A near-infrared second-region fluorine-doped carbon dot nanozyme for photothermal enhanced catalytic tumor immunotherapy and its preparation method and application

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to a near-infrared II fluorine-doped carbon dot nanozyme (F‑CDs@PEG) for photothermal-enhanced catalytic immunotherapy, as well as its preparation method and application. F‑CDs excel in NIR-II fluorescence emission for in vivo bioimaging and NIR photothermal therapy, and also possess photothermally enhanced Gox-like, POD-like, and GSH-px activities, which can be used to disrupt the energy metabolism and redox balance of tumor cells, enhance immunotherapy, and reshape the tumor microenvironment. Photothermal-enhanced immunotherapy induces tumor immunogenic cell death and further promotes the maturation of antigen-presenting cells such as DCs, thereby enhancing T cell infiltration within the tumor. In addition, F‑CDs@PEG reduces G‑CSF levels under NIR (808nm) laser irradiation, thereby inhibiting MDSC levels, alleviating T cell exhaustion, and effectively inhibiting tumor growth; the photothermal-enhanced catalytic immunotherapy mediated by F‑CDs@PEG can induce strong immune memory and have a long-term inhibitory effect on recurrent tumors.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and specifically to a near-infrared second-zone fluorine-doped carbon dot nanozyme for photothermal enhanced catalytic tumor immunotherapy, as well as a preparation method and application thereof. Background Art

[0002] Catalytic immunotherapy activates anti-tumor immune responses through catalytic reactions using biocompatible nanocatalysts. It primarily targets substrates within the tumor microenvironment, utilizing advanced catalytic materials to initiate the reaction, which is activated through stimuli such as light, ultrasound, temperature, and electric fields. This approach facilitates tumor clearance and immune regulation. Compared to traditional chemotherapy or radiotherapy, catalytic immunotherapy offers several advantages, including the use of smaller amounts of catalyst, high efficiency, and selective targeting of tumor-specific substrates. Importantly, nanocatalytic immunotherapy can reshape the immunosuppressive tumor microenvironment (TME) by targeting tumor metabolites, activating anti-tumor immunity and generating long-term immune memory. The exploration of various advanced catalytic materials has opened up new avenues for enhancing catalytic immunotherapy, aiming to boost immune responses and improve therapeutic efficacy while minimizing side effects.

[0003] Carbon dots (CDs) have attracted increasing attention in the field of biomaterials due to their excellent optical properties, low toxicity, high water dispersibility, and low production cost. They show great potential for applications in photoelectric conversion, bioimaging, biosensing, nanomedicine, and catalysis. Nanozymes based on nonmetallic carbon dots (NMDs) have attracted considerable attention due to their excellent physical and chemical properties. In recent years, many fluorescent Cdots and their hybrids have been reported to exhibit enzyme-like activities, such as peroxidase (POD), catalase (CAT), oxidase (OXD), and superoxide dismutase (SOD)-like activities. Furthermore, due to their superior photoactivity, CDs have the potential to be used as photothermal agents for photothermal therapy (PTT), which is also expected to enhance enzyme-like activity by promoting electron transfer efficiency through the thermal field. Nanozymes based on CDs can serve as multifunctional nanoplatforms for noninvasive optical diagnosis, photodynamic therapy, and catalytic therapy, as well as synergistic treatments with PTT / PDT.

[0004] Although some CD-based nanozymes have been developed in recent years, the vast majority of CD nanozymes have short emission wavelengths in the blue / green region of the spectrum, limited penetration depth, and can damage surrounding tissues. Furthermore, metal doping (i.e., Fe, Mn, Co, Ni, Cu) can enhance the NIR photoactivity and enzymatic activity of CDs. However, the fluorescence of CDs is quenched by metal ions, which limits their tracking in vitro and in vivo. The introduction of metals often raises significant concerns about long-term toxicity. Non-metallic carbon dot nanozymes that exhibit both NIR-II emission and NIR photoactivity are currently rare. New strategies for preparing NIR-II CD nanozymes with high QY and long excitation wavelengths will address the significant unmet need for cancer catalytic immunotherapy. Summary of the Invention

[0005] In response to the shortcomings of the existing technology, the present invention provides a NIR-II fluorescent F-doped CDs (F-CDs) based on non-metal doping, with high fluorescence quantum yield (QY) and photothermally enhanced multi-enzyme activity, and a preparation method thereof, and uses them in cancer catalytic immunotherapy. Through photothermally enhanced multi-enzyme activity, tumor microenvironment (TME) remodeling, immune activation and long-term immune memory are achieved, providing an innovative solution for tumor treatment.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for preparing near-infrared second-region fluorine-doped carbon dot nanozymes for photothermal enhanced catalytic immunotherapy comprises the following steps:

[0008] S1. Synthesis of crude fluorine-doped carbon dots: using p-phenylenediamine, dopamine hydrochloride, and (1S)-3,3′-difluoro[1,1′-binaphthyl]-2,2′-diol in a molar ratio of 2:2:1 as raw materials, a hydrothermal method, a solvothermal method, or a microwave-assisted method was used to synthesize crude fluorine-doped carbon dots;

[0009] S2. Separation and purification: obtaining pure fluorine-doped carbon dots by separating and purifying the crude fluorine-doped carbon dots;

[0010] S3. PEG modification: The pure fluorine-doped carbon dots are dissolved in water, and PEG is added to react fully to obtain well-water-dispersible F-CDs@PEG.

[0011] In some embodiments, in step S1, the synthesis method is a solvothermal method, and the solvent used is ethanol.

[0012] Furthermore, in step S2, the separation and purification process includes filtration, dialysis to remove impurities and freeze-drying.

[0013] Furthermore, the preparation method further comprises the steps of purifying the F-CDs@PEG: centrifugal separation, distilled water washing, and freeze drying.

[0014] Preferably, the PEG used in step S3 is PEG200.

[0015] The present invention also provides a near-infrared zone II fluorine-doped carbon dot nanozyme for photothermal enhanced catalytic immunotherapy prepared by the above preparation method and its application. The near-infrared zone II fluorine-doped carbon dot nanozyme is used to prepare tumor immunotherapy drugs.

[0016] Furthermore, the tumor is a primary tumor or a distal tumor.

[0017] Furthermore, the near-infrared second zone fluorine-doped carbon dot nanozyme is also used to prepare drugs for combined use of photothermal therapy, catalytic therapy and immunotherapy.

[0018] The beneficial effects of the present invention are:

[0019] This study developed a near-infrared II fluorine-doped carbon dot nanozyme (F-CDs@PEG) for photothermal-enhanced catalytic immunotherapy. F-CDs excel in NIR-II fluorescence emission for in vivo bioimaging and NIR photothermal therapy (PTT). They also possess photothermally enhanced glucose oxidase-like (Gox), peroxidase-like (POD), and glutathione peroxidase-like (GSH-px) activities, enabling them to disrupt the energy metabolism and redox balance of tumor cells, significantly enhancing immunotherapy and reshaping the tumor microenvironment (TME). Photothermal-enhanced immunotherapy induces tumor immunogenic cell death (ICD) and further promotes the maturation of antigen-presenting cells, such as dendritic cells (DC), thereby enhancing T cell infiltration within the tumor. Furthermore, F-CDs@PEG reduced tumor granulocyte colony-stimulating factor (G-CSF) levels under NIR (808 nm) laser irradiation, thereby suppressing the levels of immunosuppressive myeloid-derived suppressor cells (MDSCs) and alleviating T cell exhaustion, effectively inhibiting tumor growth. Its mediated photothermal-enhanced catalytic immunotherapy induced robust immune memory and had a long-term inhibitory effect on recurrent tumors. In summary, F-CDs@PEG reshaped the TME through photothermal-enhanced multi-substrate catalytic reactions, significantly hindering the progression of primary and distant tumors.

[0020] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for use in the specific embodiments or the description of the prior art. Obviously, some of the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 Schematic diagram of the synthesis of F-CDs.

[0023] Figure 2 Transmission electron microscopy images of F-CDs.

[0024] Figure 3 are the UV spectra and fluorescence spectra of F-CDs, where Figure 3 Part a shows the absorption band of F-CDs (DMSO solution) from visible light to near infrared region. Figure 3 Part b shows that F-CDs (DMSO-dissolved) exhibit obvious NIR-II emission in the range of 900 to 1400 nm.

[0025] Figure 4 This is a test diagram of the photothermal enhanced catalytic performance of F-CDs, where Figure 4 Part a shows that when methylene blue (MB) was incubated with F-CDs solution and H2O2 in NaHCO3 buffer for 10 min, the absorbance decreased significantly; Figure 4 Part b shows the absorbance of the mixture after incubation of F-CDs and GSH at different time points and temperatures; compared with 25°C, when the glucose concentration was 400mM, the Gox mimetic activity of F-CDs at 45°C and 37°C was 7 times and 3.7 times, respectively ( Figure 4 (Part c).

[0026] Figure 5 In vivo imaging of F-CDs@PEG.

[0027] Figure 6 Confocal microscopy images of tumor cell internalization and colocalization of F-CDs@PEG.

[0028] Figure 7 This is a diagram showing the effect of F-CDs@PEG on the generation of intracellular reactive oxygen species under NIR treatment.

[0029] Figure 8 The in vivo photothermal performance diagram of F-CDs@PEG.

[0030] Figure 9Figure 14 shows the tumor treatment effects after treatment with PBS, NIR, F-CDs@PEG, and F-CDs@PEG+NIR. Figure 9 As shown in part a, the tumor volumes of the control group and NIR group increased by 7.2 times and 7.1 times, respectively, and the F-CDs@PEG group increased by about 5.07 times. In contrast, F-CDs@PEG combined with laser treatment showed a significant tumor inhibitory effect, with some cases experiencing tumor regression or cessation of growth ( Figure 9 b, c).

[0031] Figure 10 This is the effect diagram of splenomegaly relief on the 14th day after treatment with PBS, NIR, F-CDs@PEG and F-CDs@PEG+NIR.

[0032] Figure 11 Figure 1 is the flow cytometric analysis of immune activation on the 14th day after treatment with PBS, NIR, F-CDs@PEG and F-CDs@PEG+NIR; Figure 11 Part (a) shows the results of analyzing immunosuppressive cell populations in tumor samples, specifically MDSCs; Figure 11 Part b shows that the percentage of MDSCs decreased in the F-CDs@PEG and F-CDs@PEG+NIR groups; by analyzing the T helper cells (CD3 + CD4 + ) and cytotoxic T lymphocytes (CTLs) (CD3 + CD8 + ) group, confirmed the activation of anti-tumor immunity ( Figure 11 Part c); CD3 of F-CDs@PEG+NIR group + CD4 + and CD3 + CD8 + The number of T cells was the highest, which was 2.3 times and 1.6 times that of the F-CDs@PEG group alone, and 2 times and 3 times that of the control group ( Figure 11 CD8 + PD-1 expression in the T cell gate ( Figure 11 The results showed that in the F-CDs@PEG and F-CDs@PEG+NIR groups, CD8 + PD-1 expression on T cells was significantly reduced, indicating that treatment alleviated T cell exhaustion ( Figure 11 (see section g in the figure).

[0033] Figure 12Figure 2 shows the flow cytometry analysis of immune memory after treatment with PBS, NIR, F-CDs@PEG and F-CDs@PEG+NIR; Flow cytometry analysis of CD44 in distant tumors + CD62L - Effector memory T cells were found in the CD4 + TEM cells ( Figure 12 The proportion of CD8 in the F-CDs@PEG+NIR laser group was significantly higher than that in the other groups; the flow cytometry results showed that the CD8 + CD44 in T cell populations + CD62L + The proportion of central memory T cells was the highest, which was 5 times and 1.5 times that of the control group and the simple F-CDs@PEG group, respectively ( Figure 12 (parts c and d). DETAILED DESCRIPTION

[0034] In order to better describe the present invention, the following is further illustrated by specific examples. The methods in the following examples are conventional methods unless otherwise specified.

[0035] Unless otherwise specified, the technical solutions described in the present invention are all conventional solutions in the field; the reagents or materials described are all from commercial channels unless otherwise specified.

[0036] The following examples are directed to the intermediate compounds and final products identified in the specification and synthesis schemes. The preparation of the compounds of the present invention is described in detail using the following examples, but the chemical reactions described are disclosed based on their general applicability to the preparation of the compounds of the present invention. Sometimes, the reactions may not be applicable to each compound within the scope of the present invention as described. Those skilled in the art will readily recognize compounds for which this will occur. In these cases, the reactions can be successfully carried out by conventional modifications known to those skilled in the art. In all preparation methods, all raw materials are known or can be easily prepared using known raw materials. All temperatures are given in degrees Celsius, and unless otherwise expressly stated, all parts and percentages are based on the amount of substance when referring to yields, and all parts are based on volume when referring to solvents and eluents.

[0037] Example 1 Preparation of F-CDs and F-CDs@PEG

[0038] Step 1: Using p-phenylenediamine, dopamine hydrochloride and (1S)-3,3′-difluoro[1,1′-binaphthyl]-2,2′-diol as raw materials (ratio: 2:2:1) and ethanol as solvent, (F-CDs) were synthesized by solvothermal method ( Figure 1 ).

[0039] Step 2: Heat at 200°C for 12 hours. After cooling to room temperature, filter through a 200 μm filter membrane. The resulting product is placed in a 1000 Da dialysis bag and stirred for three days. Finally, the pure product is collected and freeze-dried to obtain F-CDs with a yield of 48.3%. Transmission electron microscopy (TEM) images show that the F-CDs have a spherical morphology with a diameter of approximately 3 nm ( Figure 2 ).

[0040] Step 3: The prepared fluorocarbon dots (F-CDs) were dissolved in ultrapure water and sonicated for 5 minutes to prepare a 1 mg / mL dispersion. Subsequently, 0.5 mg of PEG200 was added. After stirring for 2 hours, the sample was collected by centrifugation at 12,500 rpm for 10 minutes, washed twice with distilled water, and finally freeze-dried to obtain F-CDs@PEG.

[0041] Example 2 Characteristics of F-CDs and F-CDs@PEG

[0042] 2.1 Simulated enzyme catalytic activity

[0043] The absorption spectrum of F-CDs dissolved in 10% DMSO shows absorption bands from the visible to the near-infrared region ( Figure 3 (a) Under 808 nm laser excitation, F-CDs (DMSO solution) showed obvious NIR-II emission in the range of 900 to 1400 nm ( Figure 3 Part b).

[0044] When methylene blue (MB) was incubated with F-CDs solution and H2O2 in NaHCO3 buffer for 10 min, a significant decrease in absorbance was observed ( Figure 4 (part a) of the figure). This decrease can be attributed to the efficient generation of ROS triggered by the catalytic reaction driven by F-CDs, and the catalytic activity of the simulated POD was observed to increase with increasing temperature. Glutathione (GSH) is a complex metabolite of the intracellular antioxidant mechanism that can effectively scavenge ROS produced by nanozymes, thereby reducing the therapeutic index. F-CDs can efficiently convert GSH into GSSG through an enzyme-like redox cycle. To evaluate the GSH depletion ability, we used 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) as a GSH probe to explore the GSH depletion ability of F-CDs. F-CDs were incubated with GSH at different time points and temperatures, and the absorbance of the mixture was measured ( Figure 4(Part b) As the incubation temperature increases, the absorbance of DTNB at 412 nm decreases significantly, indicating that fluorine doping and elevated temperature enhance GSH consumption. Furthermore, the catalytic activity of F-CDs at 45°C is 3.6 times higher than that at 37°C, confirming that F-CDs can deplete excess GSH in tumor cells. These results demonstrate that F-CDs can effectively serve as biocatalysts, enhancing oxidative stress in cancer cells by cyclically decomposing H₂O₂ and consuming GSH.

[0045] In addition to POD mimetic enzyme and GSH-px mimetic enzyme activity, Gox mimetic enzyme activity was further explored. Because glucose oxidase can convert glucose into H2O2 and gluconic acid. 2+ The generation of H2O2 after incubation of F-CDs with glucose was detected using xylenol orange as an indicator. The Gox catalytic activity of F-CDs increased with increasing temperature. Compared to 25°C, the Gox mimetic activity of F-CDs at 45°C and 37°C increased by 7 and 3.7 times, respectively, at a glucose concentration of 400 mM. Figure 4 (Part c) indicates that the Gox mimetic activity of F-CDs is temperature-dependent. The generated H2O2 provides more substrate for the POD-catalyzed reaction, leading to increased ROS production. These results collectively indicate that F-CDs can activate a cascade of reactions, with sustained POD-mimicking, GSH-px-mimicking, and Gox-mimicking catalytic activity, to generate more cytotoxic OH species for tumor oxidative damage.

[0046] 2.2 Efficient clearance and safety in the body

[0047] 100 μL of F-CDs@PEG solution (5 mg / mL) was injected into normal mice for NIR-II imaging. F-CDs@PEG showed bright NIR-II signals and was easy to monitor in vivo with a high signal-to-noise ratio. These signals gradually decreased over time, indicating that F-CDs@PEG has relatively efficient clearance and safety in vivo. Figure 5 shown.

[0048] 2.3 Uptake and intracellular catalysis

[0049] Further using CLSM ( Figure 6) observed the uptake behavior of F-CDs@PEG in 4T1 cells. After incubation for 24 h, F-CDs@PEG showed bright red fluorescence signals in 4T1 cancer cells. The intracellular transport of F-CDs@PEG was then evaluated by CLSM. After 4T1 cells were incubated with F-CDs@PEG for 24 h, they were co-stained with the endo / lysosome selective marker LysoTracker Green and the mitochondrial selective marker Mito-Tracker Green, respectively. The red fluorescence of F-CDs@PEG co-localized with mitochondria (PCC=35%) and lysosomes (PCC=46%) ( Figure 6 These results indicate that F-CDs@PEG can be effectively taken up by 4T1 tumor cells. Once internalized by tumor cells, they accumulate in lysosomes and mitochondria for further catalytic reactions.

[0050] 2',7'-dichlorofluorescein diacetate (DFCH-DA) was used as an indicator to detect intracellular ROS generation. CLAM images showed that Figure 7 In the study, we clearly observed the fluorescence signal of ROS in cancer cells in the F-CDs@PEG group, which was up to 5 times higher than that in the control group. In addition, under mild NIR-II laser irradiation, the nanozyme showed the strongest ROS level, which fully demonstrated that the F-CDs@PEG nanozyme can integrate the multiple functions of triple enzyme mimic activity and mild NIR-II photothermal effect to achieve efficient intracellular catalysis.

[0051] Example 3 Antitumor effect of F-CDs@PEG

[0052] 3.1 Experimental subjects:

[0053] BALB / c mice bearing 4T1 subcutaneous tumors were randomly divided into 4 groups: (1) control group; (2) NIR-I group; (3) F-CDs@PEG group; (4) F-CDs@PEG+NIR laser (808 nm, 1 W cm -2 ) group. After intratumoral injection of F-CDs@PEG in treatment groups (3) and (4), photothermal images of treated mice were collected. The tumor temperature of mice injected with normal saline showed no significant change under 808nm laser irradiation. In contrast, the tumor temperature of mice treated with F-CDs@PEG increased from 35.7℃ to 55.8℃ within 5 minutes of laser irradiation ( Figure 8 ).

[0054] 3.2 Experimental Results

[0055] Tumor volume was recorded every two days. Figure 9As shown in part a, the tumor volume of the control group and NIR group increased by 7.2 times and 7.1 times, respectively. In addition, the F-CDs@PEG group increased by about 5.07 times, indicating that the efficacy of F-CD@PEG alone in treating tumors is limited. In contrast, F-CDs@PEG combined with laser treatment showed a significant tumor inhibitory effect, with some cases showing tumor regression or cessation of growth ( Figure 9 b, c).

[0056] Splenomegaly was observed in 4T1 tumor-bearing mice in the control, NIR, and F-CDs@PEG groups. However, the F-CDs@PEG + laser irradiation group showed a significant reduction in splenomegaly, with the spleen returning to normal weight and size ( Figure 10 )

[0057] 3.3 Analysis and Summary

[0058] Immunosuppressive cell populations, particularly MDSCs ( Figure 11 The percentage of MDSCs decreased in the F-CDs@PEG and F-CDs@PEG+NIR groups ( Figure 11 The F-CDs@PEG+NIR group had the lowest MDSC level of 15.5%, demonstrating its superior immunomodulatory effect. The inhibition of MDSCs by the F-CDs@PEG+NIR group further enhanced the function and activity of effector T cells. + CD4 + ) and cytotoxic T lymphocytes (CTLs) (CD3 + CD8 + ) group, confirmed the activation of anti-tumor immunity ( Figure 11 (Part c) CD3 of F-CDs@PEG+NIR group + CD4 + and CD3 + CD8 + The number of T cells was the highest, which was 2.3 times and 1.6 times that of the F-CDs@PEG group alone, and 2 times and 3 times that of the control group. Figure 11 d, e). Reduced PD-1 expression may enhance T cell function by alleviating T cell exhaustion, lowering activation threshold, regulating immune tolerance, and changing metabolic status. + PD-1 expression in the T cell gate ( Figure 11 The results showed that in the F-CDs@PEG and F-CDs@PEG+NIR groups, CD8 + PD-1 expression on T cells was significantly reduced, indicating that treatment alleviated T cell exhaustion ( Figure 11 (see section g in the figure).

[0059] To evaluate the ability to establish an immune response against tumor metastasis, a bilateral 4T1 tumor-bearing mouse model was established. The mice were randomly divided into four groups: control group, NIR-I laser group, F-CDs@PEG group, and F-CDs@PEG+NIR laser group. Subsequently, the effector memory T cells (Tem) (CD44 + CD62L - ) cells were evaluated because they can be reactivated and expanded to kill tumor cells upon re-encountering tumor antigens. Flow cytometric analysis of CD44 + CD62L - Effector memory T cells were found in the CD4 + TEM cells ( Figure 12 The proportion of central memory T cells (Tcm) (CD44 + 、CD62L + ) is a T cell that acquires long-term memory after being activated by antigens and can be reactivated by tumor antigens to directly kill tumors. Flow cytometry results showed that F-CDs@PEG+NIR laser group CD8 + CD44 in T cell populations + CD62L + The proportion of central memory T cells was the highest, which was 5 times and 1.5 times that of the control group and the simple F-CDs@PEG group, respectively ( Figure 12 (Fig. 2c, d). No distant tumor recurrence was observed. Furthermore, the Tcm of the F-CDs@PEG group alone increased 2.5-fold compared with the control group. These results confirm that photothermal-enhanced catalytic immunotherapy with F-CDs@PEG can induce long-term immune memory responses and potentially prevent tumor recurrence.

[0060] In summary, this invention has developed a near-infrared II fluorine-doped carbon dot nanozyme, F-CDs@PEG, for photothermal-enhanced catalytic immunotherapy. F-CDs excel in NIR-II fluorescence emission for in vivo bioimaging and NIR photothermal therapy (PTT). They also possess photothermally enhanced glucose oxidase-like (Gox), peroxidase-like (POD), and glutathione peroxidase-like (GSH-px) activities, which can be used to disrupt the energy metabolism and redox balance of tumor cells, significantly enhancing immunotherapy and reshaping the tumor microenvironment (TME). Photothermal-enhanced immunotherapy induces tumor immunogenic cell death (ICD) and further promotes the maturation of antigen-presenting cells, such as dendritic cells (DC), thereby enhancing T cell infiltration within the tumor. Furthermore, F-CDs@PEG reduced tumor granulocyte colony-stimulating factor (G-CSF) levels under NIR (808 nm) laser irradiation, thereby inhibiting the expression of immunosuppressive myeloid-derived suppressor cells (MDSCs) and alleviating T cell exhaustion, effectively suppressing tumor growth. Its mediated photothermal-enhanced catalytic immunotherapy induced robust immune memory and exhibited long-term inhibitory effects on recurrent tumors. In summary, F-CDs@PEG reshaped the TME through photothermal-enhanced multi-substrate catalytic reactions, significantly hindering the progression of both primary and distant tumors.

[0061] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0062] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A method for preparing near-infrared second region fluorine-doped carbon dot nanozymes for photothermal enhanced catalytic immunotherapy, characterized in that: The steps include: S1. Synthesis of crude fluorine-doped carbon dots: using p-phenylenediamine, dopamine hydrochloride, and (1S)-3,3′-difluoro[1,1′-binaphthyl]-2,2′-diol in a molar ratio of 2:2:1 as raw materials and ethanol as solvent, a solvothermal method was used to synthesize crude fluorine-doped carbon dots. S2. Separation and purification: obtaining pure fluorine-doped carbon dots by separating and purifying the crude fluorine-doped carbon dots; S3. PEG modification: The pure fluorine-doped carbon dots were dissolved in water, and PEG200 was added for sufficient reaction to obtain F-CDs@PEG.

2. The method for preparing the near-infrared second region fluorine-doped carbon dot nanozyme for photothermal enhanced catalytic immunotherapy according to claim 1, characterized in that: In step S2, the separation and purification process includes filtration, dialysis to remove impurities and freeze-drying.

3. The method for preparing the near-infrared second region fluorine-doped carbon dot nanozyme for photothermal enhanced catalytic immunotherapy according to claim 1, characterized in that: The preparation method further comprises the steps of purifying the F-CDs@PEG: centrifugal separation, distilled water washing, and freeze drying.

4. A near-infrared second-region fluorine-doped carbon dot nanozyme for photothermal enhanced catalytic immunotherapy, characterized in that: The method is prepared according to any one of claims 1 to 3.

5. An application of near-infrared second region fluorine-doped carbon dot nanozymes for photothermal enhanced catalytic immunotherapy prepared by the preparation method according to any one of claims 1 to 3, characterized in that: The near-infrared second zone fluorine-doped carbon dot nanozyme is used to prepare drugs for treating triple-negative breast cancer.

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