Near-infrared second-region fluorine-doped carbon dot nano-enzyme for photothermal enhanced catalytic tumor immunotherapy as well as preparation method and application of near-infrared second-region fluorine-doped carbon dot nano-enzyme

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a near-infrared two-region fluorine-doped carbon dot nano-enzyme (F-CDs (at) PEG) for photo-thermal enhanced catalytic immunotherapy and a preparation method and application of the near-infrared two-region fluorine-doped carbon dot nano-enzyme (F-CDs (at) PEG). The F-CDs has excellent performance in the aspects of NIR-II fluorescence emission for in-vivo biological imaging and NIR photo-thermal therapy, has photo-thermal enhanced Gox-like, POD-like and GSH-px-like activities, and can be used for destroying energy metabolism and redox balance of tumor cells, enhancing immunotherapy and remodeling a tumor microenvironment. Photo-thermal enhanced immunotherapy induces death of tumor immunogenic cells, and further promotes maturation of antigen presenting cells such as DC, thereby enhancing T cell infiltration in tumors. Besides, the F-CDs (at) PEG reduces the G-CSF level under NIR (808nm) laser irradiation, so that the MDSC level is inhibited, T cell depletion is relieved, and tumor growth is effectively inhibited; the mediated photo-thermal enhanced catalytic immunotherapy can induce strong immune memory, and has a long-term inhibition effect on recurrent tumors.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a near-infrared II fluorine-doped carbon dot nanozyme for photothermal enhanced catalytic tumor immunotherapy, a preparation method thereof, and an application thereof. Background Art

[0002] The catalytic immunotherapy treatment method activates the anti-tumor immune response through the catalytic reaction of biocompatible nanocatalysts. It mainly targets the substrates in the tumor microenvironment, uses advanced catalytic materials for catalytic reactions, and is activated by stimuli such as light, ultrasound, temperature, and electric fields. This method helps in the clearance of tumors and immunomodulation. Compared with traditional chemotherapy or radiotherapy, catalytic immunotherapy has multiple advantages, including less amount of catalyst used, high efficiency, and selective targeting of tumor-specific substrates. More importantly, nano-catalytic immunotherapy can reshape the immunosuppressive tumor microenvironment (TME) by targeting tumor metabolites, activate anti-tumor immunity, and generate long-term immune memory. The exploration of various advanced catalytic materials has opened up new avenues for enhancing catalytic immunotherapy, aiming to boost the immune response, improve the treatment effect, and minimize side effects at the same time.

[0003] Due to their excellent optical properties, low toxicity, high water dispersibility, low production cost, etc., carbon dots have received increasing attention in the field of biomaterials. They show great application potential in fields such as optoelectronic conversion, bioimaging, biosensing, nanomedicine, and catalysis. Nanozymes based on non-metallic carbon dots have attracted much attention from researchers due to their excellent physical and chemical properties. In recent years, many fluorescent carbon dots and their hybrids have been reported to have enzyme-like activities, such as peroxidase-like (POD), catalase-like (CAT), oxidase-like (OXD), and superoxide dismutase-like (SOD) activities. On the other hand, due to their superior photoactivity, CDs have the potential to be used as photothermal agents for photothermal therapy (PTT), which is also expected to promote the electron transfer efficiency through the thermal field, thereby enhancing the enzyme-like activity. CDs-based nanozymes can be used as multifunctional nano-platforms for non-invasive optical diagnosis, photodynamic and catalytic therapy, and synergistic therapy with PTT / PDT.

[0004] Although some CD-based nanozymes have emerged in recent years, the vast majority of CD nanozymes emit at relatively short wavelengths in the blue / green region of the spectrum, with limited penetration depth and potential damage to surrounding tissues. Additionally, metal doping (i.e., Fe, Mn, Co, Ni, Cu) can enhance the NIR photoactivity and enzymatic activity of CDs. However, the fluorescence of CDs can be quenched by metal ions, which limits their in vitro and in vivo tracing. The introduction of metals often raises significant long-term toxicity concerns. Non-metallic carbon dot nanozymes with both NIR-II emission and NIR photoactivity are currently a rare occurrence. New strategies for preparing NIR-II CDs nanozymes with high QY and long excitation wavelengths will meet the major needs of cancer catalytic immunotherapy. Summary of the Invention

[0005] In view of the deficiencies in the prior art, the present invention provides a preparation method of NIR-II fluorescent F-doped CDs (F-CDs) based on non-metal doping, with high fluorescence quantum yield (QY) and photo-thermal enhanced multi-enzyme activity, and uses the same for cancer catalytic immunotherapy, achieving tumor microenvironment (TME) remodeling, immune activation, and long-term immune memory through photo-thermal enhanced multi-enzyme activity, providing an innovative solution for tumor treatment.

[0006] To achieve the above object, the present invention adopts the following technical solutions: A preparation method of a near-infrared II region fluorine-doped carbon dot nanozyme for photo-thermal enhanced catalytic immunotherapy, comprising the following steps: S1. Synthesize the crude product of fluorine-doped carbon dots: Using p-phenylenediamine, dopamine hydrochloride, and (1S)-3,3'-difluoro[1,1'-binaphthalene]-2,2'-diol in a molar ratio of 2:2:1 as raw materials, synthesize the crude product of fluorine-doped carbon dots by any one of hydrothermal method, solvothermal method, or microwave-assisted method; S2. Separate and purify: Obtain pure fluorine-doped carbon dots by separating and purifying the crude product of fluorine-doped carbon dots; S3. PEG modification: Dissolve the pure fluorine-doped carbon dots in water, add PEG and react fully to obtain F-CDs@PEG with good water dispersibility.

[0007] In some embodiments, in step S1, the synthesis method is the solvothermal method, and the solvent used is ethanol.

[0008] Further, in step S2, the process of separation and purification includes filtration, dialysis for impurity removal, and freeze-drying.

[0009] Further, the preparation method further includes a purification step for the F-CDs@PEG: centrifugal separation, washing with distilled water, and freeze-drying.

[0010] Preferably, the PEG used in step S3 is PEG200.

[0011] The present invention also provides a near-infrared second-region fluorine-doped carbon dot nanozyme for photothermal enhanced catalytic immunotherapy prepared by the above preparation method and its application. The near-infrared second-region fluorine-doped carbon dot nanozyme is applied to the preparation of tumor immunotherapy drugs.

[0012] Furthermore, the tumor is a primary tumor or a distal tumor.

[0013] Furthermore, the near-infrared second-region fluorine-doped carbon dot nanozyme is also applied to the preparation of drugs for the combined application of photothermal therapy, catalytic therapy and immunotherapy.

[0014] The beneficial effects of the present invention are as follows: The present invention develops a near-infrared second-region fluorine-doped carbon dot nanozyme F-CDs@PEG for photothermal enhanced catalytic immunotherapy. F-CDs exhibit excellent performance in NIR-II fluorescence emission for in vivo bioimaging and NIR photothermal therapy (PTT). At the same time, it has photothermal 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 enhance immunotherapy, and reshape 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 in tumors. In addition, F-CDs@PEG reduces the level of tumor granulocyte colony-stimulating factor (G-CSF) under NIR (808 nm) laser irradiation, thereby inhibiting the level of immunosuppressive myeloid-derived suppressor cells (MDSC), alleviating T cell exhaustion, and thus effectively inhibiting tumor growth; the photothermal enhanced catalytic immunotherapy mediated by it can induce strong immune memory and has a long-term inhibitory effect on recurrent tumors. In summary, F-CDs@PEG reshapes the TME through photothermal enhanced multi-substrate catalytic reactions, significantly hindering the progression of primary tumors and distant tumors.

[0015] The above description is only an overview of the technical solution of the present invention. In order to be able to understand the technical means of the present invention more clearly and implement it in accordance with the content of the specification, the following takes the preferred embodiments of the present invention and combines the drawings to illustrate in detail as follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, some of the following drawings are embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0017] Figure 1 Schematic diagram for the synthesis of F-CDs.

[0018] Figure 2 Transmission electron microscope image of F-CDs.

[0019] Figure 3 UV spectrum and fluorescence spectrum of F-CDs, where Figure 3 part a shows the absorption band of F-CDs (dissolved in DMSO) from visible light to the near-infrared region; Figure 3 part b shows that F-CDs (dissolved in DMSO) exhibit obvious NIR-II emission in the range of 900 to 1400 nm.

[0020] Figure 4 Test chart for the photo-thermal enhanced catalytic performance of F-CDs, where Figure 4 part a shows that when methylene blue (MB) is incubated with F-CDs solution and H2O2 in NaHCO3 buffer for 10 minutes, the absorbance decreases significantly; Figure 4 part b shows the absorbance of the mixture after F-CDs and GSH are incubated at different time points and temperatures; compared with 25 °C, when the glucose concentration is 400 mM, the Gox mimicking activity of F-CDs is 7 times and 3.7 times at 45 °C and 37 °C respectively ( Figure 4 part c).

[0021] Figure 5 In vivo imaging map of F-CDs@PEG.

[0022] Figure 6 Endocytosis of F-CDs@PEG by tumor cells and confocal microscopy image of co-localization.

[0023] Figure 7 Effect diagram of the generation of intracellular reactive oxygen species of F-CDs@PEG under NIR treatment.

[0024] Figure 8 In vivo photo-thermal performance map of F-CDs@PEG.

[0025] Figure 9Tumor treatment effect diagrams on the 14th day after treatment with PBS, NIR, F-CDs@PEG, and F-CDs@PEG+NIR; as Figure 9 shown in part a, the tumor volumes of the control group and the NIR group increased by 7.2-fold and 7.1-fold, respectively, and the F-CDs@PEG group increased by approximately 5.07-fold. In contrast, the combination of F-CDs@PEG and laser treatment showed significant tumor suppression, and tumor regression or growth arrest occurred in some cases ( Figure 9 parts b and c).

[0026] Figure 10 Spleen enlargement remission effect diagrams on the 14th day after treatment with PBS, NIR, F-CDs@PEG, and F-CDs@PEG+NIR.

[0027] Figure 11 Flow cytometry analysis diagrams of immune activation on the 14th day after treatment with PBS, NIR, F-CDs@PEG, and F-CDs@PEG+NIR; among them Figure 11 part a shows the results of analyzing the immunosuppressive cell population in tumor samples, especially 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 + ) populations in the tumor tissues of treated mice, anti-tumor immune activation was confirmed ( Figure 11 part c); the numbers of CD3 + CD4 + and CD3 + CD8 + T cells were the highest in the F-CDs@PEG+NIR group, 2.3-fold and 1.6-fold that of the F-CDs@PEG group alone, and 2-fold and 3-fold that of the control group ( Figure 11 parts d and e); the expression of PD-1 under the CD8 + T cell gate was evaluated ( Figure 11 part f); the results showed that the expression of PD-1 on CD8 + T cells decreased significantly in the F-CDs@PEG and F-CDs@PEG+NIR groups, indicating that the treatment alleviated T cell exhaustion ( Figure 11 part g).

[0028] Figure 12Immune memory flow cytometry analysis charts after treatment with PBS, NIR, F-CDs@PEG, and F-CDs@PEG+NIR; flow cytometry analysis of CD44 in distant tumors + CD62L - For effector memory T cells, the proportion of CD4 + Tem cells ( Figure 12 parts a and b) in the F-CDs@PEG+NIR laser group was significantly higher than that in other groups; the results of flow cytometry showed that for CD8 + T cell subsets, the proportion of CD44 + CD62L + central memory T cells was the highest in the F-CDs@PEG+NIR laser group, which was 5 times and 1.5 times that of the control group and the F-CDs@PEG alone group, respectively ( Figure 12 parts c and d). Specific implementation manners

[0029] To better describe the present invention, the following is further illustrated by specific examples. The methods in the following examples are all conventional methods unless otherwise specified.

[0030] The technical solutions described in the present invention are all conventional solutions in the art unless otherwise specified; the reagents or materials are all from commercial channels unless otherwise specified.

[0031] The following examples are directed to the intermediate compounds and final products determined in the specification and synthesis schemes. The following examples are used to describe in detail the preparation of the compounds of the present invention, but the chemical reactions described are disclosed according to 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 can easily identify the compounds for which this occurs. In these cases, the reactions can be successfully carried out by conventional improvements 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 unless otherwise explicitly indicated. When referring to yields, all parts and percentages are in terms of amount of substance. When referring to solvents and eluents, all parts are in terms of volume.

[0032] Example 1 Preparation of F-CDs and F-CDs@PEG Step 1: Using p-phenylenediamine, dopamine hydrochloride, and (1S)-3,3′-difluoro[1,1′-binaphthalene]-2,2′-diol as raw materials (ratio: 2:2:1), and using ethanol as a solvent, synthesize (F-CDs) by solvothermal method ( Figure 1 ).

[0033] Step 2: Heat at 200 °C for 12 h. After cooling to room temperature, filter with a 200 μm filter membrane, add the obtained product into a 1000 Da dialysis bag, and stir for three days. Finally, collect the pure product and freeze-dry it to obtain F-CDs with a yield of 48.3%. Transmission electron microscopy (TEM) images show that F-CDs have a spherical morphology with a diameter of approximately 3 nm ( Figure 2 ).

[0034] Step 3: Dissolve the prepared fluorocarbon dots (F-CDs) in ultrapure water, sonicate for 5 min to prepare a dispersion solution with a concentration of 1 mg / mL. Subsequently, add 0.5 mg of PEG200. After stirring for 2 h, the sample is centrifuged at 12,500 rpm for 10 min for collection, washed twice with distilled water, and finally freeze-dried to obtain F-CDs@PEG.

[0035] Example 2 Characteristics of F-CDs and F-CDs@PEG 2.1 Simulated enzyme catalytic activity The absorption spectrum of F-CDs dissolved in 10% DMSO shows an absorption band from the visible light to the near-infrared region ( Figure 3 Part a in). Under 808 nm laser excitation, F-CDs (dissolved in DMSO) show obvious NIR-II emission in the range of 900 to 1400 nm ( Figure 3 Part b in).

[0036] When methylene blue (MB) is incubated with the F-CDs solution and H2O2 in NaHCO3 buffer for 10 min, a significant decrease in absorbance is observed ( Figure 4 Part a in). This decrease can be attributed to the efficient generation of ROS triggered by the catalytic reaction driven by F-CDs, and with the increase in temperature, an enhanced catalytic activity of the simulated POD is observed. Glutathione (GSH) is a complex metabolite of the intracellular antioxidant mechanism and can effectively scavenge the ROS generated by nanoenzymes, 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. Incubate F-CDs with GSH at different time points and temperatures, and measure the absorbance of the mixture ( Figure 4Part b). With the increase of the incubation temperature, the absorbance of DTNB at 412 nm decreased significantly, indicating that fluorine doping and temperature increase enhanced the consumption of GSH, and the catalytic activity of F-CDs at 45 °C was 3.6 times that at 37 °C, confirming that F-CDs could consume the overproduced GSH in tumor cells. These results suggest that F-CDs can effectively act as a biocatalyst to increase the oxidative stress in cancer cells by cyclically decomposing H2O2 and consuming GSH.

[0037] In addition to POD mimicking enzyme and GSH-px mimicking enzyme activities, the Gox mimicking enzyme activity was further explored. Since glucose oxidase can convert glucose into H2O2 and gluconic acid. Therefore, using Fe 2+ and xylenol orange as indicators, the generation of H2O2 after incubation of F-CDs with glucose was detected. With the increase of temperature, the Gox catalytic performance of F-CDs was enhanced. Compared with 25 °C, when the glucose concentration was 400 mM, the Gox mimicking activities of F-CDs at 45 °C and 37 °C were 7 times and 3.7 times respectively ( Figure 4 Part c), indicating that the Gox mimicking activity of F-CDs is temperature-dependent. The generated H2O2 will provide more substrates for the POD catalytic reaction, resulting in the generation of more ROS. All the results together suggest that F-CDs can activate the cascade reaction and have continuous POD mimicking enzyme, GSH-px mimicking enzyme, and Gox mimicking enzyme catalytic activities to generate more cytotoxic OH species for the oxidative damage of tumors.

[0038] 2.2 High efficient clearance ability and safety in vivo 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. Over time, these signals gradually decreased, indicating that F-CDs@PEG had relatively high efficient clearance ability and safety in vivo. As Figure 5 shown.

[0039] 2.3 Uptake and intracellular catalysis Furthermore, CLSM was used ( Figure 6)The uptake behavior of F-CDs@PEG in 4T1 cells was observed. After incubation for 24 h, bright red fluorescence signals were shown in 4T1 cancer cells. Subsequently, the intracellular trafficking of F-CDs@PEG was 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 mitochondria-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 ). The above results indicate that F-CDs@PEG can be effectively taken up by 4T1 tumor cells. Once endocytosed by tumor cells, they will accumulate in lysosomes and mitochondria for further catalytic reactions.

[0040] 2′,7′-Dichlorofluorescein diacetate (DFCH-DA) was used as an indicator to detect intracellular ROS generation. CLAM images showed that in Figure 7 , we clearly observed the fluorescence signal of ROS in cancer cells of the F-CDs@PEG group, which was up to 5 times higher than that of the control group. In addition, under mild NIR-II laser irradiation, the nanozyme exhibited the strongest ROS level, which fully demonstrated that the F-CDs@PEG nanozyme can integrate multiple functions of triple mimetic enzyme activity and mild NIR-II photothermal effect to achieve efficient intracellular catalysis.

[0041] Example 3 Antitumor effect of F-CDs@PEG 3.1 Experimental subjects: 4T1 subcutaneous tumor-bearing BALB / c mice 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 ). After intratumoral injection of F-CDs@PEG in the treatment groups (3) and (4), the photothermal images of the treated mice were collected. The tumor temperature of the mice injected with normal saline did not change significantly under 808 nm laser irradiation. In contrast, the tumor temperature of the mice treated with F-CDs@PEG increased from 35.7 °C to 55.8 °C within 5 minutes of laser irradiation ( Figure 8 ).

[0042] 3.2 Experimental results The tumor volume was recorded every two days. As Figure 9As shown in part a, the tumor volumes of the control group and the NIR group increased by 7.2-fold and 7.1-fold, respectively. In addition, the F-CDs@PEG group increased by approximately 5.07-fold, indicating that the efficacy of F-CD@PEG monotherapy for tumors was limited. In contrast, the combination of F-CDs@PEG and laser treatment showed significant tumor suppression, and tumor regression or growth arrest occurred in some cases ( Figure 9 in parts b and c).

[0043] Splenomegaly was observed in 4T1 tumor-bearing mice in the control group, NIR, and F-CDs@PEG groups. However, the F-CDs@PEG + laser irradiation group showed significantly reduced splenomegaly, and the spleen returned to normal weight and size ( Figure 10 ). 3.3 Analysis and Summary Immunosuppressive cell populations, particularly MDSCs, were analyzed in tumor samples ( Figure 11 in part a). The percentage of MDSCs decreased in the F-CDs@PEG and F-CDs@PEG + NIR groups ( Figure 11 in part b), while there was no significant decrease in the simple NIR irradiation group compared with the control group. The MDSC level in the F-CDs@PEG + NIR group was the lowest, at 15.5%, showing its superior immunomodulatory effect. The inhibition of MDSCs in the F-CDs@PEG + NIR group further enhanced the function and activity of effector T cells. By analyzing the populations of T helper cells (CD3 + CD4 + ) and cytotoxic T lymphocytes (CTLs) (CD3 + CD8 + ) in the tumor tissues of treated mice, anti-tumor immune activation was confirmed ( Figure 11 in part c). The numbers of CD3 + CD4 + and CD3 + CD8 + T cells were the highest in the F-CDs@PEG + NIR group, 2.3-fold and 1.6-fold those of the F-CDs@PEG group alone, and 2-fold and 3-fold those of the control group, respectively. ( Figure 11 in parts d and e). The decrease in PD-1 expression may enhance T cell function by alleviating T cell exhaustion, reducing the activation threshold, regulating immune tolerance, and altering the metabolic state. Accordingly, the PD-1 expression under the CD8 + T cell gate was evaluated ( Figure 11 in part f). The results showed that the expression of PD-1 on CD8 + T cells was significantly reduced in the F-CDs@PEG and F-CDs@PEG + NIR groups, indicating that the treatment alleviated T cell exhaustion ( Figure 11 in part g).

[0044] 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 4 groups: control group, NIR-I laser group, F-CDs@PEG group, and F-CDs@PEG + NIR laser group. Subsequently, effector memory T (Tem) (CD44 + CD62L - ) cells in distant tumors were evaluated because they can be reactivated and expanded to kill tumor cells when they encounter tumor antigens again. Flow cytometry analysis of CD44 + CD62L - effector memory T cells in distant tumors found that the proportion of CD4 + Tem cells ( Figure 12 parts a and b in + CD62L + ) in the F-CDs@PEG + NIR laser group was significantly higher than that in other groups. Central memory T cells (Tcm) (CD44 + , CD62L + ) are T cells that acquire long-term memory after antigen activation and can be reactivated by tumor antigens to directly kill tumors. Flow cytometry results showed that the proportion of CD44 + CD62L Figure 12 central memory T cells in the CD8

[0045] T cell subset in the F-CDs@PEG + NIR laser group was the highest, which was 5 times and 1.5 times that of the control group and the F-CDs@PEG group alone, respectively ( parts c and d in Figure 12 ). No recurrence of distant tumors was seen. In addition, compared with the control group, the Tcm in the F-CDs@PEG group alone increased by 2.5 times. These results confirmed that the photothermal enhanced catalytic immunotherapy of F-CDs@PEG can induce a long-term immune memory response and may prevent tumor recurrence.In summary, the present invention has developed a near-infrared II region fluorine-doped carbon dot nanozyme F-CDs@PEG for photothermal enhanced catalytic immunotherapy. F-CDs exhibit excellent performance in NIR-II fluorescence emission for in vivo bioimaging and NIR photothermal therapy (PTT), and at the same time have photothermal 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 enhance immunotherapy, and reshape the tumor microenvironment (TME). Photothermal enhanced immunotherapy induces tumor immunogenic cell death (ICD), and further provides promotion for the maturation of antigen-presenting cells such as dendritic cells (DC), thereby enhancing T cell infiltration in tumors. In addition, F-CDs@PEG reduces the level of tumor granulocyte colony-stimulating factor (G-CSF) under NIR (808 nm) laser irradiation, thereby inhibiting the expression of immunosuppressive myeloid-derived suppressor cells (MDSC), alleviating T cell exhaustion, and effectively inhibiting tumor growth; the mediated photothermal enhanced catalytic immunotherapy can induce strong immune memory and has a long-term inhibitory effect on recurrent tumors. In short, F-CDs@PEG reshapes the TME through photothermal enhanced multi-substrate catalytic reactions, significantly hindering the progression of primary tumors and distant tumors.

[0046] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of concise description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope recorded in this specification.

[0047] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it cannot be understood as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the invention patent shall be subject to the appended claims.

Claims

1. A preparation method of near-infrared II region fluorine-doped carbon dot nanozyme 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 synthesizing crude fluorine-doped carbon dots by any one of a hydrothermal method, a solvothermal method or a microwave-assisted method; 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 are dissolved in water, and PEG is added for sufficient reaction to obtain F-CDs@PEG.

2. The preparation method of the near-infrared II region fluorine-doped carbon dot nanozyme for photothermal enhanced catalytic immunotherapy according to claim 1, characterized in that, In step S1, the synthesis method is a solvothermal method, and the solvent used is ethanol.

3. The preparation method of the near-infrared II 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.

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

5. The preparation method of the near-infrared second-region fluorine-doped carbon dot nanozyme for photothermal-enhanced catalytic immunotherapy according to claim 1, wherein, The PEG used in step S3 is PEG200.

6. A near-infrared-II 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 5.

7. Use of a near-infrared second-region fluorine-doped carbon dot nanozyme for photothermal enhanced catalytic immunotherapy prepared by the preparation method according to any one of claims 1 to 5, characterized in that, The near-infrared second zone fluorine-doped carbon dot nanozyme is used to prepare tumor immunotherapy drugs.

8. The application according to claim 7, wherein The tumor is a primary tumor or a distal tumor.

9. The application according to claim 7, wherein 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.

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