Near-infrared two-region carbon dots for nuclear magnetic resonance imaging and preparation method and application thereof

Abstract

The invention discloses a near-infrared two-region carbon dot for nuclear magnetic resonance imaging and a preparation method and application thereof, the near-infrared two-region carbon dot has rich magnetic edge state electrons, and can realize nitrogen nucleus hyperfine splitting and zero field splitting in DMSO. And due to spin averaging in an aqueous solution, a wide absorption band of 800-1300 nm is shown in a near-infrared second region, and the material has extremely fast electron relaxation time and very high photothermal conversion efficiency. The preparation method is characterized in that citric acid, formamide and formic acid are subjected to one-step solvothermal, and a complex purification procedure is not needed. Due to the existence of a magnetic edge state structure, the contrast agent becomes a T1 contrast agent with the highest relaxation rate at present. Through near-infrared light excitation, a large number of superoxide anions can be generated, and the superoxide anions are used for efficient photodynamic therapy. The carbon dots and macromolecules in blood can form a large-size protein crown, and the tumor enrichment function of the carbon dots is enhanced. On the basis of the characteristics, MR imaging guided tumor near-infrared two-region precise light treatment can be realized.

Description

Technical Field

[0001] The present invention relates to the technical field of carbon nanomaterials, and in particular, to a carbon dot in the second near-infrared region for nuclear magnetic resonance (MR) imaging, a preparation method thereof, and an application thereof. Background Art

[0002] Carbon-based materials, such as graphene and graphene nanoribbons, are hosts for spin and spin-valley qubits, and their spin-orbit coupling and hyperfine interaction (HFI) are weak, resulting in difficulty in achieving zero-field splitting (ZFS) at room temperature. Carbon dots (CDs) are zero-dimensional nanoparticles composed of a hydrophobic carbon core (insulating state) and a hydrophilic shell surface, belonging to a time-reversal symmetric fermionic system. When the time-reversal symmetry is broken by the ligand field, ZFS may occur at room temperature. Currently, extensive research on the electronic states of CNDs mainly focuses on nuclear states, surface states, and defect states, and there are basically no reports on magnetic edge states. Therefore, manipulating electron spin and migration behavior to achieve efficient HFI and ZFS is an unexplored and challenging task.

[0003] Precise cancer treatment should meet multiple criteria, including targeting tumors, having a strong tumor-killing effect, a short treatment time, and minimal side effects on the body. Currently, nanomedicine provides rich opportunities for precise cancer treatment, and researchers particularly focus on carbon-based nanomaterials that can target and treat tumors. Non-metallic CDs are favored due to their excellent biocompatibility and low cytotoxicity, and have been widely explored in cancer diagnosis and phototherapy. Combining therapeutic functions with magnetic resonance imaging (MRI) is one of the most promising goals for CDs to enter clinical applications. However, it is very challenging to achieve phototherapy or use MRI imaging in the second near-infrared (NIR-II) window. Non-metallic CDs that simultaneously achieve these two functions have not been reported.

[0004] In view of this, the present invention proposes a carbon dot in the second near-infrared region for nuclear magnetic resonance imaging, a preparation method thereof, and an application thereof. Summary of the Invention

[0005] The inventors synthesized highly paramagnetic CDs through a bottom-up method of free radical polymerization. The magnetic edge state electrons of the CDs are confined by ligands (-COOH, -C=O). These magnetic edge states exhibit ZFS due to spin-orbit coupling and dipole-dipole interactions, and produce an obvious absorption band gap in the NIR region. In dimethyl sulfoxide solvent, the CDs show nitrogen nuclear hyperfine splitting and significantly enhanced electron spin lifetime, indicating great research potential in electron spin catalysis and quantum coherence. In addition, they exhibit a fast T1 relaxation time due to spin averaging in aqueous solution, and can generate a large amount of superoxide radicals under near-infrared light irradiation, simultaneously achieving efficient MR imaging and NIR-II phototherapy. By successfully enriching in tumor tissues by forming a protein corona with serum macromolecules, we have successfully achieved precise cancer treatment. The present invention proposes a preparation method of near-infrared region II carbon dots for MR imaging with tumor-targeted therapy, and this method also provides a new way to explore the spin electron properties and mechanisms in nanomaterials.

[0006] The object of the present invention is to provide a preparation method of near-infrared region II carbon dots for MR imaging, which can generate a series of near-infrared absorptions based on magnetic edge states to solve the above technical problems.

[0007] The second object of the present invention is to provide a strategy for accelerating the relaxation of magnetic edge state electrons by spin averaging, which improves the MRI contrast effect and the near-infrared region II photothermal conversion efficiency.

[0008] The third object of the present invention is to provide a strategy for generating hyperfine splitting and zero-field splitting based on spin-orbit coupling, and to explain the source of superoxide radicals under near-infrared light irradiation.

[0009] The fourth object of the present invention is to provide a strategy for the carbon dots to spontaneously form a protein corona with macromolecules in serum, which improves the ability of tumor enrichment.

[0010] The fifth object of the present invention is to provide the application of the above carbon dots in MR imaging, tumor photothermal and photodynamic therapy, realizing precise tumor treatment.

[0011] The present invention is implemented as follows: In the first aspect, the present invention provides a preparation method of near-infrared region II carbon dots for MR imaging, including: mixing citric acid, formamide and formic acid for solvothermal reaction.

[0012] In an alternative embodiment, the mass ratio of citric acid to formamide is 1:1 - 10; preferably 1:2 - 6; Preferably, the addition amount of formic acid is to control the total concentration of citric acid and formamide in the mixed solution to be 0.05 - 1 g / mL.

[0013] In an alternative 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, it further includes: performing centrifugal separation after the solvothermal reaction, and drying the obtained solid; Preferably, freeze-drying is used for drying.

[0014] 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 volume ratio of the amount of the organic solvent to the volume of the solution after the solvothermal 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.

[0015] In a second aspect, the present invention provides a strategy for accelerating the relaxation rate of near-infrared second-region carbon dots for MR imaging, and the carbon dots are prepared by the preparation method of any one of the foregoing embodiments; Preferably, spin averaging occurs for the near-infrared second-region carbon dots in water, which is manifested as a main absorption at 550 nm and a broadband absorption at 800 - 1300 nm; the electron relaxation time in the near-infrared second region is about 1 ps; the photothermal conversion efficiency is 43.11%; Preferably, the main absorption bands of the near-infrared second-region carbon dots for the MR imaging in dimethyl sulfoxide or dimethylformamide are 580 nm, 700 nm, 820 nm, 900 nm, and 1030 nm; the absorption after 700 nm comes from the zero-field splitting of the magnetic edge state; the electron relaxation time in the near-infrared second region is about 11.2 ps; Preferably, the relaxation rate of the near-infrared second-region carbon dots for the MR imaging under a 9.4T magnetic field is not less than 270 mM - 1 s -1 , and this value is higher than currently commercial and literature-reported MRI contrast agents, and can achieve the MR imaging function of mice.

[0016] In a third aspect, a strategy based on spin-orbit coupling to enhance hyperfine splitting and zero-field splitting is provided, and the carbon dots are prepared by the preparation method of any one of the foregoing embodiments; Preferably, the near-infrared second-region carbon dots for the MR imaging can generate a nitrogen nuclear hyperfine splitting spectrum with 7 g values in DMSO solvent, and generate a near-infrared absorption peak based on zero-field splitting.

[0017] Preferably, the carbon dots in the second near-infrared region for MR imaging capture the generated photoexcited electrons with 2,2,6,6-tetramethylpiperidine-N-oxyl under near-infrared light illumination, and the reaction activity between the two is approximately 70%, demonstrating an efficient charge separation efficiency.

[0018] Preferably, the carbon dots in the second near-infrared region for MR imaging capture the generated superoxide anions with 5,5-dimethyl-1-pyrroline-N-oxide under near-infrared light illumination, demonstrating that photoexcited electrons can react with oxygen to generate superoxide anions.

[0019] In a third aspect, the present invention provides a strategy for a carbon nanodot composite material, including macromolecules such as proteins in serum and the carbon dots in the second near-infrared region for MR imaging in the foregoing embodiments.

[0020] Preferably, the carbon dots in the second near-infrared region for MR imaging can form a protein corona with biological macromolecules in mouse serum, enhancing the tumor enrichment ability and enabling tumor treatment guided by MR imaging.

[0021] Preferably, the carbon dots in the second near-infrared region for MR imaging are dissolved in mouse serum, and its mass concentration is 0-1 g / mL.

[0022] In a fourth aspect, the present invention provides the application of the carbon nanodot composite material in the foregoing embodiments in biological imaging and disease treatment.

[0023] The present invention has the following beneficial effects: By using citric acid and formamide as raw materials, highly efficient carbon dots in the second near-infrared region for MR imaging are synthesized by a one-step solvothermal method in formic acid containing both aldehyde groups and carboxyl groups, without complex purification procedures. The prepared carbon nanodots have a strong electron-withdrawing structure on the surface, which is used to confine and protect the electrons in the magnetic edge states. Due to the high paramagnetism of this carbon dot system, and the magnetic edge states can generate absorption in the second near-infrared region and ultrafast electron relaxation based on spin averaging in aqueous solution, it shows excellent performance in MR imaging and second near-infrared light therapy. The prepared carbon nanodots can form a protein corona with macromolecules such as proteins in serum, have a high fluorescence quantum yield in aqueous solution, and have the ability to enrich in tumors, enabling precise tumor treatment guided by MR imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] 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 thus 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.

[0025] Figure 1Magnetic and optical property test diagrams of carbon dots in the second near-infrared region for MR imaging provided in Embodiments 1-3 of the present invention; wherein, a and b are respectively the EPR spectra of carbon nanodots (CND) at different time points in DMSO and water. c is the absorption spectrum of CND in DMSO. d is the absorption spectra of CND and ox-CND in water; Inset: Infrared thermal imaging diagrams of CND and ox-CND at different time points under 1064 nm laser irradiation (1 W cm -2 ). e is the simulated structure of CNDs and the equatorial layer.

[0026] Figure 2 Diagram of the photoexcited state dynamics and mechanism of carbon dots in the second near-infrared region for MR imaging in Embodiment 4 of the present invention; wherein, a and b are respectively the TA spectra of CND in DMSO and H 2 O. c is the bleach signal dynamics of CND in DMSO, λpump = 700 nm, collected at 1023 nm; d is the bleach signal dynamics of CNDs in H 2 O, λpump = 550nm, collected at 1150nm; Points are experimental points, and solid lines are fitting lines. e is the EPR spectra of CND after 0 minutes and 5 minutes of NIR light irradiation. f is the energy level schematic diagram of magnetic edge states under NIR light excitation.

[0027] Figure 3 Comparison diagram of the structure and morphology of carbon dots in the second near-infrared region for MR imaging in Embodiment 5 of the present invention; Among them, a are the TEM and HRTEM (upper right inset) images of CND; Lower left inset: Particle size distribution diagram. b is the AFM image of CND; Inset: Height profile along the line. c is the FT-IR spectra of CND and ox-CND. d and e are both the hydrogen nuclear magnetic resonance spectra of CND in DMSO-d6.

[0028] Figure 4 Fluorescence and biotoxicity diagram of carbon dots in the second near-infrared region for MR imaging in Embodiment 6 of the present invention; Among them, a are the TEM and HRTEM (inset) images of CND in serum. b are the PL spectra of CND and ox-CND in serum, water and DMSO under 589 nm excitation. c are the PL decay curves (IRF = instrument response function) of CND and ox-CND in serum, water and DMSO under 510nm excitation. d is after intravenous injection of 100 µL aqueous solution of CND (100µL, 1000 µg mL −1Before and after that, PL imaging of the main organs of the mice was performed at different time points (Ex, 589 nm laser; Em, 650 nm long-pass [LP] optical filter). e is the live / dead cell imaging analysis of 4T1 cells.

[0029] Figure 5 This is the relaxation kinetics and MR imaging diagram of the carbon dots in the second near-infrared region for the MR imaging of Example 7 of the present invention; among them, a is the T -1 -weighted images of water and CND aqueous solutions (0, 100, 400, 500, and 600 μg mL 1 ) measured at 9.4 T. b is the graph of the relationship between the T 1 -weighted MR signal intensity and the CND concentration. c is the T 1 relaxation time of CND. d is the comparison graph of the relaxation rates of CND, commercial Gd-based contrast agents, and other reported contrast agents. e is the T 1 -weighted MRI for tracking the targeting ability of CND, and the circular area represents the tumor area.

[0030] Figure 6 This is the schematic diagram of the tumor treatment effect of the carbon dots in the second near-infrared region for the MR imaging of Example 8 of the present invention.

[0031] Among them, a is the infrared thermal images of the mice intravenously injected with 100 μL of PBS and CND aqueous solution (1000 μg mL -1 ) under irradiation with a 1064 nm laser at different time points at the tumor site (1 W cm -2 ). b is the fluorescence images of the tumors excised at different time points before and after intravenous injection of CNDs aqueous solution (100 μL, 1000 μg mL -1 ). c is the H&E staining of the tumor tissues before and after treatment. d is the photos of G1-G3 on the 16th day and G4 on the 90th day. f is the tumor images collected on the 16th day. e is the tumor growth curve of the 4T1 tumors in mice after different treatments (n = 5 for each group); the calculation is based on the average tumor size of each mouse (mean ± SD). g is the survival rate of G1-G4 mice. (G1: intravenously injected with PBS, G2: intravenously injected with PBS + 1064 nm laser irradiation, G3: intravenously injected with CND, G4: intravenously injected with CND + 1064 nm laser irradiation). Detailed implementation manners

[0032] 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 with the manufacturer are all conventional products that can be obtained through commercial purchase.

[0033] An embodiment of the present invention provides a method for preparing near-infrared second-region carbon dots for MR imaging, including the following steps: S1. Synthesis Citric acid, formamide, and formic acid are mixed for solvothermal reaction, and near-infrared second-region carbon dots for MR imaging are synthesized by a one-step solvothermal method without a complex purification procedure.

[0034] In the actual operation process, citric acid is dissolved in a mixed solvent of formamide and formic acid, and then placed in a reaction kettle, heated at a high temperature for solvothermal reaction to obtain a reddish-brown liquid, that is, near-infrared second-region carbon dots for MR imaging are obtained.

[0035] In some embodiments, the mass ratio of citric acid to formamide is 1:1 - 10; preferably 1:2 - 6. Further controlling the mass ratio of citric acid to formamide is beneficial to further improving the performance of the synthesized carbon nanodots, enhancing the fluorescence quantum efficiency and MR contrast effect of the carbon nanodots. Too low content of formamide will affect the hyperfine structure of the nitrogen nucleus, thereby reducing the effect of the MR contrast agent. On the contrary, too high mass of formamide will affect the formation of carbon dots and damage the surface functional groups, resulting in a decrease in the fluorescence quantum efficiency and water solubility of the carbon dots. Specifically, the mass ratio of citric acid to formamide can be 1:1, 1:4, 1:7, 1:10, etc., or any value between the above adjacent ratio values.

[0036] Furthermore, the addition amount of formic acid is to control the total concentration of citric acid and formamide in the mixed solution to be 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.

[0037] 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 carbon dots for MR imaging is improved. The temperature and reaction time will affect the degree of carbonization of the carbon dot core and the content of free radicals. Below 160 °C or too short reaction time will result in incomplete carbonization, causing a decrease in the carbon dot yield and free radical content, affecting the nuclear magnetic resonance contrast performance: above 200 °C or too long reaction time will cause excessive carbonization of the carbon dots, which will affect the water solubility and biological application of the carbon dots.

[0038] 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 adjacent temperature values above; the reaction time can be 2 h, 3 h, 4 h, 5 h, 6 h, etc., or any value between the adjacent time values above.

[0039] 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 progress of the reaction.

[0040] S2. Separation After the solvothermal reaction, centrifugal separation is carried out, and the obtained solid is dried to obtain the carbon dots in the second near-infrared region for MR imaging.

[0041] In some embodiments, an organic solvent that can promote the precipitation of carbon dots and is miscible with water is added to the system after the solvothermal reaction, 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 amount of the organic solvent to the volume of the solution after the solvothermal 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.

[0042] To increase the separation effect of the solid product, the centrifugation process is carried out in multiple times, and the number of centrifugations can be 2-4 times, such as 2 times, 3 times, 4 times. Specifically, the rotation speed of the centrifugal separation is preferably 8000-12000 revolutions per minute, and more preferably 10000 revolutions per minute. After the centrifugal separation is completed, the upper layer solution is removed, and the lower layer precipitate is freeze-dried to obtain the highly efficient carbon dots in the second near-infrared region for MR imaging.

[0043] In some embodiments, freeze-drying is used for drying to prevent the influence of high temperature on the product.

[0044] The embodiment of the present invention also provides a carbon dot in the second near-infrared region for MR imaging, which is prepared by the above preparation method. The carbon dot in the second near-infrared region for MR imaging has a high content of magnetic edge state electron structure, so that the carbon nanodot has high paramagnetism and absorption in the second near-infrared region, shows nitrogen nuclear hyperfine splitting and zero-field splitting in dimethyl sulfoxide solvent, and shows very fast electron relaxation due to spin averaging in water solvent.

[0045] Upon detection, the main absorption bands of the near-infrared II region carbon dots for MR imaging in water are a broadband absorption at 550 nm and 800 - 1300 nm, and the electron relaxation time in the near-infrared II region is about 1 ps; the main absorption peaks of the near-infrared II region carbon dots for MR imaging in dimethyl sulfoxide or dimethylformamide are at 580 nm, 700 nm, 820 nm, 900 nm, and 1030 nm; among them, the absorption after 700 nm comes from the zero-field splitting of magnetic edge states; the electron relaxation time in the near-infrared II region is about 11.2 ps; the near-infrared II region carbon dots have a relaxation rate of 270 mM under a 9.4 T magnetic field -1 s -1 ; under the excitation of a near-infrared-II region laser, the photothermal conversion efficiency is 43.11%, and a large amount of superoxide anions can be generated.

[0046] The embodiment of the present invention provides a carbon nanodot composite material, including proteins in serum and the above-mentioned near-infrared II region carbon dots. The carbon nanodot composite material has a larger particle size (30 - 80 nm) and a relatively high red fluorescence quantum yield (38%) in serum. In addition, the near-infrared II region carbon dots for MR imaging and their composite materials have high water solubility, good biocompatibility, low cytotoxicity, and can be excreted through the liver and kidneys within a short time.

[0047] In some embodiments, the near-infrared II region carbon dots for MR imaging are dissolved in mouse serum with a mass concentration of 0 - 1 g / mL, and they can effectively accumulate in tumors and be imaged.

[0048] The features and properties of the present invention are further described in detail below in conjunction with embodiments. The embodiment of the present invention provides the application of the near-infrared II region carbon dots for MR imaging in biological imaging, which can specifically be applied to the imaging of major organs throughout the body of mice as well as tumor imaging and in vivo mouse MRI tumor imaging. The specific location of tumors in the body can be judged through the fluorescence and MRI maps of the carbon dots. The enrichment principle may be due to the formation of a protein corona between the carbon dots and macromolecules in serum, which increases their particle size.

[0049] The embodiment of the present invention also provides the application of the near-infrared II region carbon dots for MR imaging in tumor treatment. Specifically, an aqueous solution of carbon dots can be injected through the tail vein, and the treatment of mouse tumors can be successfully achieved by irradiating with a near-infrared II region laser with a low light power density.

[0050] 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, and it has good application prospects in the fields of biological imaging and tumor treatment.

[0051] The features and properties of the present invention are further described in detail below in conjunction with embodiments.

[0052] Example 1 This embodiment provides a method for preparing near-infrared second-region carbon dots for MR imaging, comprising the following steps: Dissolve 1 g of citric acid in a mixed solution of 4 mL of formamide and 6 mL of formic acid. Place the liquid in 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 oven heating or heating with a parallel reactor. Add 60 mL of ethanol to the reaction solution, centrifuge at a speed of 8000 revolutions per minute, remove the upper solution, leave the lower precipitate, centrifuge 3 times, and freeze-dry the lower precipitate to obtain a black solid powder, namely the near-infrared second-region carbon dots for MR imaging, with a yield of about 5%.

[0053] Example 2 This embodiment provides a method for preparing oxidized near-infrared second-region carbon dots for MR imaging, comprising the following steps: Mix the near-infrared second-region carbon dots for MR imaging prepared in Example 1 with dimethyl sulfoxide to obtain a dimethyl sulfoxide solution of near-infrared second-region carbon dots for MR imaging with a concentration of 1 mg mL -1 , and the reaction time is 40 min. Add 60 mL of ethanol to the reaction solution, centrifuge at a speed of 8000 revolutions per minute, remove the upper solution, leave the lower precipitate, centrifuge 3 times, and freeze-dry the lower precipitate to obtain a black solid powder, namely the oxidized near-infrared second-region carbon dots for MR imaging.

[0054] Example 3 Perform electron paramagnetic resonance (EPR) spectroscopy and absorption spectroscopy analysis on the near-infrared second-region carbon dots for MR imaging obtained in Examples 1-2.

[0055] As can be seen from Figure 1 a, the near-infrared second-region carbon dots for MR imaging can produce nitrogen nuclear hyperfine splitting in DMSO. There are 7 g values representing 7 quantum states, and the rapid decrease of its ESR signal indicates a slower electron relaxation rate; as can be seen from Figure 1 b, the near-infrared second-region carbon dots for MR imaging show a broadband microwave absorption in aqueous solution due to spin averaging. The longer linewidth and slower decay rate respectively indicate a shorter electron spin lifetime and electron relaxation time; as can be seen from Figure 1 c, the magnetic edge states of the carbon dots are mainly distributed at 700 nm, 820 nm, 900 nm, and 1030 nm in the DMSO absorption band, and these absorptions are due to zero-field splitting. As can be seen from Figure 1 d, the magnetic edge states of the carbon dots show a broadband absorption in the range of 800 - 1300 nm in aqueous solution, and due to the accelerated electron relaxation, a photothermal conversion efficiency of 43.11% is exhibited. Due to the reduction of the magnetic edge state structure after oxidation, its photothermal conversion efficiency is only 22.89%. As can be seen from Figure 1 e, we speculate on the structures of the overall carbon dots and the magnetic edge states.

[0056] Example 4 The near-infrared second near-infrared carbon dots obtained in Examples 1-2 were subjected to transient absorption spectroscopy (TA), light-excited electron paramagnetic resonance (LEPR) spectroscopy, and mechanism interpretation, as Figure 2 shown.

[0057] From Figure 2 as shown in a and b, the ground state bleach signal (PB) of the near-infrared second near-infrared carbon dots for MR imaging is located at 1030 nm in DMSO solvent, and a broadband PB signal of 900-1300 nm in water. In Figure 2 as shown in c and d, it can be seen from the decay photodynamic curve that the lifetime of the magnetic edge state electrons in DMSO is about 11.2 ps, while the lifetime in aqueous solution is about 1 ps, which also indicates that its relaxation speed in water is faster. From Figure 2 as shown in e, under near-infrared light illumination, the signal of superoxide radicals was found to appear, indicating its potential for photodynamic therapy. From Figure 2 as shown in f, in DMSO, the magnetic edge state electrons tend to return to the singlet state, while in aqueous solution, they tend to sacrifice their singlet state to maintain their high spin state.

[0058] Example 5 The near-infrared second near-infrared carbon dots obtained in Examples 1-2 were subjected to transmission electron microscopy (TEM), atomic force microscopy (AFM), infrared spectroscopy (FTIR), and nuclear magnetic resonance spectroscopy (NMR) analysis, as Figure 3 shown.

[0059] As Figure 3 shown in a and b, the particle size of the near-infrared second near-infrared carbon dots for MR imaging is 2.43 ± 0.64 nm, and its height is approximately 2 nm, roughly ellipsoidal or flaky. As Figure 3 shown in c, the carboxyl level is increased after oxidation, indicating that DMSO oxidation can disrupt the ligand field on the surface of the carbon dots. As Figure 3 shown in d, the ligands undergo dehydration condensation reactions on the surface of the carbon dots, indicating that the hydrogen bonds that originally protected the magnetic edge state electrons are disrupted during the oxidation process. Due to the high reactivity of the unpaired electrons, it is easy to pair with external substances and undergo chemical reactions. Figure 3 As shown in e, the shift of the active hydrogen indicates the disruption and reconstruction of the hydrogen bonds on the surface of the carbon dots.

[0060] Example 6 The near-infrared second near-infrared carbon dots obtained in Examples 1-2 were subjected to TEM particle size, fluorescence intensity, fluorescence lifetime spectrum, in vitro organ metabolism, and live-dead cell double staining experiment analysis, as Figure 4 shown.

[0061] AsFigure 4 As can be seen from a, in serum, the near-infrared-II carbon dots for MR imaging can form large-particle protein coronas with macromolecules in the serum, and their particle size is 30 - 80 nm, providing a basis for the enrichment of carbon dots at the tumor site later. Through Figure 4 Analysis of b and c shows that in serum, both the red fluorescence intensity and lifetime of the carbon dots are increased, and their quantum yield is 37.8%, much higher than 6.2% in water. The quantum yield in DMSO solvent is approximately 56%, the fluorescence lifetime in serum is 2.99 ns, similar to 3.52 ns in DMSO, and longer than 1.41 ns in water. This indicates that the exciton transfer between carbon dots and proteins inhibits the energy dissipation of lattice vibration, proving the feasibility of its red fluorescence imaging.

[0062] As Figure 4 shown in d, a 1000 μg / mL carbon dot solution was injected into mice via the tail vein, and a 50 mW / cm 2 yellow light laser at 589 nm was used to irradiate the whole body of the mice, and then a CCD camera equipped with a 730 nm filter was used to take near-infrared fluorescence images of the mice. The image of a non-injected and non-irradiated mouse was taken as the control group, and photos were taken at 1 h, 3 h, 6 h, 12 h, 24 h, and 48 h after irradiation. The mouse organs and tumors were dissected to confirm the metabolic mode of carbon dots in the mice. It can be seen from the images that carbon dots were basically metabolized out of the body through the kidneys and liver within 48 h, indicating the biocompatibility of the near-infrared-II carbon dots for MR imaging.

[0063] As Figure 4 shown in e, 1000 μg / mL carbon dots were added to 4T1 tumor cells. After incubation for 1 h, they were incubated with a 1064 nm near-infrared laser at 1000 mW / cm 2 together with the non-irradiated control group for 24 h, and a live / dead cell double staining kit was used to test the viability of each group of tumor cells, so as to compare the inhibitory effect on tumor cells. From the results of the tumor cell killing experiment, carbon dots and the 1064 nm laser have no cytotoxicity, but the combination of the two can produce significant phototoxicity to kill tumors, providing a good basis for near-infrared-II tumor phototherapy in mice.

[0064] Example 7 Perform relaxation kinetics and MRI image analysis on the near-infrared-II carbon dots for MR imaging obtained in Examples 1 - 2, as Figure 5 shown.

[0065] As Figure 5 shown in a - c, within 1000 μg / mL, the MRI signal intensity and T1 relaxation value increase with the increase of the carbon dot concentration, and the calculated relaxation rate is 270 mM-1 s -1 As can be seen from Figure 5 d in the above, its relaxation rate is higher than that of all currently commercial and reported contrast agents. As can be seen from Figure 5 e in the above, carbon dots can be well enriched in tumors and perform MR imaging, and can be quickly metabolized out of the body within 24 hours.

[0066] Example 8 In this example, near-infrared second-region phototherapy experiments on mouse tumors were carried out on the carbon dots prepared in Example 1. As Figure 6 shown, the specific steps include: By injecting 100 μl of 1000 μg / mL carbon dots into the tail vein of mice, irradiating with a 1064 nm near-infrared laser at 1000 mW / cm 2 for 10 min. The control groups were tumor-bearing mice that were only irradiated with a 1064 nm laser on the tail vein injected with phosphate buffer alone and tumor-bearing mice that were only injected with 100 μl of 300 μg / mL carbon dots into the tail vein without irradiation. The tumor volume and mouse body weight were observed every two days before and after injection. The tumors of each group were dissected after 14 days to compare the tumor sizes. The remaining successful experimental groups were continuously observed for 90 days to confirm whether the tumors recurred.

[0067] From Figure 6 a in the above, it can be seen that the tumor tissue of mice irradiated with a 1064 nm laser can only be heated by 10 °C, but the mice injected with carbon dots into the tail vein can be heated by more than 30 °C under irradiation, which can effectively kill tumors. As Figure 6 shown in b in the above, carbon dots can be enriched at the tumor site for 1 - 24 h. As Figure 6 shown in c in the above, by comparing the H&E staining experiments of the tumor site before and after irradiation in the experimental group, the heat and superoxide radicals generated by irradiation can effectively damage the cell structure. As Figure 6 shown in the tumor growth curves and survival curves and the comparison pictures of mice and tumors in d - g in the above, the group injected with carbon dots and irradiated can successfully cure tumors and do not relapse within 90 days, and the tumor growth of other groups is basically not inhibited. During the experiment, the body weights of mice in each group remained basically unchanged. This shows that the near-infrared second-region carbon dots for MR imaging can be used as excellent tumor phototherapy reagents.

[0068] 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 in the protection scope of the present invention.

Claims

1. A method for preparing near-infrared zone II carbon dots for nuclear magnetic resonance imaging, characterized in that: include: The citric acid and formamide were mixed with formic acid to undergo a solvothermal reaction.

2. The preparation method according to claim 1, characterized in that: The mass ratio of citric acid to formamide is 1:1-10; the amount of formic acid added is to control the total concentration of citric acid and formamide 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 formamide is 1:2-6.

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

5. The preparation method according to claim 4, characterized in that: The temperature of the solvent thermal reaction is 160-200° C., and the reaction time is 4-4.5 hours.

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

7. The preparation method according to claim 6, characterized in that: The 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 anhydrous ethanol; The 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 is 2-4 times.

9. The near-infrared zone II carbon dots for nuclear magnetic resonance imaging prepared by the preparation method according to any one of claims 1 to 8, characterized in that: The near-infrared zone II carbon dots for nuclear magnetic resonance imaging have a main absorption band of 550nm and a broadband absorption of 800-1300nm in water; the electron relaxation time in the near-infrared zone II is 1ps; and the photothermal conversion efficiency is 43.11%; The near-infrared zone II carbon dots for nuclear magnetic resonance imaging can produce a nitrogen nuclear hyperfine splitting spectrum with 7 g values ​​at room temperature in dimethyl sulfoxide or dimethylformamide, and the main optical absorption bands are 580nm, 700nm, 820nm, 900nm and 1030nm; the absorption after 700nm comes from the zero-field splitting of the magnetic edge state; the electron relaxation time in the near-infrared zone II is 11.2ps; The near-infrared zone II carbon dots for nuclear magnetic resonance imaging can generate a large amount of photogenerated electrons under near-infrared light, and the photogenerated electrons can react with oxygen to generate superoxide anions; The relaxation rate of the near-infrared second-zone carbon dots for nuclear magnetic resonance imaging is not less than 270 mM under a magnetic field of 9.4 T. -1 s -1 , which can realize MR imaging function of mice.

10. Use of the carbon dots according to claim 9 in biological imaging and quantum coherence.

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