Copper-doped carbon dots and preparation method and application thereof
By preparing negatively charged carbon dots doped with monovalent copper ions, the problems of insufficient biocompatibility and antibacterial effect of bone repair materials were solved, enabling rapid healing and osteogenic differentiation of infected bone defects, and overcoming the problems of drug resistance to traditional antibiotics and low copper death catalytic activity.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- THE NAVAL MEDICAL UNIV OF PLA
- Filing Date
- 2025-11-19
- Publication Date
- 2026-06-02
Smart Images
Figure CN121376980B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical applications, specifically to a copper-doped carbon dot, its preparation method, and its application. Background Technology
[0002] The clinical treatment of bone defects caused by trauma, tumor resection, or infection remains a significant challenge. Despite the widespread use of materials including titanium alloys, bone grafts, and bone cement for bone reconstruction, the complexity of surgical procedures, difficulty in achieving proper anatomical alignment, and suboptimal biocompatibility persist. Furthermore, persistent bacterial infection and the development of intractable microbial biofilms on implant surfaces frequently jeopardize the success of bone regeneration surgeries. Therefore, the development of optimal bone repair scaffolds must simultaneously meet excellent biocompatibility standards while requiring a complex integration of osteogenic induction and antimicrobial eradication capabilities. In practice, traditional antimicrobial methods are often limited in their effectiveness in eliminating established biofilms, necessitating repeated surgical interventions. Complicating these challenges is the overuse of antibiotics, which accelerates the emergence of multidrug-resistant (MDR) pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA). In addition, the inherent limitations of small-molecule antibiotics, including systemic toxicity and adverse pharmacological effects, collectively hinder their therapeutic efficacy. As innovative alternatives to traditional antibiotics, reactive oxygen species (ROS)-based therapies, including chemokinetic, photodynamic, and sonodynamic therapies, show great potential in combating microbial infections. However, SDT and PDT, which generate ROS based on exogenous stimuli, lack high selectivity. The inevitable accumulation of sonosensitive or photosensitizing agents in normal cells also generates ROS, leading to severe toxicity to healthy tissues. Chemokinetic therapy, on the other hand, can specifically generate ROS in acidic bacterial microenvironments (BMEs) but not under neutral conditions, thus offering the advantage of bacterial-specific targeted therapy.
[0003] While CDT has potential applications in tumor-specific antibacterial therapy, the low catalytic activity of nanozymes limits its therapeutic efficacy. Therefore, directly killing bacteria using innovative forms of programmed cell death is a more promising approach. Exploring emerging programmed cell death mechanisms, such as copper death, may contribute to the development of new strategies against antiapoptotic resistance. Copper death eliminates pathogens by focusing on intracellular copper metabolism, making them less susceptible to resistance to conventional antibiotics. However, the toxicity of copper ions can impair host cells. Furthermore, glutathione present in BME can inhibit the attachment of Cu ions to liponylated proteins in the tricarboxylic acid cycle (TCA), potentially reducing the efficacy of copper-like death. Summary of the Invention
[0004] To address the aforementioned problems, this invention provides a copper-doped carbon dot, its preparation method, and its applications. The copper-doped carbon dot of this invention achieves both antibacterial and osteogenic effects by selecting a single-valent copper ion that possesses ROS-mediated copper death antibacterial properties, and by utilizing the negative charge on the surface of the carbon dot to promote osteogenic differentiation.
[0005] The specific technical solution of the present invention is as follows.
[0006] The first aspect of the present invention provides a copper-doped carbon dot for antibacterial and osteogenic purposes, which is obtained by doping monovalent copper ions onto a surface with negatively charged carbon dots.
[0007] The content of the monovalent copper ions in the copper-doped carbon dots is 3% to 10%.
[0008] This invention controls the content of monovalent copper ions to 3%~10%, ensuring antibacterial activity without excessive toxicity to osteoblasts. Too high a content would be too toxic to osteoblasts; too low a content would result in low antibacterial activity. Therefore, a content range of 3%~10% is chosen. Using divalent copper would not allow for antibacterial activity through copper death; therefore, monovalent copper must be used for doping. Its antibacterial activity is primarily achieved through copper death induced by monovalent copper and the resulting reactive oxygen species (ROS). Negatively charged carbon dots on the surface exhibit significant osteogenic activity; the negative charge allows them to bind with calcium ions, thereby promoting osteoogenesis.
[0009] In another preferred embodiment, the surface with negatively charged carbon dots is prepared by organic acid and organic amine under microwave-assisted conditions.
[0010] In another preferred embodiment, the monovalent copper ion is derived from cuprous chloride.
[0011] A second aspect of the present invention provides a method for preparing the copper-doped carbon dots for antibacterial and osteogenic purposes, comprising the following steps:
[0012] The copper-doped carbon dots were obtained by treating organic acids, organic amines and cuprous chloride in an aqueous environment under microwave conditions of 300W~700W and 160℃~200℃ for 5min~30min.
[0013] The mass ratio of organic acid, cuprous chloride and organic amine is 1g~5g : 0.05g~0.2g : 0.1mL~1mL.
[0014] In another preferred embodiment, the organic acid is citric acid and the organic amine is ethylenediamine.
[0015] The third aspect of this invention provides the application of the copper-doped carbon dots in the preparation of products for healing infectious bone defects.
[0016] In another preferred embodiment, the infectious bone defect healing product is a hydrogel containing copper-doped carbon dots.
[0017] In another preferred embodiment, the specific preparation process of the hydrogel containing copper-doped carbon dots is as follows:
[0018] The methacrylamide gelatin precursor, copper-doped carbon dots, and photoinitiator were dissolved in a buffer solution and cured under ultraviolet irradiation.
[0019] The mass ratio of methacrylamide gelatin precursor, copper-doped carbon dots, and photoinitiator is 0.5g~1g:1mg~10mg:0.01g~0.1g.
[0020] In another preferred embodiment, the photoinitiator is 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone.
[0021] The fourth aspect of this invention provides the application of the aforementioned copper-doped carbon dots in the preparation of antibacterial agents.
[0022] Compared with the prior art, the present invention has the following beneficial effects:
[0023] The copper-doped carbon dots in this invention utilize monovalent copper ions to atomize negatively charged carbon dots, resulting in copper-doped carbon dots with high concentrations of carboxyl and hydroxyl groups, exhibiting a significant negative surface charge. This triggers electrostatic interactions between the copper-doped carbon dots and bone marrow mesenchymal stem cells, thereby activating downstream pathways of the BMP / SMAD pathway. Negative charge promotes osteogenic differentiation, monovalent copper ions trigger copper-like cell death, and CDT synergistically enhances the antibacterial effect, ultimately achieving complete healing of infectious bone defects in the skull after two months. Furthermore, this invention, through comparison, found that the preparation of Cu cross-linked CD assemblies using stirred monovalent copper ions and carbon dots yielded self-assembled Cu... + @CD exhibits good antibacterial activity but lacks osteogenic properties because the doping sites of monovalent copper ions are located at the edges of the carbon dots rather than on the plane of the carbon dots. Therefore, this invention uses a microwave-assisted hydrothermal method to induce monovalent copper ions to be doped onto the surface of the carbon dots, thereby resulting in copper-doped carbon dots that simultaneously possess good antibacterial and osteogenic effects. Furthermore, the small amount of Cu generated during the preparation of copper-doped carbon dots in this invention... 2+ The presence of Cu-CDs also endows them with good GSH-px-like catalytic activity to consume glutathione (GSH) in BME, thereby avoiding the consumption of ROS generated by CDT and realizing the cascade amplification of ROS generation. Cu-CDs induce copper-like cell death, further enhancing their antibacterial and anti-biofilm activities. Attached Figure Description
[0024] Figure 1The diagram shows the structural characterization of Cu-CDs; where a is a TEM image of Cu-CDs; and b is a high-resolution transmission electron microscope image of Cu-CDs, with the circled area representing the lattice fringes.
[0025] Figure 2 Figure 1 shows the activity test results of Cu-CDs; where a is the test result of the •OH generation rate of Cu-CDs; b is the test result of the GSH consumption performance of Cu-CDs; c is the result of Cu-CDs and Cu + The chart shows the comparison of GSH consumption rates for @CD.
[0026] Figure 3 The images show the antibacterial results of Cu-CDs; where a is a photograph of MRSA-infected wounds after different treatment methods; b is a photograph of wound scars after different treatment methods; and c is a photograph of MRSA colonies after different treatment methods.
[0027] Figure 4 Figure 1 shows the osteogenic results of Cu-CDs; where a) is the cell viability of BMSCs after Cu-CDs treatment; b) is the cell viability of BMSCs after CD treatment; c) is the cell viability of Cu-CDs and Cu... + Osteogenic differentiation of BMSCs after @CD treatment. The column corresponding to 1 in the figure is the ARS staining map.
[0028] Figure 5 To encapsulate Cu-CDs and Cu in hydrogels respectively + The osteogenic differentiation results of @CDs; where a represents the osteogenic differentiation capacity and antibacterial activity of hydrogel-encapsulated Cu-CDs, and the column corresponding to 2 in the figure is the Alizarin Red staining image; b represents the hydrogel-encapsulated Cu + @CD represents osteogenic differentiation capacity and antibacterial activity; c represents the repair of infected bone defects in the skull after different treatment methods. Detailed Implementation
[0029] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments and accompanying drawings. In the description of this invention, unless otherwise specified, all reagents used are commercially available, and all methods used are conventional techniques in the art.
[0030] 2-Hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone was purchased from Sigma-Aldrich; Balb / c mice were purchased from Shanghai Slack Laboratory Animal Co., Ltd.; MRSA was purchased from the American Type Culture Collection (ATCC); and bone marrow mesenchymal stem cells were purchased from Haixing Biotechnology Co., Ltd.
[0031] Example 1
[0032] A copper-doped carbon dot for antibacterial and osteogenic purposes was prepared by microwave-assisted hydrothermal method using citric acid, ethylenediamine and cuprous chloride as raw materials. The copper-doped carbon dot is denoted as Cu-CDs.
[0033] The preparation method of the above Cu-CDs includes the following steps:
[0034] 1g of citric acid, 0.1mL of ethylenediamine and 0.05g of CuCl were dissolved in 10mL of deionized water and sonicated for 10min to obtain a mixture. The mixture was then transferred to a microwave reaction vessel and microwaved at 200℃ and 700w for 15min to obtain Cu-CDs.
[0035] The structure of Cu-CDs was characterized, and the results are as follows: Figure 1 As shown, from Figure 1 It can be seen that the carbon dots are 3nm~4nm in size, with an average particle size of 3.68nm. Under high-magnification electron microscopy, they exhibit distinct CD lattice fringes.
[0036] Comparative Example 1
[0037] Undoped CD solutions were synthesized using citric acid and ethylenediamine as raw materials, following the same steps as in Example 1, and purified by dialysis for 48 hours. Cu was prepared by metal ion coordination assembly. + @CD was used as a control sample.
[0038] First, mix 10 mL of 1 mg / mL CD solution with 1 mL of 0.05 g / mL CuCl solution, then stir at room temperature for 24 h. After washing three times by centrifugation, collect the Cu by centrifugation. + @CD.
[0039] The Cu-CDs and CDs prepared in Example 1 above were used to prepare hydrogels containing copper-doped carbon dots. The specific process is as follows.
[0040] Dissolve 10g of gelatin in 100mL of PBS solution and stir at 60℃ for 1 hour. Add 8% by weight of methacrylamide gelatin and stir for 2 hours to obtain a mixture.
[0041] The resulting mixture was dialyzed in deionized water for one week and then freeze-dried to prepare a methacrylamide gelatin precursor, denoted as GelMA precursor. 0.5 g of GelMA precursor and 0.01 g of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone were added to a PBS solution containing 1 mg of CD, and irradiated with ultraviolet light for 5 min to obtain CD / GelMA. Similarly, 0.5 g of GelMA precursor and 0.01 g of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone were added to a PBS solution containing 1 mg of Cu-CD, and irradiated with ultraviolet light for 5 min to obtain Cu-CD / GelMA.
[0042] 1. Chemical kinetics and GSH consumption performance tests of Cu-CDs antibacterial agents
[0043] The Cu-CDs prepared in Example 1 can undergo a Fenton reaction under acidic conditions to generate a large number of hydroxyl radicals (•OH). The chemokinetic properties of the Cu-CDs antibacterial agent were evaluated by using 3,3',5,5'-tetramethylbenzidine (TMB) as a •OH probe.
[0044] Cu-CDs can consume GSH. The ability of Cu-CDs antimicrobial agents to consume GSH was evaluated by using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) as a GSH probe.
[0045] The results are as follows Figure 2 As shown in the figure, copper-doped carbon dots exhibit superior chemical kinetic activity and GSH-consuming ability, significantly higher than Cu. + @CD.
[0046] 2. Antibacterial activity of Cu-CDs antibacterial agents
[0047] The antibacterial activity of Cu-CDs antibacterial agents was evaluated using a mouse wound model.
[0048] A 1 cm diameter wound was created on the back of each mouse. 100 μL of MRSA was then applied to the wound to promote bacterial growth and infection. After 24 hours, the mice were randomly divided into four groups of three. The wounds of each group were treated with 20 μL of PBS, CD, Cu-CD, or Cu, respectively. + @CD solution treatment. Additionally, wound photographs were taken on days -1, 0, 2, 4, 6, 9, and 12, and wound area was quantified using ImageJ. To further assess wound infection, swabs were collected from the wound in a "Z" pattern on days 0, 2, and 6 for bacterial culture on agar plates.
[0049] The results are as follows Figure 3As shown, compared to CDs alone, Cu-CDs have better wound repair capabilities, almost completely repairing the wound; at the same time, Cu-CDs completely eliminate MRSA bacteria in the wound.
[0050] 3. In vitro osteogenic activity of Cu-CDs antibacterial agents
[0051] To assess ALP activity, bone marrow mesenchymal stem cells (BMSCs) were seeded in six-well plates and cultured for 12 hours. Subsequently, PBS, CD, Cu-CD, and Cu were added. + @CD solution was further incubated. On days 7 and 14, the osteogenic differentiation potential of the prepared samples on bone marrow mesenchymal stem cells was assessed using an ALP assay kit.
[0052] For Alizarin Red S (ARS) staining, bone marrow mesenchymal stem cells (BMSCs) cultured for 14 days were fixed with 4% paraformaldehyde (Beyotime, China) and stained with 2% Alizarin Red (Sigma, USA). The BMSCs from different experimental groups were then imaged using a fluorescence microscope (Olympus BX53, Japan) to observe osteogenic differentiation. For quantitative analysis, the stained BMSCs were washed with 10% cetylpyridinium chloride (CPC), and the absorbance of the washing solution at 563 nm was measured.
[0053] The results are as follows Figure 4 As shown in the figure, both carbon dots and copper-doped carbon dots exhibit excellent biocompatibility and show no significant toxicity to BMSC cells; furthermore, they can both promote osteogenic differentiation of mesenchymal stem cells. Conversely, Cu+@CD does not possess osteogenic activity.
[0054] 4. Treatment of infected bone defects with Cu-CDs antibacterial agents
[0055] A 3 mm diameter defect was created in the skull of healthy 6-week-old Balb / c mice, and 20 μL of MASA solution was inoculated to establish an infected skull defect model. Then, 50 μL of methacrylamide gelatin (GelMA), CD / GelMA, and Cu-CD / GelMA hydrogel solutions were injected into the infected defect site and cured under UV light. The mice were divided into GelMA, CD / GelMA, and Cu-CD / GelMA groups. Micro-CT imaging was performed on day 0 and day 60 to assess osteogenic regeneration of the skull in each group.
[0056] The results are as follows Figure 5 As shown in the figure, the Cu-CD / GelMA group can significantly promote osteogenic differentiation of mesenchymal stem cells and achieve almost complete repair of infected bone defects.
[0057] The specific embodiments of the present invention have been described in detail above, but they are merely examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.
Claims
1. The application of copper-doped carbon dots in the preparation of infectious bone defect healing products and antibacterial agents, characterized in that, The copper-doped carbon dots refer to the copper ions doped onto the surface of negatively charged carbon dots by microwave-assisted hydrothermal method. The content of the monovalent copper ions in the copper-doped carbon dots is 3% to 10%; The surface with negatively charged carbon dots was prepared by organic acids and organic amines under microwave-assisted conditions; The monovalent copper ions are derived from cuprous chloride.
2. The application of copper-doped carbon dots according to claim 1 in the preparation of infectious bone defect healing products and antibacterial agents, characterized in that, The preparation of copper-doped carbon dots includes the following steps: The copper-doped carbon dots were obtained by treating organic acids, organic amines and cuprous chloride in an aqueous environment under microwave conditions of 300W~700W and 160℃~200℃ for 5min~30min. The mass ratio of organic acid, cuprous chloride and organic amine is 1g~5g : 0.05g~0.2g : 0.1mL~1mL.
3. The application of copper-doped carbon dots according to claim 2 in the preparation of infectious bone defect healing products and antibacterial agents, characterized in that, The organic acid is citric acid, and the organic amine is ethylenediamine.
4. The application of copper-doped carbon dots according to claim 3 in the preparation of infectious bone defect healing products and antibacterial agents, characterized in that, The infectious bone defect healing product is a hydrogel containing copper-doped carbon dots.
5. The application of copper-doped carbon dots according to claim 4 in the preparation of infectious bone defect healing products and antibacterial agents, characterized in that, The specific preparation process of the hydrogel containing copper-doped carbon dots is as follows: The methacrylamide gelatin precursor, copper-doped carbon dots, and photoinitiator were dissolved in a buffer solution and cured under ultraviolet irradiation. The mass ratio of methacrylamide gelatin precursor, copper-doped carbon dots, and photoinitiator is 0.5g~1g:1mg~10mg:0.01g~0.1g.
6. The application of copper-doped carbon dots according to claim 5 in the preparation of infectious bone defect healing products and antibacterial agents, characterized in that, The photoinitiator is 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone.