Preparation method of amino-functionalized monomolecular graphene quantum dots and photodynamic therapy application thereof
Patent Information
- Application Number
- CN202510318387.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-18
- Publication Date
- 2026-09-18
AI Technical Summary
然而，相关产品的聚集程度和较差的溶解性是导致材料浪费的相关限制因素，同时内部结构复杂，无法得到精确的相对分子质量，其安全性和有效性难以评估，无法进入实际临床应用，因此，构建一种全新的合成方法，合成具有准确结构、纯净单一的GQDs，为突破GQDs在纳米载药系统中的机制进行精准调控以及批量稳定的生产十分关键
[0025] The technical solution provided by this invention breaks through the mental barriers of conventional synthesis methods, proposing a method for preparing amino-functionalized single-molecule graphene quantum dots. These quantum dots have accurate molecular structures, with a C core. 96 sp 2 A structure with a fully conjugated graphite core and edges precisely modified with 6NH2.
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Abstract
Description
Technical Field
[0001] This invention relates to a method for preparing amino-functionalized single-molecule graphene quantum dots (NH2-GQD) and their photodynamic therapy applications. Background Technology
[0002] Graphene quantum dots (GQDs) are a class of quasi-zero-dimensional carbon nanomaterials whose central structure consists of sp... 2 Hybridized carbon atoms are arranged in a honeycomb lattice. Due to their nanoscale lateral size, quantum quantum dots (GQDs) exhibit significant quantum confinement and edge effects, endowing them with tunable band gaps and excellent optical properties, such as photoluminescence and light absorption. The nanostructure and physicochemical properties of GQDs are typically influenced by multiple factors, including size, edge configuration, and solubility. Traditional methods synthesize GQDs as mixtures with indeterminate structures and diverse compositions. Without selectivity, precise and reasonable control over the size, morphology, and edge modification of the target product is impossible, hindering in-depth research on GQDs. Furthermore, the aggregation degree of GQDs synthesized by traditional methods in solution and their poor solubility in water lead to material waste. These problems pose challenges in multiple dimensions. Therefore, there is an urgent need to develop a practical and reliable synthetic strategy to achieve precise control over the size, morphology, or functional modification of the target structure. To date, researchers have conducted in-depth studies on the precise control of the size (1-10 nm) and morphology (spherical / hexagonal) of graphene quantum dots (GQDs). Simultaneously, through chemical doping (such as N and S doping) and surface modification (amino and carboxyl functionalization), GQDs have been endowed with tunable fluorescence emission, catalytic activity, and biocompatibility, significantly expanding their applications in bioimaging and optoelectronic devices. However, the high degree of aggregation and poor solubility of these products are limiting factors leading to material waste. Furthermore, their complex internal structures prevent the acquisition of precise relative molecular masses, making it difficult to assess their safety and efficacy and hindering their clinical application. Therefore, developing a novel synthetic method to synthesize GQDs with accurate structures and pure, singular shapes is crucial for achieving precise control of the mechanisms of GQDs in nanomedicine delivery systems and for their stable, large-scale production.
[0003] Furthermore, traditional photosensitizers (such as porphyrin derivatives and phthalocyanine compounds) have many drawbacks, including poor water solubility, affecting their dispersibility and cellular uptake efficiency in the physiological environment; easy degradation under prolonged light exposure, leading to reduced reactive oxygen species (ROS) generation efficiency; non-specific distribution that may damage normal tissues and cause side effects such as photosensitivity reactions in the skin; limited responsiveness to hypoxic and acidic tumor microenvironments, reducing therapeutic efficacy; and difficulty in integrating diagnostic imaging or other therapeutic functions, limiting the application of multimodal synergistic therapy. These limitations further restrict their clinical application.
[0004] To address the shortcomings of traditional photosensitizers, such as poor water solubility and weak targeting, there is an urgent need to develop graphene quantum dots (GQDs) with precise structures and controllable molecular sizes. By expanding the π-conjugated system and functionalizing it, the performance of photodynamic therapy can be improved. Summary of the Invention
[0005] The purpose of this invention is to propose an amino-functionalized single-molecule graphene quantum dot (NH2-GQD), which overcomes the structural uncertainties and aggregation problems of traditional photosensitizers. Graphene quantum dots with precise molecular configurations are synthesized. These graphene quantum dots exhibit high performance at 520 nm (20 mW cm⁻¹). -2 Laser irradiation can generate a large amount of singlet oxygen. 1 O2). In the in vitro 4T1 cell MTT assay, the cell survival rate decreased by 80% after 1 min of light exposure compared to 1 min, demonstrating significant in vitro photodynamic therapy and excellent killing effect on cancer cells.
[0006] The technical solution adopted to realize the present invention is as follows:
[0007] An amino-functionalized single-molecule graphene quantum dot (NH2-GQD) has the following structural formula:
[0008]
[0009] Furthermore, the present invention also provides an edge-modified diphenyl ketone graphene quantum dot (AGQD), the structural formula of which is as follows:
[0010]
[0011] After hydrolysis with hydrochloric acid, the edge-modified graphene quantum dots AGQDs have good solubility in aqueous solution, which enhances the safety and clinical applicability of PDT.
[0012] The second objective of this invention is to provide a method for preparing amino-functionalized single-molecule graphene quantum dots (NH2-GQD), comprising the following steps:
[0013] Step 1: Using 1,2-bis(4-bromophenyl)ethane-1,2-dione and 1,3-diphenyl ketone as starting materials, product 1 was obtained through cyclization addition; the structural formula of product 1 is:
[0014]
[0015] Step 2: Product 1 is reacted with benzophenone imine via a substitution reaction to obtain product 2; the structural formula of product 2 is:
[0016]
[0017] Step 3: Product 2 is reacted with triynylbenzene via a Diels-Alder reaction to obtain product 3; the structural formula of product 3 is:
[0018]
[0019] Step 4: Product 3 is subjected to Scholl oxidation reaction to obtain edge-modified diphenyl ketone graphene quantum dots AGQD;
[0020] Step 5: AGQD is hydrolyzed with hydrochloric acid to obtain edge-amino-modified graphene quantum dots NH2-GQD, which are amino-functionalized single-molecule graphene quantum dots.
[0021] The specific steps of the further hydrochloric acid hydrolysis reaction are as follows:
[0022] Dissolve AGQD in 2 mL of tetrahydrofuran solution, then take HCl (2 mol L). -1 Add 9 mL to the reaction solution, stir at room temperature for 15 min, cool to precipitate, and obtain the solid product of amino-functionalized monomolecular graphene quantum dots NH2-GQD.
[0023] The third objective of this invention is the application of amino-functionalized single-molecule graphene quantum dots NH2-GQD in photodynamic therapy.
[0024] Irradiation with light of a specific wavelength induces the generation of reactive oxygen species (ROS) within cells using amino-functionalized single-molecule graphene quantum dots (NH2-GQD). Furthermore, co-incubation of NH2-GQD with tumor cells kills the tumor cells.
[0025] The technical solution provided by this invention breaks through the mental barriers of conventional synthesis methods, proposing a method for preparing amino-functionalized single-molecule graphene quantum dots. These quantum dots have accurate molecular structures, with a C core. 96 sp 2 A structure with a fully conjugated graphite core and edges precisely modified with 6NH2. Attached Figure Description
[0026] The accompanying drawings of this invention form part of the specification and are primarily used to illustrate the implementation methods and advantages of the technical solutions. The illustrated embodiments and their textual descriptions clearly reveal the key innovations, technical objectives, and advancements; however, the scope of interpretation of the drawings does not constitute a limitation on the patent claims.
[0027] It should be noted that the same symbols in the accompanying drawings correspond to the same technical components, while different symbols point to components with different functions. It should be understood that the schematic diagrams only represent the relative positions of components and the functional logic structure; specific physical dimensions and actual assembly proportions may be simplified or schematically adjusted.
[0028] In the attached diagram:
[0029] Figure 1 This is a flowchart of the synthesis process of NH2-GQD.
[0030] Figure 2 The NMR spectrum of product 5 is shown in the figure.
[0031] Figure 3 The image shows the NMR carbon spectrum of product 5.
[0032] Figure 4 The PL fluorescence spectrum of NH2-GQD is shown.
[0033] Figure 5 The image shows the in vitro ROS release effect of NH2-GQD, with a light source wavelength of 520nm.
[0034] Figure 6 The graph shows the in vitro ROS release effect of NH2-GQD at different concentrations.
[0035] Figure 7 To investigate the in vitro cytotoxicity of NH2-GQD on 4T1 cells under different excitation durations. Detailed Implementation
[0036] Embodiments of this disclosure will now be described in more detail with reference to the accompanying drawings. While some embodiments of this disclosure are shown in the drawings, it should be understood that this disclosure can be implemented in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of this disclosure. It should be understood that the accompanying drawings and embodiments of this disclosure are for illustrative purposes only and are not intended to limit the scope of protection of this disclosure.
[0037] It should also be noted that, for ease of description, only the parts relevant to the invention are shown in the accompanying drawings. Unless otherwise specified, the embodiments and features described in this disclosure can be combined with each other.
[0038] This disclosure will now be described in detail with reference to the accompanying drawings and embodiments.
[0039] Example 1:
[0040] An amino-functionalized single-molecule graphene quantum dot NH2-GQD, with the following structural formula:
[0041]
[0042] Amino-functionalized single-molecule graphene quantum dots were obtained by hydrolysis of edge-modified graphene quantum dots (AGQDs) with diphenyl ketone (DPK). The structural formula of the DPK-modified graphene quantum dots (AGQDs) is as follows:
[0043]
[0044] Example 2:
[0045] This embodiment provides a method for preparing amino-functionalized single-molecule graphene quantum dots, the overall flowchart of which is shown below. Figure 1 This includes the following steps:
[0046] Step 1: Using 1,2-bis(4-bromophenyl)ethane-1,2-dione and 1,3-diphenyl methyl ketone as raw materials, product 1 is obtained by cyclization addition.
[0047] The specific preparation method of product 1 is as follows:
[0048] Weigh the reactants 1,2-bis(4-bromophenyl)ethane-1,2-dione (1.77 g, 4.8 mmol), 1,3-diphenylacetone (1 g, 4.8 mmol), and potassium hydroxide (0.27 g, 4.8 mmol), and transfer them to a two-necked flask. Then, use a pipette to add 0.3 mL of anhydrous ethanol solution to the flask, stir for 5 min, and heat to 80 °C and reflux for 1.5 h. After the reaction is complete, wash with anhydrous ethanol, centrifuge the solution, and dry the precipitate under vacuum for 12 h to obtain product 1 (2.08 g, 84%), a dark purple solid.
[0049] Step 2: Product 1 is reacted with benzophenone imine via a substitution reaction to obtain product 2.
[0050] The specific preparation method of product 2 is as follows:
[0051] Compound 1 (0.5 g, 0.92 mmol) prepared in step 1, tris(dibenzylacetone)palladium (0.084 g, 0.092 mmol), 2,2'-bis(diphenylphosphino)-1,1'-binaphthyl (0.086 g, 0.138 mmol), and cesium carbonate (2.698 g, 8.28 mmol) were weighed and added to a 100 mL pressure-resistant flask. 30 mL of toluene and 0.928 mL of benzophenone imine were added. The reaction was carried out at 80 °C under nitrogen protection for 3 days. After the reaction solution cooled to room temperature, the solvent was removed by vacuum distillation. Then, 2 mL of tetrahydrofuran was added to the reaction flask to just dissolve the precipitate. Methanol and water (7:1 ratio) were then added to precipitate the precipitate. The bottom precipitate was collected and dried under vacuum for 12 h to obtain product 2 (0.57 g, 85%), a brown solid.
[0052] Step 3: Product 2 is reacted with triynylbenzene via a Diels-Alder reaction to obtain product 3.
[0053] The specific preparation method of product 3 is as follows:
[0054] Weigh out compound 2 (0.4 g, 0.54 mmol) and substance 1,3,5-triynebenzene (0.024 g, 0.16 mmol) prepared in step 2, and dissolve them in 10 mL of diphenyl ether. After reacting at 250 °C for 20 h, add 3 mL of methanol while hot to quench the reaction. After cooling to room temperature, add 150 mL of methanol to the solution to wash, discard the supernatant, and leave the precipitate. Dry under vacuum for 12 h to obtain product 3 (0.92 g, 74%), which is a brownish-yellow solid.
[0055] Step 4: Product 3 is subjected to Scholl oxidation reaction to obtain edge-modified graphene quantum dots AGQD.
[0056] The specific preparation method of product 4 is as follows:
[0057] Compound 3 (0.14 g, 0.061 mmol) prepared in step 3 was weighed and dissolved in 45 mL of dichloromethane solution. Nitrogen gas was continuously bubbled through the solution. Then, anhydrous ferric chloride (1.78 g, 10.98 mmol) solution dissolved in 7.5 mL of nitromethane was slowly added dropwise to the reaction solution. After stirring at room temperature for 2 h, 50 mL of methanol was added to quench the reaction. The product 4 (0.10 g, 75%) was obtained by centrifugation and was a black solid.
[0058] Step 5: AGQD is hydrolyzed with hydrochloric acid to obtain edge-amino-modified graphene quantum dots NH2-GQD, which are molecular-level amino-functionalized water-soluble graphene quantum dots.
[0059] The specific preparation method of the product NH2-GQD is as follows:
[0060] Weigh out 40 mg (0.0177 mmol) of compound 4 prepared in step 4 and dissolve it in 2 mL of tetrahydrofuran solution. Stir at room temperature, then take 2 mol / L HCl. -1 Add 9 mL of HCl to the reaction solution, stir for 15 min, then lower the reaction temperature to 0℃, and then add 2 mol / L HCl. -1 Add 5 mL of the solution and continue stirring for 20 min. Then stop the reaction, centrifuge the reaction solution, collect the precipitate, and vacuum dry for 12 h to obtain product 5 (0.2 g, 90%), which is a black solid.
[0061] like Figure 2 The image shows the NMR spectrum of product 5 synthesized by the method of this invention. The results indicate that the number of hydrogen atoms and their chemical shifts in product 5 are completely determined, confirming the successful preparation of product 5.
[0062] like Figure 3 The image shows the NMR carbon spectrum of product 5 synthesized by the method of this invention. The results show that the number of carbon atoms and chemical shifts of product 5 are completely determined, confirming the successful preparation of product 5.
[0063] like Figure 4 The figure shows the PL fluorescence spectrum of NH2-GQD synthesized according to the method of this invention. The results show that the fluorescence emission intensity reaches its maximum value when the excitation wavelength is 590 nm.
[0064] like Figure 5 The image shows the in vitro ROS release effect of NH2-GQD synthesized according to the method of this invention, with a light source wavelength of 520 nm. As the illumination time increases, the ROS content released by the NH2-GQD aqueous solution first increases and then decreases. The results indicate that NH2-GQD has the ability to release a large amount of ROS.
[0065] like Figure 6 The graph shows the in vitro ROS release effect of NH2-GQD at different concentrations, with a light source wavelength of 520 nm. As the concentration increases, the ROS release from the NH2-GQD aqueous solution first increases and then decreases. At 7.82 μmol / L... -1 The ROS capability is strongest at this time.
[0066] The graphene quantum dot preparation method provided by this invention yields products with a defined molecular structure, excellent optical properties, and potential for application in the biomedical field.
[0067] Example 3:
[0068] This embodiment demonstrates the application of amino-functionalized water-soluble graphene quantum dots in photodynamic antitumor therapy. The specific experimental content includes the following parts:
[0069] I. Verification method for the cytotoxicity of NH2-GQD:
[0070] NH2-GQD was serially diluted with RPMI-1640 medium to the following concentrations: 100, 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39, and 0.19 μmol / L. -1 Store at 4℃ protected from light. Seed 4T1 cells into 96-well plates and incubate for 24 h, then add different concentrations of sample culture medium. After incubation, add 20 μL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution (5 mg / mL) to each well. -1 After incubating for another 4 hours, the culture medium was removed, and 150 μL of DMSO and MTT were added to each well to neutralize the medium. After the procedure was completed, the absorbance at 490 nm was measured using a microplate reader. Three sets of parallel samples were measured.
[0071] like Figure 7 MTT results showed that under light-free conditions, the concentration of NH2-GQD at 25 μmol / L... -1 It has no obvious toxicity to cells.
[0072] II. Verification method for the in vitro photodynamic antitumor effect of NH2-GQD on 4T1 cells:
[0073] NH2-GQD was serially diluted with RPMI-1640 medium to the following concentrations: 100, 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39, and 0.19 μmol / L. -1 Store at 4℃ protected from light. 4T1 cells were seeded into 96-well plates and incubated for 24 h, followed by the addition of different concentrations of sample culture medium. After 24 h of incubation, the cells were irradiated with a 460 nm laser for 1, 3, 5, and 10 min, respectively. After incubation, 20 μL of MTT (3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide) solution (5 mg / mL) was added to each well. -1 After incubating for another 4 hours, the culture medium was removed, and 150 μL of DMSO and MTT were added to each well to neutralize the medium. After the procedure was completed, the absorbance at 490 nm was measured using a microplate reader. Three sets of parallel samples were measured.
[0074] like Figure 7 The MTT assay results showed that NH2-GQD concentrations at the micromolar per liter level have the ability to generate a large amount of ROS, exhibiting a time- and concentration-dependent effect. When the irradiation time was 10 min, the IC50... 50 1.27 μmol L -1 .
[0075] The above embodiments utilize the good hydrophilicity of the synthesized amino-functionalized water-soluble graphene quantum dots, which have low toxicity and good biocompatibility. However, under light irradiation, they can generate a large amount of ROS that kills cells. Therefore, they have great application potential in photodynamic therapy.
[0076] The above description is merely a selection of preferred embodiments of this disclosure and an explanation of the technical principles employed. Those skilled in the art should understand that the scope of the invention involved in the embodiments of this disclosure is not limited to technical solutions formed by specific combinations of the above-described technical features, but should also cover other technical solutions formed by arbitrary combinations of the above-described technical features or their equivalents without departing from the above-described inventive concept. For example, technical solutions formed by substituting the above-described features with (but not limited to) technical features with similar functions disclosed in the embodiments of this disclosure.
Claims
1. An amino-functionalized single-molecule graphene quantum dot (NH2-GQD), characterized in that: The structural formula is:
2. A graphene quantum dot AGQD with edge-modified diphenyl ketone, characterized in that: The structural formula is: The edge-modified diphenyl ketone graphene quantum dots AGQD are subjected to an acidification and hydrolysis reaction to obtain the amino-functionalized graphene quantum dots (NH2-GQD) of claim 1.
3. The method for preparing amino-functionalized single-molecule graphene quantum dots (NH2-GQD) according to claim 1, characterized in that... Includes the following steps: (1) Product 2 was reacted with triynylbenzene via a Diels-Alder reaction to obtain product 3, the structural formula of which is: (2) The product 3 was subjected to Scholl oxidation reaction to obtain edge-modified diphenyl ketone graphene quantum dots AGQD, the structure of which is shown in claim 2. (3) The graphene quantum dots AGQD modified with edge diphenyl ketone are hydrolyzed with hydrochloric acid to obtain the graphene quantum dots NH2-GQD modified with edge amino, which is the amino-functionalized monomolecular graphene quantum dots described in claim 1.
4. The method for preparing amino-functionalized single-molecule graphene quantum dots (NH2-GQD) according to claim 3, characterized in that: The specific steps of step (5) are as follows: Dissolve AGQD in 2 mL of tetrahydrofuran solution, then add 9 mL of HCl to the reaction solution, stir at room temperature for 15 min, cool to precipitate, and obtain the solid product of amino-functionalized water-soluble graphene quantum dots NH2-GQD.
5. An application of an amino-functionalized single-molecule graphene quantum dot (NH2-GQD), characterized in that: The amino-functionalized single-molecule graphene quantum dots (NH2-GQD) synthesized by the synthesis method described in claim 3 are used for in vitro photodynamic therapy.
6. The application of the amino-functionalized single-molecule graphene quantum dots (NH2-GQD) according to claim 5 in in vitro photodynamic therapy, characterized in that: The ability of amino-functionalized single-molecule graphene quantum dots NH2-GQD to generate reactive oxygen species (ROS), specifically singlet oxygen. 1 When co-incubated with 4T1 tumor cells and exposed to light for 10 minutes, a large amount of reactive oxygen species (ROS) is released into the cells, killing the 4T1 tumor cells.