Preparation method and application of copper selenide-platinum composite material

Abstract

This invention relates to a method for preparing and applying a copper selenide@platinum composite material. H₂SeO₃ and polyvinylpyrrolidone (PVP) are dissolved in deionized water, stirred, and then hydrazine hydrate solution is added. After stirring at room temperature, the precipitate is washed and dispersed in water to obtain a selenium nanosphere dispersion. The selenium nanosphere dispersion is mixed with PVP solution, K₂PtCl₄ solution, and anhydrous ethanol, and stirred at 50–70°C. After centrifugation and washing, the precipitate is dispersed in water to obtain a selenium@platinum nanoparticle dispersion. The selenium@platinum nanoparticle dispersion is mixed with PVP solution, copper salt solution, and ascorbic acid solution, stirred, and then H₂O₂ solution is added. After stirring and reacting, the precipitate is centrifuged and washed to obtain the copper selenide@platinum composite material. Copper selenide, as a photothermal material, has excellent photothermal conversion performance and can achieve photothermal therapy for tumors. The platinum nanoparticles grown on its surface have a radiosensitizing effect, which can further enhance the effect of radiotherapy. The copper selenide@platinum composite material prepared in this invention enables combined RT / PTT therapy for tumors.

Description

Technical Field

[0001] This invention relates to the field of radiosensitization and the synergistic treatment of tumors by photothermal therapy and radiotherapy, specifically to a method for preparing and applying a copper selenide@platinum composite material. Background Technology

[0002] Among major treatment strategies, radiotherapy (RT) is a powerful approach used for over a century. It exerts its tumor-killing effect non-invasively by directly damaging DNA and indirectly generating cytotoxic reactive oxygen species (ROS). As a classic cancer treatment method, RT has unique advantages, such as not being limited by tissue depth. However, some inherent limitations of RT restrict its long-term application, mainly in the following three aspects: 1) Ionizing radiation used to kill tumors may damage adjacent normal tissues, resulting in severe toxic side effects on the body. 2) The hypoxic nature of the tumor microenvironment (TME) may cause tumor cells to develop radiation resistance, ultimately leading to tumor eradication failure. 3) Since elevated antioxidants are a general characteristic of cancer phenotypes, the antioxidant system within cancer cells will prevent them from combating these damaging effects by quenching excess free radicals. For example, the concentration of glutathione (GSH) in cancer cells is approximately four times that in normal cells, which has the ability to inhibit ROS before they reach their target sites, ultimately significantly reducing radiation efficiency.

[0003] Over the past two decades, the rapid development of emerging advanced nanomaterials and nanobiotechnology has provided a promising opportunity for tumor radiosensitization. Various nanomaterials have been extensively studied and applied to enhance the efficacy of radiotherapy, thanks to their versatile physicochemical properties, such as good biocompatibility, inherent radiosensitive activity, high loading capacity for multiple types of drugs, and enhanced tumor tissue penetration and retention (EPR) effects.

[0004] Previously, nanomaterial-mediated radiosensitization methods mainly focused on enhancing intracellular radiation energy deposition through high Z (atomic number) nanoradiosensitizers. In recent years, numerous new nanoradiosensitizers and strategies have been proposed. These nanoradiosensitizers and strategies not only enhance ionizing radiation energy deposition but also possess the ability to catalyze ROS production, manipulate TME (such as reducing intracellular glutathione (GSH) concentration, converting low-toxicity intracellular H2O2 into highly reactive HO·, and improving tumor oxygen levels), or regulate cell cycle / signaling pathways, making cancer cells more sensitive to radiation. This significantly enhances the efficacy of radiotherapy (RT) while reducing its side effects. For example, photocatalytic semiconductor nanoparticles can be activated by X-rays to promote ROS production, thereby enhancing the radiation effect. Some nanomaterials have the ability to deplete glutathione (GSH) in tumors, thus preventing the clearance of radiation-induced ROS from glutathione (GSH) and ultimately improving treatment outcomes. Some iron (Fe) / copper (Cu)-based nanomaterials can convert intracellular H₂O₂ into highly reactive HO·, thereby damaging cancer cells and enhancing the efficacy of radiotherapy (RT). Nanomaterials with the ability to improve oxygen levels can effectively overcome tumor hypoxia, reduce radiation resistance, and ultimately improve radiation efficiency.

[0005] Another fundamental treatment strategy to improve the efficacy of radiotherapy (RT) is to combine useful therapies with RT to produce a synergistic effect. Photothermal therapy (PTT) has been reported as one of the most effective candidates for combined RT treatment. By using photothermal agents that absorb in the near-infrared (NIR) range, PTT can convert NIR light energy into heat energy for tumor ablation. On the one hand, the high temperature induced during PTT can enhance radiosensitivity by increasing intratumoral blood flow, thereby enhancing oxygenation and reversing hypoxia-induced radioresistance. The high temperature causes double-strand DNA breaks, preventing DNA repair after RT. In addition, thermotherapy can kill radiation-insensitive S-phase cancer cells, thus improving the efficiency of radiotherapy. On the other hand, due to its high penetration ability, RT can compensate for the incomplete ablation of deep tumors by NIR. Therefore, nanomaterials with photothermal ablation and radiosensitization properties hold promise for improving treatment outcomes. Inspired by this, this invention prepares nanoparticles for combined RT / PTT treatment of cancer, which can improve hypoxia at the tumor site. Summary of the Invention

[0006] To address the aforementioned issues, this invention provides a method for preparing and applying a copper selenide@platinum composite material. Utilizing copper selenide as a photothermal material, copper selenide nanospheres exhibit excellent photothermal conversion performance under near-infrared light irradiation, enabling photothermal therapy of tumors. Meanwhile, high-Z (atomic number) platinum nanoparticles grown on its surface possess radiosensitizing effects, and platinum can catalyze the decomposition of H2O2 at the tumor site, overcoming tumor hypoxia and further enhancing the efficacy of radiotherapy.

[0007] This invention is specifically achieved through the following technical solution: a method for preparing a copper selenide@platinum composite material according to this invention includes the following steps:

[0008] (1) Dissolve H2SeO3 and polyvinylpyrrolidone in deionized water and stir continuously for 10-30 min. Then add a certain amount of hydrazine hydrate solution and react at room temperature for 3-6 h under stirring. Centrifuge the product obtained from the reaction. Wash the precipitate obtained from centrifugation with deionized water and then disperse it in deionized water to obtain a 0.1-10 mg / mL selenium nanosphere dispersion.

[0009] (2) Take the selenium nanosphere dispersion prepared in step (1), mix it with polyvinylpyrrolidone aqueous solution, K2PtCl4 aqueous solution and anhydrous ethanol in a volume ratio of 6:2~6:0.01~0.1:0~50, stir and react at 50~70℃ for 5~12h. After stirring, centrifuge the obtained product, collect the precipitate obtained by centrifugation, wash it with deionized water, and then disperse it in deionized water to obtain a selenium@platinum nanoparticle dispersion of 0.1~10mg / mL;

[0010] (3) The selenium@platinum nanoparticle dispersion, polyvinylpyrrolidone aqueous solution, copper salt aqueous solution and ascorbic acid aqueous solution prepared in step (2) are added to the same container in sequence and stirred for 3 to 10 minutes. Then, hydrogen peroxide aqueous solution is added and stirred for 3 to 10 minutes. After stopping the stirring, the product is centrifuged and the precipitate obtained by centrifugation is washed with deionized water to obtain copper selenide@platinum composite material.

[0011] Preferably, in step (1), the mass ratio of H2SeO3 to polyvinylpyrrolidone is 0.774:0.2-1.2, the mass ratio of H2SeO3 to the volume of hydrazine hydrate solution is 0.774 g:1.5-3 mL, and the volume ratio of deionized water used to dissolve H2SeO3 and polyvinylpyrrolidone to the volume of hydrazine hydrate solution is 100-200:1.5-3.

[0012] Preferably, the hydrazine hydrate solution has a mass fraction of 80%.

[0013] Preferably, in step (2), the concentration of the polyvinylpyrrolidone aqueous solution is 0.01-0.1 g / mL, the concentration of the K2PtCl4 aqueous solution is 500 mmol / L, the centrifuge speed is 12000 rpm / min, and the centrifugation time is 10 min.

[0014] Preferably, in step (3), the volume ratio of the selenium@platinum nanoparticle dispersion, the polyvinylpyrrolidone aqueous solution, the copper salt aqueous solution, and the ascorbic acid aqueous solution is 1:1:0.5 to 0.7:2.

[0015] Preferably, in step (3), the concentration of the polyvinylpyrrolidone aqueous solution is 5-15 mg / mL, the concentration of the copper salt aqueous solution is 0.05-0.15 mmol / L, the concentration of the ascorbic acid aqueous solution is 0.01-5 mol / L, and the copper salt is at least one of copper chloride, copper nitrate, and copper sulfate.

[0016] Preferably, in step (3), the mass fraction of the hydrogen peroxide aqueous solution is 30%, and the volume ratio of the hydrogen peroxide aqueous solution to the selenium@platinum nanoparticle dispersion is 40-80 μL: 1 mL.

[0017] The present invention also provides a copper selenide@platinum composite material obtained by the above preparation method, wherein the copper selenide@platinum composite material has a core-shell structure, with copper selenide nanospheres as the core and platinum nanoparticles as the shell grown on the surface of copper selenide nanospheres.

[0018] The copper selenide@platinum composite material obtained by the above preparation method can be used as a radiosensitizer and photothermal material for tumor treatment. In this material, copper selenide nanospheres act as photothermal materials, and platinum nanoparticles on their surface act as radiosensitizers. The synergistic effect of the two achieves combined RT / PTT therapy for tumors.

[0019] Compared with existing technologies, this invention has significant advantages and beneficial effects. Through the above technical solution, this invention achieves considerable technological advancement and practicality, and has broad application value, possessing at least the following advantages:

[0020] (1) This invention unifies radiosensitization and photothermal therapy onto a copper selenide@platinum composite material. This composite material has a distinct core-shell structure, with copper selenide nanospheres as the core and Pt nanoparticles as the shell, grown on the surface of the copper selenide nanospheres. The copper selenide@platinum composite nanomaterial can generate a strong plasma resonance effect. Copper selenide, as a photothermal material, has excellent photothermal conversion performance, and can efficiently absorb near-infrared light and convert light energy into heat energy to achieve photothermal therapy for tumors. Meanwhile, the high Z (atomic number) platinum nanoparticles grown on its surface have a radiosensitization effect, acting as a radiosensitizer. Furthermore, platinum can catalyze the decomposition of H2O2 at the tumor site to produce highly active substances, overcoming the problem of tumor hypoxia and further improving the effect of radiotherapy.

[0021] (2) The copper selenide@platinum composite material (CS@Pt) prepared in this invention has excellent photothermal properties, with a power density of 1.0 W / cm². -2 Under irradiation with a 980nm near-infrared laser, 100μg mL -1The CS@Pt dispersion can reach a temperature of 69°C within 8 minutes. After five heating-cooling cycles, CS@Pt still exhibits highly efficient light absorption and photothermal conversion characteristics, thus demonstrating good cyclic stability. Experiments show that CS@Pt also possesses good biocompatibility and significant radiosensitization effects, enhancing X-ray damage to tumor cell DNA and inhibiting tumor cell proliferation. At a radiation dose of 6 Gy, CS@Pt achieved 98% inhibition of tumor cell proliferation, compared to only 62% in the control group without CS@Pt. The combination of CS@Pt with radiotherapy and photothermal therapy effectively inhibits tumor cell clonal activity, significantly increasing tumor cell mortality. Therefore, the CS@Pt composite material prepared in this invention possesses excellent photothermal properties, biocompatibility, and radiosensitization effects, enhancing the tumor-killing power of radiotherapy and enabling combined RT / PTT therapy for tumors.

[0022] (3) The preparation method provided by the present invention is simple and convenient, the reaction process is easy to control, the yield is high, and it is suitable for large-scale production. Attached Figure Description

[0023] Figure 1 This is a schematic diagram illustrating the preparation process of the copper selenide@platinum composite material (CS@Pt) of this invention and its application in tumor treatment.

[0024] Figure 2 These are transmission electron micrographs of CS@Pt prepared in Examples 1 and 2, where (a) corresponds to Example 1 and (b) corresponds to Example 2.

[0025] Figure 3 This is the XRD pattern of CS@Pt prepared in Example 2.

[0026] Figure 4 The CS@Pt dispersions of different concentrations prepared in Example 2 were subjected to a 1W cm⁻¹ test. -2 Temperature rise curve within 8 minutes of near-infrared laser irradiation.

[0027] Figure 5 The CS@Pt dispersion prepared in Example 2 was subjected to a 1W cm⁻¹ concentration. -2 A graph showing the temperature curve after five heating-cooling cycles under near-infrared laser irradiation.

[0028] Figure 6 This is a biocompatibility test diagram of CS@Pt prepared in Example 2 with esophageal squamous cell carcinoma cells (KYSE30) and human venous endothelial cells (HUVEC).

[0029] Figure 7 Images show the clonal formation of KYSE30 cells in the CS@Pt and Control groups under different radiation doses.

[0030] Figure 8 This is a graph showing DNA damage detection in KYSE30 cells from the CS@Pt group and the Control group under a 4 Gy radiation dose.

[0031] Figure 9 This is a fluorescence image of reactive oxygen species levels in KYSE30 cells under different treatment conditions.

[0032] Figure 10 These are images of KYSE30 cell clone formation under different treatment conditions.

[0033] Figure 11 These are images showing the liveness and death staining of KYSE30 cells under different treatment conditions.

[0034] Figure 12 This is a schematic diagram of the operation procedure for in vivo experiments on mice.

[0035] Figure 13 These are optical photographs of tumors in mice from six experimental groups that underwent different treatments.

[0036] Figure 14 This is a comparison chart of the average tumor mass in mice from six experimental groups that underwent different treatments. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] The present invention will be described in detail below with reference to specific embodiments. Unless otherwise specified, all conditions in the embodiments are performed under conventional conditions or conditions recommended by the manufacturer. Raw materials and reagents whose manufacturers are not specified are all commercially available products.

[0039] Example 1:

[0040] (1) Preparation of Se nanosphere dispersion: 774 mg H2SeO3 and 1.2 g polyvinylpyrrolidone (PVP) were dissolved in 100 mL of deionized water and stirred continuously for 10 min. Then, 2.2 mL of 80% N2H4·H2O solution was added dropwise. The reaction was carried out at room temperature for 3 h under stirring. The reaction system turned red. The system was centrifuged at 10000 r / min for 10 min. The precipitate was collected and washed three times with deionized water. The precipitate after washing was the selenium nanospheres. The obtained selenium nanospheres were dispersed in deionized water to prepare a selenium nanosphere dispersion with a concentration of 4 mg / mL.

[0041] (2) Take 3 mL of the selenium nanosphere dispersion prepared in step (1) and mix it with 3 mL of PVP aqueous solution (concentration of 0.1 g / mL) and 0.05 mL of K2PtCl4 aqueous solution (concentration of 500 mmol / L). Stir the mixture at 50 °C for 5 h. After stirring, centrifuge the product at 10000 r / min for 10 min, collect the precipitate obtained by centrifugation and wash it with deionized water. Disperse the washed precipitate in deionized water to prepare a selenium@platinum nanoparticle dispersion with a concentration of 1 mg / mL, denoted as Se@Pt dispersion.

[0042] (3) Preparation of copper selenide@platinum composite material dispersion: Take 1 mL of the Se@Pt dispersion prepared in step (2), add it to a beaker in sequence with 1 mL of PVP aqueous solution with a mass concentration of 10 mg / mL, 0.5 mL of CuCl2 aqueous solution with a concentration of 0.1 mmol / L, and 2 mL of ascorbic acid (AA) aqueous solution with a concentration of 0.1 mol / L, and stir for 5 min. After stirring, a brown solution A is obtained. Add 60 μL of H2O2 aqueous solution with a mass fraction of 30% to solution A and continue stirring for 3 min. Then centrifuge at 10000 r / min for 10 min, collect the precipitate obtained by centrifugation and wash it twice with deionized water. The precipitate after washing is the copper selenide@platinum composite material, which is a nanoparticle, denoted as CS@Pt.

[0043] Example 2:

[0044] (1) This step is the same as step (1) in Example 1;

[0045] (2) Take 3 mL of the selenium nanosphere dispersion prepared in step (1), mix it with 3 mL of PVP aqueous solution (concentration of 0.1 g / mL), 0.05 mL of K2PtCl4 aqueous solution (concentration of 500 mmol / L) and 6 mL of anhydrous ethanol, stir the mixture at 70 °C for 5 h, after stirring, centrifuge the obtained product at 10000 r / min for 10 min, collect the precipitate obtained by centrifugation and wash it with deionized water, and then disperse the washed precipitate in deionized water to prepare a selenium@platinum nanoparticle dispersion with a concentration of 1 mg / mL, denoted as Se@Pt dispersion;

[0046] (3) This step is the same as step (3) in Example 1.

[0047] Example 3:

[0048] (1) This step is the same as step (1) in Example 1;

[0049] (2) Take 3 mL of the selenium nanosphere dispersion prepared in step (1), mix it with 3 mL of PVP aqueous solution (concentration of 0.1 g / mL), 0.05 mL of K2PtCl4 aqueous solution (concentration of 500 mmol / L) and 25 mL of anhydrous ethanol, stir the mixture at 60 °C for 5 h, after stirring, centrifuge the obtained product at 10000 r / min for 10 min, collect the precipitate obtained by centrifugation and wash it with deionized water, and then disperse the washed precipitate in deionized water to prepare a selenium@platinum nanoparticle dispersion with a concentration of 1 mg / mL, denoted as Se@Pt dispersion;

[0050] (3) This step is the same as step (3) in Example 1.

[0051] Example 4:

[0052] (1) This step is the same as step (1) in Example 1;

[0053] (2) This step is the same as step (2) in Example 1;

[0054] (3) Preparation of copper selenide@platinum composite material dispersion: Take 1 mL of the Se@Pt dispersion prepared in step (2), add it to a beaker in sequence with 1 mL of PVP aqueous solution with a mass concentration of 15 mg / mL, 0.7 mL of copper nitrate aqueous solution with a concentration of 0.15 mmol / L, and 2 mL of ascorbic acid (AA) aqueous solution with a concentration of 0.5 mol / L, and stir for 8 min. After stirring, a brown solution A is obtained. Add 80 μL of H2O2 aqueous solution with a mass fraction of 30% to solution A and continue stirring for 5 min. Then centrifuge at a speed of 10000 r / min for 10 min, collect the precipitate obtained by centrifugation and wash it twice with deionized water. The precipitate after washing is the copper selenide@platinum composite material, which is nanoparticle, denoted as CS@Pt.

[0055] Figure 1 This is a schematic diagram illustrating the preparation process of the copper selenide@platinum composite material (CS@Pt) of this invention and its application in tumor treatment (taking copper chloride as an example). CS@Pt is an abbreviation for copper selenide@platinum nanocomposite material. Figure 1 As shown, Se nanospheres were prepared using N₂H₄·H₂O as a reducing agent. Then, K₂PtCl₄ was added, and the growth of Pt nanoparticles on the surface of the Se nanospheres was regulated by controlling the temperature. Subsequently, a copper source was introduced, and under the action of hydrogen peroxide, the Se nanospheres were converted into Cu. 2-x Se nanospheres (where x = 0–1). Cu 2-x Se@Pt is composed of Pt nanoparticles and Cu. 2-x The mixture consists of Se (x = 0-1) nanospheres. Copper selenide nanospheres possess excellent photothermal conversion properties, enabling photothermal therapy of tumors. High-Z platinum nanoparticles grown on their surface have a radiosensitizing effect, and platinum can catalyze the decomposition of H2O2 at the tumor site, overcoming tumor hypoxia and further enhancing the efficacy of radiotherapy. Therefore, the copper selenide@platinum composite material prepared in this invention exhibits excellent photothermal properties and radiosensitizing effects, increasing intracellular oxygen content (as is well known, tumor cells contain a large amount of H2O2, and Pt can catalyze the decomposition of H2O2 to produce oxygen), achieving combined RT / PTT therapy for tumors.

[0056] Figure 2 These are transmission electron micrographs of CS@Pt prepared in Examples 1 and 2. Figure 2 In Example 1, (a) clearly shows that CS@Pt has a distinct core-shell structure, with copper selenide nanospheres as the core and Pt densely coated on the surface of copper selenide to form a shell. Figure 2 In Figure (b), which corresponds to Example 2, the light-colored spherical objects are copper selenide nanospheres and the dark-colored granular objects are Pt nanoparticles, indicating that adding anhydrous ethanol in step (2) is beneficial for the formation of Pt nanoparticles.

[0057] Figure 3 This is the XRD pattern of CS@Pt prepared in Example 2, by... Figure 3 It can be determined that the prepared composite material is made of Cu 2-x It consists of Se (where x = 0 to 1) and Pt.

[0058] The following experiments verify the efficacy of the copper selenide@platinum composite material (CS@Pt) prepared in this invention in tumor treatment. All CS@Pt used in the following experiments are copper selenide@platinum composite materials (CS@Pt) prepared in Example 2.

[0059] Experiment 1

[0060] The copper selenide@platinum composite material prepared in Example 2 was formulated into a concentration of 0 μg / mL. -1 50 μg mL -1 100μg mL -1 The solution used had a power density of 1.0 W / cm². -2 The sample was irradiated with a 980nm near-infrared laser, and the temperature was recorded every 30 seconds using an infrared thermal imager for a total of 8 minutes. Using Origin plotting software, temperature curves of different concentrations of CS@Pt dispersions were plotted with time on the x-axis and the recorded temperature at the corresponding time on the y-axis. Figure 4 As shown, at a power density of 1.0 W / cm² -2 Within 8 minutes of irradiation with a 980 nm near-infrared laser, the heating effect of CS@Pt dispersions with different concentrations showed a concentration-dependent effect. The higher the concentration of the CS@Pt dispersion, the more significant the heating effect within the same time period; at a power density of 1.0 W / cm²... -2 Under irradiation with a 980nm near-infrared laser, 100μg mL -1 The temperature of the CS@Pt dispersion can rise to 69℃ within 8 minutes.

[0061] Experiment 2

[0062] The CS@Pt prepared in Example 2 was prepared into a 100 μg mL solution. -1 The dispersion was prepared using a power density of 1.0 W / cm³. -2The dispersion was irradiated with a 980nm near-infrared laser, and the temperature was recorded every 30 seconds using an infrared thermal imager. After recording for 2.5 minutes, the laser was turned off, the dispersion was cooled for 2 minutes, and then the laser was turned back on. During the cooling process, the temperature was recorded every 30 seconds. This operation was repeated five times. During the second to fifth laser irradiation, the temperature was recorded every 30 seconds using an infrared thermal imager, and after recording for 2 minutes, the laser was turned off. During the final cooling process, the temperature was recorded for 3 minutes. Using Origin plotting software, a curve of the five heating-cooling cycles of the CS@Pt dispersion was plotted with time on the x-axis and the recorded temperature at the corresponding time on the y-axis, as shown below. Figure 5 As shown, it can be clearly seen that after five heating-cooling cycles, CS@Pt still has efficient light absorption and photothermal conversion characteristics. The highest temperature that can be reached after the five heating-cooling cycles is not much different, reflecting that the prepared CS@Pt has good stability.

[0063] Experiment 3

[0064] Esophageal squamous cell carcinoma cells (KYSE30) are a cell line derived from human esophageal squamous cell carcinoma, and human venous endothelial cells (HUVECs) are a cell line derived from human umbilical vein endothelial cells. Both KYSE30 and HUVECs were provided by the Henan Provincial Key Laboratory of Tumor Epigenetics, First Affiliated Hospital of Henan University of Science and Technology (New District Hospital), and were purchased from the Cell Bank of the Chinese Academy of Sciences (ATCC). KYSE30 and HUVECs were cultured in 1640 medium containing 10% fetal bovine serum (FBS) and DMEM medium containing 10% FBS, respectively.

[0065] KYSE30 and HUVEC cells were seeded into 96-well plates (5000 cells per well). KYSE30 was cultured in 1640 medium containing 10% FBS, and HUVEC cells were cultured in DMEM medium containing 10% FBS. After 24 hours of culture, the KYSE30 medium was replaced with 1640 medium containing different concentrations of CS@Pt (CS@Pt concentrations were 0 μg / mL). -1 20μg mL -1 40 μg / mL -1 60μg mL -1 80μg mL -1 The HUVEC culture medium was replaced with DMEM medium containing different concentrations of CS@Pt (CS@Pt concentrations were 0 μg / mL). -1 20μg mL -1 40 μg / mL -1 60μg mL -1 80μg mL -1After culturing for another 24 hours, the culture media for KYSE30 and HUVEC were replaced with fresh 1640 medium and DMEM medium, respectively. Then, 10 μL of CCK8 reagent was added to each medium, and the cells were incubated for 2 hours. Cell viability was then detected using a microplate reader. Using Origin software, a graph was plotted with CS@Pt concentration on the x-axis and KYSE30 and HUVEC cell viability on the y-axis. Figure 6 As shown, the CS@Pt used in this experiment is the CS@Pt prepared in Example 2. Figure 6 The results show that the CS@Pt prepared in Example 2 has good biocompatibility with esophageal squamous cell carcinoma cells (KYSE30) and human venous endothelial cells (HUVEC).

[0066] Experiment 4

[0067] This experiment consisted of a CS@Pt group and a Control group. KYSE30 cells were seeded in each well of a 6-well plate (2000 cells per well). Both the CS@Pt and Control groups were initially cultured in 1640 medium containing 10% FBS. After 24 hours of culture, the medium for the CS@Pt group was replaced with medium containing 60 μg / mL of 1640 FBS. -1 The 1640 medium containing CS@Pt was used in the control group, while the medium was replaced with an equal volume of 1640 medium without CS@Pt. After culturing for 6 hours, KYSE30 cells in both groups were irradiated with different doses of X-rays. Then, the medium in both groups was replaced with fresh 1640 medium, and KYSE30 cells were cultured for another week. After one week, KYSE30 cells were fixed with 4% paraformaldehyde, stained with crystal violet, and then photographed using an exposure machine to obtain images of KYSE30 cell colony formation under different radiation doses, as shown below. Figure 7 As shown. By Figure 7 It can be seen that the proliferation of KYSE30 cells was significantly inhibited with increasing radiation dose. At the same radiation dose, the CS@Pt group showed a more significant inhibitory effect on the proliferation of KYSE30 cells compared to the Control group, indicating that the CS@Pt prepared in this invention can significantly enhance the inhibitory effect on the proliferation of KYSE30 cells.

[0068] Experiment 5

[0069] This experiment was divided into a CS@Pt group and a Control group. KYSE30 cells were seeded in each confocal dish (2000 cells per well). Both the CS@Pt and Control groups initially used 1640 medium containing 10% FBS. After 48 hours of culture, the medium for the CS@Pt group was replaced with medium containing 60 μg / mL... -1The CS@Pt group's 1640 medium was replaced with an equal volume of 1640 medium without CS@Pt. After culturing for 6 hours, the KYSE30 cells in both groups were irradiated with X-rays at a dose of 4 Gy. Then, the medium in both groups was replaced with fresh 1640 medium. Cells were stained with DNA damage detection reagents and photographed using a confocal microscope. Figure 8 The results show the DNA damage of KYSE30 cells in the CS@Pt group and the Control group at a radiation dose of 4 Gy. These results indicate that radiotherapy inhibits tumor proliferation by killing DNA, and that the CS@Pt prepared in Example 2 can enhance the damage of X-rays to cellular DNA.

[0070] Experiment 6

[0071] This experiment consisted of six experimental groups: Control group, CS@Pt group, RT group, CS@Pt+L group, CS@Pt+RT group, and CS@Pt+RT+L group. KYSE30 cells were seeded into each well of a 12-well plate (100,000 cells per well). The initial culture medium for each group was 1640 medium containing 10% FBS. After 24 hours of culture, the culture medium for the CS@Pt group, CS@Pt+L group, CS@Pt+RT group, and CS@Pt+RT+L group was replaced with medium containing 60 μg / mL... -1 The CS@Pt 1640 medium, the Control group, and the RT group were replaced with an equal volume of CS@Pt-free 1640 medium. After culturing for 6 hours, the RT group, CS@Pt+RT group, and CS@Pt+RT+L group were irradiated with X-rays at a dose of 4 Gy for radiotherapy of their KYSE30 cells. Subsequently, the CS@Pt+RT+L group was irradiated with a 980 nm near-infrared laser for 5 minutes for photothermal therapy (laser power 1 W / cm²). 2 After culturing the CS@Pt+L group for 6 hours, its KYSE30 cells were directly irradiated with a 980nm near-infrared laser for 5 minutes for photothermal therapy (laser power was 1W / cm²). 2 (No X-ray irradiation was performed). The Control group and the CS@Pt group were not irradiated with X-rays or near-infrared lasers. After all six experimental groups completed the above experimental procedures, the reactive oxygen species (ROS) expression level in each group was detected using a reactive oxygen species fluorescent probe, and images were taken using a confocal microscope. Figure 9 The above experimental steps were followed to obtain fluorescence images of reactive oxygen species (ROS) expression levels in KYSE30 cells under different treatments and in different groups. Figure 9It can be seen that the CS@Pt+RT group exhibited the most significant reactive oxygen species (ROS) expression level, indicating that Pt can catalyze the decomposition of hydrogen peroxide into water and oxygen, both of which are reactive oxygen species reactants. Furthermore, Cu... 2+ It can react with GSH, reducing the intracellular GSH content and preventing ROS from being cleared by GSH, which further increases the ROS content. However, because photothermal therapy is performed after radiotherapy, the cell morphology is completely lost after photothermal therapy, and washing the cells during subsequent staining may cause some ROS to disappear. Therefore, the ROS fluorescence signal of the CS@Pt+RT+L treatment combination will be weaker.

[0072] Experiment 7

[0073] This experiment consisted of six experimental groups: Control group, CS@Pt group, RT group, CS@Pt+L group, CS@Pt+RT group, and CS@Pt+RT+L group. KYSE30 cells were seeded into 6-well plates (2000 cells per well) in each group. The initial culture medium for each group was 1640 medium containing 10% FBS. After 24 hours of culture, the culture medium for the CS@Pt group, CS@Pt+L group, CS@Pt+RT group, and CS@Pt+RT+L group was replaced with medium containing 60 μg / mL... -1 The CS@Pt 1640 medium, the Control group, and the RT group were replaced with an equal volume of CS@Pt-free 1640 medium. After culturing for 6 hours, the RT group, CS@Pt+RT group, and CS@Pt+RT+L group were irradiated with 6 Gy X-rays for radiotherapy of their KYSE30 cells. Subsequently, the CS@Pt+RT+L group was irradiated with a 980 nm near-infrared laser for 5 minutes for photothermal therapy (laser power 1 W / cm²). 2 After culturing the CS@Pt+L group for 6 hours, its KYSE30 cells were directly irradiated with a 980nm near-infrared laser for 5 minutes for photothermal therapy (laser power was 1W / cm²). 2 (No X-ray irradiation was performed). The Control group and the CS@Pt group were not irradiated with X-rays or near-infrared lasers. After all six experimental groups completed the above experimental steps, the culture medium for all six groups was replaced with fresh 1640 medium. The cells were cultured for one week. After one week, KYSE30 cells were fixed with 4% paraformaldehyde, stained with crystal violet reagent, and then photographed using an exposure machine to obtain images of KYSE30 cell clone formation under different treatment conditions in different groups, as shown below. Figure 10As shown, compared with the Control group, CS@Pt group, and RT group, the CS@Pt+L group, CS@Pt+RT group, and CS@Pt+RT+L group all effectively inhibited the clonal formation of KYSE30 cells, indicating that both radiotherapy and photothermal therapy can effectively inhibit the clonal formation of KYSE30 cells, and the CS@Pt+RT+L group had the best therapeutic effect.

[0074] Experiment 8

[0075] This experiment consisted of six experimental groups: Control group, CS@Pt group, RT group, CS@Pt+L group, CS@Pt+RT group, and CS@Pt+RT+L group. KYSE30 cells were seeded into each well of a 12-well plate (100,000 cells per well). The initial culture medium for each group was 1640 medium containing 10% FBS. After 24 hours of culture, the culture medium for the CS@Pt group, CS@Pt+L group, CS@Pt+RT group, and CS@Pt+RT+L group was replaced with medium containing 60 μg / mL... -1 The CS@Pt 1640 medium, the Control group, and the RT group were replaced with an equal volume of CS@Pt-free 1640 medium. After culturing for 6 hours, the RT group, CS@Pt+RT group, and CS@Pt+RT+L group were irradiated with 6 Gy X-rays for radiotherapy of their KYSE30 cells. Subsequently, the CS@Pt+RT+L group was irradiated with a 980 nm near-infrared laser for 5 minutes for photothermal therapy (laser power 1 W / cm²). 2 After culturing the CS@Pt+L group for 6 hours, its KYSE30 cells were directly irradiated with a 980nm near-infrared laser for 5 minutes for photothermal therapy (laser power was 1W / cm²). 2 (No X-ray irradiation was performed). The Control group and the CS@Pt group were not irradiated with X-rays or near-infrared lasers. After all six experimental groups completed the above experimental procedures, the cells were stained with live / dead cell staining reagents and photographed using a confocal microscope. Figure 11 The above experimental steps were used to determine the liveness and death staining of KYSE30 cells under different treatments in different groups. Figure 11 This indicates that the tumor cell mortality rate was significantly increased in the CS@Pt+RT+L group (radiotherapy followed by photothermal therapy).

[0076] In vivo mouse experiments:

[0077] The AKR tumor (mouse esophageal tumor) model was established using male C57BL / 6 mice weighing approximately 16-18g and 6-8 weeks old. All animal experiments were conducted in accordance with the Laboratory Animal Guidelines of the First Affiliated Hospital of Henan University of Science and Technology and were approved by the Animal Experiment Ethics Committee. AKR tumor cells were provided by the Henan Provincial Key Laboratory of Tumor Epigenetics, New District Hospital of the First Affiliated Hospital of Henan University of Science and Technology, and purchased from the Cell Bank of the Chinese Academy of Sciences (ATCC). The culture conditions for AKR cells were consistent with those for HUVEC cells, both cultured in DMEM medium containing 10% FBS.

[0078] The mice were divided into six experimental groups, with six mice replicated in each group. The six experimental groups were: Control group, CS@Pt group, RT group, CS@Pt+RT group, CS@Pt+L group, and CS@Pt+RT+L group. The mouse tumor manipulation procedure is as follows: Figure 12 As shown, mice were fed a normal diet from day 0 to day 4. On day 5, each mouse was injected with 100 μL of AKR tumor cell suspension (in the right back). The AKR tumor cell suspension was obtained by adding AKR tumor cells to PBS buffer, and the cell concentration was 1 × 10⁻⁶. 7 / mL. Mice were fed normal diet from day 6 to day 13. On day 14, mice in the CS@Pt, CS@Pt+RT, CS@Pt+L, and CS@Pt+RT+L groups were injected with CS@Pt material at a dose of 15 mg / kg. The CS@Pt material was administered via tail vein injection in the form of a dispersion. The CS@Pt dispersion was a concentration of 5 mg / mL of CS@Pt dispersed in PBS buffer. The Control and RT groups were injected with the same volume of CS@Pt-free saline. Twelve hours after injection, the RT, CS@Pt+RT, and CS@Pt+RT+L groups were irradiated with X-rays at a dose of 6 Gy to their tumor sites (the mouse's back, specifically as shown in the image). Figure 12 The mice underwent radiotherapy (as shown in the image), followed by photothermal therapy by irradiating the tumor sites with a 980nm near-infrared laser for 5 minutes (laser power 1W / cm²). 2 ); 12 hours after injection of CS@Pt dispersion, mice in the CS@Pt+L group underwent photothermal therapy by directly irradiating their tumor sites with a 980nm near-infrared laser for 5 minutes (laser power was 1W / cm²). 2Mice in the Control and CS@Pt groups were not exposed to X-rays or near-infrared lasers. The above procedure was repeated on days 16, 18, 20, 22, and 24. Each time, mice in the CS@Pt, CS@Pt+RT, CS@Pt+L, and CS@Pt+RT+L groups were injected with 5 mg / mL PBS buffer containing CS@Pt. Mice in the Control and RT groups were injected with an equal volume of saline without CS@Pt. Twelve hours later, mice in the RT, CS@Pt+RT, and CS@Pt+RT+L groups were irradiated with X-rays as described above. Mice in the CS@Pt+RT+L group were irradiated with near-infrared lasers after X-ray irradiation. Mice in the CS@Pt+L group were irradiated with near-infrared lasers directly 12 hours after CS@Pt injection. Mice in the Control and CS@Pt groups were not exposed to X-rays or near-infrared lasers. On day 25, all mice were dissected, tumors were removed and marked, and then photographed and weighed.

[0079] Figure 13 These are optical photographs of tumors in mice in six experimental groups after the above different treatments. Each experimental group had six tumors (only three tumors were obtained in the CS@Pt+RT+L group). Figure 14 This is a comparison chart of the average tumor mass (in g) in mice from six experimental groups after the above different treatments. Figure 13 and Figure 14 It was found that the CS@Pt group did not show any inhibitory effect on tumors compared to the control group. The RT group showed a certain inhibitory effect on tumors, with tumor mass decreasing by 48.1% compared to the control group. Both the CS@Pt+RT and CS@Pt+L groups showed relatively good anti-tumor effects, with tumor mass reductions of 20.7% and 29.6% of the control group, respectively. The CS@Pt+RT+L group showed the best anti-tumor effect, with some tumors completely ablated, and the mass of the unablated tumors only 1.13% of that of the control group.

[0080] The above experiments show that the copper selenide@platinum composite material (CS@Pt) prepared in this invention has excellent photothermal properties and radiosensitizing effect, which can enhance the killing power of radiotherapy on tumors and realize the combined RT / PTT treatment of tumors.

[0081] The above description is merely an embodiment of the present invention and is not intended to limit the present invention in any way. The present invention can also have other embodiments based on the above structure and function, which will not be listed hereafter. Therefore, any simple modifications, equivalent changes, and alterations made by those skilled in the art to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for preparing copper selenide@platinum composite material, characterized in that... Includes the following steps: (1) Dissolve H2SeO3 and polyvinylpyrrolidone in deionized water and stir continuously for 10-30 min. Then add a certain amount of hydrazine hydrate solution and react at room temperature for 3-6 h under stirring. Centrifuge the product obtained from the reaction, wash the precipitate obtained by centrifugation with deionized water, and then disperse it in deionized water to obtain a 0.1-10 mg / mL selenium nanosphere dispersion. (2) Take the selenium nanosphere dispersion prepared in step (1), mix it with polyvinylpyrrolidone aqueous solution, K2PtCl4 aqueous solution and anhydrous ethanol in a volume ratio of 6:2~6:0.01~0.1:0~50, stir and react at 50~70℃ for 5~12h. After stirring, centrifuge the obtained product, collect the precipitate obtained by centrifugation, wash it with deionized water, and then disperse it in deionized water to obtain a selenium@platinum nanoparticle dispersion of 0.1~10 mg / mL. (3) The selenium@platinum nanoparticle dispersion, polyvinylpyrrolidone aqueous solution, copper salt aqueous solution and ascorbic acid aqueous solution prepared in step (2) are added to the same container in sequence and stirred for 3 to 10 min. Then, hydrogen peroxide aqueous solution is added and stirred for 3 to 10 min. After stopping the stirring, the product is centrifuged and the precipitate obtained by centrifugation is washed with deionized water to obtain copper selenide@platinum composite material.

2. The preparation method of the copper selenide@platinum composite material as described in claim 1, characterized in that... In step (1), the mass ratio of H2SeO3 to polyvinylpyrrolidone is 0.774 : 0.2~1.2, the mass ratio of H2SeO3 to the volume of hydrazine hydrate solution is 0.774 g : 1.5~3 mL, and the volume ratio of deionized water used to dissolve H2SeO3 and polyvinylpyrrolidone to the volume of hydrazine hydrate solution is 100~200 : 1.5~3.

3. The method for preparing the copper selenide@platinum composite material as described in claim 1 or 2, characterized in that... The hydrazine hydrate solution has a mass fraction of 80%.

4. The preparation method of the copper selenide@platinum composite material as described in claim 1, characterized in that... In step (2), the concentration of the polyvinylpyrrolidone aqueous solution is 0.01~0.1 g / mL, the concentration of the K2PtCl4 aqueous solution is 500 mmol / L, the centrifuge speed is 12000 rpm / min, and the centrifugation time is 10 min.

5. The method for preparing the copper selenide@platinum composite material as described in claim 1, characterized in that... In step (3), the volume ratio of selenium@platinum nanoparticle dispersion, polyvinylpyrrolidone aqueous solution, copper salt aqueous solution, and ascorbic acid aqueous solution is 1:1:0.5~0.7:

2.

6. The method for preparing the copper selenide@platinum composite material as described in claim 1 or 5, characterized in that... In step (3), the concentration of the polyvinylpyrrolidone aqueous solution is 5~15 mg / mL, the concentration of the copper salt aqueous solution is 0.05~0.15 mmol / L, the concentration of the ascorbic acid aqueous solution is 0.01~5 mol / L, and the copper salt is at least one of copper chloride, copper nitrate, and copper sulfate.

7. The method for preparing the copper selenide@platinum composite material as described in claim 1 or 5, characterized in that... In step (3), the mass fraction of the hydrogen peroxide aqueous solution is 30%, and the volume ratio of the hydrogen peroxide aqueous solution to the selenium@platinum nanoparticle dispersion is 40~80 μL : 1mL.

8. The copper selenide@platinum composite material obtained by the preparation method described in claim 1 has a core-shell structure, with copper selenide nanospheres as the core and platinum nanoparticles as the shell grown on the surface of the copper selenide nanospheres.

9. The application of the copper selenide@platinum composite material obtained by the preparation method as described in claim 1 as a radiotherapy sensitizer and photothermal material.

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