Preparation method of manganese-doped carbon dots and application thereof in bimodal imaging and photothermal therapy

Abstract

This invention relates to the field of medical imaging technology, and more particularly to a method for preparing manganese-doped carbon dots and their application in dual-modal imaging and photothermal therapy. Using reduced glutathione (GSH) as the carbon source, soluble manganese salt as the dopant source, and formamide as the solvent, this invention prepares manganese-doped fluorescent carbon dots through a one-step solvothermal reaction. These dots exhibit red fluorescence emission characteristics, a significant redshift in excitation / emission wavelength, greater penetration depth into biological tissues, making them more suitable for deep tissue and in vivo fluorescence imaging. They also possess highly efficient T1-weighted magnetic resonance imaging capabilities, achieving dual-modal imaging of fluorescence / Mn-CDs. Furthermore, the manganese-doped fluorescent carbon dots of this invention integrate red fluorescence imaging, T₁ magnetic resonance imaging, and photothermal therapy, enabling precise imaging-guided photothermal ablation, forming a complete integrated mechanism for tumor diagnosis and treatment, possessing both high biocompatibility and clinical translational potential.

Description

Technical Field

[0001] This invention relates to the field of medical imaging technology, and in particular to a method for preparing manganese-doped carbon dots and their application in dual-modal imaging and photothermal therapy. Background Technology

[0002] In modern clinical medical imaging technology, magnetic resonance imaging (MRI) has become an indispensable tool in clinical diagnosis due to its advantages such as no ionizing radiation, high soft tissue resolution, and multi-parameter imaging. Currently, the most widely used contrast agents in clinical practice are gadolinium-based chelates, represented by gadopentetate dimeglumine (Gd-DTPA). However, gadolinium ions in these contrast agents are prone to dissociation and deposition in the skin, bone, and brain tissue, and pose a risk of inducing renal systemic fibrosis in patients with renal insufficiency. Furthermore, clinical studies have confirmed that repeated use of gadolinium agents can lead to gadolinium accumulation in the deep cerebellum. Therefore, the development of novel, non-gadolinium-based MRI contrast agents with good biocompatibility and metabolic clearance is of urgent clinical need and significant research value.

[0003] Meanwhile, fluorescence imaging technology, with its real-time response, high sensitivity, and ease of operation, has become an important supplementary tool for biomedical imaging and intraoperative navigation. However, traditional organic fluorescent dyes suffer from drawbacks such as poor photostability, easy photobleaching, short wavelength, and significant biotoxicity, while most inorganic fluorescent probes are complex to prepare and have poor biodegradability, making it difficult to meet the requirements of long-term in vivo imaging and clinical translation. Therefore, developing novel fluorescent contrast agents with high biocompatibility, strong fluorescence emission, and excellent optical stability has become an important direction for promoting the development of in vivo fluorescence imaging. In actual imaging diagnosis, a multimodal imaging strategy is often used to improve diagnostic accuracy. For example, in tumor diagnosis and treatment, MRI and fluorescence imaging are often combined to simultaneously obtain macroscopic structural information and microscopic boundary details of the tumor. However, contrast agents that can simultaneously meet the requirements of MRI and fluorescence dual-modal imaging are still relatively scarce. Therefore, developing dual-modal contrast agents (probes) with both MRI and fluorescence imaging capabilities has significant research value and application prospects.

[0004] Manganese (Mn) 2+ As an essential trace element for the human body, its paramagnetism originates from 3D 5 The electronic configuration shows potential as a contrast agent for T1-weighted MRI. (The text abruptly ends here, likely due to an incomplete sentence or missing information.) 2+ Doping to prepare fluorescent carbon dots (CDs) holds promise for obtaining probes with fluorescence imaging capabilities, while simultaneously reducing free Mn. 2+The toxicity of manganese-doped carbon dots (Mn-CDs) is a concern. Currently, the main methods for preparing Mn-CDs are hydrothermal / solvothermal methods, with existing techniques often using water or ethanol as solvents and one or more of citric acid, phytic acid, or small-molecule amines as carbon sources. However, the fluorescence emission wavelengths of carbon dots prepared by existing methods are relatively short, with excitation wavelengths mostly located in the ultraviolet-blue light region, limiting their penetration depth into biological tissues and making it difficult to meet the needs of deep tissue and in vivo imaging. Furthermore, they only meet the requirements of a single MRI imaging modality and cannot simultaneously satisfy MRI and fluorescence imaging capabilities. Therefore, developing novel Mn-doped carbon dots that combine high physiological stability with long-wavelength (e.g., near-infrared) fluorescence emission characteristics is of great significance for achieving high-quality dual-modal diagnosis using both fluorescence and magnetic resonance imaging.

[0005] Furthermore, precision treatment of tumors requires not only efficient imaging techniques for accurate lesion localization but also effective image-guided therapy. Photothermal therapy (PTT), as a minimally invasive / non-invasive tumor treatment method, converts light energy into heat energy through a photothermal converter, achieving local thermal ablation of tumor cells. It offers advantages such as spatiotemporal controllability, minimal side effects, and repeatability. Based on this, constructing a substance with multiple functions including MRI imaging, fluorescence imaging, and photothermal therapy to achieve integrated tumor diagnosis and treatment is of great significance for improving treatment efficacy and reducing recurrence rates. Summary of the Invention

[0006] Based on the background technology, this invention provides a method for preparing manganese-doped carbon dots and their application in dual-modal imaging and photothermal therapy. This invention uses only glutathione as the carbon source, soluble manganese salt as the dopant source, and formamide as the solvent to construct a contrast agent with both T1-weighted magnetic resonance and red fluorescence imaging capabilities, as well as photothermal effects, through a one-step solvothermal method. This can provide a new direction for dual-modal imaging diagnosis of diseases and integrated diagnosis and treatment.

[0007] The technical solution of the present invention: A method for preparing manganese-doped fluorescent carbon dots includes the following steps: (1) Reduced glutathione and soluble manganese salt are dissolved in formamide to obtain a precursor solution; the molar ratio of reduced glutathione to manganese is (5~10):1. (2) The precursor solution is subjected to a solvothermal reaction at 180℃~200℃; (3) After purifying and drying the cooled product, manganese-doped fluorescent carbon dots can be prepared.

[0008] Furthermore, the soluble manganese salt mentioned in step (1) is selected from at least one of manganese chloride, manganese nitrate, and manganese acetate.

[0009] Furthermore, in step (3), the solvothermal reaction product is purified by dialysis.

[0010] Furthermore, in step (3), the solvothermal reaction product is dried by freeze drying.

[0011] Further, the molar concentration of the reduced glutathione in the precursor solution of step (1) is 20-30 μmol / L.

[0012] Based on the same inventive concept, the present invention also provides manganese-doped fluorescent carbon dots prepared by the aforementioned method for preparing manganese-doped fluorescent carbon dots.

[0013] Based on the same inventive concept, the present invention also provides the application of the manganese-doped fluorescent carbon dots in the preparation of imaging contrast agents according to any one of A1) to A3), wherein the contrast agent comprises: A1) Magnetic resonance imaging contrast agent; A2) Fluorescent imaging contrast agent; A3) Fluorescence-magnetic resonance dual-modal imaging contrast agent.

[0014] Based on the same inventive concept, the present invention also provides the application of the manganese-doped fluorescent carbon dots in the preparation of photothermal therapeutic agents.

[0015] Based on the same inventive concept, the present invention also provides the application of the manganese-doped fluorescent carbon dots in the preparation of MRI / fluorescence imaging-guided photothermal diagnostic and therapeutic integrated formulations.

[0016] The beneficial effects achieved by this invention are as follows: This invention uses reduced glutathione (GSH) as the carbon source, soluble manganese salt as the dopant source, and formamide as the solvent to achieve carbon core carbonization, in-situ N / S self-doping, and Mn doping through a one-step solvothermal reaction. 2+ Coordination doping was completed simultaneously, thereby preparing manganese-doped fluorescent carbon dots, sp 2 The intrinsic luminescence of the carbon nucleus, surface defect states, and Mn doping energy levels work together to give the prepared manganese-doped fluorescent carbon dots red fluorescence emission characteristics. The excitation / emission wavelengths are significantly redshifted, allowing for greater penetration depth into biological tissues, making them more suitable for deep tissue and in vivo fluorescence imaging. 2+ The successful doping of carbon dots endowed them with efficient T1-weighted magnetic resonance imaging capabilities, enabling dual-modal imaging of fluorescence / Mn-CDs. Simultaneously, the carbon framework and N / O / S functional groups enhanced the Mn... 2+ The strong coordination effect of glutathione can stabilize manganese ions and reduce free toxicity. Furthermore, the carbon dots prepared with glutathione as the carbon source retain abundant chemical groups on their surface, giving the material excellent water solubility, biocompatibility, and resistance to protein adsorption, which is beneficial for further application in bioimaging.

[0017] Furthermore, the preparation method of the present invention uses Mn 2+ The introduction of carbon and its defects can effectively increase non-radiative transition efficiency, improve photothermal conversion capacity, and achieve local heating and tumor cell ablation. The manganese-doped fluorescent carbon dots of this invention integrate red fluorescence imaging, T1 magnetic resonance imaging, and photothermal therapy. Through precise imaging positioning to guide photothermal ablation, it constitutes a complete integrated mechanism for tumor diagnosis and treatment, possessing both high biocompatibility and clinical translational potential.

[0018] This invention employs a one-step solvothermal method, using inexpensive and readily available raw materials, with mild and controllable reaction conditions, simple product purification, and easy large-scale preparation. Attached Figure Description

[0019] Figure 1 Transmission electron microscope (TEM) image of Mn-CDs prepared in Example 1 of this invention.

[0020] Figure 2 This is a particle size distribution diagram of Mn-CDs prepared in Example 1 of the present invention.

[0021] Figure 3 The X-ray photoelectron spectroscopy (XPS) full spectrum (a) and high-resolution spectra of carbon 1s (b), nitrogen 1s (c), oxygen 1s (d), sulfur 2p (e), manganese 2p (f), and manganese 3s (g) of Mn-CDs prepared in Example 1 of this invention are shown.

[0022] Figure 4 The image shows the ultraviolet absorption spectrum of Mn-CDs prepared in Example 1 of this invention.

[0023] Figure 5 The fluorescence emission spectrum of Mn-CDs prepared in Example 1 of this invention is shown.

[0024] Figure 6 This is a magnetic resonance imaging image of Mn-CDs prepared in Example 1 of the present invention.

[0025] Figure 7 The image shows the photothermal heating curve of Mn-CDs prepared in Example 1 of this invention.

[0026] Figure 8 The results of the MTT assay for phototherapy of tumor cells using Mn-CDs prepared in Example 1 of this invention are shown. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the experiments used in the following embodiments... Unless otherwise specified, all materials were purchased from commercial channels.

[0029] The reduced glutathione, manganese chloride tetrahydrate, manganese acetate tetrahydrate, manganese nitrate tetrahydrate, and formamide used in the experiment were all purchased from Shanghai Titan Technology Co., Ltd.

[0030] Example 1: Weigh 0.7 μmol of reduced glutathione and 0.07 μmol of manganese chloride tetrahydrate (MnCl2·4H2O), dissolve them in 30 mL of formamide, and stir magnetically for 30 minutes until completely dissolved. Transfer the precursor solution to a 50 mL high-pressure reactor lined with polytetrafluoroethylene, seal it, and place it in an oven at 180 °C for 8 hours. After the reaction, allow it to cool naturally to room temperature, remove the dark green solution, and dialyze it in deionized water using a dialysis bag with a molecular weight cutoff of 1000 Da for 24 hours, changing the dialysis fluid every 6 hours. After dialysis, freeze-dry the product to obtain dark green powdery manganese-doped fluorescent carbon dots (Mn-CDs).

[0031] Example 2: Weigh 0.7 μmol of reduced glutathione and 0.14 μmol of manganese acetate tetrahydrate (Mn(CH3COO)2·4H2O), dissolve them in 30 mL of formamide, and stir magnetically for 30 minutes until completely dissolved. Transfer the precursor solution to a 50 mL polytetrafluoroethylene-lined high-pressure reactor, seal it, and place it in an oven at 180 °C for 8 hours. After the reaction, allow it to cool naturally to room temperature, remove the dark green solution, and dialyze it in deionized water for 24 hours using a dialysis bag with a molecular weight cutoff of 1000 Da, changing the dialysis fluid every 6 hours. After dialysis, freeze-dry the product to obtain dark green powdery manganese-doped fluorescent carbon dots.

[0032] Example 3: Weigh 0.7 μmol of reduced glutathione and 0.14 μmol of manganese nitrate tetrahydrate (Mn(NO3)2·4H2O), dissolve them in 30 mL of formamide, and stir magnetically for 30 minutes until completely dissolved. Transfer the precursor solution to a 50 mL high-pressure reactor lined with polytetrafluoroethylene, seal it, and place it in an oven at 180 °C for 8 hours. After the reaction, allow it to cool naturally to room temperature, remove the dark green solution, and dialyze it in deionized water using a dialysis bag with a molecular weight cutoff of 1000 Da for 24 hours, changing the dialysis fluid every 6 hours. After dialysis, freeze-dry the product to obtain dark green powdery manganese-doped fluorescent carbon dots.

[0033] Example 4: Weigh 0.7 μmol g of reduced glutathione and 0.07 μmol of manganese chloride tetrahydrate (MnCl2·4H2O), dissolve them in 30 mL of formamide, and stir magnetically for 30 minutes until completely dissolved. Transfer the precursor solution to a 50 mL polytetrafluoroethylene-lined high-pressure reactor, seal it, and place it in an oven at 200 °C for 10 hours. After the reaction, allow it to cool naturally to room temperature, remove the dark green solution, and dialyze it in deionized water using a dialysis bag with a molecular weight cutoff of 1000 Da for 24 hours, changing the dialysis fluid every 6 hours. After dialysis, freeze-dry the product to obtain dark green powdery manganese-doped fluorescent carbon dots.

[0034] Performance Characterization The Mn-CDs prepared in Example 1 were accurately weighed and prepared into an aqueous solution with a concentration of 1 mg / mL, which was then ultrasonically dispersed. After appropriate dilution, the solution was dropped onto an ultrathin carbon film, dried at room temperature, and characterized by transmission electron microscopy (TEM). The test results showed that the carbon dots were uniformly dispersed quasi-spherical particles with an average particle size of 3.4 nm. Clear lattice fringes were observed in the high-resolution image, and the interplanar spacing was 0.21 nm, which is consistent with the typical structural characteristics of carbon dot nanomaterials. X-ray photoelectron spectroscopy (XPS) was used to analyze the chemical composition and surface elemental state of the product. The results showed that the carbon dots not only retained the characteristic elements of the carbon source, but also successfully achieved the Mn-CDs. 2+ Effective doping. Spectroscopic tests showed that the carbon dots exhibited a distinct characteristic absorption peak at 600-750 nm, emitting bright red fluorescence under excitation by light sources in the corresponding wavelength range. Magnetic resonance imaging (MRI) results further confirmed that these carbon dots can serve as T1-weighted imaging contrast agents, possessing both fluorescence and MRI dual-modal imaging capabilities. Photothermal heating tests indicated that Mn-CDs exhibit excellent photothermal conversion effects, showing promise for photothermal therapy of tumors; dark toxicity and phototoxicity experiments demonstrated low cytotoxicity and good photothermal therapeutic effects.

[0035] Experiment 1: Transmission electron microscopy and particle size analysis of Mn-CDs Test results are as follows Figure 1 As shown, the carbon dots are uniformly dispersed spherical particles with an average particle size of 2-5 nm. High-resolution transmission electron microscopy images show that they have clear lattice fringes and a crystal plane spacing of 0.21 nm. Figure 2 The particle size statistics show that the average size is 3.4 nm.

[0036] Experiment 2: X-ray photoelectron spectroscopy analysis of Mn-CDs Elemental composition and chemical state analysis of Mn-CDs were performed using X-ray photoelectron spectroscopy. Figure 3 As shown in Figure a, the full spectrum results indicate that Mn-CDs are mainly composed of carbon (65.0%), nitrogen (17.2%), oxygen (13.4%), sulfur (1.4%), and manganese (3.0%). The high-resolution spectrum of carbon is shown below. Figure 3 b) shows that the peak at 284.8 eV belongs to the CC / C=C bond, indicating that the carbon dots have a highly graphitized carbon core; the peaks at 286.1 eV and 287.9 ​​eV belong to the CN / C=N and CO / C=O bonds, respectively. Figure 3 As shown in c, the high-resolution spectrum of nitrogen elements indicates that nitrogen in Mn-CDs exists mainly in the forms of pyridine nitrogen (398.4 eV), pyrrole nitrogen (399.4 eV), and graphitic nitrogen (400.2 eV). The high-resolution spectrum of oxygen elements in Mn-CDs ( Figure 3 d) indicates that the carbon dot surface is rich in carboxyl groups (C=O, 531.7 eV) and hydroxyl groups (CO, 533.1 eV). Sulfur elemental spectrum ( Figure 3 e) indicates that the carbon dots contain thiol groups (R-SH, 164.0 eV). Manganese element test results ( Figure 3 f, g) indicates that it is mainly doped into carbon dots through coordination with oxygen. The peak position difference of the Mn 3s orbital further confirms that it is mainly Mn 2+ The Mn-CDs exist in their original form. X-ray photoelectron spectroscopy results show that Mn-CDs retain abundant hydrophilic groups from the carbon source, providing a structural basis for their application in the field of bioimaging.

[0037] Experiment 3: UV absorption and fluorescence spectra of Mn-CDs.

[0038] like Figure 4 As shown, Mn-CDs exhibit strong absorption peaks at 400–500 nm and 600–750 nm. Figure 5 The results show that the carbon dot can emit red fluorescence in the range of 650-750 nm when excited by 630 nm light, indicating that it can be used for near-infrared fluorescence imaging.

[0039] Experiment 4: Magnetic Resonance Imaging Signal Testing of Mn-CDs.

[0040] like Figure 6 As shown, in vitro T1-weighted imaging of Mn-CDs at different concentrations was measured using deionized water as a control. The results showed that the T1-weighted NMR image signal gradually brightened with increasing Mn-CDs concentration, indicating that it has good NMR imaging capabilities.

[0041] Experiment 5: Photothermal heating test of Mn-CDs.

[0042] like Figure 7 As shown, using deionized water as a control group, Mn-CDs were prepared into an aqueous solution with a concentration of 0.5 mg / mL and subjected to a 650 nm laser (power density 0.5 W / cm²). 2 Under irradiation, the solution temperature rose from 25°C to 45°C within 10 minutes, an increase of 20°C.

[0043] Experiment 6: Cellular dark toxicity and phototoxicity test of Mn-CDs.

[0044] like Figure 8 As shown, the cytotoxicity of Mn-CDs was studied using mouse breast cancer cells 4T1. 180 μL of 4T1 cells were seeded in 96-well plates. When the cell density reached approximately 80%, 20 μL of different concentrations (final concentrations of 0, 0.25, 0.5, 0.75, and 1 mg / mL) of Mn-CDs were added. After incubation for 24 h, the cells were washed with PBS, followed by the addition of fresh culture medium and MTT. Cell viability was measured using a microplate reader. Even at a concentration as high as 1 mg / mL without light exposure, the cells still exhibited high cell viability, indicating low cytotoxicity. The phototoxicity assay was similar to the above steps, except that after washing the cells with PBS, they were irradiated with a 650 nm laser for 8 min per well. Fresh culture medium and MTT were then added, and cell viability was measured again using a microplate reader. The results showed that at a Mn-CDs concentration of 0.5 mg / mL, cell viability significantly decreased to 30% after light exposure, confirming its excellent photothermal therapeutic effect.

[0045] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. For those skilled in the art, any modifications, equivalent substitutions, and improvements made based on the technical solutions of the present invention without departing from the spirit and essence of the present invention should be considered to fall within the scope of protection of the present invention. The scope of protection of the present invention is determined by the content of the claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method for preparing manganese-doped fluorescent carbon dots, characterized in that, Includes the following steps: (1) Reduced glutathione and soluble manganese salt are dissolved in formamide to obtain a precursor solution; the molar ratio of reduced glutathione to manganese is (5~10):

1. (2) The precursor solution is subjected to a solvothermal reaction at 180℃~200℃; (3) After purifying and drying the cooled product, manganese-doped fluorescent carbon dots can be prepared.

2. The method for preparing manganese-doped fluorescent carbon dots according to claim 1, characterized in that, In step (1) The soluble manganese salt described herein is selected from at least one of manganese chloride, manganese nitrate, and manganese acetate.

3. The method for preparing manganese-doped fluorescent carbon dots according to claim 1, characterized in that, In step (3) The products of the solvothermal reaction were purified by dialysis.

4. The method for preparing manganese-doped fluorescent carbon dots according to claim 1, characterized in that, Step (3) The solvothermal reaction products were dried by freeze drying.

5. The method for preparing manganese-doped fluorescent carbon dots according to claim 1, characterized in that, The molar concentration of reduced glutathione in the precursor solution of step (1) is 20-30 μmol / L.

6. Manganese-doped fluorescent carbon dots prepared by the method for preparing manganese-doped fluorescent carbon dots according to any one of claims 1 to 5.

7. The use of the manganese-doped fluorescent carbon dots according to claim 6 in the preparation of the imaging contrast agent in any one of A1) to A3), characterized in that, The contrast agent includes: A1) Magnetic resonance imaging contrast agent; A2) Fluorescent imaging contrast agent; A3) Fluorescence-magnetic resonance dual-modal imaging contrast agent.

8. The application of the manganese-doped fluorescent carbon dots according to claim 6 in the preparation of photothermal therapeutic agents.

9. The application of the manganese-doped fluorescent carbon dots according to claim 6 in the preparation of MRI / fluorescence imaging-guided photothermal diagnostic and therapeutic integrated formulations.

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