Carbon-based nano-composite targeting agent as well as preparation method and application thereof

Abstract

The invention provides a carbon-based nano composite targeting agent as well as a preparation method and application thereof, and relates to the technical field of nano medicine. The carbon-based nano composite targeting agent provided by the invention comprises a tumor cell nucleus targeting fluorescent nanoprobe based on graphene and methylene blue, and the tumor cell nucleus targeting fluorescent nanoprobe based on graphene and methylene blue are assembled together through intermolecular electrostatic interaction. The advantages of the graphene-based tumor cell nucleus targeting fluorescent nanoprobe and methylene blue are combined to form a novel nano tumor targeting agent, and the targeting agent can target tumor cell nucleuses, has obvious boundary distinguishing on cancer focus tissues and normal tissues, has fluorescence specific enrichment on the cancer tissues, and can be used for preparing tumor cells. The effect of guiding precise excision of the operation is achieved in solid malignant tumor treatment, the visual effect is achieved, and wide application prospects are achieved in the operation treatment of the solid malignant tumors such as breast cancer, lung cancer, gastric cancer, liver cancer, intestinal cancer and cervical cancer.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine technology, and in particular to a carbon-based nanocomposite targeting agent, its preparation method, and its application. Background Technology

[0002] Malignant tumors have become a major challenge in the global health field. Breast cancer is a common malignant tumor among women. Currently, breast cancer treatment includes surgery, chemotherapy, radiotherapy, targeted therapy, and endocrine therapy. Surgical treatment for breast cancer includes radical mastectomy, modified radical mastectomy, extended radical mastectomy, and breast-conserving surgery, with the surgical scope decreasing from large to small. The transformation of the surgical treatment philosophy for breast cancer is "from treatment with maximum tolerable trauma to precise treatment with minimal trauma." With the progress of the times and the updating of concepts, more and more patients hope to completely remove the lesion, preserve the breast, and even put forward the requirement of "scarless" treatment, that is, to remove the lesion to the maximum extent and preserve the normal tissue to the maximum extent to achieve precise surgical resection.

[0003] Under the concept of precision medicine, breast-conserving surgery is increasingly used in breast cancer surgery. The key to the success of breast-conserving surgery lies primarily in achieving a negative surgical margin. In clinical practice, to obtain a negative margin, the resection area is sometimes extended beyond the area marked with bleaching ink, resulting in the removal of excessive normal breast tissue. Modified radical mastectomy for breast cancer includes total mastectomy and ipsilateral axillary lymph node dissection (ALND), regardless of whether axillary lymph node metastasis is present.

[0004] Early-stage breast cancer often does not metastasize to lymph nodes. Lymph node metastasis first occurs in the sentinel lymph nodes, which are the first lymph nodes to show signs of metastasis. Clinically, whether early-stage breast cancer has metastasized to lymph nodes is determined by sentinel lymph node biopsy (SLNB). SLNB involves injecting dyes such as methylene blue, radionuclides, or indocyanine green into the areola or cancerous lesion during the procedure to locate the sentinel lymph nodes. SLNB represents another milestone in breast surgery, as its clinical application allows patients with negative axillary lymph nodes to avoid axillary lymph node dissection (ALND). SLNB not only provides accurate axillary lymph node status for clinical staging but also significantly reduces the incidence of lymphedema after ALND, effectively improving patients' postoperative quality of life. However, the aforementioned dyes, such as methylene blue, lack precise targeting of the tumor, cannot accurately delineate tumor boundaries, and lack the ability to differentiate between local axillary lymph node macrometastases in breast cancer. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a carbon-based nanocomposite targeting agent, its preparation method, and its application. The carbon-based nanocomposite targeting agent provided by this invention can precisely target the tumor cell nucleus and guide precise surgical resection in the treatment of solid malignant tumors.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a carbon-based nanocomposite targeting agent, comprising a graphene-based tumor cell nucleus-targeting fluorescent nanoprobe and methylene blue, wherein the graphene-based tumor cell nucleus-targeting fluorescent nanoprobe and methylene blue are assembled together by intermolecular electrostatic interactions; the graphene-based tumor cell nucleus-targeting fluorescent nanoprobe is a graphene-like single-crystal structure nanoparticle functionalized with sulfonic acid and hydroxyl groups.

[0007] Preferably, the carbon-based nanocomposite targeting agent has a size of 8-15 nm.

[0008] Preferably, the carbon-based nanocomposite targeting agent carries a negative charge of -5 to -10 mV.

[0009] This invention provides a method for preparing the carbon-based nanocomposite targeting agent described in the above technical solution, comprising the following steps: The graphene-based tumor cell nucleus-targeting fluorescent nanoprobe was self-assembled by mixing it with methylene blue liquid phase to obtain the carbon-based nanocomposite targeting agent.

[0010] Preferably, the ratio of the total mass of the graphene-based tumor cell nuclear-targeting fluorescent nanoprobe and methylene blue to the mass of methylene blue is (15~25):1.

[0011] Preferably, the ratio of the total mass of the graphene-based tumor cell nuclear-targeting fluorescent nanoprobe and methylene blue to the mass of methylene blue is 20:1.

[0012] Preferably, the self-assembly time is 2-4 hours, and the self-assembly is carried out under stirring conditions, with the stirring speed being 600-1000 rpm.

[0013] Preferably, after self-assembly, the resulting reaction solution is further subjected to filtration, dialysis, and freeze-drying in sequence; the filtration is performed using a 0.22 μm microporous membrane, and the dialysis bag used has a molecular weight cutoff of 5000 KD.

[0014] This invention provides the application of the carbon-based nanocomposite targeting agent described in the above technical solutions or the carbon-based nanocomposite targeting agent prepared by the above technical solutions in the preparation of precise tumor targeting agents.

[0015] Preferably, the tumor includes breast cancer, lung cancer, stomach cancer, liver cancer, intestinal cancer, or cervical cancer.

[0016] This invention provides a carbon-based nanocomposite targeting agent, comprising a graphene-based fluorescent nanoprobe for targeting tumor cell nuclei and methylene blue, wherein the graphene-based fluorescent nanoprobe for targeting tumor cell nuclei and methylene blue are assembled together via intermolecular electrostatic interactions. Methylene blue is clinically used for tracing and imaging in axillary sentinel lymph node biopsies for breast cancer, guiding surgical resection. However, methylene blue does not target tumor cell nuclei and cannot distinguish between normal and tumor cells. The graphene-based fluorescent nanoprobe for targeting tumor cell nuclei described in this invention has the characteristic of targeted tumor cell targeting, with the target point at the cell nucleus. This invention combines the graphene-based fluorescent nanoprobe for targeting tumor cell nuclei with methylene blue, achieving both targeting of the tumor cell nucleus and clear differentiation between cancerous and normal tissue, and fluorescence in cancerous tissue. Specific enrichment allows for precise surgical resection in the treatment of solid malignant tumors. Furthermore, methylene blue can overcome the limitation of graphene-based tumor cell nuclear-targeting fluorescent nanoprobes in providing visual guidance for surgery, achieving the same effect as carbon-based nanocomposite targeting agents. Moreover, the excitation wavelength of the carbon-based nanocomposite targeting agent is 650-680 nm, and the emission wavelength is an infrared wavelength of 650-770 nm, making it suitable for current clinical laparoscopic 4K guided surgery. Its applicability and scalability are better, facilitating the discovery of potential occult lesions. In addition, the carbon-based nanocomposite targeting agent can improve the stability of methylene blue and reduce its cytotoxicity. Attached Figure Description

[0017] Figure 1 This is a schematic diagram illustrating the synthesis process of the carbon-based nanocomposite targeting agent (GM) in the examples; Figure 2 Results of nuclear targeting tests on tumor cells (4T1 cells) using carbon-based nanoparticles (GM) and methylene blue (MB); Figure 3 The effect of carbon-based nanocomposite targeting agent (GM) and H&E on distinguishing the boundary between breast cancer lesion tissue (T) and normal tissue (N) was studied. Figure 3 In the middle section, a represents the boundary differentiation effect of GM and H&E in breast cancer lesions and normal tissues, and b represents the boundary differentiation effect of GM and H&E in axillary lymph nodes of breast cancer metastases. Figure 4 The results show the targeting properties of methylene blue (MB) on breast cancer tissue (T) and normal breast tissue (N). Figure 5 This is a TEM image of a carbon-based nanocomposite targeting agent (GM). Figure 5 The right side of the image is a magnified view of a portion of the left side. Figure 6The images show the UV and fluorescence spectra of carbon-based nanocomposite targeting agents (GM), methylene blue (MB), and graphene-based tumor cell nucleus-targeting fluorescent nanoprobes (GTTN). Figure 6 In the image, a is the ultraviolet spectrum and b is the fluorescence spectrum; Figure 7 The images show the XRD pattern and infrared spectrum of the carbon-based nanocomposite targeting agent (GM). Figure 7 In the image, a is the XRD pattern and b is the infrared spectrum; Figure 8 Zeta potential measurements of carbon-based nanocomposite targeting agent (GM), methylene blue (MB), and graphene-based tumor cell nucleus-targeting fluorescent nanoprobe (GTTN); Figure 9 The results show the fluorescence stability of the carbon-based nanocomposite targeting agent (GM). Figure 10 For the determination of MB content in GM, Figure 10 In Figure a, MB is the standard curve, and GM is the UV absorbance of GM obtained under different synthesis ratios (the ratio of the total mass of GTTN and methylene blue to the mass of methylene blue). Detailed Implementation

[0018] This invention provides a carbon-based nanocomposite targeting agent, comprising a graphene-based tumor cell nucleus-targeting fluorescent nanoprobe (GTTN) and methylene blue (MB), wherein the graphene-based tumor cell nucleus-targeting fluorescent nanoprobe and methylene blue are assembled together by intermolecular electrostatic interactions; the graphene-based tumor cell nucleus-targeting fluorescent nanoprobe is a graphene-like single-crystal structure nanoparticle functionalized with sulfonic acid and hydroxyl groups.

[0019] In this invention, the carbon-based nanocomposite targeting agent is preferably 8~15nm in size, is a spherical crystal, carries a negative charge of -5~-10mV (the negative charge makes the carbon-based nanocomposite targeting agent stable), and has blue-green fluorescence (ultraviolet excitation) and red fluorescence (infrared excitation), and its fluorescence properties are stable.

[0020] Methylene blue (MB) is an organophosphate antidote and other rescue agent. MB has a relative molecular mass of 374, is a water-soluble small molecule compound, and its absorption spectrum is 550-700 nm, with a maximum absorption wavelength of 665 nm, belonging to the red light band. MB is the most widely used photosensitizer among phenothiazines and is used as a sentinel lymph node biopsy staining agent in tumor surgery. MB has cationic properties and possesses detoxifying, analgesic, antitumor, antimicrobial, and anti-septic shock effects. Clinically, it is often used for sentinel lymph node biopsy in the axilla of breast cancer for tracing and visualization, and to guide surgical biopsy resection. However, MB does not target tumor cell nuclei and cannot distinguish between normal cells and tumor cells.

[0021] In this invention, the graphene-based tumor cell nucleus-targeting fluorescent nanoprobe is a graphene-like single-crystal structure nanoparticle functionalized with sulfonic acid and hydroxyl groups. The graphene-based tumor cell nucleus-targeting fluorescent nanoprobe (GTTN) was prepared according to the following literature: Wang, Y. Letal. A Highly Efficient Tumor-Targeting Nanoprobe with a Novel Cell Membrane Permeability Mechanism, AdvMater, which will be described in detail below. In this invention, the graphene-based tumor cell nucleus-targeting fluorescent nanoprobe (GTTN) is a carbon-based nanomaterial. Carbon-based nanomaterials are defined as materials with at least one dimension between 1 nanometer and 100 nanometers in three-dimensional space, or materials composed of these nanoscale units. These materials exhibit properties such as small size effects, quantum size effects, and surface effects, thus demonstrating unique functionality in multiple fields such as optics, electricity, thermal, magnetics, absorption, reflection, adsorption, catalysis, and bioactivity. In this invention, the GTTN is a single-crystal graphene-based fluorescent probe, which is functionalized with sulfonic acid and hydroxyl groups on its periphery. It exhibits ultraviolet fluorescence and carries a negative charge of -15 to -30 mV. It is approximately 5 nm in size, stable, and non-toxic. It has the characteristic of directional targeting of tumor cells, with the target point located in the nucleus of the tumor cells. Its disadvantages are that it does not have the function of visually guiding surgery and does not have infrared fluorescence, thus limiting its application.

[0022] This invention combines the advantages of graphene-based tumor cell nucleus-targeting fluorescent nanoprobes and methylene blue to form a novel nanotumor targeting agent. This targeting agent exhibits infrared fluorescence and targets tumor cell nuclei, enabling precise targeting of tumor lesions and metastatic lymph nodes. It can accurately distinguish between tumor lesions, normal tissue, metastatic lymph nodes, and non-metastatic lymph nodes. In the treatment of solid malignant tumors, it can guide precise surgical resection and has a visual effect, showing broad clinical application prospects.

[0023] This invention provides a method for preparing the carbon-based nanocomposite targeting agent described in the above technical solution, comprising the following steps: The graphene-based tumor cell nucleus-targeting fluorescent nanoprobe was self-assembled by mixing it with methylene blue liquid phase to obtain the carbon-based nanocomposite targeting agent.

[0024] Unless otherwise specified, all raw materials involved in this invention are commercially available products well known in the art.

[0025] In this invention, the preparation method of the graphene-based tumor cell nuclear-targeting fluorescent nanoprobe (GTTN) is as follows: Weigh 1 g of pyrene (TCI, 98% purity), add 50 mL of concentrated nitric acid (HNO3, 65-68 wt%), and reflux under magnetic stirring in an 80°C oil bath for 24 h. After the reaction stops, cool to room temperature, add 150 mL of deionized water to dilute the resulting system, and filter through a 0.22 μm microporous membrane to remove excess acid. Dissolve the resulting yellow solid in 100 mL of Na2SO3 deionized water (0.5 mol / L), stir thoroughly for 0.5 h, and transfer the resulting solution to a 150 mL high-temperature and high-pressure reactor. Heat to 130°C and hydrothermally react for 12 h. After the reaction was completed, the solution was cooled to room temperature and then transferred to a polytetrafluoroethylene reactor. The solution was then reacted in a vacuum drying oven at 200°C for 12 hours. After the reaction was completed and cooled to room temperature, the solution was filtered through a 0.22 μm microporous membrane to remove insoluble byproducts. The resulting filtrate contained GTTN and was the GTTN stock solution, which is the solution of the graphene-based tumor cell nuclear targeting fluorescent nanoprobe. It showed yellow fluorescence under ultraviolet irradiation. After further dilution with water to 300 mg / L, it showed blue-green fluorescence under ultraviolet irradiation.

[0026] In this invention, the preferred ratio of the total mass of the graphene-based tumor cell nuclear targeting fluorescent nanoprobe and methylene blue to the mass of methylene blue is (15~25):1, more preferably 20:1. The carbon-based nanocomposite targeting agent obtained under this ratio has good stability and dispersibility, high content, and high ultraviolet absorbance.

[0027] In this embodiment of the invention, the liquid phase mixing involves mixing the graphene-based tumor cell nucleus-targeting fluorescent nanoprobe solution with a methylene blue solution. The concentration of the graphene-based tumor cell nucleus-targeting fluorescent nanoprobe solution is 80 mg / mL, and the concentration of the methylene blue solution is 10 mg / mL.

[0028] In this invention, the self-assembly is performed at room temperature, and the self-assembly time is preferably 2-4 hours, which can be 2, 3, or 4 hours. The self-assembly is preferably performed under stirring conditions, and the stirring speed is preferably 600-1000 rpm. In this invention, the self-assembly involves the graphene-based tumor cell nuclear-targeting fluorescent nanoprobe and methylene blue assembling together through intermolecular electrostatic interactions.

[0029] In this invention, after self-assembly, the process preferably includes sequentially filtering, dialysis, and freeze-drying the resulting reaction solution; the filtration preferably uses a 0.22 μm microporous membrane, and the dialysis bag used has a molecular weight cutoff of 5000 KD. In this invention, the specific operation of the dialysis is preferably as follows: the filtered liquid is placed in a 5000 KD dialysis bag, then placed in pure water, and dialysis is performed under stirring (400-600 rpm), with the pure water being replaced promptly when it turns blue during dialysis. This invention removes unbound free substances and large molecular impurities or agglomerates through filtration and dialysis. In this invention, the liquid after dialysis is a solution of the carbon-based nanocomposite targeting agent. This invention does not have special requirements for the freeze-drying conditions; after freeze-drying, the carbon-based nanocomposite targeting agent powder is obtained.

[0030] This invention provides the application of the carbon-based nanocomposite targeting agent described in the above technical solutions or the carbon-based nanocomposite targeting agent prepared by the above technical solutions in the preparation of precise tumor targeting agents.

[0031] In this invention, the tumor preferably includes breast cancer, lung cancer, gastric cancer, liver cancer, colorectal cancer, or cervical cancer. Taking breast cancer as an example, the carbon-based nanocomposite targeting agent provided by this invention is targeted to breast cancer tissue and axillary metastatic lymph node tissue. It exhibits strong and weak fluorescence specific enrichment in breast cancer lesions and normal tissue (strong fluorescence specific enrichment in breast cancer lesions), with clear boundaries. It also exhibits strong and weak fluorescence specific enrichment in axillary lymph nodes metastasized to breast cancer (strong fluorescence specific enrichment for axillary lymph node cells metastasized to breast cancer), with clear boundaries. In breast cancer surgery (such as breast-conserving surgery), the carbon-based nanocomposite targeting agent provided by this invention can achieve the effect of accurately identifying lesions and metastatic lymph nodes. The carbon-based nanocomposite targeting agent provided by this invention has broad application prospects in the surgical treatment of solid malignant tumors such as breast cancer, lung cancer, gastric cancer, liver cancer, colorectal cancer, and cervical cancer.

[0032] To further illustrate the present invention, the carbon-based nanocomposite targeting agents, their preparation methods, and applications provided by the present invention are described in detail below with reference to examples, but these should not be construed as limiting the scope of protection of the present invention.

[0033] Example 1 (1) The preparation method of graphene-based tumor cell nuclear-targeting fluorescent nanoprobe (GTTN) is as follows: A bottom-up hydrothermal molecular fusion method was used for synthesis: 1 g of pyrene (TCI, 98% purity) was weighed and added to 50 mL of concentrated nitric acid (65-68 wt%). The mixture was refluxed under magnetic stirring at 80 °C in an oil bath for 24 h. After the reaction was stopped, the mixture was cooled to room temperature, and 150 mL of deionized water was added to dilute the resulting system. Excess acid was removed by filtration through a 0.22 μm microporous membrane. The resulting yellow solid was dissolved in 100 mL of 0.5 mol / L deionized Na₂SO₃ solution and stirred thoroughly for 0.5 h. The resulting solution was transferred to a 150 mL ceramic autoclave and heated to 130 °C for hydrothermal reaction for 12 h. After the reaction was completed, the solution was cooled to room temperature and then transferred to a polytetrafluoroethylene (PTFE) reactor and reacted in a vacuum drying oven at 200 °C for 12 h. After the reaction was completed and cooled to room temperature, the solution was filtered through a 0.22 μm microporous membrane to remove insoluble byproducts. The resulting solution was the graphene-based tumor cell nuclear-targeting fluorescent nanoprobe (GTTN) stock solution. 1 mL of the GTTN stock solution was dried in a 60 °C oven and weighed to determine its concentration. The GTTN stock solution concentration was 80 mg / mL. The GTTN stock solution was stored at 4 °C for later use.

[0034] Methylene blue (MB) is a commonly used clinical drug, with a strength of 10 mg / mL. It is a clear, deep blue liquid (called methylene blue (MB) solution).

[0035] (2) Preparation of carbon-based nanocomposite targeting agents (e.g.) Figure 1 (as shown) GTTN stock solution (concentration 80 mg / mL) and methylene blue solution (10 mg / mL) were mixed, with 95 mg GTTN and 5 mg methylene blue (i.e., the total mass ratio of GTTN and methylene blue to methylene blue was 20:1). The mixture was stirred at 1000 rpm for 2 hours using a magnetic stirrer, and then filtered through a 0.22 μm microporous membrane to remove excess substances. The filtered liquid was placed in a 5000 kDa dialysis bag and then added to 1000 mL of pure water. The mixture was stirred at 400 rpm for 12 hours using a magnetic stirrer, replacing the pure water as needed to prevent it from turning blue. The dialysis liquid was the carbon-based nanocomposite targeting agent (denoted as GM) stock solution. This solution could be freeze-dried, and the resulting powder stored at 4°C.

[0036] The nuclear targeting properties of carbon-based nanocomposite targeting agents (GM) and methylene blue (MB) on tumor cells (4T1 cells) were tested. The test was divided into GM group, Triton+MB group, and Triton+GM group. Specifically, 2×10 5Four T1 cells were seeded in confocal culture dishes and incubated for 24 hours. 50 μL of GM stock solution was added to the GM group, 10 μL of MB solution and 10 μL of Triton were added to the Triton+MB group, and 50 μL of GM stock solution and 10 μL of Triton were added to the Triton+GM group. After incubation for 3 minutes, images were taken under confocal imaging. Figure 2 Results of nuclear targeting assays for carbon-based nanocomposite targeting agents (GM) and methylene blue (MB) on tumor cells (4T1 cells). Figure 2 It can be seen that the carbon-based nanocomposite targeting agent (GM) has the ability to target tumor cell nuclei. In the 4T1 cell experiment, GM targeted the cell nucleus and was fluorescently enriched in the cell nucleus. In 4T1 cells with Triton cell membrane perforation, GM targeted the cell nucleus; conversely, without Triton, GM did not target the cell nucleus; in 4T1 cells with Triton cell membrane perforation, MB did not target the cell nucleus.

[0037] The targeting ability of carbon-based nanocomposite targeting agents (GM) was tested. The test method was as follows: breast cancer tissue, axillary metastatic lymph nodes and axillary non-metastatic lymph nodes were taken from the body and soaked in GM stock solution for 0.5 h. Before frozen sectioning, the tissue was rinsed 3 times with PBS and then frozen sectioned. After sectioning, the tissue was imaged under confocal microscopy. Figure 3 The study compared the effectiveness of carbon-based nanocomposite targeting agents (GM) and conventional paraffin-embedded pathological sections (H&E) in distinguishing the boundary between breast cancer lesions (T) and normal tissue (N). Figure 3 In Figure a, GM and H&E show their ability to differentiate between breast cancer lesions and normal tissue; in Figure b, GM and H&E show their ability to differentiate between axillary lymph nodes in breast cancer metastases. Figure 3 In this diagram, T represents cancerous tissue, and N represents normal tissue. Figure 3 As can be seen from Figure a, GM exhibits specific enrichment of strong and weak fluorescence in breast cancer lesions and normal tissues, with clear boundaries, similar to H&E; Figure 3 As can be seen from Figure b, GM showed strong and weak fluorescence specific enrichment and clear boundary distinction in axillary lymph nodes (L+) of breast cancer metastasis, which was the same as that of H&E. Figure 3 The results showed that the carbon-based nanocomposite targeting agent (GM) was effective in targeting breast cancer tissue and axillary lymph node metastasis. GM clearly distinguished the boundary between cancerous tissue (T) and normal tissue (N) and showed fluorescence-specific enrichment in cancerous tissue.

[0038] The targeting properties of methylene blue were tested by means of: taking breast cancer tissue from an in vitro sample, immersing the breast cancer tissue in MB solution for 0.5 h, and then imaging it under confocal imaging. Figure 4The results show the targeting properties of methylene blue (MB) on breast cancer tissue (T) and normal breast tissue (N). It can be seen that methylene blue (MB) has no targeting property for either breast cancer tissue or normal breast tissue, cannot distinguish the boundary between cancerous tissue (T) and normal tissue (N), and does not show specific fluorescence enrichment in cancerous tissue.

[0039] The physicochemical properties of the carbon-based nanocomposite targeting agent (GM) solution prepared in Example 1 are as follows: It is a spherical crystal with a size of 8-15 nm; it is a clear blue-green liquid with a negative charge of approximately -8 mV; its absorption spectrum is 650-680 nm, its emission wavelength is 650-770 nm, and it exhibits red fluorescence with stable fluorescence properties. The following are the characterizations of the physicochemical properties of GM.

[0040] Figure 5 This is a TEM image of a carbon-based nanocomposite targeting agent (GM) (GM stock solution was dropped onto a copper grid, dried, and then imaged using a transmission electron microscope). Figure 5 The image on the right is a magnified view of a portion of the left side. TEM shows that GM particles are spherical and granular, with a size of 8-15 nm.

[0041] Figure 6 The images show the UV and fluorescence spectra of carbon-based nanocomposite targeting agents (GM), methylene blue (MB), and graphene-based tumor cell nucleus-targeting fluorescent nanoprobes (GTTN). Figure 6 In the image, a is the ultraviolet spectrum, and b is the fluorescence spectrum. (From...) Figure 6 It can be seen that the ultraviolet absorption peaks are 370 nm for GTTN, 370 nm and 660 nm for GM, and 660 nm for MB; the fluorescence emission wavelengths are 472 nm for GTTN, 675 nm for MB, and 472 nm and 675 nm for GM. The results show that the GM composite material has been synthesized.

[0042] Figure 7 The images show the XRD pattern and infrared spectrum of the carbon-based nanocomposite targeting agent (GM). Figure 7 Image a shows the XRD pattern, and image b shows the infrared spectrum. XRD shows the θ peak at 25.94 on the GM surface; infrared shows the presence of OH and SO3 functional groups on the GTTN surface, as well as the 1600 cm⁻¹ peak of MB. -1 CN stretching vibration characteristic peak.

[0043] Figure 8 This image shows the zeta potential measurements of a carbon-based nanocomposite targeting agent (GM), methylene blue (MB), and a graphene-based tumor cell nucleus-targeting fluorescent nanoprobe (GTTN). GM carries a negative charge of approximately -8 mV and is assembled from GTTN and MB via charge transfer.

[0044] Figure 9 The results show the fluorescence stability of the carbon-based nanocomposite targeting agent (GM). Figure 9 The two smaller images (day 1 and day 60) show fluorescence patterns. The left image is a fluorescence spectrum at an excitation wavelength of 405 nm, while the right image is a visual image. It can be seen that the fluorescence properties of GM are stable on day 1 and day 60.

[0045] Example 2 In Example 1 (2), the ratio of the total mass of GTTN and methylene blue to the mass of methylene blue was adjusted from 20:1 to 4:1, 9:1, 12:1, 15:1, 25:1, 30:1, and 50:1, respectively, while the rest remained the same as in Example 1.

[0046] Figure 10 For the determination of MB content in GM, Figure 10 In Figure a, the standard curve of MB is shown, and in Figure b, the UV absorbance of GM obtained under different synthesis ratios (the ratio of the total mass of GTTN and methylene blue to the mass of methylene blue). Based on... Figure 10 The standard curve of a was used to determine the MB content in GM. Figure 10 As can be seen from Figure b, the synthesis ratio of 20:1 for GM is the best, and the GM obtained has the highest UV absorbance. According to the curve in Figure a, the content of MB in the prepared GM stock solution is 0.678 mg / mL, and the concentration of GM stock solution obtained at the 20:1 synthesis ratio is the highest, which is 3.67 mg / mL.

[0047] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A carbon-based nanocomposite targeting agent, characterized in that, The invention includes a graphene-based tumor cell nucleus-targeting fluorescent nanoprobe and methylene blue, wherein the graphene-based tumor cell nucleus-targeting fluorescent nanoprobe and methylene blue are assembled together by intermolecular electrostatic interactions; the graphene-based tumor cell nucleus-targeting fluorescent nanoprobe is a graphene-like single-crystal structure nanoparticle functionalized with sulfonic acid and hydroxyl groups.

2. The carbon-based nanocomposite targeting agent according to claim 1, characterized in that, The carbon-based nanocomposite targeting agent has a size of 8~15 nm.

3. The carbon-based nanocomposite targeting agent according to claim 1, characterized in that, The carbon-based nanocomposite targeting agent carries a negative charge of -5 to -10 mV.

4. The method for preparing the carbon-based nanocomposite targeting agent according to any one of claims 1 to 3, characterized in that, Includes the following steps: The graphene-based tumor cell nucleus-targeting fluorescent nanoprobe was self-assembled by mixing it with methylene blue liquid phase to obtain the carbon-based nanocomposite targeting agent.

5. The preparation method according to claim 4, characterized in that, The ratio of the total mass of the graphene-based tumor cell nuclear-targeting fluorescent nanoprobe and methylene blue to the mass of methylene blue is (15~25):

1.

6. The preparation method according to claim 5, characterized in that, The ratio of the total mass of the graphene-based tumor cell nuclear-targeting fluorescent nanoprobe and methylene blue to the mass of methylene blue is 20:

1.

7. The preparation method according to claim 4, characterized in that, The self-assembly time is 2-4 hours, and the self-assembly is carried out under stirring conditions at a stirring speed of 600-1000 rpm.

8. The preparation method according to claim 4, characterized in that, After self-assembly, the process further includes sequentially filtering, dialysis, and freeze-drying the resulting reaction solution; the filtration is performed using a 0.22 μm microporous membrane, and the dialysis bag used has a molecular weight cutoff of 5000 KD.

9. The application of the carbon-based nanocomposite targeting agent according to any one of claims 1 to 3 or the carbon-based nanocomposite targeting agent prepared by the preparation method according to any one of claims 4 to 8 in the preparation of tumor precision targeting agents.

10. The application according to claim 9, characterized in that, The tumors include breast cancer, lung cancer, stomach cancer, liver cancer, colorectal cancer, or cervical cancer.

Images