Preparation method of a biological near-infrared region one image agent targeting breast cancer cells

Abstract

This invention discloses a method for preparing biomass NIR-I-CQDs imaging agents targeting breast cancer cells. This method, adhering to the concept of green development, successfully prepares NIR-I-CQDs using biomass mango leaves as a precursor via a microwave-hydrothermal two-step method. The maximum excitation wavelength of the NIR-I-CQDs is 648 nm, the maximum emission wavelength is 728 nm, and the fluorescence quantum efficiency is 21.09%. Its structure, optical properties, stability, and biotoxicity were investigated using different techniques, demonstrating good optical properties, stability, and low biotoxicity. The prepared NIR-I-CQDs were incubated with four types of human cancer cells (MCF-7, HepG2, HeLa, and T24) and then imaged using confocal microscopy. Furthermore, the biomass NIR-I-CQDs were subcutaneously injected into tumor sites in mice modeling liver cancer, cervical cancer, bladder cancer, and breast cancer, followed by fluorescence imaging. The targeting performance of the biomass NIR-I-CQDs imaging agent against breast cancer cells was evaluated. The results showed that the mango leaf NIR-I-CQDs prepared by this invention exhibited significant targeting and affinity for breast cancer cells and breast cancer tumor sites in modeling mice. It can be used as a fluorescent imaging agent targeting breast cancer tumor cells in the field of breast cancer diagnosis and treatment.

Description

Technical Field

[0001] This invention relates to the technical field of a method for preparing biomass near-infrared one-region carbon quantum dot (NIR-I-CQDs) imaging agents that target breast cancer cells, and also to the technical field of preparing biomass NIR-I-CQDs imaging agents. Background Technology

[0002] Breast cancer is one of the most common malignant tumors among women worldwide, and its incidence continues to rise globally, seriously threatening women's physical and mental health and lives. Early diagnosis, that is, timely detection of breast cancer when it is still small, localized, and has not yet metastasized widely, is a crucial link in reducing breast cancer mortality, improving patient prognosis, and enhancing quality of life, and has irreplaceable significance. Early diagnosis of breast cancer typically involves methods such as imaging examinations, pathological confirmation, and professional physical examinations. Among these, traditional imaging diagnosis suffers from problems such as poor targeting, limited resolution, and insufficient tissue penetration depth, affecting the diagnosis of early breast cancer and missing the optimal treatment period. Near-infrared I (NIR-I, 700-900 nm) nanofluorescent probes have shown great application potential in the field of tumor in vivo imaging due to their outstanding advantages such as strong passive targeting, deep tissue penetration, low interference from biological autofluorescence, and high photosensitivity. Traditional semiconductor quantum dots (such as CdSe, PbS), although having good luminescent properties, contain heavy metals, are highly toxic, have poor biocompatibility, and their preparation process often involves toxic reagents. Carbon quantum dots (CQDs), as novel zero-dimensional carbon-based fluorescent nanomaterials, possess excellent optical properties, good biocompatibility, and ease of functionalization. However, current traditional CQDs primarily use chemical reagents as raw materials, resulting in poor environmental friendliness during preparation, and their emission wavelengths are mostly concentrated in the visible light region, making it difficult to meet the needs of deep tumor imaging. In contrast, preparing CQDs using agricultural waste such as leaves, fruit peels, and bark as precursors offers advantages such as wide availability of raw materials and low cost. This not only enables high-value conversion of agricultural and forestry waste but also reduces the environmental costs and resource consumption associated with nanomaterial preparation.

[0003] This invention utilizes agricultural waste mango leaves as raw material and employs a microwave-hydrothermal two-step method to prepare NIR-I-CQDs. The NIR-I-CQDs emit at a wavelength of 728 nm, exhibiting strong near-infrared fluorescence emission, excellent biocompatibility, and stability. Laser confocal microscopy (LSCM) imaging results after incubating the prepared NIR-I-CQDs with four types of human cancer cells—hepatocellular carcinoma (HepG2), cervical cancer (HeLa), bladder cancer (T24), and breast cancer cells (MCF-7)—showed that the NIR-I-CQDs emitted bright near-infrared red fluorescence in all four cancer cell types, with a high signal-to-noise ratio. Comparison of the fluorescence intensity within these four cell types revealed that the red fluorescence in MCF-7 cells was the brightest, significantly stronger than that in the other three cell types, indicating that the biomass NIR-I-CQDs have good targeting and affinity for breast cancer. Fluorescence imaging results of NIR-I-CQDs in model mice with liver cancer, cervical cancer, bladder cancer, and breast cancer also showed that NIR-I-CQDs significantly enhanced fluorescence in MCF-7 tumors, indicating a stronger surface targeting effect. This was reflected in the higher uptake and longer retention of NIR-I-CQDs by MCF-7 cells, and a more favorable microenvironment for fluorescence activation. These results suggest that biomass mango leaf NIR-I-CQDs can be used as a fluorescent imaging agent targeting breast cancer tumor cells in the diagnosis and treatment of breast cancer. Summary of the Invention

[0004] This invention successfully prepared NIR-I-CQDs using biomass mango leaves as a precursor via a microwave-hydrothermal two-step method. The NIR-I-CQDs emitted at a wavelength of 728 nm. Their optical properties, stability, and biotoxicity were investigated. Confocal microscopy imaging was performed after incubating the prepared NIR-I-CQDs with four types of human cancer cells: MCF-7, HepG2, HeLa, and T24. Furthermore, fluorescence imaging was conducted after subcutaneous injection of the biomass NIR-I-CQDs into tumor sites of model mice with liver cancer, cervical cancer, bladder cancer, and breast cancer to evaluate the targeting performance of the biomass NIR-I-CQDs imaging agent on breast cancer cells.

[0005] A method for preparing a biomass NIR-I-CQDs imaging agent targeting breast cancer cells, characterized in that the method comprises the following steps:

[0006] (1) Preparation of NIR-I-CQDs imaging agents

[0007] Wash fresh mango leaves with pure water, cut the leaves into pieces about 2 cm long, place them in a beaker, pour in 500 mL of anhydrous ethanol to cover the mango leaves, and stir continuously at 500 rpm / s for 12 h. Then, concentrate the ethanol supernatant at low temperature. Place the concentrated, mud-like extract in a microwave oven and heat for 2-6 min at 400-700 W to complete the initial carbonization. Add ultrapure water, transfer to a reaction vessel, and heat continuously at 160-200 ℃ for 6-10 h. After cooling to room temperature, centrifuge at 4000 rpm / s for 1 h. Extract the supernatant from the centrifugation, filter through a 0.22 µm pore size filter membrane, and then transfer to a dialysis membrane with a molecular weight cutoff of 1000 Da. Change the water every 3 h and dialyze continuously for 48 h to obtain a brown NIR-I-CQDs imaging agent solution.

[0008] (2) Biotoxicity testing

[0009] The biotoxicity of NIR-I-CQDs was studied using the MTT assay. Six culture dishes were filled with NIR-I-CQDs solution at concentrations of 0, 100, 150, 200, 250, 300, and 350 µg / mL, respectively. Then, 10-50 μL of 10%-40% MTT solution was added, followed by incubation for 14-18 h. 100 μL of dimethyl sulfoxide was added to each dish and reacted with the cells for 15 min. The culture dishes were then placed in a 570 nm enzyme-linked immunosorbent assay (ELISA) instrument for optical measurements to investigate the biotoxicity of NIR-I-CQDs.

[0010] (3) Study on the performance of breast cancer cells and modeled mouse breast cancer tumors

[0011] First, MCF-7, HeLa, T24, and HepG2 cancer cells in the logarithmic growth phase were collected and adjusted to 7.5 × 10⁻⁶ m² in DMEM medium containing 10% fetal bovine serum. 4 Cells were cultured at a density of 10 cells / mL. Cells were then seeded onto 22×24 mm sterile cell slides and incubated overnight at 37 °C with 5% CO2. After discarding the supernatant, the culture medium was replaced with serum-free medium containing NIR-I-CQDs and cultured for another 20–28 h. Cells in the coverslips were washed three times with PBS buffer, and cellular fluorescence imaging was performed using an LSCM.

[0012] Female nude mice aged 5-7 weeks were provided by Hunan Slack Jingda Experimental Animal Co., Ltd., and all animal experiments were approved by the Ethics Committee of Hezhou University. The specific method for inducing tumor formation in the model mice was as follows: 80-120 μL of a solution containing 5 × 10⁻⁶ mg / L was injected subcutaneously. 5Tumor formation was induced using suspensions of MCF-7, HepG2, HeLa, and T24 cells. When the solid tumor in the model mice reached 80 mm... 3 At that time, 10-50 μL of NIR-I-CQDs imaging agent solution was subcutaneously injected into the tumor sites of various model mice, and then fluorescence images were captured and analyzed using the Kodak In Vivo FX Pro imaging system.

[0013] Preliminary carbonization is completed by setting the microwave power to 600 W and the heating time to 4 min as described in step (1).

[0014] Preferably, the temperature set in step (1) for transferring the material into the reactor is 180°C.

[0015] Preferably, the time for continuous heating of the transfer to the reactor in step (1) is 8 h.

[0016] Preferably, the volume of the MTT solution added in step (2) is 20 μL and the content is 20%.

[0017] Preferably, the incubation time after adding the MTT solution in step (2) is 16 h.

[0018] Preferably, the culture time is 24 h after the serum-free medium containing NIR-I-CQDs is replaced in step (3).

[0019] Preferably, the volume of the injection in step (3) containing MCF-7, HepG2, HeLa and T24 cell suspensions is 100 μL.

[0020] Preferably, the volume of the NIR-I-CQDs imaging agent solution injected into the tumor site of various model mice in step (3) is 30 μL.

[0021] The present invention provides a method for preparing a biomass near-infrared imaging agent targeting breast cancer cells, which has the following advantages:

[0022] (1) NIR-I-CQDs are prepared using agricultural waste mango leaves as a precursor to achieve high-value conversion of agricultural and forestry waste, reduce costs and resource consumption, meet the "dual carbon" goal and sustainable development strategy, promote the high-value utilization of biomass resources, and practice the concept of green development.

[0023] (2) The NIR-I-CQDs prepared by microwave-hydrothermal two-step method using mango leaves have an emission wavelength of 728 nm, which is longer than that of general chlorophyll CQDs, laying the foundation for their application in imaging agents.

[0024] (3) NIR-I-CQDs prepared from mango leaves have good targeting properties for breast cancer cells, providing feasibility for their application in the field of imaging agents for breast cancer diagnosis and treatment. Attached Figure Description

[0025] Figure 1 a is a TEM image of NIR-I-CQDs, and b is a columnar particle size distribution of NIR-I-CQDs;

[0026] Figure 2 a is the FT-IR plot of NIR-I-CQDs, and b is the XRD pattern of NIR-I-CQDs;

[0027] Figure 3 a is the XPS full spectrum, b is the high-resolution spectrum of C1s and c is the spectrum of O1s;

[0028] Figure 4 a is the UV-vis diagram of NIR-I-CQDs, b is the excitation and emission spectra of NIR-I-CQDs, and c is the emission spectra of NIR-I-CQDs at different excitation wavelengths;

[0029] Figure 5 a, b, and c represent the effects of pH, NaCl solution, and xenon lamp irradiation time on the fluorescence intensity of NIR-I-CQDs, respectively.

[0030] Figure 6 Graphs showing cytotoxicity assays at different concentrations of NIR-I-CQDs;

[0031] Figure 7 LSCM images of NIR-I-CQDs after co-incubation with MCF-7, HeLa, T24 and HepG2 cancer cells;

[0032] Figure 8 Fluorescence images of mice with NIR-I-CQDs injected with breast cancer, liver cancer, cervical cancer, and bladder cancer. Detailed Implementation

[0033] The present invention will be further described in detail below with reference to specific embodiments, which do not constitute any limitation on the present invention.

[0034] I. Reagents used in the examples

[0035] Mango leaves were collected from the East Campus of Hezhou University. Fetal bovine serum (FBS), high-glucose medium (DMEM), trypsin, and 100 U / penicillin-streptomycin were purchased from Sigma-Aldrich, USA. Human normal liver cell lines (HL-7702 cells), human liver cancer cells (HepG2 cells), human bladder cancer cells (T24 cells), and human cervical cancer cells (HeLa cells) were all purchased from Dingguo Biotechnology Co., Ltd. (Beijing). Cell Counting Kit-8 (CCK-8) was purchased from Aladdin Corporation (Shanghai, China). BALB / c nude mice (5-6 weeks old) were purchased from Shanghai Laboratory Animal Co., Ltd. (Shanghai, China). Dialysis bags (MWCO: 1000 Da, pore size approximately 1.0 nm) were purchased from Shanghai Bioengineering Co., Ltd. All phosphate-buffered saline (PBS) and other chemical reagents used in the experiments were of analytical grade and were all sourced from China. Experimental water was prepared using a water purification system (Millipore, Bedford, MA).

[0036] II. Instruments used in the embodiments

[0037] The instruments used in the examples are shown in Table 1.

[0038] Table 1 Experimental Apparatus

[0039] Fluorescence photometer F380 Tianjin Gangdong Technology Co., Ltd. UV-Vis spectrophotometer Cary 60 Agilent Technologies, Inc. Transmission electron microscopy (TEM) Tecnai G2 F20 FEI Company, USA Fourier transform infrared spectrometer spectrum Two FEI Company, USA Clean bench HB-402V Hanbeak Company of South Korea Confocal laser scanning microscope LSCM710 German company Zeiss Animal live imaging device Bruker MI SE Billerica MA Company, USA

[0040] III. Sample Preparation and Detection Methods in Examples

[0041] (1) Preparation of NIR-I-CQDs imaging agents

[0042] Fresh mango leaves were washed with pure water, cut into approximately 2 cm pieces, and placed in a beaker. 500 mL of anhydrous ethanol was poured in to cover the mango leaves. The mixture was stirred continuously at 500 rpm / s for 12 h. The ethanol supernatant was then concentrated at low temperature. The concentrated, mud-like extract was placed in a microwave oven and heated at 600 W for 4 min to complete the initial carbonization. Ultrapure water was then added, and the mixture was transferred to a reaction vessel and heated continuously at 180 °C for 8 h. After cooling to room temperature, the mixture was centrifuged at 4000 rpm / s for 1 h. The supernatant was extracted and filtered through a 0.22 µm pore size filter membrane, then transferred to a dialysis membrane with a molecular weight cutoff of 1000 Da. The water was changed every 3 h, and the dialysis was continued for 48 h to obtain a brown NIR-I-CQDs imaging agent solution.

[0043] (2) Biotoxicity testing

[0044] The biotoxicity of NIR-I-CQDs was studied using the MTT assay. Six culture dishes were filled with NIR-I-CQDs solutions at concentrations of 0, 100, 150, 200, 250, 300, and 350 µg / mL, respectively. Then, 20 μL of 20% MTT solution was added, followed by incubation for 16 h. Finally, 100 μL of dimethyl sulfoxide was added to each dish and reacted with the cells for 15 min. The culture dishes were then subjected to optical measurements using a 570 nm enzyme-linked immunosorbent assay (ELISA) reader to investigate the biotoxicity of NIR-I-CQDs.

[0045] (3) Research on the targeting of breast cancer cells and the properties of breast cancer tumors

[0046] First, MCF-7, HeLa, T24, and HepG2 cancer cells in the logarithmic growth phase were collected and adjusted to 7.5 × 10⁻⁶ m² in DMEM medium containing 10% fetal bovine serum. 4 Cells were cultured at a density of 10 cells / mL. Cells were then seeded onto 22×24 mm sterile cell slides and incubated overnight at 37 °C with 5% CO2. After discarding the supernatant, the culture medium was replaced with serum-free medium containing NIR-I-CQDs and cultured for another 24 h. Cells in the coverslips were washed three times with PBS buffer, and cellular fluorescence imaging was performed using an LSCM.

[0047] Female nude mice aged 5-7 weeks were provided by Hunan Slack Jingda Experimental Animal Co., Ltd., and all animal experiments were approved by the Ethics Committee of Hezhou University. The specific method for modeling the mice is as follows: 100 μL of a solution containing 5 × 10⁻⁶ ppm was injected subcutaneously. 5 Tumorigenesis was induced using suspensions of MCF-7, HepG2, HeLa, and T24 cells. When the solid tumor reached 80 mm... 3 At that time, 30 μL of NIR-I-CQDs imaging agent solution was subcutaneously injected into the tumor site of mice, and then fluorescence images were taken and analyzed using the Kodak In Vivo FX Pro imaging system.

[0048] IV. Results and Discussion of the Examples

[0049] 1. Characterization of NIR-I-CQDs imaging agents

[0050] like Figure 1 (a) TEM images show that NIR-I-CQDs are spherical nanoparticles with a particle size range of 2.0-6.0 nm and an average particle size of 3.53 nm, and they have good dispersibility. Figure 2(b) The proportion of NIR-I-CQDs with a size between 3.0 and 4.5 nm is more than 60%, indicating that the NIR-I-CQDs synthesized from mango leaves in this invention have a uniform size distribution, which ensures that they can be used as imaging agents with stable fluorescence emission.

[0051] like Figure 2 (a) As shown in FT-IR, at 4000-400 cm -1 In the infrared spectral range, the transmission peaks of NIR-I-CQDs reveal different vibrations of chemical bonds in surface functional groups, particularly in the 1483–1388 cm⁻¹ range. -1 The obvious C characteristic peaks of NIR-I-CQDs fully demonstrate that NIR-I-CQDs are carbon-based materials. The CO stretching vibrations of NIR-I-CQDs (1206, 1114 cm⁻¹) are also evident. -1 This indicates a good carbonization structure and high water solubility. 1000 cm -1 The stretching vibration peaks of the nearby COC confirm the presence of oxygen-containing bridging bonds such as ether bonds. (3601-3021 cm⁻¹) -1 The peak at 2947 cm⁻¹ is a broad peak for the OH stretching vibration; the peak at 2947 cm⁻¹ is a broad peak for the CH stretching vibration. -1 ); C=O characteristic peak (1547 cm) -1 ); C=C skeleton vibration (1490 cm) -1 ); and OH out-of-plane bending vibration (657 cm) -1 This indicates that NIR-I-CQDs possess considerable active groups, such as -OH, -COOH, and aromatic structures. These structures can significantly improve the water solubility of NIR-I-CQDs and ensure stability during detection. Figure 2 (b) shows the XRD pattern of NIR-I-CQDs, with a broad diffraction peak near 24°, corresponding to the (002) crystal plane structure of graphite-like materials, indicating that NIR-I-CQDs have been fully graphitized.

[0052] Figure 3 (a) shows the overall XPS spectrum of the NIR-I-CQDs. Two main peaks are observed at 284.4 eV and 532.4 eV, corresponding to C1s and O1s respectively, indicating that the NIR-I-CQDs mainly contain C and O elements, with a C:O percentage ratio of 64.06:31.91. Based on the XPS partial spectrum (… Figure 3(a) It can be seen that the C 1s spectrum has a main peak at 284.9 eV, corresponding to the C=C / CC bond, and the peaks at 286.4 eV and 288.2 eV belong to the CO and C=O bonds, respectively, reflecting the sp² / sp³ hybridization state of the carbon core and the distribution of oxygen-containing functional groups on the surface; the O 1s spectrum shows a double peak at 532.2 eV and 533.8 eV, corresponding to the C=O carbonyl oxygen and CO hydroxyl oxygen, respectively, and the peaks above 533.5 eV belong to adsorbed water, revealing the hydrophilicity and surface oxidation degree of NIR-I-CQDs.

[0053] The UV absorption and fluorescence spectra of NIR-I-CQDs are shown in the figure. Figure 4 As shown in (a), a distinct UV absorption peak is observed near 312 nm, indicating that the CO bonds or other surface groups in the NIR-I-CQDs generate n-π bonds. * Transition. Under the maximum excitation wavelength of 648 nm, NIR-I-CQDs exhibit a significant maximum emission peak in the near-infrared I region at 728 nm.

[0054] Figure 4 (b) shows the fluorescence spectra of NIR-I-CQDs at different excitation wavelengths. When the excitation wavelength varies in the range of 608-668 nm, the fluorescence intensity of the emission wavelength increases with increasing excitation wavelength and decreases with increasing excitation wavelength, while the emission wavelength shows almost no redshift or blueshift. This indicates that the emission wavelength of the prepared NIR-I-CQDs does not change with the excitation wavelength, exhibiting excitation wavelength-independent emission performance. This is attributed to the consistent particle size, surface state, and defect distribution of NIR-I-CQDs, with luminescence mainly originating from a single luminescent center. Such carbon quantum dots can obtain stable monochromatic light with a single excitation source, exhibiting high color purity, minimal cross-contamination, and stable signal. Therefore, the excitation wavelength-independent emission performance of NIR-I-CQDs provides excellent photostability for the sensing and detection of food additives. To better achieve high-precision, high-stability, and high-response-speed fluorescence detection of potassium sorbate, the maximum excitation wavelength of 648 nm was selected as the optimal excitation wavelength. Calculations show that the fluorescence quantum efficiency of this quantum dot is 21.09%, a value far exceeding that of most biomass carbon quantum dots.

[62] .

[0055] 2. Fluorescence stability of NIR-I-CQDs imaging agents

[0056] To evaluate the stability of NIR-I-CQDs imaging agents, the fluorescence intensity of NIR-I-CQDs under different pH values ​​and saline environments was tested and analyzed. Figure 5(a) Under different pH values, the fluorescence intensity of NIR-I-CQDs was highest under acidic conditions and decreased significantly under alkaline conditions, but the F / F0 ratio did not fall below 80%, indicating that NIR-I-CQDs have certain acid-base stability. The salt stability of NIR-I-CQDs is as follows: Figure 5 As shown in (b), the fluorescence intensity variation is almost negligible within the salt concentration range of 80-200 nmol / L, indicating its applicability in bioimaging under biological salinity conditions. Figure 5 In (c), the fluorescence stability of NIR-I-CQDs under continuous xenon lamp irradiation was recorded, and the change was also within 20%, which indicates that the prepared NIR-I-CQDs have good photostability and resistance to photobleaching. These properties make NIR-I-CQDs particularly suitable as imaging agents for long-term fluorescence imaging, dynamic tracking, and high-brightness displays, which is one of its core advantages over traditional fluorescent dyes.

[0057] 3. Cytotoxicity of NIR-I-CQD imaging agents

[0058] To investigate the biological applications of NIR-I-CQDs, we performed cytotoxicity experiments on the HepG2 cell line. Figure 6 As can be seen, when the concentration of NIR-I-CQDs reaches 500 µg / mL, the cell viability can still reach over 90%, indicating that NIR-I-CQDs have low cytotoxicity and good biocompatibility. Furthermore, it has great application potential in in vitro live-cell imaging and long-term cell tracking.

[0059] 4. Targeting breast cancer cells and breast cancer tumors

[0060] To further and visually verify the targeting ability, cell biocompatibility, and intracellular fluorescence imaging performance of the mango leaf NIR-I-CQDs imaging agent for breast cancer cells, and to clarify its feasibility as a novel green imaging agent for breast cancer cell visualization, this invention provides important cellular-level experimental evidence for the subsequent application of NIR-I-CQDs imaging agents in the biomedical field. The mango leaf NIR-I-CQDs imaging agent was co-incubated with four types of cancer cells—HepG2, HeLa, T24, and MCF-7—and subjected to LSCM fluorescence imaging. The results are as follows: Figure 7As shown in the image, NIR-I-CQDs can penetrate the membranes of these cancer cells without hindrance and locate in the cytoplasm, with a relatively high uptake. They emit red fluorescence in all four cancer cell types, resulting in a high signal-to-noise ratio in the imaging. Comparing the fluorescence intensity within these four cell types, the red fluorescence in MCF-7 cells was the brightest, significantly stronger than that in the other three cell types. This indicates that breast cancer cells have good targeting and affinity for NIR-I-CQDs, resulting in higher uptake, longer retention, and a more favorable microenvironment for fluorescence activation. Furthermore, even after 30 minutes of re-image capture, the red fluorescence in all four cell types did not weaken, indicating good fluorescence stability of NIR-I-CQDs in the four cancer cell types. Bright-field images of the four cancer cell types show that they have normal morphology, with no large numbers of dead, shrunken, or ruptured cells, indicating that NIR-I-CQDs are relatively safe for various cancer cell types.

[0061] To further verify the targeting ability of NIR-I-CQDs to breast cancer tumor cells, this invention also injected the NIR-I-CQDs imaging agent solution into mouse models of breast cancer, liver cancer, cervical cancer, and bladder cancer for in vivo fluorescence imaging. Figure 8 As can be seen, the red fluorescence intensity at the tumor site in the breast cancer model mouse was significantly stronger than that at the tumor site in other model mice. This further indicates that NIR-I-CQDs also exhibit good targeting ability for in vivo breast cancer tumor cells. Therefore, they demonstrate the strongest EPR effect, highest cellular uptake, and most suitable microenvironment activation at the tumor site in the breast cancer model mouse, resulting in NIR-I-CQDs enrichment and fluorescence activation that are far stronger than in liver cancer, cervical cancer, and bladder cancer model mice. Therefore, these mango leaf NIR-I-CQDs can be used as a fluorescent imaging agent targeting breast cancer tumor cells in the field of breast cancer diagnosis and treatment.

Claims

1. A method for preparing a biomass NIR-I-CQDs imaging agent targeting breast cancer cells, characterized in that, The method includes the following steps: (1) Preparation of NIR-I-CQDs imaging agents Wash fresh mango leaves with pure water, cut the leaves into pieces about 2 cm long, place them in a beaker, pour in 500 mL of anhydrous ethanol to cover the mango leaves, stir continuously at 500 rpm / s for 12 h, then concentrate the ethanol extract at low temperature, place the concentrated extract into a mud-like state in a microwave reactor, set the microwave power to 400-700 W and heat for 2-6 min to complete the initial carbonization, then add ultrapure water, transfer to a reaction vessel, set the temperature to 160-200 ℃ and heat continuously for 6-10 h, cool to room temperature, centrifuge at 4000 rpm / s for 1 h, extract the supernatant after centrifugation, filter with a 0.22 µm pore size filter membrane, and then transfer to a dialysis membrane with a molecular weight cutoff of 1000 Da, change the water every 3 h, dialyze continuously for 48 h to obtain a brown NIR-I-CQDs imaging agent solution; (2) Biotoxicity testing The biotoxicity of NIR-I-CQDs was studied using the MTT assay. Six culture dishes were filled with NIR-I-CQDs solutions at concentrations of 0, 100, 150, 200, 250, 300, and 350 µg / mL, respectively. Then, 10-50 μL of 10%-40% MTT solution was added, followed by incubation for 14-18 h. 100 μL of dimethyl sulfoxide was added to each dish and reacted with the cells for 15 min. The culture dishes were then placed in a 570 nm enzyme-linked immunosorbent assay (ELISA) instrument for optical measurements to investigate the biotoxicity of NIR-I-CQDs. (3) Study on the performance of breast cancer cells and modeled mouse breast cancer tumors MCF-7, HeLa, T24, and HepG2 cancer cells in the logarithmic growth phase were collected and adjusted to 7.5 × 10⁻⁶ m² in DMEM medium containing 10% fetal bovine serum. 4 Cells were seeded at a density of 100 cells / mL and then incubated overnight at 37 °C with 5% CO2 on sterile 22 × 24 mm cell slides. The supernatant was discarded, and the cells were replaced with serum-free medium containing NIR-I-CQDs and cultured for another 20–28 h. Cells in the coverslips were washed three times with PBS buffer, and cellular fluorescence imaging was performed using an LSCM. Female nude mice aged 5-7 weeks were provided by Hunan Slack Jingda Experimental Animal Co., Ltd. All animal experiments were approved by the Ethics Committee of Hezhou University. The specific method for tumor formation in the model mice was as follows: 80-120 μL of a solution containing 5 × 10⁻⁶ ppm was injected subcutaneously. 5 Tumor formation was induced using suspensions of MCF-7, HepG2, HeLa, and T24 cells. When the solid tumor in the model mice reached 80 mm... 3 At that time, 10-50 μL of NIR-I-CQDs imaging agent solution was subcutaneously injected into the tumor sites of various model mice, and then fluorescence images were taken and analyzed using the Kodak InVivo FX Pro imaging system.

2. The preparation method according to claim 1, characterized in that, In step (1), the microwave power is set to 600W and the heating time is 4 minutes to complete the initial carbonization.

3. The preparation method according to claim 2, characterized in that, The temperature set in step (1) for transferring the material to the reactor is 180°C.

4. The preparation method according to claim 3, characterized in that, The time for continuous heating in the reactor as described in step (1) is 8 h.

5. The preparation method according to claim 4, characterized in that, In step (2), the volume of the added MTT solution is 20 μL and the content is 20%.

6. The preparation method according to claim 5, characterized in that, The incubation time after adding the MTT solution in step (2) is 16 h.

7. The preparation method according to claim 6, characterized in that, In step (3), the culture time is 24 h after replacing the medium with serum-free medium containing NIR-I-CQDs.

8. The preparation method according to claim 7, characterized in that, In step (3), the volume of the injected suspension containing MCF-7, HepG2, HeLa and T24 cells is 100 μL.

9. The preparation method according to claim 8, characterized in that, In step (3), the volume of the NIR-I-CQDs imaging agent solution injected into the tumor sites of various model mice is 30 μL.

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