Preparation method of copper and calcium ion co-doped carbon dots and application of copper and calcium ion co-doped carbon dots in tumor targeted therapy
By heating and reacting folic acid, citric acid, urea, inorganic salts containing calcium ions and inorganic salts containing copper ions under specific conditions, a carbon doping of copper and calcium ions is formed, which solves the problem that copper and calcium ions are difficult to dopant into carbon doping in the prior art, and efficient targeting and killing of tumor cells is achieved.
Patent Information
- Application Number
- CN202510210959.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-25
- Publication Date
- 2025-05-30
AI Technical Summary
The prior art is difficult to successfully dopant copper and calcium ions into smaller carbon dots, resulting in insufficient precise targeting in tumor-targeted therapy.
By heating and reacting folic acid, citric acid, urea, inorganic salts containing calcium ions and inorganic salts containing copper ions under specific proportions and conditions, a carbon dot co-doped copper and calcium ions is formed. The process is carried out under vacuum, incorporating metal ions into the carbon dot structure by in-situ dehydration and carbonization.
Effective doping of copper and calcium is achieved, the targeting and killing efficiency of carbon dots in tumor cells is improved, and it is not harmful to normal tissue cells.
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Figure CN120057895A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of nano-drugs, and in particular relates to a method for preparing copper and calcium ion co-doped carbon dots and an application thereof in tumor targeted therapy. Background Art
[0002] Squamous cell carcinoma is a type of malignant tumor that originates from squamous epithelial cells. It can occur in multiple tissues and organs such as the head and neck, lungs, esophagus and skin, and is highly invasive. Although significant progress has been made in the diagnosis and treatment of squamous cell carcinoma, its morbidity and mortality remain high worldwide. Therefore, it is particularly urgent to explore new and effective treatment methods. In the field of tumor treatment, metal elements have shown broad application prospects. Among them, copper death, as a new type of regulated cell death different from traditional programmed cell death, has attracted widespread attention. At the same time, calcium ions have also become a new research hotspot because they can induce mitochondrial dysfunction through calcium overload and then induce cell apoptosis. However, although copper death and calcium overload-mediated apoptosis have shown great potential as emerging cancer treatment strategies, they still face the limitation of insufficient precise targeting, which greatly limits their practical application in clinical practice.
[0003] As an emerging type of carbon-based nanomaterials, carbon dots show broad application prospects in the biomedical field due to their unique size advantages, good cell permeability, significant fluorescence properties and adjustable surface functionalization. Carbon dots with specific physiological functions can be synthesized by selecting the type of precursor and optimizing the synthesis conditions. However, copper and calcium, two elements with large atomic radii, are difficult to successfully incorporate into smaller carbon dots. This lattice mismatch problem is a key technical problem that needs to be solved in the preparation of nanomedicines. In response to this challenge, the present invention proposes a method for preparing copper and calcium ion co-doped carbon dots and its application in tumor targeted therapy. Summary of the invention
[0004] The purpose of the present invention is to provide a method for preparing copper and calcium ion co-doped carbon dots and their application in tumor targeted therapy, aiming to solve the problems raised in the above background technology.
[0005] The purpose of the present invention is achieved through the following technical solutions:
[0006] A copper and calcium ion co-doped carbon dot is prepared by the following method:
[0007] Dissolve folic acid, citric acid, urea, inorganic salts containing calcium ions, and inorganic salts containing copper ions in pure water. After the raw materials are completely dissolved by heating at 120 °C for 60 - 80 min, the product reacts at 200 - 300 °C for 60 min in a vacuum environment to obtain a brown, fluffy, porous precursor containing carbon dots. After purification, a carbon dot solution co-doped with copper and calcium ions is obtained;
[0008] Among them, the mass ratio of folic acid, citric acid, urea, inorganic salts containing calcium ions, and inorganic salts containing copper ions is 1:2.5:5:(0.01 - 20):(0.01 - 10).
[0009] Furthermore, the mass ratio of folic acid, citric acid, urea, inorganic salts containing calcium ions, and inorganic salts containing copper ions is 1:2.5:5:5:2.5.
[0010] Furthermore, the inorganic salts containing copper ions include but are not limited to copper chloride dihydrate, copper oxide, copper sulfate, etc.; the inorganic salts containing calcium ions include but are not limited to calcium chloride, calcium carbonate, calcium nitrate, etc.
[0011] Furthermore, in the carbon dots co-doped with copper and calcium ions, the mass proportions of copper and calcium elements are respectively above 6% and 15%.
[0012] Furthermore, the specific operation of the purification is as follows:
[0013] First, dissolve the precursor with dilute hydrochloric acid, centrifuge at 10000 rpm at room temperature for 10 min, and then dialyze with a 100 - 500 Da dialysis bag in pure water for 72 h to obtain a light yellow carbon dot solution after purification.
[0014] An application of the carbon dots co-doped with copper and calcium ions as described above, characterized in that it is applied to the preparation of a drug targeting tumor cells positive for folate receptors.
[0015] An application of the carbon dots co-doped with copper and calcium ions as described above, characterized in that it is applied to the preparation of an antitumor drug.
[0016] Compared with the prior art, the beneficial effects of the present invention are:
[0017] The synthesis process of the present invention is simple and low-cost, and retains the function of folic acid in the raw materials to target folate receptors, thus facilitating the precise targeting of carbon dots to tumor cells. After precisely targeting tumor cells, copper ions and calcium ions enter the tumor cells, and through the synergistic effect of the copper death mechanism mediated by copper ions and apoptosis mediated by calcium overload, precise killing of tumor cells is achieved without harm to normal tissue cells. Description of the Drawings
[0018] Figure 1Photograph of the crude product containing carbon dots prepared in Example 1 under sunlight.
[0019] Figure 2 Characterization of the properties of the carbon dot solution in Example 2; where (a) is the photoluminescence spectrum of the carbon dot solution; (b) are photographs of the carbon dot solution under sunlight and under ultraviolet light; (c) is the EDS spectrum of the carbon dots.
[0020] Figure 3 Results of the targeting detection of carbon dots in Example 3; where (a) is the enrichment of carbon dots in SCC7 tumor cells; (b) is the enrichment of carbon dots in SCC7 cells after adding folic acid; (c) is the enrichment of carbon dots in L929 cells.
[0021] Figure 4 Results of the in vitro anti-tumor activity evaluation of carbon dots in Example 4; where (a) is the cell viability of SCC7 tumor cells after treatment with carbon dots; (b) is the cell viability of normal cells L929 after treatment with carbon dots.
[0022] Figure 5 Mechanism of carbon dot-induced tumor cell death and anti-tumor effect in vivo in Example 5; where (a) is the immunofluorescence image of SCC7 tumor cells after treatment with carbon dots, in which the DLAT oligomer antibody is labeled red and the cell nucleus is stained blue with DAPI; (b) is the Western blot detection of the expression of iron-sulfur cluster proteins LIAS and FDX1; (c) is the confocal laser scanning microscope image of detecting the Ca 2+ level in SCC7 cell mitochondria at different times using Rhod-2; (d) is the Western blot detection of the expression of key apoptosis-related proteins, including Bax, Caspase-9, Caspase-3, and Bcl-2; (e) are tumor images of nude mice bearing tumors 12 days after tail vein injection of PBS and carbon dots.
[0023] Figure 6 Mechanism diagram of the present invention. Detailed implementation manners
[0024] For a clearer understanding of the technical features, objectives, and beneficial effects of the present invention, the technical solutions of the present invention are described in detail below, but it should not be construed as a limitation on the scope of implementation of the present invention.
[0025] Under a vacuum heating environment, the precursor forms a thin-walled foam structure, confining copper ions and calcium ions within and preventing their diffusion. As the temperature rises, the precursor undergoes in-situ dehydration and carbonization, integrating the metal ions into the carbon dot structure, thereby achieving effective doping of copper and calcium. Copper ion- and calcium ion-codoped carbon dots target folate receptor-positive cells while combining the copper ion-mediated cuproptosis mechanism and the calcium overload-mediated apoptosis mechanism to synergistically achieve precise killing of tumor cells without harming normal tissue cells. The specific mechanism of the present invention is as Figure 6 shown.
[0026] The following describes the specific implementation of the present invention in detail with specific examples.
[0027] Example 1: This example provides a copper and calcium ion-codoped carbon dot (Cu / Ca-CDs), which is prepared by the following method:
[0028] Weigh 0.2 g of folic acid, 0.5 g of citric acid, 1.0 g of urea, 0.5 g of copper chloride dihydrate, and 1.0 g of calcium chloride, dissolve them in 3 mL of pure water, heat at 120 °C for 70 min to dissolve, and under vacuum conditions, react the product at 250 °C for 60 min in a vacuum environment to obtain a brown, fluffy, porous spherical precursor containing carbon dots ( Figure 1 ). Dissolve the precursor with dilute hydrochloric acid and centrifuge at 10,000 rpm for 10 min at room temperature to remove larger agglomerated particles. Subsequently, use a 100 - 500 Da dialysis bag to dialyze in pure water for 72 h to obtain a pale yellow carbon dot solution after purification.
[0029] Example 2: The pale yellow carbon dot solution can emit green fluorescence under ultraviolet light excitation ( Figure 2 b) in it), showing a single emission peak at 530 nm ( Figure 2 a) in it). Through the characterization of EDS energy spectrum, it is observed that the elemental composition of the synthesized carbon dots includes elements C, N, O, Cl, Ca, and Cu, where the mass ratio of Cu element is 6.5%, and the mass ratio of Ca element is 15.7% ( Figure 2 c) in it), and this finding confirms the doping of copper and calcium elements in the carbon dots, and the obtained carbon dots are copper and calcium ion-codoped carbon dots.
[0030] Example 3: Prepare a copper and calcium ion-codoped carbon dot solution according to the steps of Example 1 and conduct a targeting detection. The results show that after SCC7 cells are co-incubated in a culture medium containing the carbon dot solution for 60 min, obvious green fluorescence is shown. After 120 min, the fluorescence intensity increases significantly, indicating that the cells have taken up a large amount of carbon dots ( Figure 3In a). In contrast, when SCC7 cells were co-incubated with a culture medium containing carbon dots and folic acid, there was almost no green fluorescence at 60 min, and the fluorescence intensity was still very weak at 120 min ( Figure 3 In b). After mouse fibroblast L929 cells (which lack folate receptor expression on the cell membrane surface) were co-incubated with a culture medium containing carbon dots for 60 min, no obvious fluorescence was observed, and even at 120 min, the fluorescence intensity did not increase significantly ( Figure 3 In c). This result indicates that due to the lack of folate receptors, it is difficult for carbon dots to enter normal cells, further confirming the high specificity of the prepared carbon dots for tumor cells, thus highlighting their potential in tumor-targeted therapy.
[0031] Example 4: A carbon dot solution co-doped with copper and calcium ions was prepared according to the steps of Example 1, and the in vitro anti-tumor activity of the carbon dots was evaluated by the CCK-8 assay. The results showed that the carbon dots had a concentration-dependent cytotoxic effect on tumor cells, and the cell mortality rate increased with the increase in concentration. When the carbon dot concentration reached 400 μg / mL, the survival rate of SCC7 tumor cells was significantly reduced to about 30% ( Figure 4 In a). In contrast, the cytotoxic effect of the carbon dots on normal L929 cells showed obvious differences. At low concentrations, the carbon dots showed weak toxicity to L929 cells and had no obvious concentration dependence ( Figure 4 In b). These results indicate the strong killing effect of the carbon dots on tumor cells.
[0032] Example 5: A carbon dot solution co-doped with copper and calcium ions was prepared according to the steps of Example 1. The copper death pathway was verified by detecting the oligomerization of DLAT in tumor cells after treatment with the carbon dot solution by immunofluorescence staining technology and Western blot analysis of the loss of iron-sulfur cluster proteins. Through immunofluorescence staining technology, obvious red fluorescence signals were observed in the tumor cells of the carbon dot group, indicating that the carbon dots significantly promoted the oligomerization of DLAT ( Figure 5 In a). The Western blot results showed that the iron-sulfur cluster proteins FDX1 and LIAS in SCC7 tumor cells treated with the carbon dots were significantly reduced ( Figure 5 In b), which further confirmed that the carbon dots might induce the copper death pathway by interfering with the function of iron-sulfur cluster proteins. To explore whether the carbon dots also induced apoptosis of tumor cells through the calcium overload mechanism, we used the Rhod-2 calcium probe to detect the intracellular calcium ion level after treatment with the carbon dot solution. The results showed that the red fluorescence intensity gradually increased with time, indicating that the calcium ions in the carbon dots were gradually released into the tumor cells ( Figure 5In c). This finding provides direct evidence that carbon dots induce apoptosis through the calcium overload mechanism. In addition, the expression levels of key apoptosis-related proteins were analyzed by Western blot, including the pro-apoptotic proteins Bax, Caspase-9, and Caspase-3, and the anti-apoptotic protein Bcl-2. The results showed that carbon dot treatment significantly upregulated the expression of Bax, Caspase-9, and Caspase-3 and downregulated the expression of Bcl-2( Figure 5 In d). It is suggested that carbon dots induce tumor cell death through a synergistic mechanism of cuproptosis and calcium overload-induced apoptosis. Carbon dots were further applied to a tumor-bearing nude mouse animal model to evaluate their anti-tumor effect in vivo. Compared with tumor-bearing nude mice treated with PBS, the volume of tumors in tumor-bearing nude mice in the carbon dot group was significantly reduced, effectively killing tumor cells and resulting in tumor ablation( Figure 5 In e). This result not only verified the anti-tumor activity of carbon dots in vitro but also provided strong in vivo evidence for their potential application in tumor treatment.
[0033] Example 6: This example provides a copper- and calcium-ion co-doped carbon dot, which is prepared by the following method:
[0034] Weigh 0.2 g of folic acid, 0.5 g of citric acid, 1.0 g of urea, 1.0 g of copper(II) chloride dihydrate, and 1.0 g of calcium chloride and dissolve them in 3 mL of pure water. Heat at 120 °C for 70 min to dissolve. Under vacuum conditions, the product reacts at 250 °C for 60 min in a vacuum environment to obtain a fluffy brown spherical precursor containing carbon dots. Dissolve the precursor with dilute hydrochloric acid and centrifuge at 10,000 rpm for 10 min at room temperature to remove larger agglomerated particles. Subsequently, use a 100 - 500 Da dialysis bag to dialyze in pure water for 72 h, and a pale yellow carbon dot solution is obtained after purification.
[0035] Example 7: This example provides a copper- and calcium-ion co-doped carbon dot, which is prepared by the following method:
[0036] Weigh 0.2 g of folic acid, 0.5 g of citric acid, 1.0 g of urea, 0.5 g of copper(II) chloride dihydrate, and 0.5 g of calcium chloride and dissolve them in 3 mL of pure water. Heat at 120 °C for 70 min to dissolve. Under vacuum conditions, the product reacts at 250 °C for 60 min in a vacuum environment to obtain a fluffy brown spherical precursor containing carbon dots. Dissolve the precursor with dilute hydrochloric acid and centrifuge at 10,000 rpm for 10 min at room temperature to remove larger agglomerated particles. Subsequently, use a 100 - 500 Da dialysis bag to dialyze in pure water for 72 h, and a pale yellow carbon dot solution is obtained after purification.
[0037] The above are only the preferred embodiments of the present invention. It should be noted that for those skilled in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, which should also be regarded as the protection scope of the present invention, and these will not affect the implementation effect of the present invention and the practicality of the patent.
Claims
1. A copper and calcium ion co-doped carbon dot, characterized in that: Prepared by the following method: Folic acid, citric acid, urea, inorganic salts containing calcium ions and inorganic salts containing copper ions are dissolved in pure water. After the raw materials are completely dissolved by heating at 120°C for 60-80 minutes, the product is reacted at 200-300°C for 60 minutes under a vacuum environment to obtain a brown, fluffy and porous precursor containing carbon dots. After purification, a carbon dot solution co-doped with copper and calcium ions is obtained. Among them, the mass ratio of folic acid, citric acid, urea, inorganic salt containing calcium ions and inorganic salt containing copper ions is 1:2.5:5:(0.01-20):(0.01-10).
2. The copper and calcium ion co-doped carbon dots according to claim 1, characterized in that: The mass ratio of the folic acid, citric acid, urea, the inorganic salt containing calcium ions and the inorganic salt containing copper ions is 1:2.5:5:5:2.
5.
3. The copper and calcium ion co-doped carbon dots according to claim 1, characterized in that: The mass proportions of copper and calcium elements in the copper and calcium ion co-doped carbon dots are 6% and 15% or more respectively.
4. The copper and calcium ion co-doped carbon dots according to claim 1, characterized in that: The specific operation of the purification is as follows: First, the precursor was dissolved in dilute hydrochloric acid, centrifuged at 10000 rpm at room temperature for 10 min, and then dialyzed in pure water for 72 h using a 100-500 Da dialysis bag to obtain a light yellow carbon dot solution after purification.
5. An application of the copper and calcium ion co-doped carbon dots according to any one of claims 1 to 4, characterized in that: Used to prepare drugs targeting folate receptor-positive tumor cells.
6. An application of the copper and calcium ion co-doped carbon dots according to any one of claims 1 to 4, characterized in that: Used in the preparation of drugs for treating tumors.