Carbon dot with near-infrared second-zone photo-thermal electric catalytic activity and preparation method and application thereof

Abstract

The invention discloses a carbon dot with near-infrared second-zone photo-thermal electric catalytic activity and a preparation method and application thereof, and the carbon dot has rich self-trapping excitons and can generate a large number of hydroxyl radicals under near-infrared second-zone photo-thermal-thermal-electric triggering. In the process from local low temperature to high temperature, the carbon dots show an electron-like behavior, and the mobility of a photo-generated electron stream is higher than that of a photo-generated hole stream, so that efficient charge separation is caused, and the thermoelectric catalytic performance is excellent. Under the dual action of hydroxyl radicals generated by near-infrared second-area photo-thermal and thermoelectric catalysis, the carbon dots can effectively induce pyroptosis of cells and can be applied to immunotherapy of tumors. The preparation method of the carbon dots with the near-infrared second-area photo-thermal electric catalytic activity is simple and easy to implement, the price is low, large-scale batch preparation is easy, and the carbon dots have excellent application prospects in the fields of biomedicine, environmental governance and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of carbon nanomaterials, and in particular, to a near-infrared second-region optothermocatalytic active carbon dot, a preparation method thereof, and an application thereof. Background Art

[0002] Pyroptosis is a pro-inflammatory programmed cell death mode, first defined by Cookson et al. in 2001. Its typical morphological features include the formation of plasma membrane pores, cell swelling caused by osmotic imbalance, and the leakage of cytoplasmic contents. This process is driven by the proteolytic cleavage of members of the gasdermin (GSDM) protein family mediated by caspase, and the generated N-terminal fragments form plasma membrane pores through oligomerization. Along with the release of inflammatory cytokines such as interleukin-1β (IL-1β) and IL-18, pyroptosis can reshape the tumor microenvironment and activate systemic anti-tumor immunity, thus becoming an important strategy for cancer immunotherapy. Although existing studies have induced pyroptosis by means of pharmaceutical preparations, bacterial components, or externally field-activated reactive oxygen species (ROS) nanomaterials, problems such as the potential biosafety hazards of transition metal-based nanosystems and perovskite materials, and the defects of low dispersibility and poor photostability of organic photosensitizers still restrict their clinical applications. There is an urgent need to develop pyroptosis trigger agents (PTAs) with precise activation characteristics, biocompatibility, and microenvironment responsiveness.

[0003] The 2023 Nobel Prize in Chemistry-winning achievement of quantum dot (QDs) technology has opened up a new dimension for biomedical research. Among them, carbon quantum dots (CQDs) have shown unique advantages in the field of nanomedicine due to their excellent biocompatibility and multifunctional optoelectronic properties. Based on their tunable photoluminescence, photothermal, and photocatalytic properties, CQDs have achieved breakthroughs in the fields of bioimaging and phototherapy, but their optothermocatalytic potential has not been fully explored. Limited by low hot carrier mobility and charge separation efficiency, the application of CQDs in optothermocatalytically induced pyroptosis still faces challenges. Summary of the Invention

[0004] The inventors constructed a CQD system with NIR-II absorption characteristics and a photothermal conversion efficiency of 40% through free radical polymerization. By precisely regulating the thermal gradient field of self-trapped excitons (STE), we achieved directional migration and continuous charge separation of high-mobility hot electron flow, promoting the generation of hydroxyl radicals (reactive oxygen species). This free radical flow achieved controllable cell pyroptosis by activating the reactive oxygen species (ROS) / mitochondria / caspase-1 / GSDMD pathway, showing significant therapeutic advantages in tumor-bearing mouse models. The immunogenic cascade induced by pyroptosis activated systemic anti-tumor immunity and completely inhibited tumor recurrence. This strategy implements precise intervention on primary cancer lesions and provides a new paradigm for the development of personalized whole-cell vaccines. This work not only established self-trapped exciton regulation as a universal design principle for thermoelectric nanomaterials, but also created a new model of non-invasive immunotherapy based on NIR-II light activation.

[0005] The object of the present invention is to provide a method for preparing near-infrared second-zone photothermal and electrocatalytic activated carbon dots, which have near-infrared second-zone absorption and efficient thermocatalytic performance, so as to solve the above technical problems.

[0006] The second objective of the present invention is to provide a technology for generating hydroxyl radicals by photothermally regulating self-trapped excitons in the second near-infrared region, and to explain the reasons for its efficient thermoelectric catalytic activity.

[0007] The third purpose of the present invention is to provide a solution for inducing cell pyroptosis by thermoelectric catalysis and to explore the mechanism of pyroptosis.

[0008] The fourth objective of the present invention is to provide a strategy for spontaneously forming a protein corona with carbon dots and macromolecules in serum, thereby improving the ability of tumor enrichment.

[0009] The fifth purpose of the present invention is to provide the application of the above-mentioned carbon dots in photothermal and electrocatalytic therapy and photoimmunotherapy of tumors, so as to achieve precise tumor treatment.

[0010] The present invention is achieved in that: In a first aspect, the present invention provides a method for preparing near-infrared second-zone photothermal electrocatalytic activated carbon dots, comprising: mixing citric acid and urea with formic acid to conduct a solvothermal reaction.

[0011] In an optional embodiment, the mass ratio of citric acid to urea is 1:1-10; preferably 1:2-6; Preferably, the amount of formic acid added is such that the total concentration of citric acid and urea in the mixed solution is controlled to be 0.05-1 g / mL.

[0012] In an optional embodiment, the temperature of the solvothermal reaction is 150-250° C., and the reaction time is 2-6 h; Preferably, the temperature of the solvothermal reaction is 160 - 200 °C, and the reaction time is 4 - 4.5 h; In an alternative embodiment, the method further includes: performing centrifugal separation after the solvothermal reaction, and drying the obtained solid; Preferably, freeze-drying is used for drying.

[0013] In an alternative embodiment, centrifugal separation is carried out by adding an organic solvent that can promote the precipitation of carbon dots and is miscible with water to the system after the solvothermal reaction, and then centrifuging to precipitate the solid; Preferably, the organic solvent is absolute ethanol; Preferably, the ratio of the amount of the organic solvent used to the volume of the solution after the reaction is 1 - 4:1; Preferably, the centrifugation process is carried out in multiple times; more preferably, the number of centrifugations is 2 - 4 times.

[0014] Preferably, the application of the carbon dots with photothermal electrocatalytic activity in the second near-infrared region in bioimaging.

[0015] In a second aspect, the present invention provides a strategy for generating hydroxyl radicals by regulating self-trapped excitons with photothermal in the second near-infrared region. The carbon dots are prepared by the preparation method of any one of the foregoing embodiments; the application of the carbon dots in the photothermal-thermoelectric catalytic water reaction to generate hydroxyl radicals.

[0016] Preferably, the carbon dots in the second near-infrared region exhibit a main absorption at 560 nm and a broadband absorption at 800 - 1300 nm in water; the electron relaxation time in the second near-infrared region is about 13 ps; the photothermal conversion efficiency is 40%; the fluorescence quantum efficiency of red light is about 20%, and the fluorescence lifetime is 2.05 ns.

[0017] Preferably, the carbon dots with photothermal electrocatalytic activity in the second near-infrared region can generate a large amount of hydroxyl radicals as the photothermal temperature increases, and can kill tumor cells; Preferably, the migration speed of the hot electron flow of the carbon dots with photothermal electrocatalytic activity in the second near-infrared region is much greater than that of the hot hole flow, so efficient charge separation is generated.

[0018] In a third aspect, a scheme for inducing pyroptosis by thermoelectric catalysis is provided for the first time. The carbon dots are prepared by the preparation method of any one of the foregoing embodiments; Preferably, the carbon dots with photothermal electrocatalytic activity in the second near-infrared region can efficiently induce pyroptosis and have the function of activating the body's immunity.

[0019] Preferably, the carbon dots with photothermal electrocatalytic activity in the second near-infrared region achieve pyroptosis through the classical ROS / mitochondria / caspase-1 / GSDMD pathway.

[0020] Fourthly, the present invention provides a strategy for carbon nanodot composites, including macromolecules such as proteins in serum and the near-infrared II region photothermal electrocatalytic active carbon dots in the foregoing embodiments.

[0021] Preferably, the near-infrared II region photothermal electrocatalytic active carbon dots can form a protein corona with biological macromolecules in mouse serum, enhancing the tumor enrichment ability and enabling targeted tumor treatment.

[0022] Preferably, the near-infrared II region photothermal electrocatalytic active carbon dots are dissolved in mouse serum, and their mass concentration is 0-1 g / mL.

[0023] Fifthly, the present invention provides the application of the carbon nanodot composites in the foregoing embodiments in tumor photothermal electrocatalytic therapy and immunotherapy.

[0024] The present invention has the following beneficial effects: By using citric acid and urea as raw materials, highly efficient near-infrared II region photothermal electrocatalytic active carbon dots are synthesized by a one-step solvothermal method in formic acid containing both aldehyde groups and carboxyl groups. No complex purification procedures are required. The prepared carbon nanodots have a strong electron-withdrawing structure on their surface, which is used to confine and protect self-trapped excitons. Since self-trapped excitons have high reactivity and can produce near-infrared II region absorption and ultrafast electron relaxation in aqueous solution, they have excellent near-infrared II region photothermal properties. Due to the different migration rates of hot carriers, these carbon dots can generate hydroxyl radicals as the photothermal temperature rises and induce the pyroptosis of tumor cells. The prepared carbon nanodots can form a protein corona with macromolecules such as proteins in serum, have the ability to enrich in tumors, can achieve targeted tumor photothermal electrocatalytic therapy, and activate the body's immunity to avoid tumor recurrence. Description of the Drawings

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0026] Figure 1Schematic diagram of the synthesis and structure of near-infrared II region optothermocatalytic active carbon dots provided in Example 1 of the present invention; a: Transmission electron microscope (TEM) and high-resolution TEM (HRTEM) (upper right inset) images of CQDs, lower left inset: particle size distribution diagram. b: Atomic force microscope (AFM) image of CQDs, inset: height profile along the line. c: Full X-ray photoelectron spectroscopy (XPS) spectrum of CQDs. High-resolution d: C 1s, e: O 1s, and f: N 1s XPS spectra of CQDs. g: Raman spectrum of CQDs. h: Electron paramagnetic resonance (EPR) spectra of CQDs in the solid state and aqueous solution. a.u., arbitrary unit.

[0027] Figure 2 Schematic diagram of the photoexcited state dynamics and optothermocatalytic mechanism of near-infrared II region optothermocatalytic active carbon dots in Example 3 of the present invention; a: 2D pseudo-color map of the TA spectrum of CQDs in aqueous solution, pump wavelength is 550 nm (1 kHz, 100 fs). b: Bleaching signal dynamics of CQDs collected at 520 nm, 600 nm, 750 nm, and 1000 nm when λ pump = 550 nm in aqueous solution. The dots are experimental points and the solid lines are fitting lines. c: Energy level diagram of CQDs excited at λ pump = 550 nm. d: Temperature changes of aqueous solutions of CQDs with different concentrations and pure water under irradiation of a 1064 nm laser at 1 W cm -2 . e: Temperature changes of a 500 μg ml -1 aqueous solution of CQDs under irradiation of a 1064 nm laser with different power densities. f: Temperature curve of a 500 μg ml -1 aqueous solution of CQDs after 5 optothermal heating cycles under irradiation of a 1064 nm laser (0.6 W cm -2 ). g: EPR spectra of CQDs in aqueous solution after being irradiated with NIR-II light at different temperatures. h: Energy level diagram and optothermocatalytic process under NIR-II photo-thermal excitation. μ e , electron mobility. μ h , hole mobility. J e , current density.

[0028] Figure 3 Fluorescence and biosafety assessment diagram of near-infrared II region optothermocatalytic active carbon dots in Example 4 of the present invention; a: Cell viability of different cell groups incubated with CQDs at different concentrations. b: Cellular uptake of CQDs by EMT6 cells after incubation for 0, 30, 60, and 120 minutes in the Mchery-1 channel. c: Blood circulation of mice administered with CQDs (%ID g -1). Changes over time. d: Fluorescent imaging of major organs of mice at different time points before and after intravenous injection of 100 µL aqueous solution of CQDs (1000 µg mL -1 ), (589 nm laser; 650 nm long-pass optical filter). e: Biochemical tests of mice in the CQDs group on days 0, 1, 15, and 60, ALT (U / L): alanine aminotransferase; AST (U / L): aspartate aminotransferase; ALP (U / L): alkaline phosphatase; BUN (mmol / L): blood urea nitrogen; CR (mmol / L): creatinine; CK (mmol / L): creatine kinase test, n = 3, two-way ANOVA multiple comparison test, n.s. indicates no statistical difference between groups. f: Calcein AM and PI assays of EMT6 cells treated with CQDs (500 μg mL -2 ) after irradiation with a 1064 nm laser (1 W cm -1 ) for 10 minutes.

[0029] Figure 4 Schematic diagram of the photothermal electrocatalytic cell pyroptosis and pyroptosis mechanism of the near-infrared II region photothermal electrocatalytic active carbon dots in Example 5 of the present invention; a: Phase contrast imaging analysis of CQDs-induced pyroptosis of EMT6 cells (arrows represent pyroptotic cells). b: Immunoblot showing the cleavage of GSDMD by caspase-1, c-Cas1, cleaved Caspase-1; GSDMD-F, full-length GSDMD; GSDMD-N, N-terminal cleavage products of GSDMD, respectively. c: LDH and d: IL-1β release in EMT6 cells treated with PBS (Group I), NIR-II light (Group II), CQDs in the dark (Group III), and CQDs after NIR-II irradiation (Group IV) (CQDs: 500 µg mL -1 , NIR-II: 0.8 W cm -2 , 10 minutes). e: CLSM images of CRT exposed on the surface of EMT6 cells under different treatment conditions (CQDs: 500 µg mL -1 , NIR-II: 0.8 W cm -2 , 10 minutes), CRT: calreticulin. Detailed implementation manners

[0030] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Those not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments not indicated by the manufacturer are all conventional products that can be obtained through commercial purchase.

[0031] An embodiment of the present invention provides a method for preparing near-infrared second-region optoelectrothermal catalytic active carbon dots, comprising the following steps: S1. Synthesis Mix citric acid and urea with formic acid for a solvothermal reaction, and synthesize near-infrared second-region optoelectrothermal catalytic active carbon dots by a one-step solvothermal method without complex purification procedures.

[0032] In actual operation, dissolve citric acid and urea in a formic acid solvent, then put it into a reaction kettle, heat it at a high temperature for a solvothermal reaction to obtain a reddish-brown liquid, that is, near-infrared second-region optoelectrothermal catalytic active carbon dots.

[0033] In some embodiments, the mass ratio of citric acid to urea is 1:1 - 10; preferably 1:2 - 6. Further controlling the mass ratio of citric acid to urea is beneficial to further improving the performance of the synthesized carbon nanodots and enhancing the fluorescence quantum efficiency of the carbon nanodots. Specifically, the mass ratio of citric acid to urea can be 1:1, 1:2, 1:4, 1:6, 1:10, etc., or any value between the above adjacent ratio values.

[0034] Furthermore, the addition amount of formic acid is controlled such that the total concentration of citric acid and urea in the mixed solution is 0.05 - 1 g / mL, such as 0.05 g / mL, 0.1 g / mL, 0.2 g / mL, 0.3 g / mL, 0.4 g / mL, 0.5 g / mL, 1 g / mL, etc., or any value between the above adjacent concentration values.

[0035] In some embodiments, the temperature of the solvothermal reaction is 150 - 250 °C, and the reaction time is 2 - 6 h; preferably, the temperature of the solvothermal reaction is 160 - 200 °C, and the reaction time is 4 - 4.5 h. By further optimizing the reaction temperature and time, the yield of near-infrared second-region optoelectrothermal catalytic active carbon dots is improved.

[0036] Specifically, the temperature of the solvothermal reaction can be 150 °C, 170 °C, 190 °C, 210 °C, 230 °C, 250 °C, etc., or any value between the above adjacent temperature values; the reaction time can be 2 h, 3 h, 4 h, 5 h, 6 h, etc., or any value between the above adjacent time values.

[0037] Specifically, the solvothermal reaction is carried out in a reaction kettle, preferably in a steel-lined polytetrafluoroethylene reaction kettle, and the heating reaction is preferably carried out under closed conditions to prevent other factors from interfering with the reaction.

[0038] S2. Separation After the solvothermal reaction, carry out centrifugal separation, and dry the obtained solid to obtain near-infrared second-region optoelectrothermal catalytic active carbon dots.

[0039] In some embodiments, after the solvothermal reaction, an organic solvent that can promote the precipitation of carbon dots and is miscible with water is added to the system, and then centrifugation is carried out to precipitate the solid. Specifically, the organic solvent is selected from anhydrous ethanol, anhydrous methanol, etc., and can be one or more; preferably anhydrous ethanol, which is easily available and non-toxic. The volume ratio of the organic solvent to the volume of the solution after the reaction is 1-4:1 (such as 1:1, 2:1, 3:1, 4:1, etc.), and it is appropriate to use an excessive amount of the organic solvent (such as a volume ratio of 2-3:1) to fully separate the solid product.

[0040] To enhance the separation effect of the solid product, the centrifugation process is carried out in multiple steps. The number of centrifugation times can be 2-4 times, such as 2 times, 3 times, or 4 times. Specifically, the rotation speed of the centrifugation is preferably 8000-12000 revolutions per minute, and more preferably 10000 revolutions per minute. After the centrifugation is completed, the upper layer solution is removed, and the lower layer precipitate is freeze-dried to obtain carbon dots with near-infrared II region photothermal electrocatalytic activity.

[0041] In some embodiments, freeze-drying is used for drying to prevent the influence of high temperature on the product.

[0042] The embodiments of the present invention also provide a kind of carbon dots with near-infrared II region photothermal electrocatalytic activity, which are prepared by the above preparation method. The carbon dots with near-infrared II region photothermal electrocatalytic activity have a high content of self-trapped exciton structure, enabling the carbon nanodots to have near-infrared II region absorption and photothermal electrocatalytic performance. In an aqueous solvent, due to spin averaging, the electron relaxation is very fast, about 13 ps, and it has a 40% near-infrared photothermal performance.

[0043] After detection, the main absorption bands of the carbon dots with near-infrared II region photothermal electrocatalytic activity in water are 560 nm and a broadband absorption in the range of 800-1300 nm. The red fluorescence quantum efficiency is about 20%, and the fluorescence lifetime is 2.05 ns. The main absorption peaks of the carbon dots with near-infrared II region photothermal electrocatalytic activity in dimethyl sulfoxide or dimethylformamide are at 580 nm, 700 nm, 820 nm, 910 nm, and 1030 nm. Among them, the absorption after 700 nm comes from the zero-field splitting of self-trapped excitons. The red fluorescence quantum efficiency is about 70%, and the fluorescence lifetime is 5.79 ns. Under the excitation of near-infrared-II region laser, the hydroxyl radicals will increase with the increase of the photothermal temperature.

[0044] An embodiment of the present invention provides a carbon nanodot composite material, which includes proteins in serum and the above-mentioned carbon dots in the second near-infrared region. The carbon nanodot composite material has a larger particle size (30 - 80 nm) and a relatively high red fluorescence quantum yield in serum (about 40%). In addition, the carbon dots with photo-thermoelectric catalytic activity in the second near-infrared region and their composite materials have high water solubility, good biocompatibility, low cytotoxicity, and can be excreted through the liver and kidneys in a short time.

[0045] In some embodiments, the carbon dots with photo-thermoelectric catalytic activity in the second near-infrared region are dissolved in mouse serum, and their mass concentration is 0 - 1 g / mL. They can effectively accumulate in tumors and be imaged.

[0046] The following further describes the features and properties of the present invention in detail with reference to embodiments. An embodiment of the present invention provides the application of carbon dots with photo-thermoelectric catalytic activity in the second near-infrared region in biological imaging. Specifically, it can be applied to the red light imaging of the main organs of the whole body of mice and tumors. The tumor imaging can determine the specific location of tumors in the body through the red fluorescence of the carbon dots. The enrichment principle may be that the carbon dots form a protein corona with macromolecules in the serum, resulting in an increase in their particle size.

[0047] An embodiment of the present invention also provides the application of carbon dots with photo-thermoelectric catalytic activity in the second near-infrared region in tumor treatment. Specifically, an aqueous solution of carbon dots can be injected into the tail vein, and the treatment of mouse tumors can be successfully achieved by irradiating with a near-infrared region laser with a low light power density. At the same time, efficient pyroptosis is achieved in the tumor, and the body's immunity is activated to achieve tumor immunotherapy.

[0048] In addition, the preparation method of the carbon dots is simple, the price is low, and it is easy to be prepared in large batches. It has good application prospects in the fields of biological imaging and tumor treatment.

[0049] The following further describes the features and properties of the present invention in detail with reference to embodiments.

[0050] Example 1 This example provides a preparation method of carbon dots with photo-thermoelectric catalytic activity in the second near-infrared region, including the following steps: Dissolve 1 g of citric acid and 2 g of urea in 10 mL of formic acid solvent, put the liquid into a 20 mL polytetrafluoroethylene high-pressure reaction kettle, heat and react. The reaction temperature is 180 °C, the reaction time is 4 hours, and the heating method is heating in an oven or using a parallel reactor. After the reaction, 60 mL of ethanol is added to the solution, and centrifugation is carried out at a speed of 8000 revolutions per minute. The upper layer solution is removed, and the lower layer precipitate is left. Centrifugation is carried out 3 times, and the lower layer precipitate is freeze-dried to obtain a black solid powder, namely carbon dots with photo-thermoelectric catalytic activity in the second near-infrared region.

[0051] Example 2 The near-infrared second-region optothermocatalytic active carbon dots obtained in Example 1 were characterized in terms of morphology and structure to explore the process of self-trapped exciton formation, and the results are as Figure 1 shown. Figure 1 In a: Transmission electron microscopy (TEM) and high-resolution TEM (HRTEM) (inset in the upper right corner) images of CQDs, inset in the lower left corner: particle size distribution diagram. b: Atomic force microscopy (AFM) image of CQDs, inset: height profile along the line. c: Full X-ray photoelectron spectroscopy (XPS) spectrum of CQDs. High-resolution d: C 1s, e: O 1s, and f: N 1s XPS spectra of CQDs. g: Raman spectrum of CQDs. h: Electron paramagnetic resonance (EPR) spectra of CQDs in the solid state and aqueous solution. a.u., arbitrary unit.

[0052] As Figure 1 shown in a of, TEM shows that the average particle size of CQDs is 3.33 ± 0.54 nm, and high-resolution TEM shows graphitized lattice fringes of 0.21 nm and core lattice defects. As Figure 1 shown in b of, atomic force microscopy (AFM) confirms its spherical morphology (height about 3 nm).

[0053] Figure 1 It can be seen from c of that the full XPS spectrum shows elements of C (285.3 eV), N (400.2 eV), and O (531.5 eV). As Figure 1 shown in d-f of, the high-resolution C 1s spectrum resolves peaks of C=C (284.5 eV), C=N (285.2 eV), C-O (286.8 eV), C=O (288.5 eV), and 290.6 eV; the O 1s spectrum shows C=O (531 eV), C-O / O-H (532 eV), and O=C-O (533.7 eV); the N 1s spectrum contains pyridine nitrogen (399.4 eV), pyrrole nitrogen (400.1 eV), and graphitic nitrogen (401.2 eV). The 290.6 eV peak of C 1s originates from the transition of STEs to higher energy levels under X-ray excitation, confirming the existence of STEs.

[0054] Figure 1 It can be seen from g of that the Raman spectrum shows that the intensity ratio of the D band (1360 cm -1 -1) to the G band (1580 cm -1 -1) is ID / IG = 1.17, and the 2D band (2700 cm -1 -1) is broadened, indicating disorder in the sp 2 domain and lattice distortion. The D*, A, and D’ peaks at 1277, 1468, and 1625 cm -1 -1 confirm that the structural defects break the time-reversal symmetry, providing a basis for the formation of STEs. As Figure 1As shown in Fig. h, the EPR spectrum shows g = 1.997, confirming the existence of a large number of unpaired electrons, providing evidence for the formation of STEs.

[0055] Example 3 The near-infrared second-region optothermocatalytic active carbon dots obtained in Example 1 were subjected to transient absorption spectroscopy (TA) and optothermocatalytic mechanism interpretation, as Figure 2 shown. Figure 2 Fig. a: 2D false-color map of the TA spectrum of CQDs in aqueous solution, with a pump wavelength of 550 nm (1 kHz, 100 fs). b: Kinetic curves of the bleaching signals of CQDs collected at 520 nm, 600 nm, 750 nm, and 1000 nm when λ pump = 550 nm in aqueous solution. The dots are experimental points, and the solid lines are fitting lines. c: Energy level diagram of CQDs excited at λ pump = 550 nm. d: Temperature changes of CQD aqueous solutions with different concentrations and pure water under irradiation with a 1064 nm laser at 1 W cm -2 . e: Temperature changes of a 500 μg ml -1 CQD aqueous solution under irradiation with a 1064 nm laser at different power densities. f: Temperature curve of a 500 μg ml -1 CQD aqueous solution after 5 optothermal heating cycles under irradiation with a 1064 nm laser (0.6 W cm -2 ). g: EPR spectra of CQDs in aqueous solution after NIR-II light irradiation at different temperatures. h: Energy level diagram and optothermocatalytic process under NIR-II optothermal excitation. μ e , electron mobility. μ h , hole mobility. J e , current density.

[0056] As Figure 2 shown in Fig. a, the transient absorption spectrum of the CQDs aqueous solution shows a ground state bleach signal (GSB) in the range of 450 - 650 nm, a ground state bleach signal at 950 - 1100 nm, and a photoinduced absorption signal (PIA) in the range of 700 - 880 nm, corresponding to the STEs state. Figure 2 The kinetic curve in Fig. b shows that the core / surface state energy transfers to the STEs state. The STEs state rapidly rises at 750 nm (formation of lattice distortion) and then slowly decays (exciton localization inhibits recombination). As Figure 2 shown in the energy level schematic diagram in Fig. c, and the decay time at 1000 nm is 13 ps, proving its potential for near-infrared II-region optothermal conversion.

[0057] As Figure 2As shown in d-e of the figure, the aqueous solution of CQDs (500 μg / mL) was irradiated with NIR-II laser for 10 minutes, and the temperature increased by 41.7 °C, and the photothermal conversion efficiency reached 40%. The increase in concentration and laser power can significantly improve the temperature rise effect. As Figure 2 As shown in f of the figure, after 5 cycles, the photothermal performance did not decay, proving its good photothermal stability.

[0058] As Figure 2 shown in g of the figure, the EPR spectrum shows that the temperature gradient promotes the generation of hydroxyl radicals. Combining the 13 ps ultrafast relaxation time and the difference in hot carrier migration, the Figure 2 mechanism shown in h of the figure is proposed: the temperature gradient changes the Fermi distribution, and the electrons at the hot end preferentially migrate to the cold end to achieve charge separation. The holes react with water to generate hydroxyl radicals. These results lay the foundation for thermoelectric catalytic therapy.

[0059] Example 4 The biosafety performance of the near-infrared II photothermoelectric catalytic active carbon dots obtained in Example 1 was evaluated, as Figure 3 shown. Figure 3 a in the figure: Cell viability of different cell groups incubated with different concentrations of CQDs. b: Cellular uptake of CQDs by EMT6 cells after incubation for 0, 30, 60, and 120 minutes in the Mchery-1 channel. c: Blood circulation of CQD-administered mice (%ID g -1 ) over time. d: Fluorescence imaging of major organs of mice at different time points before and after intravenous injection of 100 μL of CQD aqueous solution (1000 μg mL -1 ), (589 nm laser; 650 nm long-pass optical filter). e: Biochemical tests of mice in the CQDs group on days 0, 1, 15, and 60, ALT (U / L): alanine aminotransferase; AST (U / L): aspartate aminotransferase; ALP (U / L): alkaline phosphatase; BUN (mmol / L): blood urea nitrogen; CR (mmol / L): creatinine; CK (mmol / L): creatine kinase test, n = 3, two-way ANOVA multiple comparison test, n.s. indicates no statistical difference between groups. f: Calcein AM and PI assays of EMT6 cells treated with CQDs (500 μg mL -2 ) after irradiation with a 1064 nm laser (1 W cm -1 ) for 10 minutes.

[0060] As Figure 3 shown in a of the figure, in vitro CCK-8 experiments showed that when the concentration of CQDs reached 1000 μg / mL, it had no significant effect on the survival rates of 4T1, EMT6, and AC16 cells. As Figure 3As shown in b, the uptake of CQDs by EMT6 cells gradually increased within 2 h, and CQDs could successfully enter the cells. As Figure 3 As shown in c, the pharmacokinetic determination showed a blood half-life of 118.5 minutes. As Figure 3 As shown in d, after intravenous injection of an aqueous solution of CQDs (100 μL, 1000 μg / mL), in vivo fluorescence imaging showed that it mainly accumulated in the liver and kidneys, and the accumulation at the tumor site reached the peak after 6 h, demonstrating that CQDs could target tumors. The signal completely disappeared after 48 h, proving that CQDs could be rapidly metabolized out of the body. As Figure 3 As shown in e, there were no statistically significant differences in the biochemical indexes (ALT, AST, ALP, BUN, CR, CK, LDH) compared with the control group, and these results further demonstrated the biosafety of the near-infrared II region photothermal electrocatalytic active carbon dots.

[0061] As Figure 3 shown in the live / dead cell staining in f, green fluorescence indicates live cells and red fluorescence indicates dead cells. After irradiation with a 1064 nm laser (1 W / cm 2 ), significant red fluorescence was presented in EMT6 cells, confirming that CQDs had the ability of near-infrared II region photothermal killing.

[0062] Example 5 Analysis of pyroptosis induction and mechanism of the near-infrared II region photothermal electrocatalytic active carbon dots obtained in Example 1, as Figure 4 shown. Figure 4 a in the figure: Phase contrast imaging analysis of pyroptosis of EMT6 cells induced by photocatalytic CQDs (arrows represent pyroptotic cells). b: Immunoblot showing the cleavage of GSDMD by caspase-1, c-Cas1, cleaved Caspase-1; GSDMD-F, full-length GSDMD; GSDMD-N, N-terminal cleavage products of GSDMD, respectively. c: LDH and d: IL-1β release in EMT6 cells treated with PBS (group I), NIR-II light (group II), CQDs in the dark (group III), and CQDs after NIR-II irradiation (group IV) (CQDs: 500 μg mL -1 , NIR-II: 0.8 W cm -2 , 10 minutes). e: CLSM images of CRT exposed on the surface of EMT6 cells under different treatment conditions (CQDs: 500 μg mL -1 , NIR-II: 0.8 W cm -2 , 10 minutes), CRT: calreticulin.

[0063] As Figure 4As shown in a, in the CQDs treatment group (Group IV) excited by near-infrared II region light, EMT6 cells showed the characteristic of large vesicular swelling of the plasma membrane, which was significantly different from the control group. Figure 4 As shown in b, Western blot analysis showed that the expression levels of cleaved caspase-1 (c-caspase1) and N-terminal gasdermin D (N-GSDMD) in Group IV increased, confirming that CQDs induced pyroptosis through the classical c-cas1-GSDMD pathway.

[0064] Figure 4 Analysis of the cell lysates in c-d showed that the LDH concentration and IL-1β content in the supernatant of Group IV increased to 7 times that of the control group. Figure 4 Confocal microscopy imaging in e showed that only the cell membrane surface of Group IV presented the characteristic red fluorescence signal of calreticulin (CRT), confirming the characteristics of immunogenic cell death. Experimental data showed that the pyroptosis process induced by CQDs could release damage-associated molecular patterns such as CRT, activating the innate and adaptive immune response pathways.

[0065] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A preparation method of carbon dots with near-infrared second-region optothermoelectric catalytic activity, characterized in that, Comprising: Mix citric acid and urea with formic acid and carry out a solvothermal reaction.

2. The preparation method according to claim 1, characterized in that, The mass ratio of citric acid to urea is 1:1 - 10; the addition amount of formic acid is to control the total concentration of citric acid and urea in the mixed solution to be 0.05 - 1 g / mL.

3. The preparation method according to claim 2, characterized in that, The mass ratio of citric acid to urea is 1:2 - 6.

4. The preparation method according to claim 1, characterized in that, The temperature of the solvothermal reaction is 150 - 250 °C, and the reaction time is 2 - 6 h.

5. The preparation method according to claim 4, wherein The temperature of the solvothermal reaction is 160 - 200 °C, and the reaction time is 4 - 4.5 h.

6. The preparation method according to claim 1, characterized in that, This method further comprises: carrying out centrifugal separation after the solvothermal reaction, and drying the obtained solid.

7. The preparation method according to claim 6, characterized in that, Drying is carried out by freeze-drying.

8. The preparation method according to claim 6, characterized in that, The centrifugal separation is to add an organic solvent that can promote the precipitation of carbon dots and is miscible with water to the system after the solvothermal reaction, and then centrifuge to precipitate the solid; The organic solvent is absolute ethanol; The volume ratio of the amount of the organic solvent to the volume of the solution after the solvothermal reaction is 1 - 4:1; The number of centrifugation times is 2 - 4 times.

9. The carbon dots with near-infrared second-window optoelectrochemical catalytic activity prepared by the preparation method according to any one of claims 1-8, characterized in that, The main absorption bands of the carbon dots with near-infrared second-region photothermoelectric catalytic activity in water are 560 nm and 800 - 1300 nm; The carbon dots with near-infrared II photothermal electrocatalytic activity have a photothermal conversion efficiency of 40% under the irradiation of a 1064 nm laser with a power of 1 W cm -2 . The carbon dots with near-infrared second-region photothermoelectric catalytic activity can promote the separation of electrons and holes with the increase of temperature, and the photo-generated holes react with water to generate hydroxyl radicals.

10. Use of the carbon dots according to claim 9 in the production of hydroxyl radicals by near-infrared second-region photothermal and thermoelectric catalytic water reaction.

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