A copper-cerium rare earth alloy nanocomposite, a preparation method thereof and application of the nanocomposite in inducing copper death and synergistic radiotherapy sensitization

Abstract

This invention discloses a copper-cerium rare-earth alloy nanocomposite, its preparation method, and its application in inducing copper death and synergistically enhancing radiosensitivity. The rare-earth nanomaterial, denoted as R&CC@ICG, comprises solid Cu and Ce rare-earth nanoparticles (i.e., CC) and a cyclic polypeptide c (RGDfK) and a fluorescent dye ICG located on the surface of the Cu and Ce-containing solid rare-earth nanoparticles. This material exhibits good biocompatibility and, in vivo, significantly enhances the sensitivity of tumor cells to radiation by inducing copper death, thereby increasing the tumor-killing ability of radiotherapy. It addresses the problem of tumor recurrence caused by some radiosensitive tumor cells, and has significant clinical implications for improving patient prognosis.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a copper-cerium rare-earth alloy nanocomposite, its preparation method, and its application in induced copper death and synergistic radiosensitization. Background Technology

[0002] Radiation therapy is an important component of comprehensive treatment for colorectal cancer (CRC). However, radiation resistance greatly limits the effectiveness of radiation therapy, making it difficult to eliminate tumors. Increasing the radiation dose is necessary to better control the tumor. Because colorectal cancer has moderate sensitivity to radiation and a hypoxic tumor microenvironment, higher and potentially harmful radiation doses are usually required to achieve sufficient tumor-killing effects.

[0003] Currently, an effective strategy to address these limitations is to enhance the efficacy of radiotherapy (RT) by introducing radiosensitizers into the tumor microenvironment (TME). Nitroimidazole compounds, which have been reported as radiosensitizers, exhibit dose-limiting toxicity, making it impossible to achieve sufficient drug concentrations for sensitization throughout the entire course of fractionated treatment. While platinum-based drugs offer good radiosensitization, they also have significant toxic side effects.

[0004] Therefore, improving the radiosensitivity of tumors and enhancing their biosafety are urgent technical issues that need to be addressed to overcome the bottlenecks in radiotherapy for colorectal cancer. Summary of the Invention

[0005] This invention provides a copper-cerium rare-earth alloy nanocomposite, its preparation method, and its application in inducing copper death and synergistic radiosensitization. This material exhibits good safety, effectively induces copper death, and demonstrates excellent radiosensitization effects. After modification with PEG2000-NH2 and coupling with c(RGDfK), it possesses superior tumor-targeting capabilities. It can serve as a targeting probe that specifically accumulates in tumor tissue, actively being taken up by tumor cells, inducing copper death in tumor cells, and significantly increasing the sensitivity of tumor cells to radiation. This enhances the tumor-killing ability of radiotherapy and solves the problem of tumor recurrence caused by some radiosensitive tumor cells.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A copper-cerium rare earth alloy nanocomposite (denoted as R&CC@ICG) comprises solid Cu and Ce rare earth nanoparticles (abbreviated as CC) and a cyclic polypeptide c (RGDfK) and a fluorescent dye located on the surface of the CC.

[0007] According to an embodiment of the present invention, the cyclic polypeptide c (RGDfK) is coupled to the CC surface by modifying the CC with PEG2000-NH2.

[0008] According to an embodiment of the present invention, the particle size of the copper-cerium rare earth alloy nanocomposite is 20~100 nm, for example 30~80 nm, and exemplarily 40 nm.

[0009] According to an embodiment of the present invention, the content of Ce element in the copper-cerium rare earth alloy nanocomposite is 5-25%, for example 10-20%, and exemplary values ​​are 5%, 6%, 7%, 8%, 9%, 10%, 10.25%, 11%, 12%, 13%, 14%, 15%, 18%, 20%, 25%, or any number between any two numbers.

[0010] According to an embodiment of the present invention, the content of Cu element in the copper-cerium rare earth alloy nanocomposite is 30-80%, for example 40-70%, and exemplary values ​​are 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 65.62%, 70%, or any number between any two numbers.

[0011] According to an embodiment of the present invention, the content of the cyclic polypeptide c (RGDfK) in the copper-cerium rare earth alloy nanocomposite is 1-20%, for example 5-15%, and exemplary values ​​are 1%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 11.25%, 12%, 13%, 14%, 15%, 20%, or any number between any two numbers.

[0012] According to an embodiment of the present invention, the fluorescent dye is indocyanine green (ICG); further, the content of indocyanine green (ICG) in the copper-cerium rare earth alloy nanocomposite is 1~20%, for example 5~15%, exemplary of 1%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20% or any number between any two numbers.

[0013] According to an embodiment of the present invention, the copper-cerium rare earth alloy nanocomposite is degraded in an aqueous solution at pH 6.5 ± 0.2.

[0014] According to an embodiment of the present invention, the Cu element in the copper-cerium rare earth alloy nanocomposite includes Cu. 2+ and Cu + The existence form of Ce element contains Ce 3+ and Ce 4+ The form of existence.

[0015] The present invention also provides a method for preparing the above-mentioned copper-cerium rare earth alloy nanocomposite, the method comprising the following steps: (1) Cerium salt, copper salt and hexamethylenetetramine were reacted in an aqueous solvent to obtain solid rare earth nanoparticles containing Cu and Ce (denoted as CC). (2) The CC and ICG are reacted in an aqueous solution by stirring to obtain the product CC@ICG; (3) The CC@ICG and PEG2000-NH2 are reacted in an alcohol solvent to obtain CC@ICG&NH2 nanoparticles; (4) The CC@ICG&NH2 nanoparticles, cyclic polypeptide c(RGDfK), EDC and NHS are reacted in an aqueous solvent to obtain the copper-cerium rare earth alloy nanocomposite (denoted as R&CC@ICG).

[0016] According to an embodiment of the present invention, the mass ratio of the cerium salt to the copper salt is 1:(1.5-5), for example 1:(2-3).

[0017] According to embodiments of the present invention, the copper salt is a water-soluble copper salt, such as cerium chloride hexahydrate (CuCl2•6H2O); and / or, the cerium salt is a water-soluble cerium salt, such as cerium chloride hexahydrate (CeCl3•6H2O).

[0018] According to an embodiment of the present invention, step (1) includes: first dissolving cerium salt (e.g., CeCl3•6H2O) and copper salt (e.g., CuCl2•6H2O) powders in ultrapure water, placing them in an oil bath at 90~110℃ (e.g., 100℃) and stirring continuously for 20~40 min, then adding hexamethylenetetramine, reacting for 5~40 min, and after post-processing, obtaining the CC.

[0019] In an exemplary scheme, step (1) includes: dissolving 30 mg CeCl3•6H2O and 70 mg CuCl2•6H2O powder in 60 mL of ultrapure water and placing it in a 100 mL round-bottom flask. Finally, the flask is placed in an oil bath at 100°C and stirred continuously at 300 r / min. After 30 min, the solution becomes clear and transparent. Subsequently, 100 mg of hexamethylenetetramine is added to the above mixed aqueous solution, and the temperature is maintained at 100°C. The magnetic stirring speed is 300 r / min, and the reaction continues for 5 to 30 min. Then, the solution is cooled to room temperature, and the reaction solution is centrifuged in a centrifuge to collect the precipitate (5000 r / min). The precipitate is then washed three times with ultrapure water, and the obtained product is collected to obtain the CC.

[0020] According to an embodiment of the present invention, step (2) includes: dissolving the CC and ICG powders in an aqueous solution and placing them at 35~40℃ (e.g., 37℃) with magnetic stirring for 12~24h, and then washing with water to obtain CC@ICG.

[0021] In an exemplary scheme, step (2) includes: dissolving 100 mg of CC and 10 mg of ICG powder from step (1) in 10 mL of aqueous solution and placing it at 37°C under magnetic stirring (300 r / min). After stirring for 18 h, the solution is washed three times with ultrapure water (5000 r / min), and then the precipitated sample is freeze-dried to obtain the product CC@ICG.

[0022] According to an embodiment of the present invention, the alcohol solvent is (anhydrous) ethanol.

[0023] According to an embodiment of the present invention, step (3) includes: ultrasonically dispersing the CC@ICG in an alcohol solvent, adding PEG2000-NH2 in the alcohol solvent at 70~80℃, stirring the reaction for 8~15 h, and washing with water to obtain CC@ICG-NH2 nanoparticles.

[0024] In an exemplary scheme, step (3) includes: dissolving 100 mg of the CC@ICG prepared above in 30 mL of anhydrous ethanol, dispersing it, heating it in an oil bath at 75°C, stirring at 300 rpm, then adding 100 mg of PEG2000-NH2 and stirring for 12 h, and then washing it with ultrapure water by centrifugation to obtain CC@ICG-NH2 nanoparticles.

[0025] According to an embodiment of the present invention, the mass ratio of the CC@ICG-NH2 nanoparticles to the cyclic polypeptide c(RGDfK) is (5~15):1, for example 10:1.

[0026] According to an embodiment of the present invention, step (4) includes: reacting the CC@ICG-NH2 nanoparticles, cyclic polypeptide c(RGDfK), EDC and NHS in an aqueous solvent at 2~6℃ for 10~15 h to obtain the R&CC@ICG.

[0027] In an exemplary scheme, step (4) includes: dissolving 100 mg CC@ICG-NH2 and 10 mg c(RGDfK) peptide in ultrapure water, adding 20 mg EDC and NHS, stirring overnight at 4°C, washing three times with ultrapure water to obtain Ce-based rare earth nanocomposite (i.e., R&CC@ICG), precipitating and storing at room temperature.

[0028] This invention also provides the application of the above-mentioned rare earth nanomaterials as tumor-targeting probes and / or radiosensitizers. Preferably, the copper-cerium rare earth alloy nanocomposite can be used for efficient tumor enrichment, synergistic radiosensitization, or real-time bioimaging.

[0029] According to an embodiment of the present invention, the tumor-targeting probe is a colorectal cancer-targeting probe.

[0030] According to an embodiment of the present invention, the radiosensitizer is a radiosensitizer for colorectal cancer.

[0031] The beneficial effects of this invention are: The inventors discovered that elements with high atomic numbers, such as cerium, can produce a dose-enhancing effect after being exposed to ionizing radiation, promoting tumor cell apoptosis. This is because these elements have a larger X-ray photon capture cross section and Compton scattering effect, thus allowing for the preparation of formulations that are more efficient than other radiosensitizers.

[0032] The inventors also discovered that radiotherapy can induce copper death, and that copper ion carriers can synergistically induce copper death in conjunction with radiotherapy by increasing intracellular copper accumulation, significantly overcoming radiotherapy resistance. Based on these findings, the inventors believe that using copper-containing radiosensitizers to induce copper death is an effective treatment strategy.

[0033] This invention develops a simple one-step method for synthesizing multifunctional cerium- and copper-doped nanospheres (CCs) with nanostructures. After encapsulating ICG on the CC surface and coupling it with a cRGD peptide (R&CC@ICG), this nanoplatform can precisely identify colorectal cancer and trigger the rapid release of ICG, Cu, and Ce ions in a simulated tumor microenvironment (pH = 6.5). Combined with radiotherapy, it mediates copper death in mitochondria, improving radiosensitivity and synergistically enhancing the efficacy of radiotherapy, and inhibiting lung metastasis. The Fenton-like reaction of the bimetallic ions and radiotherapy jointly generate a large amount of ROS, inducing apoptosis and arresting the cell cycle in the G2 / M phase, inhibiting cell proliferation, which has important clinical significance for improving patients' quality of life and prognosis. Furthermore, the R&CC@ICG material exhibits good biocompatibility, providing possibilities for its further clinical translation. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the R&CC@ICG synthesis of the present invention; Figure 2 High-resolution transmission electron microscope (TEM) images of CC after adding hexamethylenetetramine for 5, 10, 15, and 30 minutes in Experiment 1 of this invention; Figure 3 This is a transmission electron microscope image of CC in a weakly alkaline environment in Experiment 1 of this invention; Figure 4 The images are TEM images of CC@ICG after incubation for different times in biological buffer solutions at pH 6.5 and pH 7.2 in Experiment 1 of this invention. Figure 5 High-resolution transmission electron microscopy (HRTEM) images of CC and CC@ICG from Experiment 1 of this invention; Figure 6 This is the SEM image of CC@ICG from Experiment 1 of this invention; Figure 7 The image and elemental distribution diagram of high-angle annular dark-field scanning transmission electron microscope for Experiment 1 CC of this invention are shown below. Figure 8 The image shown is an energy dispersive spectroscopy (EDS) image of Experiment 1 CC of this invention; Figure 9 This is a partial detection result of Experiment 1 of the present invention, wherein, A is the X-ray photoelectron energy spectrum of CC prepared in Experiment 1; B is the fine X-ray photoelectron energy spectrum of CC prepared in Experiment 1; C represents the fine spectrum of Cu2P X-ray photoelectron energy of CC prepared in Experiment 1; Figure 10 The UV absorption spectra of CC@ICG, R&CC@ICG and c(RGDfK) in Experiment 1 of this invention are shown below. Figure 11 The high-resolution transmission electron microscope (TEM) used in Experiment 1 of this invention is R&CC@ICG. Figure 12 For Experiment 2 of this invention, the cell targeting of R&CC@ICG is detected, wherein: A is a graph showing the quantitative analysis of the average fluorescence intensity of MC38 cells in Experiment 2 after co-incubation with PBS, CC@ICG and R&CC@ICG for 0.5, 1, 2 and 4 hours, respectively, by flow cytometry. B shows representative fluorescence microscopy images of MC38 cells from Experiment 2 after incubation with CC@ICG and R&CC@ICG for 0.5, 1, 2, and 4 hours. C is a quantitative analysis of the average fluorescence intensity of each group of fluorescence microscopy images of MC38 cells incubated with CC@ICG and R&CC@ICG for 0.5, 1, 2 and 4 hours in Experiment 2; D is a flow cytometry quantitative analysis of MC38 cells in Experiment 2 with R&CC@ICG and Blocking (after incubation with R&CC@ICG pretreated with c(RGDfK) for 4 hours). E shows representative fluorescence microscopy images of MC38 cells from Experiment 2 after treatment with R&CC@ICG and Blocking (4 hours after pretreatment with c(RGDfK)); F is a quantitative analysis of the average fluorescence intensity of each group of MC38 cells in Experiment 2 after being pretreated with R&CC@ICG and Blocking (4 hours after c(RGDfK) pretreatment). Figure 13Experiment 3 in this embodiment of the invention was conducted to detect the effect of R&CC@ICG-mediated dual-enhanced copper death and radiotherapy, in which the PBS group (G1), R&CC@ICG group (G2), 4Gy group (G3), and R&CC@ICG+4Gy group (G4) were included: Experiment A shows the Western blot analysis of the expression levels of DLAT monomer, POLD1, ACO2, HSP70, LIAS, and FDX1 in MC38 cells after treatment with CuCl2 and different concentrations of R&CC@ICG. Experiment B uses Western blotting to analyze the expression levels of DLAT monomer, POLD1, ACO2, HSP70, LIAS and FDX1 in MC38 cells after treatment with PBS, R&CC@ICG, 4Gy and R&CC@ICG+4Gy. C represents the digital staining images of clones formed in Experiment 3 with or without R&CC@ICG treatment at different radiation doses (0, 2, 4, and 6 Gy). D is a quantitative analysis diagram of the digital staining images formed by cloning in Experiment 3 with or without R&CC@ICG treatment at different radiation doses (0, 2, 4 and 6 Gy). E shows immunofluorescence images of γ-H2AX in MC38 cells cultured under different treatments under 4 Gy irradiation in Experiment 3. F is a quantitative analysis of γ-H2AX immunofluorescence images of MC38 cells cultured under different treatments under 4 Gy irradiation in Experiment 3. Figure 14 Experiment 4 of this invention was used to detect the in vitro antitumor activity of R&CC@ICG combined with radiotherapy, which included the PBS group (G1), R&CC@ICG group (G2), 4Gy group (i.e., radiotherapy alone RT group, G3), and R&CC@ICG+4Gy group (i.e., R&CC@ICG+radiotherapy RT group, G4): Experiment A uses flow cytometry to analyze the apoptosis of MC38 cells treated with a combination of PBS, R&CC@ICG, 4Gy, and R&CC@ICG+4Gy irradiation. B is a quantitative analysis diagram of cell apoptosis in each group using flow cytometry in Experiment 4; C represents the live and dead staining fluorescence microscopy images of MC38 cells after different treatments in Experiment 4; D represents the intracellular ROS levels of MC38 cells treated in each group, as determined by flow cytometry in Experiment 4. E represents the intracellular ROS levels of MC38 cells treated in each group, detected by fluorescence microscopy in Experiment 4. F is a graph showing the quantitative analysis of GSH consumption in MC38 cells after treatment with different concentrations of R&CC@ICG in Experiment 4. G is a graph showing the quantitative analysis of cell cycle detection by flow cytometry of MC38 cells under different treatments in Experiment 4. Figure 15 For Experiment 5 of this invention, in vivo tumor targeting of R&CC@ICG in a MC38 subcutaneous xenograft mouse model was performed, wherein: A shows continuous near-infrared II (NIR-II) fluorescence imaging at different time points after intravenous injection of CC@ICG (top image) and R&CC@ICG (bottom image) into mice in Experiment 5. B is a quantitative analysis of continuous near-infrared II (NIR-II) fluorescence imaging at different time points after intravenous injection of CC@ICG and R&CC@ICG into mice in Experiment 5. C represents the representative near-infrared II (NIR-II) fluorescence images of the R&CC@ICG targeted group and the Blocking group (i.e., the c(RGDfK) blocking group) 48 hours after R&CC@ICG injection in Experiment 5. D is a quantitative analysis of the near-infrared II (NIR-II) fluorescence images of the R&CC@ICG targeting group and the Blocking group (i.e., the c(RGDfK) blocking group) 48 hours after injection of R&CC@ICG in Experiment 5. Figure 16 This invention relates to Experiment 6, which describes the detection of the synergistic radiosensitization effect of copper death induced by R&CC@ICG in a subcutaneous xenograft mouse model. The experimental groups included: PBS group (G1), R&CC@ICG group (G2), 6Gy group (G3), and R&CC@ICG+6Gy group (G4). A is a schematic diagram of the in vivo antitumor therapy experimental design for Experiment 6; B shows the in vitro tumor images of mice on day 14 after different treatments, including PBS, R&CC@ICG, 6Gy, and R&CC@ICG+6Gy combined irradiation; C represents the relative tumor growth curve of each tumor-bearing mouse after combined irradiation treatment with PBS, R&CC@ICG, 6Gy, and R&CC@ICG+6Gy in Experiment 6. D represents the growth curve of the tumor over time after combined irradiation with PBS, R&CC@ICG, 6Gy, and R&CC@ICG+6Gy in Experiment 6. E represents the tumor weight of mice on day 14 after combined irradiation with PBS, R&CC@ICG, 6Gy, and R&CC@ICG+6Gy in Experiment 6. F represents the weight monitoring of mice in Experiment 6 after being treated with a combination of PBS, R&CC@ICG, 6Gy, and R&CC@ICG+6Gy irradiation, every two days. G represents the tumor tissue from experiment 6 that was treated with a combination of PBS, R&CC@ICG, 6Gy, and R&CC@ICG+6Gy irradiation for 14 days. The tissue was then subjected to H&E staining, immunohistochemistry, and immunofluorescence analysis (TUNEL, KI67, LIAS, ACO2, DLAT). Figure 17 Experiment 6 in this embodiment of the invention evaluates the in vivo biocompatibility of R&CC@ICG, including the PBS group (G1), R&CC@ICG group (G2), 6Gy group (G3), and R&CC@ICG+6Gy group (G4): A shows the H&E staining results of mouse organ tissues for animal-level safety of R&CC@ICG in Experiment 6. BC is a hematologic analysis of the animal-level safety of R&CC@ICG in Experiment 6; Figure 18 To detect the synergistic radiosensitization effect of copper death induced by R&CC@ICG in a mouse model of MC38 orthotopic tumor in this invention, Experiment 7 of this embodiment included the following groups: PBS group (G1), R&CC@ICG group (G2), 6Gy group (G3), and R&CC@ICG+6Gy group (G4). A represents the small animal in vivo imaging monitoring of mice in each group at 0, 5, 10, and 15 days after different treatments including PBS, R&CC@ICG, 4Gy, and R&CC@ICG+6Gy combined irradiation in Experiment 7. B is a quantitative analysis of small animal in vivo imaging monitoring at 0, 5, 10, and 15 days after mice in Experiment 7 were treated with different treatments including PBS, R&CC@ICG, 4Gy, and R&CC@ICG+6Gy combined irradiation. C shows in vitro images of in situ colorectal tumors in mice from each group on day 14 after different treatments including PBS, R&CC@ICG, 6Gy, and R&CC@ICG+6Gy combined irradiation in Experiment 7. D represents the weight of ex vivo tumors in each group after 15 days of combined irradiation with PBS, R&CC@ICG, 6Gy, and R&CC@ICG+6Gy in Experiment 7. Representative histological images of in situ colorectal cancer sites in Experiment E7 after combined irradiation with PBS, R&CC@ICG, 6Gy, and R&CC@ICG+6Gy; Figure 19Experiment 7 of this invention was conducted to detect the anti-lung metastasis function of R&CC@ICG, which included the PBS group (G1), R&CC@ICG group (G2), 6Gy group (G3), and R&CC@ICG+6Gy group (G4). A represents the gross images of lung tissue collected from each group after different treatments, including PBS, R&CC@ICG, 6Gy, and R&CC@ICG+6Gy combined irradiation in Experiment 7. B represents representative H&E staining images of lung tissue collected from each group after different treatments, including PBS, R&CC@ICG, 6Gy, and R&CC@ICG+6Gy combined irradiation in Experiment 7. C is a quantitative analysis of lung tumor lesions in mice in each group on day 14 after different treatments including PBS, R&CC@ICG, 6Gy, and R&CC@ICG+6Gy combined irradiation in Experiment 7. Detailed Implementation

[0035] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0036] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0037] In this invention, cRGD, cRGD(fK) and c(RGDfK) represent the same cyclic polypeptide.

[0038] Example 1 This invention discloses a copper-cerium alloy rare-earth nanoprobe (i.e., R&CC@ICG) that induces copper death and synergistically enhances radiosensitivity. The synthetic route is as follows: Figure 1 As shown. The probe is R&CC@ICG, which is obtained by using hydrated rare earth chlorate (CeCl3•xH2O) and hydrated cuprate (CuCl2•xH2O) as precursors to obtain copper-cerium rare earth alloy particles, and then further loading fluorescent molecules on the surface, and finally coupling the cyclic polypeptide c (RGDfK) externally through PEG2000-NH2.

[0039] A method for preparing copper-cerium alloy rare-earth nanoprobes, comprising the following steps: S1. First, dissolve 30 mg CeCl3•6H2O and 70 mg CuCl2•6H2O powder in 60 mL of ultrapure water and place it in a 100 mL round-bottom flask. Finally, place it in an oil bath at 100℃ and stir continuously at 300 r / min. After 30 min, the solution is clear and transparent.

[0040] S2. Subsequently, 100 mg of hexamethylenetetramine was added to the above mixed aqueous solution, and the temperature was maintained at 100℃ with a magnetic stirring speed of 300 r / min. The reaction was continued for 30 min. After cooling to room temperature, the reaction solution was centrifuged (5000 r / min) to collect the precipitate, and then washed three times with ultrapure water. The obtained product was collected to obtain CC. At the same time, three other types of small balls were prepared with reaction times of 5 min, 10 min, and 15 min.

[0041] S3. Dissolve 100 mg of CuCe and 10 mg of ICG powder from step S2 in 10 mL of aqueous solution and place at 37 °C with magnetic stirring (300 r / min). After stirring for 18 h, wash the solution three times with ultrapure water (5000 r / min). Then freeze-dry the precipitate sample to obtain the product CC@ICG.

[0042] S4. Dissolve 100 mg of the prepared CC@ICG in 30 mL of anhydrous ethanol, disperse it, heat it in an oil bath at 75 °C, stir at 300 rpm, then add 100 mg of PEG2000-NH2 and continue stirring for 12 h. After washing with ultrapure water by centrifugation, CC@ICG-NH2 nanoparticles are obtained.

[0043] S5. Dissolve 100 mg CuCe@ICG-NH2 and 10 mg c(RGDfK) peptide in ultrapure water, add 20 mg EDC and NHS, stir overnight at 4°C, and wash three times with ultrapure water to obtain copper-cerium alloy rare earth nanocomposite (i.e., R&CC@ICG). After precipitation, store at room temperature. This copper-cerium alloy rare earth nanoprobe can be used to induce tumor copper death and synergistically enhance radiosensitivity.

[0044] The prepared R&CC@ICG probe was characterized and tested, revealing a relatively uniform particle size, good safety profile, and specific targeting ability for rectal cancer cells. R&CC@ICG exhibited better tumor enrichment compared to CC@ICG and could be inhibited by excess c(RGDfK). In the NIR-II region, the probe accurately distinguished between colorectal tumors and normal tissue. Its application in fluorescent surgical navigation for colorectal cancer enabled accurate and timely assessment of surgical margins, reducing the positive margin rate and thus addressing the challenge of local recurrence. Furthermore, the probe completely degraded into ~5 nm nanoparticles under physiological conditions, further demonstrating its good biosafety. This targeting probe, which specifically aggregates in tumor tissue, significantly enhances the sensitivity of tumor cells to radiation, holding significant clinical importance for improving patients' quality of life and prognosis.

[0045] Experiment 1: Characterization of R&CC@ICG 1. Morphology and Size Detection of CC and R&CC@ICG: Small amounts of CC reacted with hexamethylenetetramine for 5 min, 10 min, 15 min, and 30 min respectively were dissolved in ultrapure water. 10 μl of each solution was dropped onto an ultrathin carbon film and dried in a 40°C oven. TEM was used at 100 kV to observe the morphology, size, and dispersibility of the samples. Clear, uncontaminated fields of view were photographed and stored. Furthermore, the morphology, size, and dispersibility of CC and CC@ICG were observed under a high-resolution transmission electron microscope (HRTEM) at 200 kV.

[0046] 2. Observation of CC@ICG morphology using scanning electron microscopy: Dissolve a certain amount of CC@ICG in ultrapure water, and add 10 μl of the solution to a copper grid. Dry the grid in an oven at 40°C. Attach the copper grid to conductive adhesive and use a scanning electron microscope (SEM) at 100 kV to observe the morphology, size, and dispersion of the sample. Take clear, uncontaminated photos and save them.

[0047] 3. Degradation of CC@ICG in different pH buffer solutions: Appropriate amounts of CC@ICG were dissolved in aqueous solutions at pH 7.2 and pH 6.5, respectively. After incubation for 0, 12, 24, and 36 hours, 10 μl of the solution was dropped onto an ultrathin carbon film and dried in an oven at 40°C. TEM was used to observe the morphological changes of the samples at 100 kV, and photographs were taken and stored at appropriate fields of view.

[0048] 4. Compositional analysis was conducted based on the different X-ray photon characteristic energies of different elements: A certain amount of CC was dissolved in ultrapure water, and 10 μl of the solution was dropped onto a copper grid and dried in an oven at 40°C. The morphology, size, and dispersion were imaged using a high-angle annular dark-field scanning transmission electron microscope (HADDF) at 200 kV. Then, an energy dispersive spectroscopy (EDS) instrument was used to analyze the morphology, size, dispersion, and composition of the sample.

[0049] 5. Valence state analysis of Cu and Ce: 100 mg of CC was used to test the full spectrum of the sample and the fine spectra of Cu2p and Ce3d using X-ray photoelectron spectroscopy (XPS).

[0050] 6. Determination of UV absorption peaks: cRGD, CC@ICG and R&CC@ICG were dispersed in ultrapure water, and the UV absorption peaks of the three solutions were detected using a UV-Vis-NIR spectrophotometer. The similarities and differences of each absorption peak were compared to further evaluate the coupling status of c(RGDfK) peptide.

[0051] Experiment 1 test results: such as Figure 2As the incubation time increased from 10 minutes to 30 minutes, the diameter of CC nanocarriers significantly increased, decreasing from 25 nm to 80 nm. Furthermore, in a weakly alkaline environment, the surface of the CC nanocarriers underwent further etching, resulting in a rougher surface morphology, such as... Figure 3 Observation under magnification using transmission electron microscopy (TEM) Figure 5 ), and it was found that after loading ICG, CC@ICG ( Figure 5 The morphology and dispersion of the right image remained unchanged. Furthermore, according to the SEM image (…), Figure 6 The alloyed CC@ICG nanospheres exhibited greater water solubility and higher crystallinity, with a rougher interface. The elemental distribution of CC was characterized using high-angle annular dark-field scanning electron microscopy (HAADF-STEM), and the elemental distribution images (…) are shown in the images. Figure 7 ) and energy dispersive spectroscopy (EDS) Figure 8 All of them show that Cu, Ce, O and N elements coexist. Figure 4 As shown, in an aqueous solution at pH 6.5, CC@ICG degrades into small particles after 12 h and is essentially degraded after 36 h; in an aqueous solution at pH 7.2, CC@ICG retains its intact morphology after 36 h; this demonstrates its degradation performance in the extracellular matrix of tumor cells. Figure 9 In the full XPS spectrum of CC, Cu 2p and Ce 3d peaks are present throughout the entire XPS spectrum. Figure 9 (A); The Cu2p fine spectrum shows two characteristic peaks of Cu²⁺ at 934.2 eV and 942.17 eV, while the peaks of Cu⁺ appear at 932.8 eV and 952.5 eV ( ). Figure 9 In B), the Cu element in CC is mainly Cu. 2+ and Cu + Cu exists in the form of + The form exists in Cu 2+ The existing form has a stronger ability to induce copper death; the fine spectrum of Ce3d shows that the outer cerium oxide has Ce. 3+ and Ce 4+ Two forms exist ( Figure 9 The conversion between the valence states of C and Ce is beneficial for catalyzing Fenton-like reactions within tumors. For example... Figure 10 As shown, compared to CC@ICG, R&CC@ICG has a UV absorption peak near 258 nm, and c(RGDfK) also has an absorption peak at this point, confirming that c(RGDfK) has been successfully connected. Figure 11 The TEM image shows that the morphology and dispersion of R&CC@ICG did not change significantly compared to CC@ICG.

[0052] Experiment 2: Cell targeting assay of R&CC@ICG 1. MC38 cells were plated and cultured for 24 h. The final product probes from steps S3 and S5 were added, and the concentration of the final product was adjusted to be consistent, with a concentration of 150 μg / mL. PBS was used as a control. After incubation for different times (0.5, 1, 2, 4 h), the cells of each group were collected and flow cytometry was used to detect the average fluorescence intensity of each group of cells.

[0053] 2. After culturing MC38 cells on slides for 24 h, the two probes mentioned above in steps S3 and S5 were added respectively, with a final concentration of 150 μg / mL. After incubation for different times (0.5, 1, 2, 4 h), the slides were collected, fixed, stained with DAPI, mounted, photographed and observed under an upright fluorescence microscope, and the fluorescence intensity was counted.

[0054] 3. MC38 cells were plated and divided into a PBS control group, a probe group, and a c(RGDfK) blocking group. The PBS control group received a final concentration of 150 μg / mL of probe R&CC@ICG, while the c(RGDfK) blocking group received an equal volume of R&CC@ICG along with a final concentration of 40 μg / mL of free c(RGDfK). After 4 h of incubation, cells from each group were collected and flow cytometry was used to detect the mean fluorescence intensity of each group.

[0055] 4. MC38 cells were cultured on slides for 24 h and divided into a probe group and a c(RGDfK) blocking group. The probe R&CC@ICG was added to the probe group at a final concentration of 150 μg / mL, while the c(RGDfK) blocking group received an equal amount of R&CC@ICG along with free c(RGDfK) at a final concentration of 40 μg / mL. After 4 h of incubation, the slides were collected, fixed, stained with DAPI, mounted, and photographed under an upright fluorescence microscope. The fluorescence intensity was then counted.

[0056] Experiment 2 test results: such as Figure 12 Figure A shows the flow cytometry quantitative analysis results of MC38 cells incubated with PBS, CC@ICG, and R&CC@ICG at different time points. Figure 12 The fluorescence microscopy results of MC38 cells incubated with CC@ICG and R&CC@ICG for 4 h (scale bar = 100 μm) and corresponding fluorescence statistics are shown. MC38 cells exhibit higher uptake of R&CC@ICG, which can be blocked by excess c(RGDfK). Figure 12 (D, E, and F) demonstrate that the synthesized R&CC@ICG probe has specific targeting ability for colorectal cancer cells.

[0057] Experiment 3: Detection of R&CC@ICG-induced copper death in tumor cells and radiosensitization 1. Western blot assay to evaluate the ability of R&CC@IC to induce copper death: MC38 cells were plated, and 10 mg of the final product probe from step S5 was weighed and ultrasonically dispersed in 5 mL of deionized water to obtain a solution with a concentration of 2 mg / mL. At the same time, a solution containing the same molar mass of CuCl2 was prepared. After the cells adhered, different volumes of R&CC@ICG (0, 100, 150, 200, 250, 400 ug / mL) and CuCl2 (75 ug / mL) solutions were added and incubated for 24 h. Cells from each group were collected for subsequent Western blot analysis.

[0058] 2. Western blot experiment to evaluate the ability of R&CC@ICG combined with RT to induce copper death: MC38 cells were plated and cultured for 24 h. After cell attachment, they were divided into 4 groups: PBS (5 mL of culture medium alone), R&CC@ICG (15 mL of culture medium containing 150 μg / mL R&CC@ICG), RT alone, and R&CC@ICG + RT. Cells were treated for 12 h, washed 3 times with culture medium, and then irradiated with 4 Gy of radiotherapy. They were then cultured in a 37°C cell culture incubator containing 5% CO2 for 12 h. Cells from each group were collected for subsequent Western blot analysis.

[0059] 3. Colony formation assay to evaluate the radiosensitizing properties of R&CC@ICG: MC38 cells were plated and cultured for 24 h. After cell adhesion, they were divided into four groups: PBS (5 mL of culture medium alone), R&CC@ICG (15 mL of culture medium containing 150 μg / mL R&CC@ICG), radiotherapy alone (RT), and R&CC@ICG + radiotherapy (RT). Cells were treated for 4 h, washed three times with culture medium, and then irradiated with radiotherapy doses of 0-2-4-6 Gy. They were then cultured at 37°C in a cell culture incubator containing 5% CO2 for 10-14 days. After culturing, cell colonies were gently washed with PBS, fixed with 4% paraformaldehyde, stained with 0.5% crystal violet for 20 min, washed, and dried at room temperature. Colonies of more than 50 cells were counted under a microscope, and the viability fraction was calculated.

[0060] 4. Fluorescence microscopy detection of intracellular γ-H2AX changes after R&CC@ICG combined with radiotherapy: MC38 cells were cultured on slides for 24 h. After cell adhesion, they were divided into four groups: PBS (5 mL culture medium alone), R&CC@ICG (15 mL culture medium containing 150 μg / mL R&CC@ICG), radiotherapy alone (RT), and R&CC@ICG + radiotherapy (RT). Cells were treated for 12 h, washed three times with culture medium, and then irradiated with a 4 Gy radiotherapy dose. They were then cultured at 37°C in a 5% CO2 incubator for 12 h. The culture medium was removed, and the cells were washed with PBS and fixed with fixative. After washing, immunostaining blocking solution was added, and the cells were blocked at room temperature for 20 minutes. The immunostaining blocking solution was removed, and rabbit γ-H2AX monoclonal antibody was added. After incubation at room temperature for 1 hour, the cells were washed with washing solution, and then anti-rabbit 488 was added and incubated at room temperature for 1 hour. Finally, nuclear staining solution (DAPI) was added, and the cells were stained at room temperature for 5 minutes. Add an appropriate amount of anti-fluorescence quenching mounting solution, cover with a coverslip, and observe under a fluorescence microscope.

[0061] Test results of Experiment 3: such as Figure 13 In this study, MC38 cells were treated with different concentrations of R&CC@ICG and CuCl2, and the expression of copper death-related marker proteins was detected. The results showed significant DLAT oligomerization in MC38 cells, with a decrease in DLAT monomers. Furthermore, compared with the blank control group, the expression of key proteins in copper death, ACO2, LIAS, FDX1, and POLD1, was downregulated, while HSP70 expression was upregulated, a trend consistent with the positive control group (CuCl2). Further investigation was conducted to determine the incidence of copper death in each group, whether or not R&CC@ICG was combined with RT. Figure 13 The results of the B-cell assay showed that the levels of DLAT, ACO2, LIAS, FDX1, and POLD1 were significantly reduced, especially in the R&CC@ICG+RT group of MC38 cells, which exhibited stronger copper death and better post-radiotherapy effects. Figure 13In a study, colony counts and survival fractions were performed on PBS (2 mL of culture medium alone), R&CC@ICG (2 mL of culture medium containing 150 μg / mL R&CC@ICG), radiotherapy alone (RT), and R&CC@ICG+RT radiotherapy groups. The results showed that at the same radiation dose, the colony count and survival fraction in the R&CC@ICG+RT group were significantly lower than those in the radiotherapy alone group, and the colony count decreased with increasing radiotherapy dose, demonstrating that the synthesized R&CC@ICG probe has excellent radiosensitizing and killing ability against tumor cells. When DNA is damaged, cells rapidly initiate a series of cell signaling events involving post-translational phosphorylation. Once DNA double-strand breaks (DSBs) occur, histone H2AX undergoes rapid phosphorylation, forming γH2AX foci (lesions). Figure 13 MC38 cells treated with PBS, R&CC@ICG, 4 Gy, and R&CC@ICG+4 Gy were stained with γ-H2AX. Fluorescence microscopy and statistical analysis showed that the fluorescence intensity of the R&CC@ICG+RT radiotherapy group was significantly higher than that of the radiotherapy-only group and the probe-only group, demonstrating that R&CC@ICG-induced copper death can effectively promote radiotherapy-induced DNA damage, thereby sensitizing cells to radiotherapy.

[0062] Experiment 4: Detection of in vitro antitumor activity of R&CC@ICG combined with radiotherapy 1. Flow cytometry analysis of the effect of R&CC@ICG combined with radiotherapy on apoptosis: MC38 cells were plated and cultured for 24 h. After cell adhesion, they were divided into four groups: PBS (2 mL culture medium alone), R&CC@ICG (2 mL culture medium containing 150 μg / mL R&CC@ICG), radiotherapy alone (4 Gy), and R&CC@ICG + radiotherapy (4 Gy). Cells were treated for 4 h, washed three times with culture medium, and then irradiated with a 4 Gy radiotherapy dose. They were then cultured at 37°C in a 5% CO2 incubator for another 24 h. After digestion, centrifugation, and washing with physiological saline, the cells were collected and dispersed in Binding Buffer. Annexin V-FITC was added at room temperature, the cells were gently mixed, and stained in the dark for 15 min. Propidium iodide staining solution was then added, the cells were gently mixed, and stained for 5 min. The fluorescence intensity of Annexin V-FITC produced by apoptosis was detected by flow cytometry and statistically analyzed.

[0063] 2. Fluorescence microscopy detection of the effect of R&CC@ICG combined with radiotherapy on cell apoptosis: MC38 cells were seeded in 96-well plates and, after cell attachment, divided into four groups: PBS (2 mL of culture medium alone), R&CC@ICG (2 mL of culture medium containing 150 μg / mL R&CC@ICG), radiotherapy alone (4 Gy), and R&CC@ICG + radiotherapy (4 Gy). Cells were treated for 4 h, washed three times with culture medium, and then irradiated with a 4 Gy radiotherapy dose. The cells were then incubated at 37°C in a 5% CO2 cell culture incubator for 12 h. The culture medium was removed, and the cells were washed once with PBS. 100 μL of Calcein AM / PI detection working solution was added to each well, and the cells were incubated at 37°C in the dark for 30 min. After incubation, the staining effect was observed under a fluorescence microscope (Calcein AM is green fluorescence, Ex / Em=494 / 517nm; PI is red fluorescence, Ex / Em=535 / 617nm), and the average fluorescence intensity of each group was analyzed after taking pictures.

[0064] 3. Flow cytometry and microscopy analysis of changes in intracellular ROS production after combined R&CC@ICG radiotherapy: MC38 cells were plated and cultured for 24 h. After cell adhesion, they were divided into four groups: PBS (2 mL culture medium alone), R&CC@ICG (2 mL culture medium containing 150 μg / mL R&CC@ICG), radiotherapy alone (4 Gy), and R&CC@ICG + radiotherapy (4 Gy). Cells were treated for 4 h, washed three times with culture medium, and then irradiated with a 4 Gy radiotherapy dose. They were then cultured at 37°C in a 5% CO2 incubator for 12 h. For flow cytometry analysis, DCFH-DA was diluted 1:1000 with probe dilution buffer to a final concentration of 10 μM / L. After collection, cells were suspended in diluted DCFH-DA at a concentration of one to twenty million cells / mL and incubated at 37ºC for 30 minutes. After incubation, cells were washed three times with PBS solution and then detected and analyzed by flow cytometry.

[0065] For fluorescence microscopy detection, 1 mL of the fluorescent probe DCFH-DA diluted 1:1000 was added to adherent cells in each treatment group and incubated for 30 min. After incubation, the staining solution in the well was carefully removed, and an appropriate amount of PBS was slowly added along the well wall to wash the cell spheres once and then replaced with complete culture medium. The fluorescence staining effect was then observed under a fluorescence microscope, and the trend of the change in the average fluorescence intensity of each group was analyzed after taking pictures.

[0066] 4. Detection of the effect of R&CC@ICG on GSH levels: After MC38 cells were plated and cultured for 24 h, the medium was replaced with R&CC@ICG medium containing (PBS, 50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL) for 12 h. Cells were washed with PBS, collected, and protein removal reagent M solution was added. Each group of cells underwent two rapid freeze-thaw cycles using liquid nitrogen and a 37°C water bath, followed by incubation at 4°C for 5 minutes and centrifugation. The supernatant was used for the determination of total glutathione. A separate sample of the prepared total glutathione content was added to diluted GSH removal auxiliary solution, followed by GSH removal working solution. The mixture was incubated at 25°C for 60 minutes, and then 0.5 mg / mL NADPH solution was added and mixed thoroughly. Immediately after 30 minutes, the absorbance of A412 was measured using a microplate reader along with the standard. The GSH content can be calculated based on the measured total glutathione and GSSG content. The calculation formula is: GSH = Total Glutathione - GSSG × 2.

[0067] 5. Effect of R&CC@ICG on Cell Cycle: MC38 cells were plated and cultured for 24 h. After cell adhesion, they were divided into four groups: PBS (2 mL culture medium alone), R&CC@ICG (2 mL culture medium containing 150 μg / mL R&CC@ICG), radiotherapy alone (4 Gy), and R&CC@ICG + radiotherapy (4 Gy). Cells were treated for 4 h, washed three times with culture medium, and then irradiated with a 4 Gy dose of radiotherapy. They were then cultured at 37°C in a 5% CO2 incubator for 12 h. The cell culture medium was collected into centrifuge tubes for later use. Cells were digested and the collected cell culture medium was added. All adherent cells were pipetted off and gently dispersed. The cells were collected again into centrifuge tubes. Centrifugation was performed for 5 minutes, and the supernatant was discarded. Approximately 1 mL of ice-cold PBS was added to resuspend the cells. The cells were centrifuged again, and the supernatant was aspirated. Then, 1 mL of ice-cold 70% ethanol was added for fixation at 4°C for 2 hours, followed by centrifugation for 5 minutes to collect the cells. Add 0.5 mL of propidium iodide staining solution to each cell sample, slowly and thoroughly resuspend the cell pellet, incubate at 37°C in the dark for 30 minutes, and perform flow cytometry detection and statistical analysis after staining.

[0068] The test results of Experiment 4: Figure 14 The apoptosis rates of cells in the following groups were compared: AB, PBS (2 mL of culture medium alone), R&CC@ICG (2 mL of culture medium containing 150 μg / mL R&CC@ICG), radiotherapy alone (RT alone), and R&CC@ICG + radiotherapy RT. The results showed that the apoptosis rate in the R&CC@ICG + radiotherapy group was as high as 15.6%, significantly higher than the 9.9% in the radiotherapy alone group and the 5.0% in the probe alone group. Figure 14The cell live / dead staining images shown in C demonstrate that the synthesized R&CC@ICG probe has the ability to mediate copper death and enhance radiosensitization in tumor cells. Figure 14 The study compared intracellular ROS production in PBS (2 mL of culture medium alone), R&CC@ICG (2 mL of culture medium containing 150 μg / mL R&CC@ICG), radiotherapy alone, and R&CC@ICG + radiotherapy groups. Flow cytometry and fluorescence imaging results showed that the R&CC@ICG + radiotherapy group produced significantly more reactive oxygen species than the radiotherapy alone and probe alone groups. Simultaneously, R&CC@ICG demonstrated the ability to deplete glutathione, such as... Figure 14 As shown in Figure F, the synthesized R&CC@ICG probe demonstrates excellent killing ability against tumor cells. Cell cycle arrest is considered a key strategy in many anticancer drugs; flow cytometry was used to detect the cell cycle distribution of MC38 cells after treatment with different groups. Figure 14 As shown in Figure G, compared with the other three groups, the cell cycle of the R&CC@ICG+RT (4 Gy) group was significantly altered, exhibiting G2 / M phase-induced cell cycle arrest. This demonstrates that the synthesized R&CC@ICG probe possesses excellent in vitro antitumor activity against tumor cells.

[0069] Experiment 5: In vivo tumor targeting detection of R&CC@ICG in MC38 subcutaneous xenograft mouse model Six MC38 subcutaneous xenograft mice were randomly divided into CC@ICG and R&CC@ICG groups. Each group of mice received an injection of the corresponding probe via tail vein. NIR-II images of the mice were acquired using a NIR-II small animal imaging device at different time points (1, 6, 12, 24, 36, 48, 72, and 96 h) after injection. The signal-to-background ratio (SNR) differences between the groups at different time points were analyzed using PSLViewer software. Additionally, six more MC38 subcutaneous xenograft mice were randomly divided into an R&CC@ICG group and a Blocking group. The Blocking group received an injection of 80 mg / kg cRGD in PBS solution 30 minutes before the injection of the same concentration of R&CC@ICG probe. NIR-II images of the mice were acquired using a NIR-II small animal imaging device, and the SNR differences between the groups at different time points were analyzed using PSLViewer software.

[0070] Experiment 5 Test Results: NIR-II fluorescence images of tumor sites at different time points (1, 6, 12, 24, 36, 48, 72, 96 h) after injection of CC@ICG (top) and R&CC@ICG (bottom). Figure 15 (A) and corresponding time point TBR quantitative analysis ( Figure 15As shown in Figure B), after tail vein injection of the probe in MC38 tumor-bearing mice, the TBR of the R&CC@ICG group was higher than that of the CC@ICG group at all time points, and the TBR of the R&CC@ICG group reached its highest point at 48 h. This demonstrates that R&CC@ICG has a better tumor enrichment effect than CC@ICG. Figure 15 In the Blocking group, which was pre-injected with c(RGDfK) into CD4, fluorescence imaging 48 hours after injection of the R&CC@ICG probe showed a significant decrease in signal intensity compared to the R&CC@ICG group. These results demonstrate that R&CC@ICG has excellent tumor enrichment efficiency.

[0071] Experiment 6: Investigating the efficacy and biosafety of R&CC@ICG-mediated copper death combined with radiosensitization in a subcutaneous xenograft mouse model. Sixteen mice with subcutaneous xenograft tumors (tumor volume 50 mm) were used. 3 Mice were randomly divided into four groups: a control group (PBS), an R&CC@ICG group, a PBS + RT (6 Gy) group, and an R&CC@ICG + RT (6 Gy). Each group received two rounds of treatment according to a predetermined protocol: for the first six days, mice received either a 200 μL injection of PBS or a 200 μL injection of R&CC@ICG (5 mg / kg) nanoprobe via tail vein daily, and on days 2 and 4, mice were irradiated with 0 or 6 Gy into the abdomen, respectively. Radiotherapy was performed using a support with a lead collimator to protect the head, neck, and chest of the mice and prevent radiation interference to normal tissues. Every two days after radiotherapy, the long diameter (L) and short diameter (W) of the tumor were measured using calipers, and the volume was calculated according to the formula: "Volume = L × W". 2 Tumor volume was calculated using a 2" method, and body weight changes were measured and recorded every two days. On day 14, blood was collected from mice to detect blood biochemical indicators such as creatinine, blood urea nitrogen, and alanine aminotransferase, as well as blood cytological indicators such as white blood cells, red blood cells, and platelets. Mice in each group were immediately euthanized by cervical dislocation after blood collection, tumors were removed, and white light images were taken. The final weight of the tumors was weighed and recorded. Finally, the heart, liver, kidney, brain, lung, and spleen were collected for H&E staining. The pathological state of the organs and tissues in each group of mice was observed. Finally, tumors from each group of mice were subjected to immunohistochemistry and immunofluorescence staining for copper death markers such as LIAS, ACO2, and DLAT, as well as proliferation indicators KI67 and TUNEL, to detect their expression levels in small animal tumor tissues, in order to assess their ability to induce copper death and synergistically enhance radiosensitivity in vivo.

[0072] Experiment 6 test results: such as Figure 16 The treatment plan shown in Figure A will be followed. Figure 16Tumor size increased rapidly in the BD, PBS groups, or R&CC@ICG-only treatment groups. Therefore, R&CC@ICG treatment alone is unlikely to completely eradicate the tumor. Compared with radiotherapy alone, the R&CC@ICG + radiotherapy group showed a significant tumor growth inhibition effect. Final tumor size and weight ( Figure 16 The presence of E in the middle also strongly supports the radiosensitizing effect of R&CC@ICG. For example... Figure 16 In the R&CC@ICG + radiotherapy group, compared with the other three groups, the expression of LIAS, ACO2, and DLAT was significantly reduced, confirming the successful induction of copper death and improving radiosensitivity. Furthermore, compared with the untreated group, the proportion of Ki67-positive cells was lower and the proportion of TUNEL-positive cells was higher in all treatment groups. The significant decrease in Ki67 and the significant increase in TUNEL indicate that R&CC@ICG combined with radiotherapy can effectively inhibit tumor proliferation. These data suggest that R&CC@ICG-induced copper death has promising applications in enhancing radiotherapy sensitivity. Figure 17 HE staining of A and Figure 17 Hematological analysis by BC showed that no significant in vivo toxicity or organ damage, or weight loss caused by R&CC@ICG was observed within 14 days after radiotherapy. Figure 16 (Middle F).

[0073] Experiment 7: Investigating the in vivo performance of R&CC@ICG against colorectal cancer and lung metastasis. 1. First, administer a subcutaneous injection of 3×10 6 A subcutaneous tumor mouse model was constructed using MC38-luc cells, and the tumors were allowed to grow to 500-600 mm. 3 Subcutaneous xenografts were dissected and minced to the size of rice grains. Mice were then anesthetized with isoflurane and surgically implanted under sterile conditions. The abdomen was incised to expose the cecum. The rice-grain-sized xenografts were attached to the ileocecal region using bio-adhesive. Seven days after inoculation, all mice were intraperitoneally injected with d-fluorescein (150 mg kg⁻¹), and the development of orthotopic colorectal cancer was assessed using a small animal biofluorescence imaging system. Subsequently, mice were randomly divided into four groups of six mice each: control group (PBS), R&CC@ICG group, PBS + RT (6 Gy) group, and R&CC@ICG + RT (6 Gy). For the first six days, mice were injected daily via tail vein with R&CC@ICG (5 mg / kg) nanoprobes, and the abdomen was irradiated with 0 or 6 Gy on days 2 and 4, respectively. Small animal in vivo imaging was performed every 5 days until the end of monitoring on day 15. Mice were sacrificed, and the tumors in the ileocecal region were collected, photographed, weighed, and subsequently stained with hematoxylin and eosin (H&E).

[0074] 2. Lung metastasis inhibition experiment: Sixteen female C57BL / 6J mice (4-6 weeks old) were injected via the tail vein with 100 μl containing 1 × 10⁻⁶ mmol / L. 6 A colorectal cancer lung metastasis model was established using PBS solution with MC38 cells (cell viability >90%). Seven days after inoculation, mice were randomly divided into a control group (PBS), an R&CC@ICG group, a PBS + RT (6 Gy) group, and an R&CC@ICG + RT (6 Gy) group. For the first six days, mice were injected daily via tail vein with either 200 μl of PBS or 200 μl of R&CC@ICG (5 mg / kg) nanoprobe. The experiment ended on day 14, after which mice were sacrificed and lung tissue was collected. The lung tissue was photographed, and the number of tumor nodules was quantified. Subsequently, histopathological analysis of the lung tissue was performed.

[0075] Experiment 7 test results: such as Figure 18 In vivo imaging of small animals (A-type) showed that tumors continued to grow in the control group, while tumor growth was inhibited in the R&CC@ICG and RT groups alone. The R&CC@ICG+RT group exhibited the most significant tumor inhibition, highlighting the synergistic effect of R&CC@ICG through copper death-mediated radiosensitization. Semi-quantitative data confirmed these findings, showing significant differences between the treatment groups. Figure 18 In the case of B, after a 15-day experiment, the in situ colorectal tumor was isolated and further confirmed by H&E staining. Figure 18 (Middle E). Compared with all other groups, the tumor volume in the R&CC@ICG+RT group was significantly reduced, which further highlights the effectiveness of R&CC@ICG combined with radiotherapy in the treatment of colorectal cancer. Figure 18 Medium CD).

[0076] The therapeutic effect of R&CC@ICG in the MC38-luc lung metastasis model was then evaluated. After 14 days of treatment, all mice were euthanized, and lung tissue was collected to visually represent the tumor nodules on the front and back of the lungs in each group. Figure 19 (A) Figure 19 The nodule count in the BC tumor study showed an average of 96 nodules in the control group, 50 nodules in the R&CC@ICG group, 16 nodules in the RT group, and only 2 nodules in the R&CC@ICG+RT group. These results indicate that R&CC@ICG combined with RT helps reduce the tumor burden of colorectal cancer lung metastases.

[0077] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A copper-cerium rare-earth alloy nanocomposite, characterized in that, The copper-cerium rare earth alloy nanocomposite comprises solid Cu and Ce rare earth nanoparticles, as well as cyclic polypeptide c (RGDfK) and fluorescent dye located on the surface of the solid Cu and Ce rare earth nanoparticles.

2. The copper-cerium rare-earth alloy nanocomposite according to claim 1, characterized in that, The cyclic polypeptide c (RGDfK) is coupled to the surface of the Cu and Ce solid rare earth nanoparticles by modifying PEG2000-NH2 on the Cu and Ce solid rare earth nanoparticles.

3. The copper-cerium rare-earth alloy nanocomposite according to claim 1, characterized in that, In the copper-cerium rare earth alloy nanocomposite, the Cu element includes Cu. 2+ and Cu + The existence form of Ce element contains Ce 3+ and Ce 4+ The form of existence.

4. The copper-cerium rare-earth alloy nanocomposite according to claim 1, characterized in that, The particle size of the copper-cerium rare earth alloy nanocomposite is 20~100 nm.

5. The copper-cerium rare-earth alloy nanocomposite according to claim 1, characterized in that, The content of Ce element in the copper-cerium rare earth alloy nanocomposite is 5-25%, the content of Cu element in the copper-cerium rare earth alloy nanocomposite is 30-80%, and the content of the cyclic polypeptide c (RGDfK) in the copper-cerium rare earth alloy nanocomposite is 1-20%.

6. The copper-cerium rare-earth alloy nanocomposite according to claim 1, characterized in that, The fluorescent dye is indocyanine green (ICG). Preferably, the content of indocyanine green (ICG) in the copper-cerium rare earth alloy nanocomposite is 1-20%.

7. The method for preparing the copper-cerium rare-earth alloy nanocomposite according to any one of claims 1-6, characterized in that, The preparation method includes the following steps: (1) Cerium salt, copper salt and hexamethylenetetramine were reacted in an aqueous solvent to obtain solid rare earth nanoparticles containing Cu and Ce (denoted as CC). (2) The CC and ICG are reacted in an aqueous solution by stirring to obtain the product CC@ICG; (3) The CC@ICG and PEG2000-NH2 are reacted in an alcohol solvent to obtain CC@ICG&NH2 nanoparticles; (4) The CC@ICG&NH2 nanoparticles, cyclic polypeptide c(RGDfK), EDC and NHS are reacted in an aqueous solvent to obtain the copper-cerium rare earth alloy nanocomposite (denoted as R&CC@ICG).

8. The use of the rare earth nanomaterials according to any one of claims 1-6 as tumor-targeting probes; Preferably, the tumor-targeting probe is a colorectal cancer-targeting probe.

9. The use of the rare earth nanomaterials according to any one of claims 1-6 as radiosensitizers; Preferably, the radiosensitizer is a radiosensitizer for colorectal cancer.

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