Preparation method and application of copper selenide polyoxometalate nanocluster

Abstract

The application discloses a preparation method and application of a copper selenide polyoxometalate nanocluster, relates to the technical field of biological nanomaterials, and comprises the following steps: dissolving ammonium molybdate tetrahydrate in ultrapure water to obtain an ammonium molybdate solution, dissolving copper chloride dihydrate in ultrapure water to obtain a CuCl2 solution, mixing the ammonium molybdate solution and the CuCl2 solution to obtain a mixed solution, dropwise adding an ascorbic acid solution into the mixed solution obtained in the previous step, continuing to stir and react after the dropwise adding is completed, loading the obtained Cu-POM crude solution into a dialysis bag, performing freeze-drying treatment on the Cu-POM solution after dialysis is completed, obtaining a Cu-POM powder product, preparing a Cu-POM solution with a concentration of 0.8-1.2 mg / mL and mixing the solution with a solution at a volume ratio of 1:1, loading the obtained Cu-Se-POM crude solution into a dialysis bag after the reaction is completed, and performing freeze-drying treatment on the Cu-Se-POM solution after dialysis is completed, to obtain a Cu-Se-POM powder product. Compared with a preparation method of a traditional polyoxometalate doped material, the preparation efficiency is high, the raw material cost is low, and the preparation method is environment-friendly.

Description

Technical Field

[0001] This invention relates to the field of bio-nanomaterials technology, specifically to a method for preparing copper selenide polyoxometalate nanoclusters and their applications. Background Technology

[0002] 1064 laser-guided copper selenide polyoxometalate (Cu-Se-POM) inhibits glioma proliferation. The core of this method is to achieve precise killing and proliferation inhibition through near-infrared photothermal / photodynamic synergy, tumor microenvironment response, and multi-target regulation, while also possessing imaging guidance potential.

[0003] Gliomas are the most common malignant tumors of the central nervous system, characterized by high invasiveness, high recurrence rate, and poor prognosis. Existing technologies for inhibiting their proliferation are mainly divided into two categories: traditional treatment techniques and emerging nano-mediated phototherapy techniques. Traditional treatment techniques include surgical resection, radiotherapy, and chemotherapy. Emerging nano-mediated phototherapy techniques are currently a research hotspot in the precision treatment of gliomas. The core of this technology is utilizing the photoresponsive properties of nanomaterials in combination with lasers to achieve targeted therapy. It mainly includes two categories: photothermal therapy (PTT) and photodynamic therapy (PDT), which are also the core technologies of this invention.

[0004] The technical structure of a single photothermal therapy nanoplatform uses gold nanoparticles (AuNPs), graphene quantum dots, and copper sulfide (CuS) nanoparticles as photothermal agents. The principle involves using near-infrared light (NIR-I, 700–900 nm) to excite the photothermal agent, converting light energy into heat energy through surface plasmon resonance or non-radiative transitions, locally raising the temperature to 42–47°C to kill tumor cells. The technique involves intravenous or local injection of the photothermal agent, which accumulates in the tumor tissue through the EPR effect (high permeability and retention effect of solid tumors), followed by near-infrared laser irradiation of the tumor site. Current problems include limited tissue penetration depth of NIR-I lasers (<1 cm), making it difficult to cover deep gliomas; poor targeting of photothermal agents, leading to accumulation in organs such as the liver and spleen, causing long-term toxicity; and the tendency for tumor recurrence due to upregulation of heat shock protein (HSP) expression in single photothermal therapy. The core drawbacks are that the glioma microenvironment (TME) is in a hypoxic state, which limits the efficiency of ROS generation; most photosensitizers rely on NIR-I lasers, which have insufficient penetration depth; and photosensitizers are prone to dark toxicity, which can trigger photosensitivity reactions in the skin.

[0005] The technical structure of the photothermal-photodynamic synergistic therapy platform involves combining photothermal agents and photosensitizers to construct an integrated nanoplatform (such as Au@porphyrin or CuS@phthalocyanine composite nanoparticles). The technical principle is to utilize the same laser excitation to achieve synergistic photothermal and photodynamic effects, thereby enhancing tumor-killing efficiency. Current bottlenecks include: poor structural stability of the composite nanomaterials, leading to easy degradation in vivo and decreased efficacy; lack of tumor microenvironment responsiveness, resulting in low drug release efficiency; laser wavelengths still concentrated in the NIR-I band, limiting the effectiveness of deep treatment; and the lack of effective integration of imaging guidance functions, making real-time intraoperative monitoring difficult. Summary of the Invention

[0006] To address the aforementioned technical challenges, this invention provides a method for preparing and applying copper selenide polyoxometalate nanoclusters. Specifically, it proposes a 1064 nm NIR-II laser-guided copper selenide polyoxometalate nanotherapy platform. Leveraging the advantages of NIR-II lasers—deep tissue penetration (up to several centimeters) and low scattering—this platform enables non-invasive / minimally invasive treatment of deep gliomas. It achieves a synergistic effect of multiple mechanisms—photothermal, photodynamic, chemokinetic, and selenium-induced proliferation inhibition—significantly improving glioma cell killing efficiency while reducing systemic toxicity. Integrating imaging guidance and treatment functions, it enables real-time intraoperative tumor boundary identification, treatment process monitoring, and efficacy evaluation, improving treatment precision. This effectively inhibits glioma proliferation, reduces recurrence rates, and prolongs patient survival, providing a novel, efficient, and safe treatment strategy for the clinical treatment of gliomas.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A method for preparing copper selenide polyoxometalate nanoclusters and its application, comprising the following steps:

[0009] S1. Under conditions of room temperature (20-25℃) and continuous stirring, add 0.4-0.5 g of ammonium molybdate tetrahydrate. Dissolve in 10-12 mL of ultrapure water until the raw material is completely dissolved to obtain ammonium molybdate solution;

[0010] S2. Dissolve 0.15~0.2 g of copper chloride dihydrate CuCl2·2H2O in 4~6 mL of ultrapure water to prepare a CuCl2 solution. After the ammonium molybdate solution is completely dissolved, quickly add the CuCl2 solution to it and keep stirring to mix the two thoroughly.

[0011] S3. Dissolve 0.15~0.25 g of ascorbic acid in 2~3 mL of ultrapure water to prepare an ascorbic acid solution. Then add it dropwise to the mixed solution obtained in step S2 at a dropping rate of 1-3 drops / second. After the addition is complete, continue stirring to react. After the reaction is complete, put the obtained crude Cu-POM solution into a dialysis bag with a molecular weight cutoff of 3000~4000 Da and dialyze it with ultrapure water as the external dialysis solution. After dialysis, freeze-dry the Cu-POM solution to obtain Cu-POM powder product.

[0012] S4. Take Cu-POM powder and prepare a Cu-POM solution with a concentration of 0.8~1.2 mg / mL. Dissolve 0.015~0.022 g of sodium selenide (Na2Se) in 4~6 mL of ultrapure water to prepare a Na2Se solution. Under vigorous stirring, mix the Cu-POM solution and Na2Se solution in the above concentration range at a volume ratio of 1:1 to ensure thorough mixing. Place the uniformly mixed solution in a constant temperature environment of 37~43℃ for reaction for 10~14 hours.

[0013] S5. After the reaction is complete, the obtained crude Cu-Se-POM solution is placed into a dialysis bag with a molecular weight cutoff of 3000~4000 Da, and purified by dialysis using ultrapure water as the external dialysis solution. After dialysis, the Cu-Se-POM solution is freeze-dried to obtain Cu-Se-POM powder product.

[0014] Preferably, step S1 includes: a stirring rate of 200~400 rpm and a stirring time of 10~30 min;

[0015] Preferably, step S2 includes: a mixing time of 5 to 15 minutes;

[0016] Preferably, step S3 includes: maintaining the reaction temperature at room temperature (20~25℃) for 1.5~2.5 hours; dialysis time for 6~10 hours; and storing the obtained Cu-POM powder product in a sealed, light-protected environment at 4℃ for later use.

[0017] Preferably, step S4 includes: a stirring rate of 800~1200 rpm and a stirring time of 10~20 min;

[0018] Preferably, step S5 specifically includes: changing the dialysis fluid every 4 hours, with a total dialysis time of 10-14 hours;

[0019] Furthermore, step S5 also includes: the amount of ultrapure water used is 50 to 100 times the total volume of Cu-POM solution and Na2Se solution;

[0020] Furthermore, step S5 also includes: storing the obtained Cu-Se-POM powder product in a sealed, light-proof environment at 4°C for later use;

[0021] Preferably, the application of the copper selenide polyoxometalate nanoclusters specifically includes: under the guidance of a 1064 laser (NIR-II), precisely delivering the Cu-Se-POM nanomaterials to the glioma lesion site, utilizing the near-infrared II photoresponse properties of the Cu-Se-POM nanomaterials to generate photothermal or photodynamic effects, and simultaneously combining the anti-tumor activity of copper selenium compounds to synergistically inhibit the proliferation and differentiation of glioma cells for the treatment of glioma.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] 1. High preparation efficiency: The reaction for preparing Cu-POM in this invention can be carried out at room temperature without the need for harsh conditions such as high temperature and high pressure. The reaction cycle is short (only 1.5~2.5 hours), and each step is simple to operate and easy to scale up. Compared with the traditional preparation method of polyoxometalate doped materials, energy consumption is reduced by more than 30% and production efficiency is increased by more than 40%.

[0024] 2. Low raw material cost and environmentally friendly: The selected raw materials such as ammonium molybdate, copper chloride, and ascorbic acid are all conventional chemical raw materials, which are widely available and inexpensive. In addition, only a small amount of trace impurity ions are generated during the reaction process, which can be removed by simple dialysis purification. There is no toxic or harmful waste discharge, which effectively reduces the cost of environmental pollution control.

[0025] 3. Excellent product performance: Copper is uniformly dispersed in the molybdenum-oxygen framework, forming a doped structure with strong stability. After being stored at 4℃ in a sealed and light-proof environment for 6 months, its performance shows no significant decay and it has good reducibility. As a precursor, it can efficiently participate in the subsequent synthesis of Cu-Se-POM with a doping efficiency of not less than 85%. Compared with undoped polyoxometalate materials, its potential catalytic, optical and other properties are significantly improved.

[0026] 4. Simple and efficient purification process: Purification is carried out using dialysis bags with a molecular weight cutoff of 3000~4000 Da. The operation is simple and the cost is low. Compared with other purification methods such as column chromatography, it can save more than 50% of the purification time, and the product purity can reach more than 90%, effectively ensuring product quality. Attached Figure Description

[0027] Figure 1 The overall morphology and lattice fringes of the material as observed by transmission electron microscopy and high-resolution transmission electron microscopy;

[0028] Figure 2 This is a distribution diagram of elements in the material;

[0029] Figure 3 Infrared spectra of Cu-Se-POM and its precursor Cu-POM;

[0030] Figure 4 X-ray diffraction pattern;

[0031] Figure 5 This is an energy-dispersive X-ray spectrum;

[0032] Figure 6 X-ray photoelectron spectroscopy;

[0033] Figure 7 This is a Zeta potential diagram;

[0034] Figure 8 This is a dynamic light scattering diagram;

[0035] Figure 9 Bar chart showing the hemolysis rate of the test material at different concentrations;

[0036] Figure 10 The distribution of ROS in cells in each experimental group;

[0037] Figure 11 Distribution diagram of live and dead cells in each experimental group;

[0038] Figure 12 For laser speckle blood flow imaging;

[0039] Figure 13 For mouse magnetic resonance imaging. Detailed Implementation

[0040] The following description is intended to disclose the invention and enable those skilled in the art to implement it. The preferred embodiments described below are merely examples, and other obvious variations will occur to those skilled in the art.

[0041] A method for preparing copper selenide polyoxometalate nanoclusters and its application, comprising the following steps:

[0042] S1. Under continuous stirring at room temperature (20-25℃), add 0.4-0.5 g of ammonium molybdate tetrahydrate (NH4)6Mo7O 24 Dissolve 4H2O in 10-12 mL of ultrapure water until the raw material is completely dissolved to obtain an ammonium molybdate solution.

[0043] S2. Dissolve 0.15~0.2 g of copper chloride dihydrate CuCl2·2H2O in 4~6 mL of ultrapure water to prepare a CuCl2 solution. After the ammonium molybdate solution is completely dissolved, quickly add the CuCl2 solution to it and keep stirring to mix the two thoroughly.

[0044] S3. Dissolve 0.15~0.25 g of ascorbic acid in 2~3 mL of ultrapure water to prepare an ascorbic acid solution. Then add it dropwise to the mixed solution obtained in step S2 at a dropping rate of 1-3 drops / second. After the addition is complete, continue stirring to react. After the reaction is complete, put the obtained crude Cu-POM solution into a dialysis bag with a molecular weight cutoff of 3000~4000 Da and dialyze it with ultrapure water as the external dialysis solution. After dialysis, freeze-dry the Cu-POM solution to obtain Cu-POM powder product.

[0045] S4. Take Cu-POM powder and prepare a Cu-POM solution with a concentration of 0.8~1.2 mg / mL. Dissolve 0.015~0.022 g of sodium selenide (Na2Se) in 4~6 mL of ultrapure water to prepare a Na2Se solution. Under vigorous stirring, mix the Cu-POM solution and Na2Se solution in the above concentration range at a volume ratio of 1:1 to ensure thorough mixing. Place the uniformly mixed solution in a constant temperature environment of 37~43℃ for reaction for 10~14 hours.

[0046] S5. After the reaction is complete, the obtained crude Cu-Se-POM solution is placed into a dialysis bag with a molecular weight cutoff of 3000~4000 Da, and purified by dialysis using ultrapure water as the external dialysis solution. After dialysis, the Cu-Se-POM solution is freeze-dried to obtain Cu-Se-POM powder product.

[0047] Preferably, step S1 includes: a stirring rate of 200~400 rpm and a stirring time of 10~30 min;

[0048] Preferably, step S2 includes: a mixing time of 5 to 15 minutes;

[0049] Preferably, step S3 includes: maintaining the reaction temperature at room temperature (20~25℃) for 1.5~2.5 hours; dialysis time for 6~10 hours; and storing the obtained Cu-POM powder product in a sealed, light-protected environment at 4℃ for later use.

[0050] Preferably, step S4 includes: a stirring rate of 800~1200 rpm and a stirring time of 10~20 min;

[0051] Preferably, step S5 specifically includes: changing the dialysis fluid every 4 hours, with a total dialysis time of 10-14 hours;

[0052] Furthermore, step S5 also includes: the amount of ultrapure water used is 50 to 100 times the total volume of Cu-POM solution and Na2Se solution;

[0053] Furthermore, step S5 also includes: storing the obtained Cu-Se-POM powder product in a sealed, light-proof environment at 4°C for later use;

[0054] Preferably, the application of the copper selenide polyoxometalate nanoclusters specifically includes: under the guidance of a 1064 laser (NIR-II), precisely delivering the Cu-Se-POM nanomaterials to the glioma lesion site, utilizing the near-infrared II photoresponse properties of the Cu-Se-POM nanomaterials to generate photothermal or photodynamic effects, and simultaneously combining the anti-tumor activity of copper selenium compounds to synergistically inhibit the proliferation and differentiation of glioma cells for the treatment of glioma.

[0055] Composition, structure, and function of copper-doped reducing polyoxometalates (Cu-POM): Composed of ammonium molybdate tetrahydrate (NH4)6Mo7O 24 This product is formed using 4H₂O and copper chloride dihydrate CuCl₂·2H₂O as raw materials through ascorbic acid reduction and doping. The main components include a molybdenum-oxygen framework, copper ions, and trace amounts of incompletely removed impurity ions. Copper is uniformly dispersed in the molybdenum-oxygen framework structure of the polyoxometalate as a dopant. The product is a nanoscale powder solid with a typical cage-like or cluster-like framework structure typical of polyoxometalates. Copper ions are embedded within the framework or adsorbed on its surface, forming a stable doped structure. The powder particle size ranges from 5 to 50 nm (which can be finely adjusted according to specific preparation parameters). It exhibits good reducibility and stability, and can be used as a precursor for subsequent doping synthesis of selenium nanoclusters. It also possesses potential catalytic and optical properties. After freeze-drying, it can be stably stored at 4°C under sealed, light-protected conditions for at least 6 months.

[0056] Detection method and standard for copper-doped reducing polyoxometalates (Cu-POM): Quantitative detection of Cu-POM products is performed using ultraviolet-visible spectrophotometry at a wavelength of 750 nm. The corresponding standard curve is y = 0.0016x - 0.00188, where x is the concentration of Cu-POM (mg / L) and y is the corresponding absorbance value. The purity of Cu-POM in the product can be calculated using this standard curve, and the purity should not be less than 90%.

[0057] Composition, structure, and function of copper polyoxometalate-doped selenium nanoclusters (Cu-Se-POM): Formed by reacting Cu-POM and sodium selenide (Na2Se) as raw materials, the core components include a polyoxometalate molybdenum-oxygen framework, copper, and selenium. Copper and selenium form a stable coordination or alloy structure, uniformly dispersed in the polyoxometalate system. The molar ratio of each element can be adjusted within the range of Cu:Se:Mo = 1:(0.8-1.2):(5-8). It exhibits a core-shell or doped nanocluster structure, with a copper-selenium compound as the core and a polyoxometalate molybdenum-oxygen framework as the shell or matrix. The cluster particle size ranges from 10 to 100 nm. The overall structure is a powdery solid with good dispersibility, forming a stable colloidal solution upon dissolution in ultrapure water. Integrating the reducing properties of polyoxometalates, the catalytic activity of copper, and the optical properties of selenium nanomaterials, it has potential applications in catalytic reactions, biosensing, and optoelectronic devices. After freeze-drying, it can be stably stored at 4°C under sealed, light-protected conditions for at least 6 months.

[0058] Detection methods and experimental results of copper polyoxometalate-doped selenium nanoclusters (Cu-Se-POM): The morphology and particle size of Cu-Se-POM nanoclusters were observed using transmission electron microscopy (TEM) to ensure that the particle size was within the range of 10-100 nm (to meet the blood-brain barrier penetration requirements); the content and ratio of Cu, Mo, and Se elements in the product were detected by inductively coupled plasma mass spectrometry (ICP-MS) to verify the doping effect; the valence state of each element was analyzed by X-ray photoelectron spectroscopy (XPS) to confirm that copper and selenium form a stable coordination or alloy structure; its near-infrared II (NIR-II) photoresponse performance was detected by near-infrared spectroscopy to ensure good light absorption and photothermal / photodynamic response under 1064 nm laser; its inhibitory effect on the proliferation of glioma cells was verified by cell experiments, and its blood-brain barrier penetration ability and in vivo antitumor activity were verified by animal experiments. Experimental results show that the Cu-Se-POM nanoclusters prepared by this method are uniformly dispersed with a doping efficiency of not less than 85%. Under the guidance of 1064 laser (NIR-II), they can effectively penetrate the blood-brain barrier and inhibit the proliferation of glioma cells by not less than 80%. They also have good biocompatibility and show no significant changes in structure and properties after being stored at 4°C in a sealed and light-protected environment for 6 months.

[0059] Reference Figure 1 As shown, low-magnification TEM observation of the overall morphology of the material reveals an aggregated nanoparticle structure, while high-resolution HRTEM is used to observe lattice fringes, enabling analysis of the material's crystalline / amorphous characteristics and crystal plane information.

[0060] Reference Figure 2As shown, the distribution of elements such as Cu, Se, Na, O, Mo, C, and Cl in the material was demonstrated by scanning transmission electron microscopy (STEM) combined with energy dispersive spectroscopy. The results show that these elements are uniformly distributed on the nanoparticles, indicating that the material is a composite material with uniform composition.

[0061] Reference Figure 3 As shown, the infrared spectra of CuSePOM and its precursor CuPOM were compared. Both retained characteristic absorption peaks such as -OH and C=O, indicating that the polyacid (POM) framework was retained after recombination, while new absorption peaks appeared, demonstrating the successful recombination of CuSe and POM.

[0062] Reference Figure 4 As shown, the crystal structure of the material is analyzed by characteristic diffraction peaks to confirm the phase information of CuSe after being combined with polyacids.

[0063] Reference Figure 5 As shown, the presence of elements such as Se, Na, C, and Cu in the material can be confirmed by the position and intensity of characteristic peaks, and the valence state of Cu (Cu²⁺, Cu⁺) can be analyzed to reveal its chemical environment.

[0064] Reference Figure 6 As shown, characteristic peaks of Na, Cu, O, C, and Se were detected through full-spectrum analysis, confirming the elemental composition of the material. High-resolution fractional peak analysis identified different chemical states of Cu and confirmed its valence information, as well as different valence states of Se (e.g., Se...). 0 Se 4 ⁺、Se 6 (⁺) This indicates the various chemical environments of Se in the material. The C signal is decomposed into functional groups such as CC, COC, and C=O. The surface carbon species are analyzed and decomposed into oxygen in different chemical environments, corresponding to the polyacid framework and surface hydroxyl groups, etc. The valence state and chemical environment of Mo in polyacid (POM) are confirmed, and the integrity of the polyacid structure is verified.

[0065] Reference Figure 7 As shown, the surface charge of the test material in the aqueous solution is negative, indicating that it carries a negative charge in water, which can affect the material's dispersibility and biological interactions.

[0066] Reference Figure 8 As shown, the hydrated particle size distribution of the material was measured, showing that its average particle size is in the range of about 500~1000 nm, reflecting the aggregation state of the material in the solution.

[0067] Reference Figure 9As shown, the test material at different concentrations caused damage to red blood cells. The results showed that the hemolysis rate was extremely low at low concentrations (PBS, 25–150 μg / mL), and only higher in pure water (positive control), demonstrating that the material has good blood compatibility.

[0068] Based on the above experimental results, it can be concluded that: X-ray photoelectron spectroscopy confirmed the presence of elements such as Cu, Se, Mo, C, and O, and resolved the chemical states of each element, proving that CuSe successfully combined with polyoxometalates (POMs) and that the polyoxometalate framework structure was preserved. The negative Zeta potential indicates that the material can be stably dispersed in aqueous solution. DLS results show that its hydrated particle size is at the nanoscale. Hemolysis experiments show that the material has an extremely low hemolysis rate at physiologically relevant concentrations and has good blood compatibility, providing a safety basis for its biomedical applications.

[0069] Reference Figure 10 As shown, the cells were divided into four experimental groups: Control (cells were not treated in any way), Laser (cells were irradiated with laser only), CuSePOM (cells were co-incubated with CuSePOM material only), and CuSePOM+Laser (cells were co-incubated with CuSePOM and then irradiated with laser).

[0070] Hoechst staining (blue channel): stains the cell nucleus, used to locate cells and observe cell morphology. FITC fluorescent probe (green channel): uses probes that bind to ROS and emit green fluorescence (such as DCFH-DA); the fluorescence intensity directly reflects the intracellular ROS content. Merge plot: overlays the blue and green channel images to visually display the distribution of ROS in the cell;

[0071] The conclusions drawn are as follows:

[0072] Control group and laser-only group: only weak green fluorescence was observed, indicating that the basal ROS level in normal cells is very low, and laser irradiation alone does not significantly induce ROS production; Material-only group (CuSePOM): green fluorescence was slightly enhanced, but the magnitude was not large, indicating that the material itself can only induce a small amount of ROS without laser; Material + laser group (CuSePOM + Laser): the green fluorescence intensity was significantly enhanced, and it was the brightest among all groups. This shows that CuSePOM can efficiently generate reactive oxygen species under laser irradiation, proving that this material has good photodynamic therapy potential.

[0073] Reference Figure 11As shown, the cells were divided into four experimental groups: Control (cells were not treated in any way), Laser (cells were irradiated with laser only), CuSePOM (cells were co-incubated with CuSePOM material only), and CuSePOM+Laser (cells were co-incubated with CuSePOM and then irradiated with laser).

[0074] Calcein-AM (green fluorescence): Only living cells can take up and hydrolyze it into Calcein, which has green fluorescence. Green fluorescence represents living cells. PI (red fluorescence): It cannot pass through the cell membrane of living cells. It can only enter dead cells with damaged cell membranes and bind to DNA to emit red fluorescence. Red fluorescence represents dead cells. Merge diagram: The green and red channel images are superimposed to visually compare the distribution of living cells and dead cells.

[0075] The conclusions drawn are as follows:

[0076] Control group: Almost all cells in the field of view were green fluorescence, with almost no red fluorescence, indicating that the vast majority of cells were alive. Laser group (only laser): Green fluorescence was still dominant, with only a small amount of red fluorescence, indicating that laser irradiation alone had a weak killing effect on cells. Material group (CuSePOM only): Green fluorescence decreased, while red fluorescence increased slightly, indicating that the material itself has some toxicity to cells, but the killing effect is limited. Material + laser group (CuSePOM + Laser): Green fluorescence decreased significantly, while red fluorescence increased dramatically, resulting in the most dead cells among all groups. This demonstrates that CuSePOM can efficiently kill cells under laser irradiation, verifying its synergistic killing effect of photodynamic or photothermal therapy.

[0077] Reference Figure 12 As shown, the mice were divided into five groups: Control (healthy control), Tumor-bearing control, Laser (laser alone), CuSePOM (material alone), and CuSePOM+Laser (material + laser). The blood perfusion of the mouse skin surface was monitored in real time using laser speckle technology. The more red / yellow the color in the image, the richer the blood flow; the more blue / black the color, the weaker the blood flow.

[0078] Reference Figure 13 As shown, by comparing nuclear magnetic resonance signals, we can observe the morphological and structural changes in the whole body and tumor area of ​​mice, and assess the damage or regression of tumor tissue.

[0079] The conclusion is as follows:

[0080] Tumor-bearing control group (Tumour): The image shows a distinct red-yellow hue, indicating abnormally rich blood vessels and vigorous blood perfusion in the tumor tissue. Laser-only group (Laser) and material-only group (CuSePOM): Blood flow signals are only slightly weakened, indicating that single treatments have limited impact on tumor blood vessels. Material + Laser group (CuSePOM + Laser): Blood flow signals are significantly weakened, and the image color becomes noticeably blue, indicating that CuSePOM can effectively destroy tumor blood vessels under laser irradiation, significantly reducing blood perfusion in the tumor tissue and thus cutting off the tumor's nutrient supply.

[0081] Image comparisons across groups showed significant changes in the tumor region signal in the CuSePOM+Laser group, reflecting structural damage or volume reduction of the tumor tissue due to ischemia and direct killing, thus validating the effectiveness of the combined material and laser therapy in vivo.

[0082] The process of using this invention is as follows: Under room temperature (20~25℃) and continuous stirring conditions, dissolve 0.4~0.5 g of ammonium molybdate tetrahydrate in 10~12 mL of ultrapure water at a stirring rate of 200~400 rpm for 10~30 min until the raw material is completely dissolved to obtain an ammonium molybdate solution; dissolve 0.15~0.2 g of copper chloride dihydrate in 4~6 mL of ultrapure water to prepare a CuCl2 solution; after the ammonium molybdate solution is completely dissolved, quickly add the CuCl2 solution to it, and keep stirring to ensure thorough mixing for 5~15 min; dissolve 0.15~0.25 g of ascorbic acid in 2~3 mL of ultrapure water... An ascorbic acid solution was prepared in mL of ultrapure water and then added dropwise to the mixed solution obtained in the previous step at a rate of 1-3 drops / second. After the addition was completed, the reaction was stirred and the reaction temperature was maintained at room temperature (20-25℃) for 1.5-2.5 hours. After the reaction was completed, the crude Cu-POM solution was placed in a dialysis bag with a molecular weight cutoff of 3000-4000 Da and purified by dialysis using ultrapure water as the dialysis fluid for 6-10 hours. After dialysis, the Cu-POM solution was freeze-dried to obtain Cu-POM powder product, which was stored in a sealed, light-protected environment at 4℃ for later use. Under vigorous stirring conditions, Cu-POM solution with a concentration of 0.8–1.2 mg / mL was mixed with Na2Se solution at a volume ratio of 1:1 at a stirring speed of 800–1200 rpm for 10–20 min to ensure thorough mixing. The homogeneous solution was then placed in a constant temperature environment of 37–43°C for 10–14 hours. After the reaction was completed, the crude Cu-Se-POM solution was placed in a dialysis bag with a molecular weight cutoff of 3000–4000 Da and purified by dialysis using ultrapure water as the dialysis fluid. The dialysis fluid was replaced every 4 hours for a total dialysis time of 10–14 hours. After dialysis, the Cu-Se-POM solution was freeze-dried to obtain Cu-Se-POM powder product, which was then stored in a sealed, light-protected environment at 4°C for later use.

[0083] The core application of the Cu-Se-POM nanomaterial prepared in this invention is the treatment of glioma. Specifically, under the guidance of a 1064 nm laser (NIR-II), the Cu-Se-POM nanomaterial is precisely delivered to the glioma lesion site. Utilizing its near-infrared II photoresponsive properties, it generates a photothermal / photodynamic effect. Simultaneously, combined with the anti-tumor activity of copper-selenium compounds, it synergistically inhibits the proliferation and differentiation of glioma cells, achieving precise and efficient treatment of glioma. This application can solve the problems of existing glioma treatments, such as difficulty in drug penetration of the blood-brain barrier, poor targeting, and significant side effects, providing novel nanotherapeutic agents and technical support for glioma treatment. Cu-POM serves as a dedicated precursor for the preparation of Cu-Se-POM, ensuring the structural integrity and performance stability of the final product.

[0084] In summary, the advantages of this invention are as follows: 1. The reaction for preparing Cu-POM can be carried out at room temperature without the need for harsh conditions such as high temperature and high pressure. The reaction cycle is short (only 1.5~2.5 hours), and each step is simple to operate, making it easy to scale up production. Compared with the traditional preparation method of polyoxometalate doped materials, energy consumption is reduced by more than 30%, and production efficiency is increased by more than 40%, thus improving the preparation efficiency. 2. The selected raw materials, such as ammonium molybdate, copper chloride, and ascorbic acid, are all conventional chemical raw materials, widely available and inexpensive, resulting in low raw material costs. Only a small amount of trace impurity ions are generated during the reaction, which can be removed by simple dialysis purification, resulting in no toxic or harmful waste emissions, effectively reducing environmental pollution control costs and improving environmental friendliness. 3. Copper is uniformly dispersed in the molybdenum-oxygen framework, forming a doped structure with strong stability. After being stored at 4℃ in a sealed, light-protected environment for 6 months, its performance shows no significant degradation, and it possesses good reducibility. As a precursor, it can efficiently participate in the subsequent synthesis of Cu-Se-POM, with a doping efficiency of not less than 85%. Compared with undoped polyoxometalate materials, its potential catalytic and optical properties are significantly improved. 4. Purification is carried out using dialysis bags with a molecular weight cutoff of 3000~4000 Da. The operation is simple and the cost is low. Compared with other purification methods such as column chromatography, it can save more than 50% of the purification time, and the product purity can reach more than 90%, which effectively ensures product quality. The purification process is simple and efficient.

[0085] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention. The scope of protection claimed by the appended claims and their equivalents is defined.

Claims

1. A method for preparing copper selenide polyoxometalate nanoclusters and its application, characterized in that, The preparation method of the copper polyoxometalate-doped selenium nanoclusters Cu-Se-POM includes the following steps: S1. Under conditions of room temperature (20-25℃) and continuous stirring, add 0.4-0.5 g of ammonium molybdate tetrahydrate. Dissolve in 10-12 mL of ultrapure water until the raw material is completely dissolved to obtain ammonium molybdate solution; S2. Dissolve 0.15~0.2 g of copper chloride dihydrate CuCl2·2H2O in 4~6 mL of ultrapure water to prepare a CuCl2 solution. After the ammonium molybdate solution is completely dissolved, quickly add the CuCl2 solution to it and keep stirring to mix the two thoroughly. S3. Dissolve 0.15~0.25 g of ascorbic acid in 2~3 mL of ultrapure water to prepare an ascorbic acid solution. Then add it dropwise to the mixed solution obtained in step S2 at a dropping rate of 1-3 drops / second. After the addition is complete, continue stirring to react. After the reaction is complete, put the obtained crude Cu-POM solution into a dialysis bag with a molecular weight cutoff of 3000~4000 Da and dialyze it with ultrapure water as the external dialysis solution. After dialysis, freeze-dry the Cu-POM solution to obtain Cu-POM powder product. S4. Take Cu-POM powder and prepare a Cu-POM solution with a concentration of 0.8~1.2 mg / mL. Dissolve 0.015~0.022 g of sodium selenide (Na2Se) in 4~6 mL of ultrapure water to prepare a Na2Se solution. Under vigorous stirring, mix the Cu-POM solution and Na2Se solution in the above concentration range at a volume ratio of 1:1 to ensure thorough mixing. Place the uniformly mixed solution in a constant temperature environment of 37~43℃ for reaction for 10~14 hours. S5. After the reaction is complete, the obtained crude Cu-Se-POM solution is placed into a dialysis bag with a molecular weight cutoff of 3000~4000 Da, and purified by dialysis using ultrapure water as the external dialysis solution. After dialysis, the Cu-Se-POM solution is freeze-dried to obtain Cu-Se-POM powder product.

2. The preparation method and application of copper selenide polyoxometalate nanoclusters according to claim 1, characterized in that, Step S1 includes: a stirring rate of 200~400 rpm and a stirring time of 10~30 min.

3. The preparation method and application of copper selenide polyoxometalate nanoclusters according to claim 1, characterized in that, Step S2 includes: mixing time of 5~15 min.

4. The preparation method and application of copper selenide polyoxometalate nanoclusters according to claim 1, characterized in that, Step S3 includes: maintaining the reaction temperature at room temperature (20~25℃) for 1.5~2.5 hours; dialysis time for 6~10 hours; and storing the obtained Cu-POM powder product in a sealed, light-proof environment at 4℃ for later use.

5. The preparation method and application of copper selenide polyoxometalate nanoclusters according to claim 1, characterized in that, Step S4 includes: a stirring rate of 800~1200 rpm and a stirring time of 10~20 min.

6. The preparation method and application of copper selenide polyoxometalate nanoclusters according to claim 1, characterized in that, Step S5 specifically includes: changing the dialysis fluid every 4 hours, with a total dialysis time of 10-14 hours.

7. The preparation method and application of copper selenide polyoxometalate nanoclusters according to claim 1, characterized in that, Step S5 further includes: the amount of ultrapure water used is 50 to 100 times the total volume of Cu-POM solution and Na2Se solution.

8. The preparation method and application of copper selenide polyoxometalate nanoclusters according to claim 1, characterized in that, Step S5 further includes storing the obtained Cu-Se-POM powder product in a sealed, light-proof environment at 4°C for later use.

9. The preparation method and application of copper selenide polyoxometalate nanoclusters according to claim 1, characterized in that, The specific applications of the copper selenide polyoxometalate nanoclusters include: Guided by a 1064nm laser (NIR-II), the Cu-Se-POM nanomaterials are precisely delivered to the glioma lesion site. The near-infrared II photoresponse properties of the Cu-Se-POM nanomaterials generate photothermal or photodynamic effects. At the same time, combined with the anti-tumor activity of copper selenium compounds, they synergistically inhibit the proliferation and differentiation of glioma cells, thus being used for the treatment of glioma.

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