8 of treatment, mice were again injected via tail vein with 100 μL of PBS, CDs (10 mg / kg), or supra-CDs (10 mg / kg). Twelve hours later, the mice in the L, CDs+L, and supra-CDs groups received 10 min of white light irradiation, and tumor volume was continuously monitored. On day 16 of treatment, mice were sacrificed, and their tumors were dissected, weighed, and photographed. The animal experiments involved in this embodiment have been approved by the Ethics Committee of the Laboratory Animal Research Center of Southern University of Science and Technology, with approval number SUSTC-JY2019092.

[0131] Figure 10 The results showed that neither light exposure nor non-light exposure of supra-CDs had an inhibitory effect on tumor growth. CDs under light exposure could slow down the growth rate of tumors, while supra-CDs under light exposure could completely inhibit tumor growth.

[0132] In summary, this invention primarily provides a donor-acceptor type carbon dot assembly, its preparation method, and its applications. The donor-acceptor type carbon dot assembly supra-CDs is obtained by co-melting CDs with DCHQ molecules containing electron-withdrawing groups. supra-CDs consist of a core formed by aggregated CDs and an interface composed of DCHQ. The introduction of the electron-withdrawing molecule DCHQ promotes electron transfer within supra-CDs. Under light irradiation, excited electrons transfer from the core of supra-CDs to the interface and further react with oxygen in the surrounding environment to generate superoxide radicals; while long-lived photogenerated holes are stabilized in the core of supra-CDs, and these photogenerated holes can further oxidize some important biological substrates. Under the combined action of ultrasonic radicals and photogenerated holes, supra-CDs can effectively kill tumors under light irradiation. Using mouse breast cancer cells (4T1) as a tumor cell model and BALB / c mice subcutaneously loaded with 4T1 cells as an animal model, experimental results demonstrate the excellent anti-tumor therapeutic effect of supra-CDs.

[0133] Example 10

[0134] This example tested the antibacterial rate of different concentrations of supra-CDs against Staphylococcus aureus under light conditions. The specific operation was as follows: Staphylococcus aureus was cultured in a 37°C incubator using fresh Luria-Bertani (LB) medium until it reached the logarithmic growth phase. Then, it was washed three times with sterile PBS, centrifuged at 10,000 rpm for 2 min, and the absorbance at 600 nm was measured and adjusted to 1. Then, 400 μL of bacterial suspension containing different concentrations of supra-CDs was added to each well of a 48-well plate and irradiated with white light for 10 min. Supra-CDs at a concentration of 0 served as a control group. The plates were then incubated at 37°C for 120 min. Subsequently, 100 μL of the bacterial suspension was spread on LB agar plates, incubated overnight at 37°C, and the colony count was observed and counted.

[0135] The antibacterial rate is calculated as follows: Antibacterial rate = (number of colonies in the control group - number of colonies in the experimental group) / number of colonies in the control group × 100%.

[0136] The results are as follows Figure 11 As shown, the antibacterial rate gradually increased after white light irradiation with increasing supra-CDs concentration, exhibiting a dose-dependent effect. When supra-CDs reached 500 ppm, its antibacterial rate reached 90%.

Claims

1. A method for preparing a carbon dot assembly, characterized in that: The carbon dots are reacted with an electron acceptor material to obtain the carbon dot assembly. The mass ratio of the carbon dots to the electron acceptor material is 1:(0.1-3); The reaction is a solvothermal reaction; The temperature of the solvothermal reaction is 40–200°C; the time of the solvothermal reaction is 0.5–24 h. The electron acceptor material is 2,3-dicyanohydroquinone.

2. The method according to claim 1, characterized in that: The carbon dots were prepared by a solvothermal reaction of citric acid and urea. The mass ratio of citric acid to urea is 1:(1-5); The solvothermal reaction temperature is 120–200°C, and the reaction time is 0.5–24 h. The solvent is at least one selected from N,N-dimethylformamide, dimethyl sulfoxide, formic acid, acetic acid, acetone, and N-methylpyrrolidone.

3. A carbon dot assembly, prepared by the preparation method according to any one of claims 1 to 2.

4. The application of the carbon dot assembly according to claim 3 in 1) to 3): 1) Preparation of reactive oxygen species; 2) Preparation of bactericides; 3) Preparation of anti-tumor drugs.

5. The application according to claim 4, characterized in that: The bactericide is a photocatalytic bactericide; The antitumor drug is a photocatalytic drug.

6. A bactericide comprising the carbon dot assembly of claim 3; The bactericide also includes other bactericidal components; The bactericide also includes pharmaceutically acceptable carriers or excipients.

7. An antitumor drug comprising the carbon dot assembly of claim 3; The drugs also include other anti-tumor drugs; The drug also includes pharmaceutically acceptable excipients; The tumors include breast cancer.

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