Two-photon boron-containing carbon dot materials, their preparation methods and applications

Abstract

This invention discloses a two-photon boron-containing carbon dot material, its preparation method, and its application. The method includes the following steps: S1, dissolving 2,5-dihydroxyterephthalohydrazide and boric acid in ethanol, and sonicating to obtain a mixture; S2, transferring the mixture to a reaction vessel and heating it for reaction; S3, after the reaction is complete, filtering the product through a filter membrane; S4, dialyzing the filtrate, and freeze-drying the dialysate to obtain the two-photon boron-containing carbon dot material. This invention successfully achieves the synergistic integration of high boron content and two-photon long-wavelength excitation fluorescence function in a single carbon dot system, providing a novel therapeutic nanoprobe with both real-time fluorescence tracing of deep tissues and precise boron delivery functions for boron neutron capture therapy, showing significant application prospects in the precise diagnosis and treatment of malignant tumors.

Description

Technical Field

[0001] This invention relates to the field of biomedical nanomaterials technology, and particularly to a novel boron-containing carbon dot nanomaterial. The core of this invention lies in developing a novel boron-containing carbon dot nanomaterial that combines two-photon fluorescence emission capability with high boron content, enabling real-time fluorescence tracing of deep tissues and precise boron dose monitoring during BNCT treatment. The technical field of this invention encompasses the integrated application of boron-containing carbon dot design and synthesis, fluorescence imaging-guided tumor diagnosis, and boron neutron capture therapy. Background Technology

[0002] Cancer is one of the leading causes of death worldwide, and its treatment remains a significant challenge for all countries. To date, numerous cancer treatments have been developed, including chemotherapy, radiotherapy, photodynamic therapy, photothermal therapy, and immunotherapy. Among these radiotherapy methods, boron neutron capture therapy (BNCT) is a promising approach. It is based on the irradiation of non-radioactive materials with low-energy thermal neutrons. 10 The nuclear reaction that occurs when boron generates high-linear-energy-transfer particles can selectively kill boron-containing tumor cells without harming normal cells. Simultaneously, during BNCT treatment, real-time tracking of the distribution and metabolism of boron drugs in the body, and precise identification of the time point of maximum boron accumulation at the tumor site to match neutron irradiation, is crucial for improving efficacy and reducing side effects. Fluorescence imaging, with its advantages of high sensitivity, low cost, and non-invasiveness, has become an effective means of integrating diagnosis and treatment with BNCT. Therefore, developing novel boron-carrying reagents that combine high boron content with excellent fluorescence imaging capabilities is an important direction for promoting the precision development of BNCT.

[0003] Boron-containing carbon dots (BCDs), as an emerging carbon-based fluorescent nanomaterial, have become a breakthrough in solving the dual challenges of boron delivery and real-time tracking in brain-brain tumor catalysis (BNCT) due to their ease of synthesis, excellent fluorescence properties, good water solubility, and low biotoxicity. Studies applying BCDs to BNCT have been reported. For example, Li et al. synthesized BCDs using glucose and BPA as precursors via a one-step hydrothermal method and further encapsulated them with macrophage-derived exosomes, developing a boron delivery system capable of crossing the blood-brain barrier. This system demonstrated superior BNCT efficacy compared to the clinical drug BPA in an orthotopic mouse model of glioma (Adv. Funct. Mater. 2021, 31, 2100969). In his doctoral dissertation, "Research on Boron-Containing Carbon Dots for Boron Neutron Capture Therapy" (Northeast Normal University, 2025), Zhong Tianyuan systematically explored the strategy of synthesizing BCDs using boric acid as the boron source and various organic molecules as carbon sources. By selecting different carbon precursors, he achieved gradual regulation of the boron content, fluorescence wavelength, and tumor targeting of BCDs. Some of the BCDs achieved significant in vivo BNCT tumor-suppressing effects in mouse models such as melanoma.

[0004] Currently, boron-containing carbon dots (BCDs) used in BNCT (Brain-Neuro-Tissue Therapy) are mainly prepared using a one-step hydrothermal or solvothermal method. The preparation strategy typically uses boron-containing small molecules (such as boric acid) as the boron source and organic small molecules (such as citric acid, p-phenylenediamine, folic acid, etc.) as the carbon source for doping synthesis. This synthesis method is relatively simple, with controllable experimental conditions, and allows adjustment of the final carbon dot's fluorescence emission wavelength, surface functional groups, and boron content by varying the types and proportions of precursors. Based on this preparation strategy, BCDs have made some progress in BNCT therapies. Reported BCDs have been able to achieve fluorescent tracking of tumors and deliver boron to tumor cells via endocytosis, achieving a certain degree of BNCT therapeutic effect. However, in the currently reported BCD systems, the fluorescence emission is mostly in single-photon excitation mode, with emission wavelengths mainly concentrated in the blue to green light range. Near-infrared emission capability is insufficient, resulting in limited penetration depth in deep tissue imaging and significant background autofluorescence interference, making it difficult to meet the requirements for high signal-to-noise ratio real-time imaging of deep tumors in BNCT treatment.

[0005] While existing techniques for preparing boron-containing carbon dots (BCDs) using hydrothermal / solvothermal synthesis strategies have achieved preliminary results, the resulting BCDs mostly exhibit single-photon fluorescence properties, with excitation and emission wavelengths largely located in the ultraviolet-visible region. This leads to strong absorption and scattering by biological tissues, resulting in insufficient penetration. Although some reported cases of CDs being encapsulated in exosomes to improve tumor retention time, their intrinsic fluorescence emission wavelengths are still insufficient to achieve high-quality in vivo imaging of deep tissues. Furthermore, existing BCD systems do not fully utilize the advantages of two-photon fluorescence imaging. Two-photon excitation uses near-infrared light as a light source, offering advantages such as deeper tissue penetration, lower optical damage, and lower background interference; however, there are currently no reports of applying two-photon BCDs to integrated BNCT diagnosis and treatment research. These technical limitations restrict the application potential of existing boron-containing carbon dot materials in the precision diagnosis and treatment of BNCT.

[0006] Therefore, it is now necessary to improve existing technologies to provide more reliable solutions. Summary of the Invention

[0007] This invention aims to address the technical problems of existing boron-containing carbon dots used in boron neutron capture therapy, such as short excitation wavelength, lack of long-wavelength excitation characteristics, insufficient penetration into deep tissues, and low signal-to-noise ratio in imaging. The invention develops a novel boron-containing carbon dot material that combines high boron content with two-photon long-wavelength excitation fluorescence function, providing a novel nano-therapeutic probe for real-time, high signal-to-noise ratio fluorescence tracing and precise boron dose monitoring of deep tumors during BNCT treatment.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In its first aspect, the present invention provides a method for preparing a two-photon boron-containing carbon dot material, comprising the following steps: S1. Dissolve 2,5-dihydroxyterephthalohydrazide and boric acid in ethanol, and sonicate to obtain a mixture; S2. Transfer the mixture to a reaction vessel and heat to react; S3. After the reaction is complete, the product is filtered through a filter membrane. S4. Dialyze the filtrate, freeze-dry the dialysate, and obtain a two-photon boron-containing carbon dot material.

[0009] Preferably, step S1 specifically involves dissolving 2,5-dihydroxyterephthalohydrazide and boric acid in ethanol at a molar ratio of (0.5-2):(3-12), and then dispersing the mixture using ultrasound to obtain a mixture.

[0010] Preferably, step S2 specifically involves transferring the mixture into a reaction vessel and heating it at 160-220°C for 3-12 hours.

[0011] Preferably, step S3 specifically involves: after the reaction is completed, naturally cooling to room temperature and filtering using a 0.2-0.3 μm filter membrane.

[0012] Preferably, step S4 specifically involves: dialyzing the filtrate in an ethanol-water solution using a dialysis bag with a capacity of 100-1000 Da for 12-48 hours, then freeze-drying the dialysate from the dialysis bag to obtain a two-photon boron-containing carbon dot material.

[0013] Preferably, the preparation method of the two-photon boron-containing carbon dot material includes the following steps: S1. Take 0.5-2 mmol of 2,5-dihydroxyterephthalohydrazide and 3-12 mmol of boric acid, dissolve them in 10-40 mL of ethanol, and sonicate for 5-30 minutes to obtain a mixture. S2. Transfer the mixture to a stainless steel high-pressure reactor lined with polytetrafluoroethylene and heat it at 160-220℃ for 3-12 hours. S3. Cool to room temperature and filter the product through a 0.2-0.3 μm filter membrane; S4. Dialyze the filtrate in an ethanol-water solution composed of ethanol and deionized water in a volume ratio of (0.5-2):(0.5-2) using a 100-1000 Da dialysis bag for 12-48 hours. Replace the ethanol-water solution every 4-8 hours. Take the dialysis solution in the dialysis bag and freeze-dry it for 12-48 hours to obtain the two-photon boron-containing carbon dot material.

[0014] Preferably, the preparation method of the two-photon boron-containing carbon dot material includes the following steps: S1. Take 1 mmol of 2,5-dihydroxyterephthalohydrazide and 6 mmol of boric acid, dissolve them in 20 mL of ethanol, and sonicate for 15 minutes to obtain a mixture. S2. Transfer the mixture to a 50 mL stainless steel high-pressure reactor lined with polytetrafluoroethylene and heat it at 200°C for 6 hours. S3. Cool to room temperature and filter the product through a 0.22 μm filter membrane; S4. Dialyze the filtrate in an ethanol-deionized water solution (ethanol and deionized water in a 1:1 volume ratio) using a 1000 Da dialysis bag for 24 hours, changing the ethanol-deionized water solution every 6 hours. Take the dialysate from the dialysis bag and freeze-dry it at -50 °C for 24 hours to obtain a two-photon boron-containing carbon dot material.

[0015] In a second aspect, the present invention provides a two-photon boron-containing carbon dot material, characterized in that it is prepared by the method described above.

[0016] A third aspect of the present invention provides the use of the two-photon boron-containing carbon dot material as described above in the preparation of boron neutron capture therapeutic drugs or formulations.

[0017] A fourth aspect of the present invention provides the use of the two-photon boron-containing carbon dot material as described above in the preparation of tumor fluorescence imaging tracers.

[0018] The beneficial effects of this invention are: Compared with existing technologies, the two-photon boron-containing carbon dot material prepared by this invention has at least the following significant advantages: 1. The two-photon boron-containing carbon dot material of this invention utilizes a specific precursor combination (2,5-dihydroxyterephthalohydrazide and boric acid) and is successfully prepared under optimized hydrothermal synthesis conditions (200℃, 6 hours) to produce a boron-containing carbon dot material that combines high boron content with two-photon long-wavelength excitation fluorescence characteristics. This material produces bright visible fluorescence with a maximum emission wavelength of 520 nm. More importantly, it possesses two independent excitation channels at 390 nm and 740 nm. The long-wavelength excitation peak at 740 nm is located in the near-infrared region, which can effectively avoid the strong absorption and scattering of short-wavelength light by biological tissues, significantly improve the penetration ability of excitation light in deep tissues, and greatly reduce the tissue autofluorescence background. This enables high signal-to-noise ratio imaging of deep tissues, overcoming the shortcomings of existing boron neutron capture therapy boron-containing carbon dots that are mostly limited to short-wavelength single-photon excitation, have insufficient tissue penetration, and poor imaging quality.

[0019] 2. In the two-photon boron-containing carbon dot material of the present invention, the boron content of 2.5 wt% can meet the basic load requirements of boron-carrying reagents for boron neutron capture therapy; and the preparation process of this material is simple, the conditions are mild and controllable, and it can be completed by two raw materials through a one-step hydrothermal reaction, which is suitable for large-scale production.

[0020] 3. This invention successfully integrates high boron content and two-photon long-wavelength excitation fluorescence function in a single carbon dot system, providing a novel diagnostic and therapeutic nanoprobe that combines real-time fluorescence tracing of deep tissues with precise boron delivery for boron neutron capture therapy. It has important application prospects in the precise diagnosis and treatment of malignant tumors. Attached Figure Description

[0021] Figure 1 Fourier transform infrared spectra of B-CDs prepared in Example 1; Figure 2 The B content determination chart of B-CDs prepared in Example 1; Figure 3 Excitation spectra of B-CDs prepared in Example 1; Figure 4 Emission spectra of B-CDs prepared in Example 1. Detailed Implementation

[0022] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0023] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. For examples where specific conditions are not specified, conventional conditions or conditions recommended by the manufacturer are followed. For reagents or instruments whose manufacturers are not specified, they are all commercially available products.

[0025] Example 1 A two-photon boron-containing carbon dot material is prepared by the following method: S1. Accurately weigh 1 mmol of 2,5-dihydroxyterephthalohydrazide and 6 mmol of boric acid, dissolve them in 20 mL of ethanol, and sonicate for 15 minutes to obtain a transparent mixture. S2. Transfer the mixture to a 50 mL stainless steel high-pressure reactor lined with polytetrafluoroethylene and heat it in an oven at 200°C for 6 hours. S3. Allow the product to cool naturally to room temperature, filter it through a 0.22 μm filter membrane, and collect the filtrate. S4. Dialyze the filtrate in an ethanol-deionized water solution (ethanol and deionized water in a 1:1 volume ratio) using a 1000 Da dialysis bag for 24 hours. Replace the ethanol-deionized water solution every 6 hours. Take the dialysate from the dialysis bag and freeze-dry it at -50 °C for 24 hours to obtain a brown, fluffy powder, which is a two-photon boron-containing carbon dot material, denoted as B-CDs.

[0026] Performance testing 1. Figure 1 Fourier transform infrared spectra of B-CDs show that the amide carbonyl group was successfully retained on the carbon dot surface, boron atoms were incorporated through the formation of COB bonds and contain a small amount of aromatic edges, the material surface is enriched with amino and hydroxyl groups, and has good hydrophilicity and potential for further functionalization.

[0027] 2. The B content of B-CDs was determined using ICP-OES, and the results are as follows: Figure 2 As shown, the boron content in these BCDs is 2.5 wt%. This provides a sufficient boron source for B-CDs in BNCT treatment research.

[0028] 3. Prepare a 0.1 mg / mL ethanol solution of B-CDs. Measure the three-dimensional fluorescence spectrum using a fluorescence spectrophotometer. The experimental results are as follows: Figure 3 and Figure 4 As shown, the maximum emission wavelength of B-CDs is located at 520 nm, and the fluorescence intensity measured at this emission wavelength is 8089 (au). B-CDs exhibit two independent excitation peaks at 390 nm and 740 nm, respectively. When excited at 390 nm, the fluorescence emission intensity at 520 nm is 3012; when excited at 740 nm, the fluorescence emission intensity at 520 nm is 182.8. This result indicates that these boron-containing carbon dots can be excited not only by conventional UV-Vis light but also by 740 nm near-infrared long-wavelength light, producing the same 520 nm fluorescence emission, exhibiting typical two-photon excitation / upconversion fluorescence characteristics.

[0029] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details.

Claims

1. A method for preparing a two-photon boron-containing carbon dot material, characterized in that, Includes the following steps: S1. Dissolve 2,5-dihydroxyterephthalohydrazide and boric acid in ethanol, and sonicate to obtain a mixture; S2. Transfer the mixture to a reaction vessel and heat to react; S3. After the reaction is complete, the product is filtered through a filter membrane. S4. Dialyze the filtrate, freeze-dry the dialysate, and obtain a two-photon boron-containing carbon dot material.

2. The method for preparing two-photon boron-containing carbon dot materials according to claim 1, characterized in that, Step S1 specifically involves dissolving 2,5-dihydroxyterephthalohydrazide and boric acid in ethanol at a molar ratio of (0.5-2):(3-12), dispersing them by ultrasonication to obtain a mixture.

3. The method for preparing two-photon boron-containing carbon dot materials according to claim 1, characterized in that, Step S2 specifically involves transferring the mixture to a reaction vessel and heating it at 160-220°C for 3-12 hours.

4. The method for preparing two-photon boron-containing carbon dot materials according to claim 1, characterized in that, Step S3 specifically involves: after the reaction is complete, allowing the mixture to cool naturally to room temperature and then filtering it using a 0.2-0.3 μm filter membrane.

5. The method for preparing two-photon boron-containing carbon dot materials according to claim 1, characterized in that, Step S4 specifically involves dialyzing the filtrate in an ethanol-water solution using a 100-1000 Da dialysis bag for 12-48 hours, then freeze-drying the dialysis solution from the dialysis bag to obtain a two-photon boron-containing carbon dot material.

6. The method for preparing two-photon boron-containing carbon dot materials according to claim 1, characterized in that, Includes the following steps: S1. Take 0.5-2 mmol of 2,5-dihydroxyterephthalohydrazide and 3-12 mmol of boric acid, dissolve them in 10-40 mL of ethanol, and sonicate for 5-30 minutes to obtain a mixture. S2. Transfer the mixture to a stainless steel high-pressure reactor lined with polytetrafluoroethylene and heat it at 160-220℃ for 3-12 hours. S3. Cool to room temperature and filter the product through a 0.2-0.3 μm filter membrane; S4. Dialyze the filtrate in an ethanol-water solution composed of ethanol and deionized water in a volume ratio of (0.5-2):(0.5-2) using a 100-1000 Da dialysis bag for 12-48 hours. Replace the ethanol-water solution every 4-8 hours. Take the dialysis solution in the dialysis bag and freeze-dry it for 12-48 hours to obtain the two-photon boron-containing carbon dot material.

7. The method for preparing two-photon boron-containing carbon dot materials according to claim 6, characterized in that, Includes the following steps: S1. Take 1 mmol of 2,5-dihydroxyterephthalohydrazide and 6 mmol of boric acid, dissolve them in 20 mL of ethanol, and sonicate for 15 minutes to obtain a mixture. S2. Transfer the mixture to a 50 mL stainless steel high-pressure reactor lined with polytetrafluoroethylene and heat it at 200°C for 6 hours. S3. Cool to room temperature and filter the product through a 0.22 μm filter membrane; S4. Dialyze the filtrate in an ethanol-deionized water solution (ethanol and deionized water in a 1:1 volume ratio) using a 1000 Da dialysis bag for 24 hours, changing the ethanol-deionized water solution every 6 hours. Take the dialysate from the dialysis bag and freeze-dry it at -50 °C for 24 hours to obtain a two-photon boron-containing carbon dot material.

8. A two-photon boron-containing carbon dot material, characterized in that, It is prepared by the method described in any one of claims 1-7.

9. The use of the two-photon boron-containing carbon dot material as described in claim 8 in the preparation of boron neutron capture therapeutic drugs or formulations.

10. The application of the two-photon boron-containing carbon dot material as described in claim 8 in the preparation of tumor fluorescence imaging tracers.

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