Copper-doped carbon dot nano-enzyme as well as preparation method and application thereof

Abstract

The invention relates to a copper-doped carbon dot nano-enzyme and a preparation method and application thereof.The preparation method of the copper-doped carbon dot nano-enzyme comprises the steps that spinach powder serves as a precursor, CuCl2. 2H2O serves as a doping agent, and a copper-doped carbon dot nano-enzyme solution is prepared in a dilute H2SO4 solvent through a one-step solvothermal method; and centrifuging the obtained product, filtering to remove insoluble substances, dialyzing to remove impurities, and freeze-drying to obtain the copper-doped carbon dot nano-enzyme. The preparation method is simple and low in cost, the prepared copper-doped carbon dot nano-enzyme has peroxidase-like activity, glutathione oxidase-like activity and photo-thermal performance, H2O2 can be catalyzed to generate various active oxygen, glutathione overexpressed by tumor cells is consumed, and diagnosis and treatment of the tumor cells are achieved. The ROS therapy, the chemical kinetics therapy and the photothermal therapy are combined, and tumor ablation is remarkably achieved.

Description

Technical Field

[0001] This invention relates to the fields of fluorescent nanomaterials and biomedicine, specifically to a copper-doped carbon dot nanozyme, its preparation method, and its application. Background Technology

[0002] In recent years, with the continuous deepening of oncology research, innovative treatment strategies such as photothermal therapy (PTT), chemokinetic therapy (CDT), photodynamic therapy (PDT), and immunotherapy have shown significant advantages. Among them, PTT has attracted widespread attention due to its non-invasiveness, high spatial selectivity, and minimal damage to normal tissues. Its mechanism involves using photothermal agents to convert light energy into heat energy, inducing local high temperatures in the tumor, thereby specifically killing tumor cells. CDT utilizes hydrogen peroxide (H2O2) overexpressed in the tumor microenvironment (TME) to generate highly toxic hydroxyl radicals (·OH) through Fenton or Fenton-like reactions, achieving highly efficient and specific clearance of tumor cells.

[0003] Glutathione (GSH) is a potent antioxidant and detoxifier, playing a crucial role in maintaining cellular redox balance. However, overexpression of GSH at tumor sites, while promoting tumor proliferation, metastasis, and maintaining redox homeostasis, also scavenges reactive oxygen species (ROS) generated by glutathione glutathione (CDT), significantly reducing the effectiveness of CDT. Therefore, reducing intracellular GSH levels is an important strategy for improving the efficacy of CDT.

[0004] Cu 2+ It is a widely studied GSH consumable, and has been extensively investigated in the field of anti-tumor therapy. (Except for Cu) 2+ In addition, GSH can be depleted by other metal ions and enzyme-active nanomaterials. Compared with natural enzymes, nanozymes have advantages such as high catalytic efficiency, stable activity, and convenient preparation, and have wide applications in sensing, food safety, and biomedicine. Carbon dots (CDs), as an emerging fluorescent carbon nanomaterial, have the characteristics of wide availability of raw materials, simple preparation, good biocompatibility, and excellent photochemical performance. Their abundant functional groups and high specific surface area are conducive to the anchoring and synergistic effect of metal active sites. In recent years, hybrid nanozyme systems constructed by CDs and metals have been widely studied in the fields of catalysis and tumor therapy. The construction of such composite materials not only makes full use of the synergistic catalytic effect between CDs and metal components, but also achieves optimization of catalytic performance through careful structural design. However, research on developing the catalytic performance of CDs by directly doping them with metals is relatively limited, and the technology is not yet mature.

[0005] It should be noted that the above content falls within the inventor's technical knowledge and does not necessarily constitute prior art. Summary of the Invention

[0006] The purpose of this invention is to solve the problems existing in the prior art and provide a copper-doped carbon dot nanozyme, its preparation method and application. The preparation method of this invention has the advantages of being simple, having a wide range of raw material sources and low requirements for preparation conditions. The copper-doped carbon dot nanozyme obtained has multi-enzyme activity and photothermal properties, and can be used as a multifunctional nanomedicine for multimodal synergistic enhancement of tumor therapy.

[0007] The present invention achieves the above objectives by adopting the following technical solutions: This invention provides a copper-doped carbon dot nanozyme, comprising carbon quantum dots prepared using spinach powder as a precursor and copper chloride dihydrate (molecular formula CuCl2·2H2O) as a dopant. The copper-doped carbon dot nanozyme exhibits peroxidase (POD)-like activity, glutathione oxidase (GSHOx)-like activity, and photothermal properties, and can catalyze the production of various reactive oxygen species (ROS) from H2O2.

[0008] A method for preparing copper-doped carbon dot nanozymes includes the following steps: S1. Spinach powder and CuCl2·2H2O are ultrasonically dispersed into a dilute H2SO4 solution to prepare a mixed solution; S2. Place the mixture obtained in step S1 into a hydrothermal reactor and carry out a hydrothermal reaction. S3. Centrifuge and filter the product obtained in step S2 to remove insoluble matter and obtain a clear yellow solution. Dialyze the solution with a dialysis bag to remove impurities and obtain a copper-doped carbon dot nanozyme solution. S4. The copper-doped carbon dot nanozyme solution obtained in step S3 is freeze-dried to obtain the target copper-doped carbon dot nanozyme.

[0009] Preferably, in step S1, the mass-to-volume ratio of the spinach powder, CuCl2·2H2O, and dilute H2SO4 is 0.1~0.5 g : 20~50 mg : 10~50 mL.

[0010] Preferably, in step S1, the concentration of the dilute H2SO4 is 0.1 ~ 1.0 mol / L.

[0011] Preferably, in step S2, the temperature of the hydrothermal reaction is 120 ~ 200 ℃ and the time is 2 ~ 6 h.

[0012] Preferably, in step S3, the molecular weight cutoff of the dialysis bag is 500-1000 Da, and the dialysis time is 12-24 h.

[0013] This invention provides a copper-doped carbon dot nanozyme prepared by the above method.

[0014] This invention provides an application of copper-doped carbon dot nanozymes in the preparation of antitumor drugs. The prepared multifunctional nanomedicines (antitumor drugs) are used for multimodal synergistic enhancement of tumor therapy.

[0015] The present invention, by employing the above method, can bring the following beneficial effects: (1) This invention uses spinach powder as a precursor and CuCl2·2H2O as a dopant to prepare a copper-doped carbon dot nanozyme solution in dilute H2SO4 solvent using a one-step solvothermal method. After impurity removal and freeze-drying, the copper-doped carbon dot nanozyme is obtained. The preparation method of this invention is simple and low in cost. (2) The prepared copper-doped carbon dot nanozyme has peroxidase-like (POD) activity, glutathione oxidase-like (GSHOx) activity, and photothermal properties. It can catalyze the production of various reactive oxygen species (ROS) from H2O2 and consume glutathione (GSH) overexpressed by tumor cells, thereby achieving the diagnosis and treatment of tumor cells. This invention combines ROS therapy, chemokinetic therapy (CDT), and photothermal therapy (PTT) to significantly achieve tumor ablation. Attached Figure Description Figure 1 Transmission electron microscope image and size distribution diagram of the copper-doped carbon dot nanozyme prepared in this invention; Figure 2 The infrared spectrum of the copper-doped carbon dot nanozyme prepared in this invention; Figure 3 X-ray photoelectron spectroscopy (XPS) of the copper-doped carbon nanoparticle nanozyme prepared in this invention. Figure 4 The UV-Vis absorption spectrum, fluorescence excitation and emission spectrum, and images of the copper-doped carbon dot nanozyme solution under sunlight and UV light are shown for the copper-doped carbon dot nanozyme prepared in this invention. Figure 5 Temperature change over time of the copper-doped carbon dot nanozyme solution prepared for this invention under laser irradiation; Figure 6 Detection of peroxidase-like activity of copper-doped carbon nanoparticles prepared in this invention Figure 7 The types of reactive oxygen species and their ESR spectra generated in the catalytic reaction of the copper-doped carbon dot nanozyme prepared in this invention. Figure 8 For the in vitro GSH consumption detection of the copper-doped carbon dot nanozyme prepared in this invention; Figure 9 Detection of GSH consumption in vivo for the copper-doped carbon dot nanozyme prepared in this invention; Figure 10 Cytotoxicity of the copper-doped carbon dot nanozyme prepared in this invention; Figure 11Intracellular ROS detection of the copper-doped carbon dot nanozyme prepared in this invention; Figure 12 Analysis of the hemolytic behavior of the copper-doped carbon dot nanozyme prepared in this invention; Figure 13 The changes in mouse body weight and tumor volume during the treatment process of this invention; Figure 14 The organ indices of mice in each group after treatment according to this invention; Figure 15 This invention employs hematoxylin and eosin (H&E) staining to perform histopathological analysis of anatomical tissues from mice in different treatment groups. Detailed Implementation

[0016] To more clearly illustrate the overall concept of the present invention, a detailed description will be provided below with reference to the accompanying drawings and examples.

[0017] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and therefore the scope of protection of the invention is not limited to the specific embodiments disclosed below.

[0018] The various embodiments in this specification are described in a progressive manner. The same or similar parts between the various embodiments can be referred to each other. Each embodiment focuses on describing the differences from other embodiments.

[0019] Example 1 A method for preparing copper-doped carbon dot nanozymes with multi-enzyme activity and photothermal properties includes the following steps: S1. 0.2 g spinach powder and 0.035 g CuCl2·2H2O were ultrasonically dispersed in 20 mL of 0.3 M H2SO4 to prepare a mixed solution; S2. Place the mixture obtained in step S1 into a hydrothermal reactor and heat it at 160 °C for 4 h. S3. The product obtained in step S2 is centrifuged at 10,000 r / min for 10 min to remove insoluble matter and obtain a clear yellow solution. After filtration, the solution is dialyzed with a dialysis bag with a molecular weight cutoff of 500-1000 Da for 12 h to obtain a copper-doped carbon dot nanozyme solution. S4. Freeze-dry the copper-doped carbon dot nanozyme solution obtained in step S3 to obtain copper-doped carbon dot nanozyme.

[0020] Example 2 A method for preparing copper-doped carbon dot nanozymes with multi-enzyme activity and photothermal properties includes the following steps: S1 prepared a mixture by ultrasonically dispersing 0.2 g spinach powder and 0.05 g CuCl2·2H2O in 20 mL 0.3 M H2SO4; S2. Place the mixture obtained in S1 in a hydrothermal reactor and heat it at 180 °C for 4 h. S3. The product obtained in S2 was centrifuged at 10,000 r / min for 10 min to remove insoluble matter and obtain a clear yellow solution. After filtration, the solution was dialyzed with a dialysis bag with a molecular weight cutoff of 500-1000 Da for 18 h to obtain a copper-doped carbon dot nanozyme solution. S4. Freeze-dry the copper-doped carbon dot nanozyme solution obtained in S3 to obtain copper-doped carbon dot nanozyme.

[0021] Example 3 A method for preparing copper-doped carbon dot nanozymes with multi-enzyme activity and photothermal properties includes the following steps: S1. Disperse 0.3 g spinach powder and 0.05 g CuCl2·2H2O by ultrasonication in 30 mL 0.3 M H2SO4 to prepare a mixed solution; S2. Place the mixture obtained in S1 in a hydrothermal reactor and heat it at 200 °C for 5 h. S3. The product obtained in S2 was centrifuged at 10,000 r / min for 10 min to remove insoluble matter and obtain a clear yellow solution. After filtration, the solution was dialyzed with a dialysis bag with a molecular weight cutoff of 500-1000 Da for 24 h to obtain a copper-doped carbon dot nanozyme solution. S4. Freeze-dry the copper-doped carbon dot nanozyme solution obtained in S3 to obtain copper-doped carbon dot nanozyme.

[0022] To verify the feasibility of the preparation method of the present invention and the performance of the copper-doped carbon dot nanozyme obtained, the following verification was carried out using Example 1 as an example: (1) Characterization of the structure and optical properties of the copper-doped carbon nanoparticle nanozyme prepared in Example 1 like Figure 1 The image shown is a transmission electron microscope image of the prepared copper-doped carbon dot nanozyme. Figure 1 (a) and size distribution diagram ( Figure 1 (See Figure (b)). As can be seen from the figure, the copper-doped carbon nanoparticles have a quasi-spherical morphology, are uniformly dispersed, and have an average size of 2.83 ± 0.36 nm.

[0023] like Figure 2 The image shows the infrared spectrum of the prepared copper-doped carbon dot nanozyme. From the image, it can be concluded that compared to undoped carbon dots, the infrared spectrum of the copper-doped carbon dot nanozyme is significantly better at 1116 cm⁻¹. -1and 473 cm -1 The new peaks appearing nearby originate from the stretching vibrations of Cu-N and Cu-O, indicating that the carboxyl, hydroxyl, and amino groups interact with Cu. 2+ Coordination proves the successful doping of copper ions.

[0024] like Figure 3 The image shows the X-ray photoelectron spectroscopy (XPS) spectrum of the prepared copper-doped carbon nanozymes. From the image, it can be concluded that the copper-doped carbon nanozymes are mainly composed of five elements: C, N, O, S, and Cu.

[0025] like Figure 4 The image shows the UV-Vis absorption spectrum, fluorescence excitation and emission spectra of the prepared copper-doped carbon dot nanozymes, as well as images of the copper-doped carbon dot nanozyme solution under sunlight and UV light. The images show that the copper-doped carbon dot nanozymes exhibit significant absorption at 200 nm and 280 nm, and a broad absorption band in the 600-1000 nm range. Furthermore, the absorbance increases with increasing concentration of the copper-doped carbon dot nanozymes. The optimal excitation location for the copper-doped carbon dot nanozymes is 360 nm, and the optimal emission location is 460 nm. The solution appears pale yellow under sunlight and exhibits blue fluorescence under UV light.

[0026] (2) Photothermal performance analysis of copper-doped carbon nanoparticles prepared in Example 1 1.0 mL of different concentrations (0~1.0 mg / mL) -1 Copper-doped carbon dot nanozyme solutions were prepared at different laser power densities (0.25~1.00 W / cm²). -2 The photothermal performance of copper-doped carbon nanoparticle nanozymes was measured by irradiating them with a 660 nm laser for 15 min. Pure water was used as a control, and the experiment was conducted under the same conditions. Temperature changes were recorded every 1 min, and the photothermal conversion efficiency was calculated.

[0027] like Figure 5 As shown in the figure, the temperature change of the copper-doped carbon dot nanozyme solution over time is shown in (Figure (a) shows the temperature change of the copper-doped carbon dot nanozyme solution at a constant concentration under laser irradiation at different laser power densities, and Figure (b) shows the temperature change of copper-doped carbon dot nanozymes at different concentrations under laser irradiation at a constant laser power density). It can be seen from the figure that the temperature change of the copper-doped carbon dot nanozyme solution exhibits a significant dependence on laser power density. Figure 5 (a) and concentration dependence ( Figure 5 (See Figure (b)). The concentration of the copper-doped carbon nanoparticle nanozyme was 1.0 mg / mL. -1 Laser irradiation for 15 min (660nm, 1.0 W cm⁻¹) -2In the case of copper-doped carbon nanoparticles, the highest temperature rises to 49 ℃, which is sufficient to destroy the tumor, while the temperature of pure water only rises to 29.2 ℃. This indicates that copper-doped carbon nanoparticles can effectively absorb red light and convert light energy into heat energy to achieve tumor ablation. The photothermal conversion efficiency of copper-doped carbon nanoparticles is 29.2%.

[0028] (3) Detection of peroxidase-like (POD) activity of copper-doped carbon nanozymes prepared in Example 1 The peroxidase-like (POD) activity of copper-doped carbon dot nanozymes was investigated by catalytic oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) using H2O2. Copper-doped carbon dot nanozymes (0.14 mg / mL) were used as the catalyst. -1 H2O2 (0.1 mM) and TMB (0.25 mM) were added to a total volume of 2 mL of HAc-NaAc buffer solution with pH = 4, and the reaction was carried out at 40 °C for 15 min. The UV-Vis absorption and corresponding color changes of the solution at 652 nm were recorded.

[0029] like Figure 6 The figure shows the peroxidase (POD)-like activity of the prepared copper-doped carbon dot nanozyme. As can be seen from the figure, only when the copper-doped carbon dot nanozyme, TMB, and H2O2 coexist does the UV-Vis absorption spectrum of the solution show a significant absorption peak at 652 nm, and the solution color turns blue, indicating that the copper-doped carbon dot nanozyme possesses excellent POD-like activity.

[0030] (4) Determination of the types of reactive oxygen species (ROS) generated in the catalytic reaction of the copper-doped carbon nanozyme prepared in Example 1 and their electron spin spectroscopy (ESR). To determine the type of ROS generated during the catalytic reaction, isopropanol (IPA), p-benzoquinone (PBQ), and sodium azide (NaN3) were used as ·OH and O2, respectively. •- and 1 O2 scavengers were used to detect the types of ROS generated in the catalytic reaction. 5,5-Dimethyl-1-pyrrolidone-N-oxide (DMPO) and 2,2,6,6-tetramethylpiperidine (TEMP) were used as scavengers to capture ·OH / O2. •- and 1 O2.

[0031] like Figure 7 As shown, the types of reactive oxygen species (ROS) generated in the catalytic reaction and their ESR spectra are as follows. It can be seen from the figure that, compared with the blank group (without ROS scavengers), the POD-like activity of the copper-doped carbon dot nanozyme significantly decreased after the addition of IPA, PBQ, and NaN3. Figure 7Figure (a) shows that copper-doped carbon nanoparticles generated a large amount of ·OH and O2 during the catalytic oxidation of H2O2. •- and 1 O2. When copper-doped carbon nanoparticles, H2O2, and a scavenger (DMPO or TEMP) are present simultaneously, the ESR spectrum shows the presence of ·OH and O2. •- and 1 O2 signal peak ( Figure 7 Figure (b) further confirms that ·OH and O2 •- and 1 The generation of O2.

[0032] (5) Detection of glutathione oxidase (GSHOx) activity of the copper-doped carbon nanozyme prepared in Example 1: Using 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) as a probe, the glutathione (GSH) consumption capacity of copper-doped carbon dot nanozymes was measured, and their glutathione oxidase-like (GSHOx) activity was investigated. In the control group, a mixture of DTNB (30 μM) and different concentrations of GSH (2 mL) was incubated in PBS buffer (pH 7.4) for 5 min, and the absorbance at 412 nm was recorded. In the experimental group, copper-doped carbon dot nanozymes (0.14 mg / mL) were incubated... -1 The solution was reacted with GSH (50 μM) in PBS (pH 7.4) for 1 h, and then DTNB (30 μM) was added and reacted for 5 min. The absorbance of the solution at 412 nm was measured.

[0033] like Figure 8 The figure shows the glutathione oxidase (GSHOx) activity of the prepared copper-doped carbon dot nanozyme. As can be seen from the figure, pure DTNB has a characteristic absorption peak at 325 nm, while a new absorption peak appears at 412 nm after the addition of GSH. When the copper-doped carbon dot nanozyme is present, the absorbance at 412 nm decreases significantly, indicating that the copper-doped carbon dot nanozyme consumes GSH and exhibits glutathione oxidase (GSHOx)-like activity.

[0034] (6) Detection of GSH consumption in vivo of copper-doped carbon nanoparticles prepared in Example 1 Cancer cells (HeLa) were seeded into 35 mm culture dishes (3 × 10⁻⁶). 4 (1 cell / dish) until cells adhered. Then, the cells were incubated with copper-doped carbon nanoparticles for 24 h, and the GSH level of HeLa cells was measured using a GSH detection kit.

[0035] like Figure 9The figure shows the in vivo GSH consumption of copper-doped carbon nanozymes. As can be seen from the figure, the GSH level in HeLa cells incubated with copper-doped carbon nanozymes was reduced by 14% compared to the control group, confirming that copper-doped carbon nanozymes can effectively reduce the intracellular GSH level in tumor cells, thereby disrupting intracellular oxidative defense mechanisms and enhancing the therapeutic effect on tumors.

[0036] (7) Cytotoxicity test of the copper-doped carbon nanoparticle nanozyme prepared in Example 1 In vitro cytotoxicity was determined using the CCK-8 assay. First, normal cells (HL-7702) and cancer cells (HeLa) were seeded into 96-well plates (1×10⁻⁶ cells / wells). 4 Cells / well were placed in a cell culture incubator and cultured for 24 h to allow them to adhere. Then, 200 μL of culture medium containing different concentrations of copper-doped carbon nanoparticles was added, and the cells were incubated for another 24 h. After incubation, the cells were washed three times with PBS buffer (pH 7.4). The light-treated group was then subjected to 5 min of light treatment (660 nm, 1.0 W cm⁻¹). -2 Then, 180 μL of fresh culture medium and 20 μL of LCK-8 solution were added to each well. After incubation for 40 min, the absorbance of each well at 450 nm was recorded using a microplate reader, and the cell viability was calculated.

[0037] like Figure 10 The figure shows the cytotoxicity of copper-doped carbon nanoparticle nanozymes (Figure (a) shows the cytotoxicity of copper-doped carbon nanoparticle nanozymes to normal cells (HL-7702) and cancer cells (HeLa), and Figure (b) shows the cytotoxicity of copper-doped carbon nanoparticle nanozymes to cancer cells with and without laser irradiation). It can be seen from the figure that when the concentration of copper-doped carbon nanoparticle nanozymes reaches as high as 500 μg / mL... -1 At that time, the survival rate of HL-7702 cells reached 60%, while the survival rate of HeLa cells was only 25%. Figure 10 Figure (a) shows that copper-doped carbon dot nanozymes have a high selective killing ability against cancer cells. To further verify the photothermal therapy (PTT) effect of copper-doped carbon dot nanozymes in vitro, a 660 nm laser was introduced as an exogenous stimulus. After irradiation with the 660 nm laser, the survival rate of HeLa cells co-incubated with copper-doped carbon dot nanozymes further decreased. The copper-doped carbon dot nanozyme concentration was 500 μg / mL. -1 At that time, the survival rate of HeLa cells decreased to 15% ( Figure 10 Figure (b) shows that PTT treatment further exacerbated cancer cell death.

[0038] (8) Detection experiment of intracellular reactive oxygen species (ROS) of copper-doped carbon nanozymes prepared in Example 1 Intracellular ROS were measured using the 2',7'-dichlorodihydrofluorescein diacetate (DCFH DA) probe. HeLa cells or HL-7702 cells were cultured at 3 × 10⁻⁶ cells / year. 5 Cells were seeded at a density of 1 cell / dish in 35 mm cell culture dishes and cultured for 24 h to allow cell adhesion. Then, 1 mL of culture medium containing copper-doped carbon nanoparticles was added, and the cells were incubated at 37 °C for 6 h. The light-treated group underwent 5 min of light exposure (660 nm, 1.0 W cm⁻¹). -2 The cells were then washed with PBS, incubated with medium containing 10 μM DCFH-DA for 30 min, and then the intracellular ROS signal was observed using a fluorescence microscope after PBS washing.

[0039] like Figure 11 The figure shows the intracellular ROS detection results of copper-doped carbon nanoparticle nanozymes. As can be seen from the figure, compared with untreated HeLa cells and HL-7702 cells, HeLa cells treated with copper-doped carbon nanoparticle nanozymes exhibited bright green fluorescence, verifying the ROS generation capacity of copper-doped carbon nanoparticle nanozymes at the cancer cell level. Furthermore, HeLa cells co-treated with copper-doped carbon nanoparticle nanozymes and a 660 nm laser showed even stronger green fluorescence, indicating that the photothermal effect further promoted intracellular ROS generation.

[0040] (9) In vitro hemolysis experiment of copper-doped carbon nanoparticle nanozyme prepared in Example 1 The blood compatibility of copper-doped carbon nanodot nanozymes was evaluated using an in vitro hemolysis assay. Fresh whole blood was obtained from healthy BALB / c mice. Red blood cells were separated from the serum by centrifugation at 1000 rpm for 5 min at 4 °C. After washing three times with PBS, the purified red blood cells were diluted 20-fold. Then, 0.2 mL of the diluted red blood cell suspension was diluted to 1 mL with PBS containing different concentrations of copper-doped carbon nanodot nanozymes. After incubation at 37 °C for 3 h, the suspension was centrifuged at 1000 rpm for 3 min. The absorbance of the supernatant at 540 nm was recorded by photographing and the hemolysis rate was calculated. Red blood cells treated with deionized water and PBS were used as positive and negative controls, respectively.

[0041] like Figure 12 The figure shows the hemolysis results of copper-doped carbon dot nanozymes. As can be seen from the figure, the concentration of copper-doped carbon dot nanozymes is as high as 500 μg / mL. -1 At that time, the hemolysis rate was only 1.72%, indicating that copper-doped carbon dot nanozymes have good blood compatibility.

[0042] (10) In vivo antitumor therapy experiment of copper-doped carbon nanozyme prepared in Example 1 4T1 tumor-bearing mice were randomly divided into three groups (n=3): (1) saline group, (2) copper-doped carbon nanoparticle nanozyme group, and (3) copper-doped carbon nanoparticle nanozyme + light irradiation group. When the tumor volume increased to approximately 70 mm... 3 Mice received local injections every two days for a total of four times. The copper-doped carbon nanoparticle nanozyme + light irradiation group received 660 nm laser irradiation for 15 minutes 30 minutes after each injection. Body weight and tumor size were monitored every two days starting from the first administration. Finally, all mice were euthanized, and key tissues (heart, liver, spleen, lung, kidney, and tumor) were collected for H&E staining, pathological analysis, and organ index calculation.

[0043] like Figure 13 The figures show the changes in body weight and tumor volume in mice during treatment. Figure (a) shows the average tumor volume after different treatments, (b) shows the average body weight after different treatments, (c) shows a photograph of the tumor removed at the end of treatment, and (d) shows the average tumor weight after different treatments. After 12 days of treatment, the tumor volume of mice injected with saline increased significantly, while the tumor volume of mice injected with copper-doped carbon nanoparticle nanozymes decreased by 55%. Under 660 nm laser irradiation, the tumor volume of mice injected with copper-doped carbon nanoparticle nanozymes completely disappeared after 12 days of treatment. During the treatment period, there were no significant abnormal fluctuations in mouse body weight. Compared with the control group without tumor transplantation, there were no significant differences in the indices of the heart, liver, spleen, lungs, and kidneys in the copper-doped carbon nanoparticle nanozyme treatment group and the copper-doped carbon nanoparticle nanozyme + light irradiation treatment group (e.g., ...). Figure 14 As shown in the figure, this indicates that copper-doped carbon dot nanozymes have no obvious toxic side effects.

[0044] like Figure 14 The figure shows the organ indices of mice in each group after treatment, with tumor-free mice serving as the control group. Figure 15 Histopathological analysis of anatomical tissues (tumor, heart, liver, spleen, lung, and kidney) in mice from different treatment groups was performed using hematoxylin and eosin (H&E) staining (Control group mice represent mice not inoculated with tumor cells). Results showed that compared to the saline group, the spleen index decreased in the copper-doped carbon nanoparticle nanozyme treatment group and the copper-doped carbon nanoparticle nanozyme + phototherapy group, indicating that both groups could alleviate tumor-induced splenomegaly. Splenomegaly may be a signal of poor treatment efficacy. Based on the spleen index results, the spleen index of the mice in the copper-doped carbon nanoparticle nanozyme treatment group and the copper-doped carbon nanoparticle nanozyme + phototherapy group was close to that of the healthy mice in the control group, demonstrating excellent tumor treatment efficacy.

[0045] H&E staining of major organs in mice revealed acute and chronic inflammation in the livers of all groups. Mild extramedullary hematopoiesis was observed in the spleens of mice in the copper-doped carbon nanoparticle nanozyme treatment group and the copper-doped carbon nanoparticle nanozyme + phototherapy group, while significant extramedullary hematopoiesis was observed in the spleens of mice in the saline group. Local pulmonary septal widening and acute and chronic inflammation were also observed in mice in the saline group. Tumor sections from all groups showed significant necrosis, with the tumor volume in the saline group being significantly larger than that in the copper-doped carbon nanoparticle nanozyme treatment group.

[0046] The above results demonstrate that the preparation method of this invention is feasible, and the prepared copper-doped carbon nanodot nanozymes exhibit good biocompatibility, high stability, and good water solubility. They possess peroxidase-like (POD) activity, glutathione oxidase-like (GSHOx) activity, and photothermal properties. They can catalyze the production of various reactive oxygen species (ROS) from H₂O₂ and consume glutathione (GSH) overexpressed by tumor cells, thus achieving synergistic therapy against tumor cells. This suggests potential applications in cancer treatment.

[0047] The above specific embodiments should not be construed as limiting the scope of protection of the present invention. For those skilled in the art, any alternative improvements or modifications made to the embodiments of the present invention shall fall within the scope of protection of the present invention.

[0048] Any aspects of this invention not described in detail are well-known to those skilled in the art.

Claims

1. A copper-doped carbon dot nanozyme, characterized in that, This includes carbon quantum dots prepared using spinach powder as a precursor and copper chloride dihydrate as a dopant.

2. The method for preparing a copper-doped carbon dot nanozyme according to claim 1, comprising the following steps: S1. Spinach powder and CuCl2·2H2O are ultrasonically dispersed into a dilute H2SO4 solution to prepare a mixed solution; S2. Place the mixture obtained in step S1 into a hydrothermal reactor and carry out a hydrothermal reaction. S3. Centrifuge and filter the product obtained in step S2 to remove insoluble matter and obtain a clear yellow solution. Dialyze the solution with a dialysis bag to remove impurities and obtain a copper-doped carbon dot nanozyme solution. S4. The copper-doped carbon dot nanozyme solution obtained in step S3 is freeze-dried to obtain the target copper-doped carbon dot nanozyme.

3. The method for preparing copper-doped carbon dot nanozymes according to claim 2, characterized in that, In step S1, the mass-to-volume ratio of the spinach powder, CuCl2·2H2O and dilute H2SO4 is 0.1~0.5 g : 20~50 mg : 10~50 mL.

4. The method for preparing a copper-doped carbon dot nanozyme according to claim 3, characterized in that, In step S1, the concentration of the dilute H2SO4 is 0.1~1.0 mol / L.

5. The method for preparing a copper-doped carbon dot nanozyme according to claim 4, characterized in that, In step S2, the temperature of the hydrothermal reaction is 120 ~ 200 ℃, and the time is 2 ~ 6 h.

6. The method for preparing a copper-doped carbon dot nanozyme according to claim 5, characterized in that, In step S3, the molecular weight cutoff of the dialysis bag is 500-1000 Da, and the dialysis time is 12-24 h.

7. Copper-doped carbon dot nanozymes prepared by the preparation method according to any one of claims 2-6.

8. The application of the copper-doped carbon nanodot nanozyme according to claim 1 in the preparation of antitumor drugs.

9. The application of the copper-doped carbon nanodot nanozyme according to claim 7 in the preparation of antitumor drugs.

Images