Copper-doped carbon dots as well as preparation method and application thereof

Abstract

The invention relates to the field of biological medicine application, and provides copper-doped carbon dots for antibiosis and osteogenesis as well as a preparation method and application of the copper-doped carbon dots, and the copper-doped carbon dots are obtained by doping monovalent copper ions on the surfaces of carbon dots with negative charges through a microwave-assisted hydrothermal method; the content of the monovalent copper ions in the copper-doped carbon dots is 3%-10%. The copper-doped carbon dots have ROS generation mediated copper-like death-like antibacterial performance, and show obvious surface negative charges, so that electrostatic interaction between the copper-doped carbon dots and mesenchymal stem cells can be triggered, downstream activation of a BMP / SMAD pathway is triggered, and the BMP / SMAD pathway is activated. The negative charges promote osteogenic differentiation, Cu-triggered copper-like death and the synergistic antibacterial effect of CDT, so that the infectious bone defect of the skull can be completely healed after 2 months.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and particularly relates to a copper-doped carbon dot and a preparation method and application thereof. BACKGROUND

[0002] The clinical treatment of bone defects caused by trauma, tumor resection or infection remains a great challenge. Although materials including titanium alloys, bone grafts and bone cements are widely used for bone reconstruction, complex surgical procedures, difficulty in achieving proper anatomical alignment and suboptimal biocompatibility performance still present challenges. In addition, persistent bacterial infection and the development of stubborn microbial biofilm on the surface of implants often jeopardize the success of bone regeneration surgery. Therefore, the development of an optimal bone repair scaffold must meet the excellent biocompatibility standard while requiring the complex integration of osteoinductive and antibacterial eradication capabilities. In practical applications, traditional antibacterial methods often have limited effect in eliminating established biofilms, requiring repeated surgical intervention. What makes these challenges even more complicated is that the overuse of antibiotics accelerates the emergence of multi-drug resistant (MDR) pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA). In addition, the inherent defects of small molecule antibiotics, including systemic toxicity and adverse pharmacological effects, collectively hinder their therapeutic effects. As an innovative alternative to traditional antibiotics, reactive oxygen species (ROS)-based treatment modalities, including chemical, photodynamic and sonodynamic therapy, show great potential in combating microbial infections. However, SDT and PDT based on exogenous stimulation to generate ROS do not have high selectivity. The inevitable accumulation of sonosensitizers or photosensitizers in normal cells can also generate ROS in normal cells, thereby causing serious toxic side effects to healthy tissues. Chemical dynamic therapy can specifically generate ROS in acidic bacterial microenvironments (BMEs) but not in neutral conditions, thus having the advantage of bacterium-specific targeted therapy.

[0003] Although CDT has potential applications in tumor-specific antibacterial therapy, the low catalytic activity of nanoscale enzymes limits the therapeutic effect of CDT. Therefore, directly killing bacteria using innovative forms of programmed cell death is a more promising way. Exploration of emerging programmed cell death mechanisms (such as copper death) can help develop new strategies to combat anti-apoptotic resistance. Copper death eliminates pathogens by focusing on intracellular copper metabolism, making it less likely to cause conventional antibiotic resistance. However, the toxicity of copper ions can damage host cells. In addition, glutathione present in the BME can hinder Cu ions from attaching to the lipoylated proteins of the tricarboxylic acid cycle (TCA), which can lead to reduced efficacy of copper-like death. SUMMARY

[0004] In order to solve the above problems, the application provides a copper-doped carbon dot and a preparation method and application thereof.The copper-doped carbon dot in the application has ROS generation mediated copper-like death antibacterial performance by selecting monovalent copper ions, and the negative charge on the surface of the carbon dot has the ability to promote osteogenic differentiation, thereby realizing antibacterial and osteogenic dual effects.

[0005] The technical scheme of the application is as follows.

[0006] The application provides a copper-doped carbon dot for antibacterial and osteogenic purposes, which refers to monovalent copper ions doped on the surface of a surface-negative carbon dot. The content of the monovalent copper ions in the copper-doped carbon dot is 3% to 10%.

[0007] The application controls the content of the monovalent copper ions to be 3% to 10%, so as to ensure antibacterial performance and also not have high toxicity to osteoblasts.If the content is too high, the toxicity to osteoblasts is too great;if the content is too low, the antibacterial activity is low, and therefore the content range of 3% to 10% is selected.If divalent copper is used, antibacterial activity cannot be generated through copper death, and therefore monovalent copper must be selected for doping.The antibacterial activity is mainly realized through copper death and generated ROS caused by monovalent copper.The surface-negative carbon dot shows significant osteogenic activity, and the negative charge can be combined with calcium ions, thereby promoting osteogenesis.

[0008] In another preferred embodiment, the surface-negative carbon dot is prepared from an organic acid and an organic amine under microwave-assisted conditions.

[0009] In another preferred embodiment, the monovalent copper ions are from cuprous chloride, The application provides a preparation method of the copper-doped carbon dot for antibacterial and osteogenic purposes, which comprises the following steps: The copper-doped carbon dot is obtained by treating an organic acid, an organic amine and cuprous chloride in a water environment under microwave conditions with a power of 300 W to 700 W and a temperature of 160 DEG C to 200 DEG C for 5 min to 30 min. The mass ratio of the organic acid, cuprous chloride and organic amine is 1 g to 5 g: 0.05 g to 0.2 g: 0.1 mL to 1 mL.

[0010] In another preferred embodiment, the organic acid is citric acid, and the organic amine is ethylenediamine.

[0011] The application provides an application of the copper-doped carbon dot in preparing an infectious bone defect healing product.

[0012] In another preferred embodiment, the infectious bone defect healing product is a hydrogel containing the copper-doped carbon dot.

[0013] In another preferred embodiment, the hydrogel containing copper-doped carbon dots is prepared specifically as follows: The methacrylated gelatin precursor, copper-doped carbon dots and photoinitiator are dissolved in a buffer solution, and cured under ultraviolet irradiation to obtain; The mass ratio of the methacrylated gelatin precursor, copper-doped carbon dots and photoinitiator is 0.5g~1g:1mg~10mg:0.01g~0.1g.

[0014] In another preferred embodiment, the photoinitiator is 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone.

[0015] The fourth aspect of the present application provides the use of the copper-doped carbon dots in preparing an antibacterial agent.

[0016] Compared with the prior art, the present application has the following beneficial effects: The copper-doped carbon dots in the present application utilize monovalent copper ions to dope on the surface of negatively charged carbon dots, so that the obtained copper-doped carbon dots have high concentrations of carboxyl and hydroxyl groups, and exhibit obvious surface negative charge. This can induce the electrostatic interaction between the copper-doped carbon dots and the bone marrow mesenchymal stem cells, then trigger the downstream activation of the BMP / SMAD pathway, the osteogenic differentiation promoted by negative charge, the copper-like death triggered by monovalent copper ions and the synergistic antibacterial effect of CDT, thereby realizing the complete healing of the infected bone defect of the skull after 2 months. And the present application finds that, after stirring, the monovalent copper ions and the carbon dots prepare Cu cross-linked CD assemblies, and the self-assembled Cu + CDs only have good antibacterial activity, but do not have osteogenic performance, because the doping sites of monovalent copper ions are on the edge of the carbon dots rather than on the plane of the carbon dots. Therefore, the present application makes the monovalent copper ions dope on the surface of the carbon dots by a microwave-assisted hydrothermal method, so that the obtained copper-doped carbon dots have good antibacterial and osteogenic effects. In addition, the presence of a small amount of Cu 2+ generated in the process of preparing the copper-doped carbon dots also endows the Cu-CDs with good GSH-px mimetic catalytic activity, so as to consume glutathione (GSH) in BME, thereby avoiding the consumption of ROS produced by CDT, and realizing the cascade amplification of ROS generation. Cu-CDs induce copper-like death, further enhancing the antibacterial and antibiofilm activity. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 FIG. 1 is a schematic diagram of the structural characterization of Cu-CDs; wherein a is a TEM picture of Cu-CDs; b is a high-resolution transmission electron microscope picture of Cu-CDs, and the circled part in the picture is a crystal lattice fringe.

[0018] Figure 2 The active experiment results of Cu-CDs; wherein, a is the test results of the production rate of •OH of Cu-CDs; b is the test results of the GSH consumption performance of Cu-CDs; c is the GSH consumption rate of Cu-CDs and Cu + @CD.

[0019] Figure 3 The antibacterial results of Cu-CDs; wherein, a is the photos of MRSA infected wounds after different treatments; b is the traces of wounds after different treatments; c is the photos of MRSA colonies after different treatments.

[0020] Figure 4 The osteogenesis results of Cu-CDs; wherein, a is the cell survival rate results of BMSCs after Cu-CDs treatment; b is the cell survival rate results of BMSCs after CD treatment; c is the osteogenic differentiation of BMSCs after Cu-CDs and Cu + @CD treatment, and the corresponding column 1 in the figure is the ARS staining chart.

[0021] Figure 5 The osteogenic differentiation results of Cu-CDs and Cu + @CD wrapped by hydrogel; wherein, a is the osteogenic differentiation ability and antibacterial activity of Cu-CDs wrapped by hydrogel, and the corresponding column 2 in the figure is the alizarin red staining chart; b is the osteogenic differentiation ability and antibacterial activity of Cu + @CD wrapped by hydrogel; c is the repair of infected bone defects of skull after different treatments. DETAILED DESCRIPTION

[0022] In order to enable those skilled in the art to better understand the technical solutions of the present application and to implement them, the present application will be further described below in conjunction with specific embodiments and drawings. In the description of the present application, if not specially stated, the reagents used are commercially available, and the methods used are conventional techniques in the art.

[0023] 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone was purchased from Sigma-Aldrich; Balb / c mice were purchased from Shanghai Slek Experimental Animal Co., Ltd.; MRSA was purchased from ATCC American Type Culture Collection; bone marrow mesenchymal stem cells were purchased from Haixing Biotechnology Co., Ltd.

[0024] Example 1 A copper-doped carbon dot for antibacterial and osteogenic purposes, using citric acid, ethylenediamine and cuprous chloride as raw materials, copper-doped carbon dots were prepared by microwave-assisted hydrothermal method, denoted as Cu-CDs.

[0025] The preparation method of the above-mentioned Cu-CDs, comprising the following steps: Dissolve 1g of citric acid, 0.1mL of ethylenediamine and 0.05g of CuCl in 10mL of deionized water and sonicate for 10min to obtain a mixture; transfer the mixture to a microwave reaction vessel and microwave at 200℃ and 700w for 15min to obtain Cu-CDs.

[0026] 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.

[0027] Comparative Example 1 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 1. Chemical kinetics and GSH consumption performance tests of Cu-CDs antibacterial agents The Cu-CDs prepared in Example 1 can undergo Fenton reaction to generate a large amount of hydroxyl radicals (·OH) under acidic conditions. The chemical kinetic performance of Cu-CDs antibacterial agent was evaluated by using 3,3',5,5'-tetramethylbenzidine (TMB) as a ·OH probe.

[0033] The Cu-CDs can consume GSH, and the ability of Cu-CDs antibacterial agent to consume GSH was evaluated by using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) as a GSH probe.

[0034] The results are shown in Figure 2 From the figure, it can be seen that the copper-doped carbon dots have excellent chemical kinetic activity and GSH consumption ability, which are significantly higher than those of Cu + @CD.

[0035] 2. Antimicrobial activity of Cu-CDs antibacterial agent The antimicrobial activity of Cu-CDs antibacterial agent was evaluated by a mouse wound model.

[0036] A 1-centimeter-diameter wound was formed on the back of the mouse. Then 100 μL of MRSA was applied to the wound to promote bacterial growth and infection. After 24 h, the mice were randomly divided into four groups, with 3 mice in each group. Each group of wounds was treated with 20 μL of PBS, CD, Cu-CD, and Cu + @CD solution, respectively. In addition, wound photos were taken on days -1, 0, 2, 4, 6, 9, and 12, and the wound area was quantified using ImageJ. To further evaluate wound infection, swabs were collected from the wound in a "Z" pattern on days 0, 2, and 6 to obtain bacterial culture on plates.

[0037] The results are shown in Figure 3 Compared with CD alone, Cu-CDs have better wound repair ability, which almost completely repairs the wound; at the same time, Cu-CDs completely eliminate MRSA bacteria in the wound.

[0038] 3. In vitro osteogenic activity of Cu-CDs antibacterial agent To detect ALP activity, bone marrow mesenchymal stem cells (BMSCs) were seeded in a six-well plate and cultured for 12 hours. Then, PBS, CD, Cu-CD, and Cu + @CD solution was further incubated. On days 7 and 14, the prepared samples were evaluated for their osteogenic differentiation potential on bone marrow mesenchymal stem cells using an ALP assay kit.

[0039] For alizarin red S (ARS) staining, 14-day-old bone marrow mesenchymal stem cells were fixed with 4% paraformaldehyde (Beyotime, China) and stained with 2% alizarin red (Sigma, USA). Then the bone marrow mesenchymal stem cells of different experimental groups were imaged using a fluorescence microscope (Olympus BX53, Japan) to observe the osteogenic differentiation effect. For quantitative analysis, the stained BMSC cells were washed with 10% cetylpyridinium chloride (CPC) and the absorbance of the washing solution at 563 nm was measured.

[0040] Results are shown in Figure 4 As can be seen from the figure, carbon dots and copper-doped carbon dots have excellent biocompatibility and no obvious toxicity to BMSC cells; and they can both promote the osteogenic differentiation of mesenchymal stem cells. In contrast, Cu+@CD does not have osteogenic activity.

[0041] 4. Infection bone defect treatment of Cu-CDs antibacterial agent A 3mm diameter defect was made on the skull of a healthy 6-week-old Balb / c mouse, inoculated with 20μL of MASA solution to establish an infected skull defect model. Then 50μL of methacrylated gelatin (GelMA), CD / GelMA and Cu-CD / GelMA hydrogel solution was injected into the infected defect site and cured under ultraviolet light, divided into GelMA group, CD / GelMA group and Cu-CD / GelMA group. Micro-CT imaging was performed at day 0 and day 60 to evaluate the osteogenic regeneration of the skull of mice in each group.

[0042] Results are shown in Figure 5 As can be seen from the figure, the Cu-CD / GelMA group can significantly promote the osteogenic differentiation of mesenchymal stem cells and achieve almost complete repair of the infected bone defect.

[0043] The above describes the specific embodiments of the present application in detail, but it is only as an example, the present application is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modification and substitution of the present application are also within the scope of the present application. Therefore, any equivalent transformation and modification without departing from the spirit and scope of the present application should be covered within the scope of the present application.

Claims

1. Copper-doped carbon dots for antibacterial and osteogenic applications, characterized in that, is prepared by microwave-assisted hydrothermal method, and monovalent copper ions are doped on the surface of the carbon dots with negative charges on the surface; The content of the monovalent copper ions in the copper-doped carbon dots is 3% to 10%.

2. The copper-doped carbon dots for antibacterial and osteogenic applications as claimed in claim 1, wherein, The carbon dots with negative charges on the surface are prepared under the condition of microwave assistance by using organic acid and organic amine.

3. The copper-doped carbon dots according to claim 2, characterized in that, The monovalent copper ions are from cuprous chloride.

4. A method for the preparation of copper-doped carbon dots for antibacterial and osteogenic applications as claimed in claim 3, wherein the said method comprises the steps of: The method comprises the following steps: The copper-doped carbon dots are obtained by treating organic acid, organic amine and cuprous chloride in a water environment under the condition of microwave with a power of 300 W to 700 W and a temperature of 160 DEG C to 200 DEG C for 5 min to 30 min. The mass ratio of the organic acid, cuprous chloride and organic amine is 1 g to 5 g: 0.05 g to 0.2 g: 0.1 mL to 1 mL.

5. The method for the preparation of copper-doped carbon dots for antibacterial and osteogenic applications as claimed in claim 4, wherein, The organic acid is citric acid, and the organic amine is ethylenediamine.

6. The copper-doped carbon dots of claim 3 are used in the preparation of an infectious bone defect healing product.

7. Use according to claim 6, characterized in that, The infectious bone defect healing product is a hydrogel containing copper-doped carbon dots.

8. Use according to claim 6, characterized in that, The hydrogel containing copper-doped carbon dots is prepared as follows: Methacrylated gelatin precursor, copper-doped carbon dots and photoinitiator are dissolved in a buffer solution, and a hydrogel is obtained by solidification under ultraviolet irradiation. The mass ratio of the methacrylated gelatin precursor, copper-doped carbon dots and photoinitiator is 0.5 g to 1 g: 1 mg to 10 mg: 0.01 g to 0.1 g.

9. Use according to claim 8, characterized in that, The photoinitiator is 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone.

10. The copper-doped carbon dots of claim 3 are used in the preparation of an antibacterial agent.

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