A bimetallic nanoassembly and its preparation method and application

Abstract

The present invention provides a bimetallic nanoassembly, a preparation method thereof, and an application thereof. The method comprises: A) mixing a phenolic polymer with a surfactant solution; B) mixing the solution obtained in step A) with a hafnium chloride solution; C) mixing the solution obtained in step B) with a cross-linking agent and a copper sulfate solution; D) adjusting the pH of the solution obtained in step C) to 7 to 9, centrifuging and precipitating, and obtaining an Hf-Cu polyphenol nanoassembly. The coordination-mediated bimetallic HfCu-polyphenol self-assembly method provided by the present invention is simple, environmentally friendly, inexpensive, and has good biocompatibility. It provides a synthesis strategy for radionuclide-labeled HfCu-bimetallic nanoassembly structures that can improve tumor hypoxia. It also provides new ideas for the diagnosis and treatment of clinical internal and external radiotherapy tumors.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomaterials, and in particular to a bimetallic nanoassembly, a preparation method and an application thereof. Background Art

[0002] Radiation therapy, also known as radiotherapy, is one of the most effective strategies currently used in clinical cancer treatment. The basic principle of radiotherapy is that high-energy X-rays or gamma-rays directly interact with the DNA of cells, causing DNA damage, or indirectly react with water molecules to generate reactive oxygen free radicals (ROS) (such as superoxide, hydrogen peroxide, hydroxyl radicals, etc.). ROS can damage DNA or other cellular components and induce cell apoptosis and necrosis. However, radiotherapy alone is usually not enough to completely eliminate tumor cells. The main reasons include: (1) Hypoxia is an important feature of solid tumors. The imbalance between oxygen supply and demand leads to tumor hypoxia, and the free radicals generated by radiotherapy will be reduced by the large number of sulfhydryl groups in the hypoxic environment, resulting in a reduction in tumor cell DNA damage caused by radioresistance, which in turn leads to tumor invasion and metastasis, immune escape, etc., reducing the efficacy of radiotherapy; (2) Tumors have low radiation absorption of high-energy rays. Radiotherapy often requires high-dose radiation, which inevitably damages normal tissues around the tumor. In addition, the high expression of glutathione (GSH) free radical scavengers in the tumor microenvironment (TME) also tends to reduce the effect of radiotherapy.

[0003] The study found that compared with the radiosensitizers represented by iodine-based small molecules used in clinical practice, inorganic nanomaterials with high Z atomic numbers such as Au, Pt, TaO x , HfO2 (clinical phase III radiosensitizer), Hf-MOF, etc. have the ability to induce radiation dose enhancement effect in tumors, can partially concentrate ionization energy in the tumor, and are radiosensitizers that can improve and sensitize the efficacy of radiotherapy. Among them, Hf-based nanoradiotherapy sensitizers have the advantages of good radiotherapy sensitization effect, good biosafety and high CT signal enhancement ability, and their synthesis cost is much lower than that of precious metal nanoradiotherapy sensitizers. However, the ultra-small size of Hf-based nanoradiotherapy sensitizers is easily metabolized from the tumor and has a very short retention time in the tumor. The size of the Hf-based radiotherapy sensitizers currently used in clinical practice, such as HfO2, is about 50nm. This size has limited penetration and diffusion depth in the tumor, thus restricting its radiotherapy sensitization effect. Therefore, it is imperative to simply and low-cost construct an Hf-based radiotherapy sensitization strategy that can penetrate deep into the tumor, retain for a long time, and significantly increase free radicals in the tumor to enhance radiotherapy. This strategy requires mitigating intratumoral hypoxia while simultaneously generating multiple free radicals to enhance radiosensitization and reduce side effects on normal tissues. A combined therapeutic strategy of size-reduced / disassembled engineered nanomedicines responsive to the biological microenvironment offers a novel approach to addressing these challenges.

[0004] NO is a typical endogenous gaseous signaling molecule with concentration-dependent physiological properties. For example, when the heart is stimulated by ischemia and hypoxia, NO within a certain dose range has physiological mechanisms such as anti-inflammatory, anti-apoptotic, anti-oxidative stress, and promotion of angiogenesis, which has an important protective effect on the heart. NO is also an effective radiosensitizer, and its main radiosensitization modes are twofold: 1) A certain dose of NO can react with superoxide anions to generate reactive nitrogen radicals (RNS), which are more likely to cause tumor cell apoptosis than ROS; 2) NO can restore abnormal tumor blood vessels to normal, dilate tumor blood vessels, and thus increase blood perfusion and oxygen supply. In order to fully exert the function of NO, the dose and duration of NO treatment need to be strictly controlled, otherwise it will lead to completely opposite results. However, there is currently a lack of NO delivery methods that can meet both requirements.

[0005] L-arginine (L-Arg) is currently considered an effective alternative to NO precursors due to its excellent biosafety. However, as a water-soluble small molecule, L-Arg is difficult to efficiently load into nanomedicines. Furthermore, its inefficient catalytic NO production in vivo is characterized by a short half-life (less than 5 seconds) and diffusion distance (20-160 microns). Efficient loading and controlled intratumoral release are urgently needed. Studies have shown that ROS generated during tumor treatments such as radiotherapy and photodynamic therapy can further efficiently catalyze the decomposition of L-Arg, enabling photoinduced NO gas therapy to alleviate tumor hypoxia. Phenolic structures, such as gallic acid, tannic acid, and catechol, exhibit strong affinity for a wide range of materials through noncovalent interactions (e.g., hydrogen bonds, π–π interactions, coordination bonds, and electrostatics), particularly with specific metals. Phenolic self-assembled nanosystems are excellent drug carriers and have been used in photodynamic therapy, immunotherapy, radiodynamic therapy, and their combination therapies. However, most of the reported metal polyphenol assembly systems are single metals, and there are few reports on bimetallic polyphenol nanoassembly systems. In particular, there are currently no reports on bimetallic-polyphenol nanoself-assembled radiotherapy sensitizers for deep tumor penetration and improvement of hypoxia. Summary of the Invention

[0006] To address the above technical issues, the present invention provides a bimetallic nanoassembly, its preparation method, and application. The coordination-mediated bimetallic HfCu-polyphenol self-assembly method provided by the present invention is simple, environmentally friendly, inexpensive, and biocompatible. It provides a synthetic strategy for radionuclide-labeled HfCu-bimetallic nanoassembly structures that can improve tumor hypoxia and offers new insights into the clinical diagnosis and treatment of tumors undergoing internal and external radiotherapy.

[0007] Specifically, the first aspect of the present invention provides a method for preparing a bimetallic nanoassembly, comprising:

[0008] A) mixing a phenolic polymer with a surfactant solution;

[0009] B) mixing the solution obtained in step A) with a hafnium chloride solution;

[0010] C) mixing the solution obtained in step B), a cross-linking agent, and a copper sulfate solution;

[0011] D) adjusting the pH of the solution obtained in step C) to 7-9, and centrifuging to obtain Hf-Cu polyphenol nanoassemblies.

[0012] The present invention provides a simple, environmentally friendly, low-cost, and biocompatible coordination-mediated bimetallic HfCu-polyphenol self-assembly method. This method yields uniformly sized HfCu-polyphenol nanoparticles with a pH-controlled size range of 95 to 150 nm. Loaded with the NO donor L-arginine (HLC), these self-assembled nanoparticles can disassemble in response to the tumor microenvironment and achieve deep intratumoral penetration and retention. Furthermore, NO gas therapy improves tumor hypoxia, while copper apoptosis synergizes with radiosensitization to effectively inhibit tumors and reduce side effects on normal tissues.

[0013] Furthermore, step B) further comprises mixing the solution obtained in step A) with an aqueous hafnium chloride solution and then adding an L-arginine solution, preferably stirring for 2 to 5 minutes after adding the L-arginine solution.

[0014] In the present invention, in step D), the solution obtained in step C) is adjusted to a pH of 7 to 9, preferably a pH of 8, using NaOH or an aqueous ammonia solution.

[0015] In the present invention, by adjusting the pH to 7-9 with NaOH solution or ammonia solution, especially at pH 8, the bimetallic nanoassembly system can have an optimal particle size of about 100 nm. Reducing the pH value can increase the particle size.

[0016] Preferably, the Hf-Cu polyphenol nanoassembly is obtained by centrifugation at a rotation speed of 8000-15000 rpm and washing the precipitate.

[0017] Preferably, the pH is adjusted to 8 with a NaOH solution, the mixture is centrifuged at 8000-15000 rpm, and the precipitate is washed to obtain the Hf-Cu polyphenol nanoassembly; preferably, the pH is adjusted to 8 with a 0.5-0.6 M NaOH solution, the mixture is stirred, centrifuged at 8000-15000 rpm for 2-4 minutes, and the precipitate is washed with deionized water.

[0018] Preferably, step D) further comprises mixing the Hf-Cu polyphenol nanoassembly with deionized water, adding BSA solution, centrifuging, and washing to obtain BSA-modified HfCu-polyphenol nanoparticles.

[0019] More preferably, in step D), the BSA-modified HfCu-polyphenol nanoparticles are further subjected to radionuclide 99 Tc labeling.

[0020] In the present invention, the HLC nano self-assembled particles modified with protein BSA have high nuclide 99 The Tc labeling rate is 88.23%. This radionuclide-labeled nanomedicine can achieve SPECT imaging-guided tracing of deep intratumoral penetration effects and its internal and external tumor radiotherapy. At the same time, it reduces the dose of X-rays used during radiotherapy, thereby reducing side effects on normal tissues.

[0021] Preferably, the radionuclide labeling comprises: using acetic acid-sodium acetate buffer to prepare the BSA-modified HfCu-polyphenol nanoparticles into HLC@BSA solution, adding stannous chloride solution and sodium pertechnetate solution, ultrasonically dispersing and then centrifuging and washing to obtain radionuclide-labeled bimetallic Hf-Cu nano self-assembly HLC@BSA@ 99m Tc.

[0022] Preferably, the Hf-Cu polyphenol nanoassembly is mixed with deionized water and then added with BSA solution, stirred in the dark for 12 to 24 hours, and then centrifuged at 8000 to 10000 rpm for 3 to 4 minutes. The precipitate is washed with deionized water to obtain BSA-modified HfCu-polyphenol nanoparticles, which are then subjected to radionuclide 99 Tc labeling, BSA modification and nuclide 99 Tc-labeled bimetallic HfCu-polyphenol self-assembled particles HLC@BSA. Further preferably, the mass-to-volume ratio of the Hf-Cu polyphenol nanoassembly, deionized water, and BSA solution is 10-20 mg:1-2 mL:1-2 mL; and the concentration of the BSA solution is 10-20 mg / mL.

[0023] Preferably, in step D), the radionuclide labeling comprises: preparing the BSA-modified HfCu-polyphenol nanoparticles into an HLC@BSA solution using an acetic acid-sodium acetate buffer, adding a stannous chloride solution and a sodium pertechnetate solution, and performing ultrasonic dispersion and subsequent centrifugal washing.

[0024] More preferably, the acetic acid-sodium acetate buffer is an acetic acid-sodium acetate buffer with a pH of 4.6 to 5.0; the HLC@BSA solution is a 1 to 2 mg / mL HLC@BSA solution; the concentration of the stannous chloride solution is 2 to 3 mg / mL; the activity of the sodium pertechnetate solution is 5 to 8 mCi; the volume ratio of the HLC@BSA solution to the stannous chloride solution is 1 to 2 mL:20 to 30 μL; and the volume ratio of the stannous chloride solution to the sodium pertechnetate solution is 1 to 2:20 to 28. Preferably, ultrasonic dispersion is performed for 10 to 30 minutes, followed by centrifugation at 8,000 to 12,000 rpm for 5 to 10 minutes, and the precipitate is added with deionized water and centrifuged and washed again.

[0025] Preferably, the phenolic polymer comprises tannic acid; and / or the cross-linking agent is formaldehyde solution.

[0026] More preferably, the mass ratio of poloxamer, tannic acid and hafnium chloride is 0.15-0.3:1-2:0.32-0.64.

[0027] In the present invention, the molar ratio of Hf and Cu can be controlled by the feed ratio of raw materials; preferably, in the hafnium chloride aqueous solution and the copper sulfate solution, the molar ratio of hafnium chloride to copper sulfate is (0.00005-0.0001):(0.00002-0.00004), preferably 0.0001:0.00002.

[0028] In the second aspect, the bimetallic nanoassembly provided by the present invention is Hf-Cu-polyphenol nanoparticles; preferably BSA modified with nuclides 99 Tc-labeled bimetallic Hf-Cu-polyphenol self-assembled particles;

[0029] Preferably, the molar ratio of Hf to Cu is 2.6:1;

[0030] And / or, the particle size of the bimetallic nanoassembly is 95-150 nm.

[0031] The bimetallic nanoassembly provided by the present invention is preferably prepared by the above-mentioned method for preparing the bimetallic nanoassembly.

[0032] In a third aspect, the present invention provides a bimetallic nanoassembly prepared by the above-mentioned method for preparing a bimetallic nanoassembly or the use of the above-mentioned bimetallic nanoassembly in preparing tumor therapeutic agents, antibacterial agents or environmental management agents.

[0033] Preferably, the tumor therapeutic agent includes a radiosensitizer.

[0034] In a fourth aspect, the present invention provides a tumor treatment device comprising an X-ray laser and a bimetallic nanoassembly prepared by the above-mentioned bimetallic nanoassembly preparation method or the above-mentioned bimetallic nanoassembly.

[0035] The beneficial effects of the present invention are at least as follows: the present invention synthesizes bimetallic Hf-Cu-polyphenol nanoparticles HC by self-assembly at room temperature through a coordination-mediated strategy, wherein the ratio of Hf and Cu can be controlled by the feed ratio of the raw materials. This method is simple, green, and environmentally friendly, and can synthesize a large amount of bimetallic-polyphenol nano self-assembly system HC with good biocompatibility at one time. Then, the NO donor L-Arg is efficiently loaded through hydrogen bonding and benzene ring conjugation to obtain an HLC nano self-assembly structure. The dose of NO gas that is controllably catalytically released from L-Arg under the action of X-rays can improve tumor hypoxia, while copper death and synergistic radiosensitization can effectively inhibit tumors, reduce the X-ray dose used, and thus reduce side effects on normal tissues. In addition, the dual-modality imaging technology based on single-photon emission computed tomography / computed tomography (SPECT) can comprehensively utilize the functional information obtained by SPECT imaging and the anatomical structure information obtained by CT imaging. With the help of nano-contrast agents, it has been used for rapid, efficient, and non-invasive diagnostic research on a variety of tumors and cardiovascular diseases. Radionuclide technetium 99 Tc can release r-rays, has an ideal half-life (6.02h), requires a small dose, and is non-toxic. Its chemical and biological properties are suitable for labeling most medical imaging drugs. 99 Tc chelation, designed 99 Tc-labeled HLC (HLC@BSA@ 99m Tc). This radionuclide-labeled bimetallic HLC nanoassembly can not only enhance the therapeutic effect of internal radiotherapy with radioisotopes, but also improve external radiotherapy with hypoxia combined with copper-sensitized Hf elements, generating multiple free radicals and enabling deep intratumoral penetration tracking guided by SPECT / CT imaging, effectively inhibiting tumors. At the same time, it reduces the X-ray dose used, thereby alleviating side effects on normal tissues. In summary, the present invention provides a synthetic strategy for radionuclide-labeled HfCu-bimetallic nanoassembly structures that can improve tumor hypoxia, and it also provides new ideas for the clinical diagnosis and treatment of tumors using internal and external radiotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0037] Figure 1 A flow chart for preparing a bimetallic nanoassembly provided in an embodiment of the present invention;

[0038] Figure 2 The morphology and elemental distribution of HB and HL provided in Example 1 of the present invention are as follows: (a) is a transmission electron microscopy (TEM) image of Hf-BSA (HB), with a particle diameter of approximately 2-3 nm; (b) and (c) are scanning electron microscopy (SEM) and TEM images, respectively, of Hf-Cu polyphenol nanoassemblies (HC), with an estimated particle size of approximately 95 nm; (d) is an energy spectrum image of the Hf-Cu polyphenol nanoassemblies; (e) is a high-angle dark field transmission HHAADF and elemental energy spectrum distribution image of the Hf-Cu polyphenol nanoassemblies;

[0039] Figure 3 TEM images of the disassembly of HLC in a simulated tumor microenvironment provided in Example 2 of the present invention: (a) and (b) are TEM images of Hf-Cu polyphenol and L-Arg nanoassembly HLC after treatment with a simulated tumor microenvironment solution (0.1 M H2O2 + PBS 5.5) for 4 h and 12 h, respectively;

[0040] Figure 4 X-ray photoelectron spectroscopy (XPS) diagrams of HB and HLC provided in Examples 1 and 2 of the present invention: (a) is the X-ray photoelectron spectrum diagram of Hf-BSA (HB) and HLC; (b)-(f) are the detailed X-ray photoelectron spectra of C, O, Hf, Cu, and N elements in HLC, respectively;

[0041] Figure 5 (a) HLC cloning effect diagram of 4T1 breast tumor cells (b) and statistical data on the survival rate of 4T1 breast tumor cells in different treatment groups provided in Example 2 of the present invention;

[0042] Figure 6 Dose-dependent effect curves of X-ray radiosensitization provided in Example 2 of the present invention: (a) shows colony formation of 4T1 breast tumor cells treated with Hf-Cu polyphenol nanoassemblies (HLC) at different X-ray irradiation doses; (b) shows the survival rate of 4T1 mouse breast tumor cells treated with HLC at different X-ray irradiation doses, HLC concentration: 50 μg / mL.

[0043] Figure 7 This is a graph showing the dose-dependent toxic effects of HB and HLC on tumor cells provided in Examples 1 and 2 of the present invention, with an incubation time of 24 h;

[0044] Figure 8 HB@BSA@ provided in Examples 1 and 3 of the present invention 99mTc(a) and HLC@BSA@ 99m In vivo SPECT / CT imaging of Tc (b) and distribution maps (c) and (d). DETAILED DESCRIPTION

[0045] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0046] Where specific techniques or conditions are not specified in the examples of the present invention, the techniques or conditions described in the literature in the field or in accordance with the product specifications were used. For devices, instruments, reagents, etc. used, where the manufacturer is not specified, all are conventional products that can be purchased through regular channels. The raw materials used in the present invention are all readily available in the domestic product market.

[0047] Some embodiments of the present invention provide a method for preparing a bimetallic nanoassembly, the process of which is as follows: Figure 1 As shown. The radionuclide-labeled radiotherapy sensitizer provided in the embodiment of the present invention is an L-arginine-modified HfCu-polyphenol nano-engineered self-assembled particle, referred to as HLC, with a size of about 100 nm. It is obtained by a simple metal coordination-mediated self-assembly synthesis method at a mild ambient temperature, which does not require a complicated operation process. It is simple, green, environmentally friendly, and has a high yield. This strategy not only has a simple assembly function, but also has an intelligent responsive disassembly / disassembly property. This is mainly because HLC also has a degradable disassembly efficacy that responds to the micro-acidic environment of the tumor. After intravenous administration, it has a better high permeability and long retention (EPR) effect than the ultra-small Hf-BSA nanoparticles of 2 to 3 nm, and can be disassembled from 100 nm to a smaller size under the action of the acidic tumor microenvironment with high expression of H2O2 and the free radicals generated by radiotherapy. This new responsive disassembly strategy increases the deep penetration depth of HLC into the tumor. Moreover, the polyphenols used are plant extracts that are commonly found in nature, and the cross-linked polymer products are safe. Furthermore, their abundant hydrogen bonds and π–π conjugated functional groups are crucial prerequisites for metal coordination and nanoparticle self-assembly. These biodegradable, self-assembled nanoparticles are easily metabolized in the body, have no toxic side effects, and exhibit high biosafety. Therefore, HC / HLC particles are expected to serve as a model system for the preparation of engineered nanoparticles and basic research in nanoscience, opening up diverse applications, functions, and properties, such as biomedicine and environmental remediation, while offering a wide range of tunable possibilities.

[0048] Based on the high Z atomic number of Hf in HLC and its high proportion of self-assembly, it can enhance the radiotherapy effect. In combination with the free radicals generated by X-ray radiotherapy, it can controllably release NO from the NO donor L-arginine in HLC, thereby improving the effect of tumor hypoxia and sensitizing radiotherapy. Most importantly, due to the effective coordination of the Cu element in the bimetallic element, HLC exerts the copper death effect to further enhance radiotherapy and effectively inhibit tumors. Furthermore, the BSA-modified HLC is easy to be radionuclide 99 Tc labeling, and the labeling rate is high, this 99 Tc-labeled HLC nano-self-assemblies can not only enhance the therapeutic effect of internal radiotherapy with radioisotopes, but also improve external radiotherapy with hypoxia combined with copper-sensitized Hf elements, generate multiple free radicals, and realize SPECT imaging-guided tumor diagnosis and treatment and the tracing of its deep intratumoral penetration effects, which has potential application prospects in biology and clinical medicine.

[0049] Example 1

[0050] Synthesis of Bimetallic HfCu-Polyphenol (HC) Nanoparticles via Coordination-Mediated Self-Assembly Strategy

[0051] Prepare an aqueous solution of hafnium chloride by adding 0.032g of hafnium chloride (HfCl4) to 4mL of deionized water. Add 0.015g of poloxamer (Pluronic F127) to a mixture of 14mL of deionized water and 2mL of anhydrous ethanol. After sonication, add 0.1g of tannic acid powder and stir for 2 minutes until the tannic acid is completely dissolved, resulting in a pale white solution. Add the entire amount of the prepared aqueous hafnium chloride solution dropwise to the pale white solution, which will further turn white and form flocculent particles. Add 0.2mL of formaldehyde solution (37wt%) dropwise to the solution, stir for 1 minute, and then add 1mL of copper sulfate solution (0.5wt% CuSO4, 5mg) dropwise. Continue stirring for 5 minutes. The molar ratio of Hf to Cu in the raw materials is 5:1. Finally, adjust the solution to pH 8 with 0.5M NaOH and continue stirring for 1 hour. After stirring, the resulting solution was centrifuged at 10,000 rpm for 4 minutes, and the resulting precipitate was washed three times with deionized water to obtain pure Hf-Cu polyphenol nanoparticles (HC), which were freeze-dried for later use.

[0052] Comparative Example 1

[0053] Hf-BSA Nanoclusters (HB) and Their Nuclides 99 Synthesis of Tc-labeled Hf-BSA nanoclusters

[0054] Dissolve 0.012g of hafnium chloride (HfCl4) powder in 2mL of deionized water until uniformly dispersed. Add this solution to 2mL of 10mg / mL BSA solution and stir for 2 minutes. Adjust the pH to 8 by adding 0.5M NaOH solution dropwise. Stir the solution in the dark for 12 hours. After stirring, transfer the solution to a dialysis bag and dialyze for 48 hours, changing the water every 24 hours, to obtain Hf-BSA nanoclusters (HB).

[0055] Prepare a 1 mg / mL Hf-BSA solution using an acetic acid-sodium acetate buffer system at pH 4.6. Take 1 mL of this solution and add 20 μL of stannous chloride solution (2 mg / mL, SnCl2) and mix thoroughly. Then, add 280 μL of sodium pertechnetate solution (activity: 5 mCi) to the solution and ultrasonically disperse it for 30 minutes. After ultrasonication, centrifuge at 12,000 rpm for 5 minutes and collect the supernatant. Wash the pellet three times with 1 mL of deionized water by centrifugation, collecting the supernatant after each wash. Afterwards, measure the activity of both the supernatant and the pellet in a measuring well.

[0056] Example 2

[0057] Self-assembled L-arginine-loaded bimetallic HfCu-polyphenol nanoparticles (HLC)

[0058] Prepare a hafnium chloride aqueous solution by adding 0.032g of hafnium chloride (HfCl4) to 4mL of deionized water. Add 0.015g of poloxamer (Pluronic F127) to a mixture of 14mL of deionized water and 2mL of anhydrous ethanol and sonicate until the poloxamer dissolves. Once dissolved, add 0.1g of tannic acid powder and stir for 2 minutes until the tannic acid is completely dissolved and the solution turns pale white. Add the prepared hafnium chloride aqueous solution dropwise to the pale white solution, which will further turn white and form flocculent particles. Add 5mg of L-arginine to the solution and stir for 2 minutes. Add 0.2mL of formaldehyde solution (37wt%) dropwise to the solution, stir for 1 minute, then add 1mL of copper sulfate solution (0.5wt% CuSO4, 5mg) dropwise and continue stirring for 5 minutes. The molar ratio of Hf to Cu in the raw materials is 5:1. Finally, the pH was adjusted to 8 with 0.5 M NaOH solution and stirring was continued for 1 h. The resulting solution was centrifuged at 10,000 rpm for 4 min, and the precipitate was washed three times with deionized water to obtain pure Hf-Cu polyphenol nanoassembly (HLC). According to the ICP-MS test results, the molar ratio of Hf to Cu in the obtained assembly HC was 2.6:1.

[0059] Example 3

[0060] BSA modification and nuclide 99Tc-labeled bimetallic HfCu-polyphenol self-assembled nanoparticles

[0061] 20 mg of HfCu-polyphenol nanoparticles (HLC) prepared by the method of Example 2 was added to 2 mL of deionized water and ultrasonically dispersed. This solution was then added to 2 mL of a 20 mg / mL BSA solution and stirred in the dark for 24 h. After stirring, the resulting solution was centrifuged at 10,000 rpm for 4 min, and the precipitate was washed once with deionized water to obtain BSA-modified HfCu-polyphenol nanoparticles (HLC@BSA).

[0062] Prepare a 1mg / mL HLC@BSA solution using acetic acid-sodium acetate buffer at pH 4.6. Take 1mL of the above solution, add 20μL of stannous chloride solution (2mg / mL, SnCl2) and mix thoroughly. Add 280μL of sodium pertechnetate solution (activity: 5mCi) to the above solution. After ultrasonic dispersion for 30 minutes, centrifuge at 12000rpm for 5 minutes and collect the supernatant. Add 1mL of deionized water and continue centrifugation and wash three times. Collect the supernatant after each wash. After the end, measure the activity of the supernatant and precipitate in the measurement well to obtain 99 Tc-labeled HLC@BSA (HLC@BSA@ 99m Tc) and stored at 4°C.

[0063] Example 4

[0064] The same method as in Example 2 was used, except that the pH of the 0.5M NaOH solution was adjusted to 7.5 instead of 8. The BSA modified nuclide was prepared by the method in Example 3. 99 Tc-labeled bimetallic HfCu-polyphenol self-assembled particles.

[0065] Example 5

[0066] The same method as in Example 4 was used, except that the amount of hafnium chloride was changed from 0.032 g to 0.016 g, and the molar ratio of hafnium to copper was changed from 5:1 to 2.5:1, to obtain a bimetallic-polyphenol self-assembly system with a varying ratio of Hf to Cu. The molar ratio of Hf to Cu in the obtained product HC was calculated to be 1.1:1 by ICP-MS. Then, the method of Example 3 was used to prepare BSA modified with nuclides. 99 Tc-labeled bimetallic HfCu-polyphenol self-assembled particles.

[0067] The morphology and element distribution diagram of HB and HL provided in Example 1 of the present invention ( Figure 2): (a) is a transmission electron microscope (TEM) image of Hf-BSA (HB), with a particle diameter of approximately 2-3 nm; (b) and (c) are scanning electron microscope (SEM) and TEM images of Hf-Cu polyphenol nanoassemblies (HC), respectively, with an estimated particle size of approximately 95 nm; (d) is an energy spectrum image of the Hf-Cu polyphenol nanoassemblies; (e) is a high-angle dark field transmission HHAADF and elemental energy spectrum distribution image of the Hf-Cu polyphenol nanoassemblies; Figure 2 It is proved that Hf and Cu bimetallic elements co-exist in the self-assembled HC nanoparticles.

[0068] TEM image of HLC disassembly in a simulated tumor microenvironment provided in Example 2 of the present invention ( Figure 3 ): (a) and (b) are TEM images of Hf-Cu polyphenol and L-Arg nanoassembly HLC after treatment with simulated tumor microenvironment solution (0.1 M H2O2 + PBS, pH 5.5) for 4 h and 12 h, respectively; Figure 3 The images showed that the spherical morphology of HLC particles in the simulated tumor microenvironment began to collapse at 4 hours. After 12 hours, the nanoparticles lost their spherical morphology, expanded and twisted. It was preliminarily demonstrated that Hf-Cu polyphenol nanoassemblies can respond to disassembly in the acidic tumor microenvironment with high H2O2 expression, thereby being more conducive to deep penetration and retention in the tumor.

[0069] The X-ray photoelectron spectroscopy (XPS) diagrams of HB and HLC provided in Examples 1 and 2 of the present invention ( Figure 4 ): (a) is the X-ray photoelectron spectra of Hf-BSA (HB) and HLC; Figures (b)-(f) are the X-ray photoelectron spectra of C, O, Hf, Cu, and N elements in HLC, respectively; Figure 4 The XPS fine structure spectrum demonstrates that peaks for Hf, O, C, N, and Cu are clearly present in the Hf-Cu polyphenol nanoassembly HLC structure. The N peak is primarily derived from the nitrogen in L-Arg. The Hf peak is stronger than the Cu peak, indicating that the Hf content is higher than the Cu content.

[0070] The cloning effect diagram of 4T1 breast tumor cells by HLC provided in Example 2 of the present invention (a) and the statistical data of the survival rate of 4T1 breast tumor cells in different treatment groups (b) are shown in FIG. Figure 5; The 4T1 breast tumor cell cloning effect diagram of HLC provided in Example 2 of the present invention (a) is that 1000 4T1 breast tumor cells per well are seeded in a 6-well plate, and different groups are treated according to corresponding requirements. The experimental conditions selected are: the X-ray irradiation dose is 4Gy; the concentration of the material is 50μg / ml. By counting the number of clones formed, the cell cloning rate can be calculated. Compared with the Control+X-ray, HB+X-ray, HC+X-ray and control groups (Control group, HB group, HLC group), the cell survival rate of the HLC+X-ray group is lower, indicating that the HLC+X-ray group has better killing ability for tumor cells; (b) The statistical values ​​of the survival rate of 4T1 breast tumor cells in different treatment groups also prove that the HLC+X-ray group has a stronger killing ability for tumor cells than other groups, and the cell survival rate of the HLC+X-ray group is less than 22%.

[0071] The dose-dependent effect diagram of X-ray radiotherapy sensitization provided in Example 2 of the present invention ( Figure 6 ): (a) Colony formation diagram of 4T1 breast tumor cells treated with Hf-Cu polyphenol nanoassemblies HLC at different X-ray irradiation doses; (b) Survival rate of 4T1 mouse breast cancer cells treated with HLC at different X-ray irradiation doses, HLC concentration: 50 μg / mL; Figure 6 It can be seen that with the increase of radiation dose, the killing effect of Hf-Cu polyphenol nanoassembly HLC on tumor cells is significantly enhanced. Cloning experiments show that the HLC+X-ray group can effectively reduce the radiation dose, and its sensitization ratio (SER10) is 1.29. The dose-dependent toxic effect diagram of HB and HLC on tumor cells provided in Examples 1 and 2 of the present invention ( Figure 7 ); (a) is the cell survival rate of 4T1 mouse breast tumor cells after incubation with different concentrations of HB and HLC for 24 hours. After incubation for 24 hours, the cell survival rate can still reach more than 98% when the concentration of HB is in the range of 50μg / mL. However, when the concentration is in the range of 100-200μg / mL, HLC shows a certain killing effect on 4T1 tumor cells, which may be that copper death has a certain tumor killing effect. In the HB group, since there is no Cu element, only Hf exists, and there is no tumor cell killing effect at a concentration of 200μg / mL. This further illustrates that the presence of Cu in the Hf-Cu bimetallic system is also important, and the synergy of the two metal elements Hf-Cu has potential anti-tumor effects.

[0072] HB@BSA@ provided in Examples 1 and 3 of the present invention 99m Tc(a) and HLC@BSA@ 99mIn vivo SPECT / CT imaging and distribution maps of Tc (b) (c) and (d) ( Figure 8 ). Used to demonstrate the deep intratumoral penetration and retention effect, passive targeting effect, and distribution and metabolic pathway of HLC disassembly under the response of tumor microenvironment: (a) HB@BSA@ 99m SPECT / CT images of Tc 1h, 3h, and 6h after intravenous (iv) and intratumoral (it) injection. 99 The labeling rate of Tc-labeled HB is 90.99%); (b) HLC@BSA@ 99m SPECT / CT images 1h, 3h, and 6h after Tc intravenous and intratumoral injection ( 99 The labeling rate of Tc-labeled HLC was 88.23%); the results showed that after intratumoral administration, HLC@BSA@ 99m Tc has higher intratumoral penetration and retention than HB@BSA@ 99m Tc, HB@BSA@ 99m At 6 h, part of Tc leaked out of the tumor and entered the liver and spleen metabolic pathway, but HLC@BSA@ 99m No obvious imaging signals were found in the liver and spleen of the Tc group. (cd) Gamma counter quantification of HB@BSA@ of different sizes and compositions 6 hours after administration by two different routes of administration (including intravenous iv and intratumoral it). 99m Tc and HLC@BSA@ 99m The distribution of Tc in the main organs and tumors of living mice. 99m Tc disassembles in the tumor microenvironment, and the passive targeting (EPR effect) and intratumoral permeability of the 1h group are obvious. As the time is extended to 6h, there is still obvious retention in the tumor site, which is significantly better than HB@BSA@ 99m The intratumoral penetration of Tc. After 6 hours of intratumoral administration, HB@BSA@ 99m Tc and HLC@BSA@ 99m Tc group showed enhanced retention and penetration in the tumor, and HLC@BSA@ 99m The intratumoral retention intensity of Tc group was higher than that of HB@BSA@ 99m In general, the quantitative results of gamma counting were consistent with those of SPECT / CT imaging.

[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for preparing a bimetallic nanoassembly, characterized in that: include: A) mixing a phenolic polymer with a surfactant solution; B) mixing the solution obtained in step A) with a hafnium chloride solution; C) mixing the solution obtained in step B), a cross-linking agent, and a copper sulfate solution; D) adjusting the pH of the solution obtained in step C) to 7-9, and centrifuging to obtain Hf-Cu polyphenol nanoassemblies; Step B) further comprises mixing the solution obtained in step A) with an aqueous solution of hafnium chloride and then adding an L-arginine solution; The phenolic polymer includes tannic acid; the surfactant is poloxamer; and the cross-linking agent is formaldehyde solution.

2. The method for preparing a bimetallic nanoassembly according to claim 1, wherein: In step D), the solution obtained in step C) is adjusted to a pH of 7-9 with NaOH solution or ammonia solution.

3. The method for preparing a bimetallic nanoassembly according to claim 2, wherein: In step D), the solution obtained in step C) is adjusted to pH 8 with NaOH solution or ammonia solution.

4. The method for preparing a bimetallic nanoassembly according to claim 3, wherein: Step D) further comprises mixing the Hf-Cu polyphenol nanoassembly with deionized water, adding BSA solution, centrifuging, and washing to obtain BSA-modified HfCu-polyphenol nanoparticles.

5. The method for preparing a bimetallic nanoassembly according to any one of claims 1 to 4, characterized in that: Step D) also includes subjecting the BSA-modified HfCu-polyphenol nanoparticles to radionuclide 99 Tc labeling.

6. The method for preparing a bimetallic nanoassembly according to claim 5, wherein: The radionuclide labeling comprises: using acetic acid-sodium acetate buffer to prepare the BSA-modified HfCu-polyphenol nanoparticles into an HLC@BSA solution, adding a stannous chloride solution and a sodium pertechnetate solution, ultrasonically dispersing and then centrifuging and washing to obtain a radionuclide-labeled bimetallic Hf-Cu nano self-assembly.

7. The method for preparing a bimetallic nanoassembly according to any one of claims 1 to 4, characterized in that: In the hafnium chloride aqueous solution and the copper sulfate solution, the molar ratio of hafnium chloride to copper sulfate is (0.00005~0.0001): (0.00002~0.00004).

8. The method for preparing a bimetallic nanoassembly according to claim 7, wherein: In the hafnium chloride aqueous solution and the copper sulfate solution, the molar ratio of hafnium chloride to copper sulfate is 0.0001:0.00002.

9. A bimetallic nanoassembly, characterized in that: The bimetallic nanoassembly is the Hf-Cu polyphenol nanoassembly according to claim 1.

10. The bimetallic nanoassembly according to claim 9, characterized in that: The bimetallic nanoassembly is BSA modified and nuclide 99 Tc-labeled bimetallic Hf-Cu-polyphenol self-assembled particles; the molar ratio of Hf and Cu is 2.6:1; And / or, the particle size of the bimetallic nanoassembly is 95-150 nm.

11. Use of the bimetallic nanoassembly prepared by the method for preparing a bimetallic nanoassembly according to any one of claims 1 to 8 or the bimetallic nanoassembly according to claim 9 or 10 in preparing drugs for tumor treatment, antibacterial treatment or environmental management.

12. A tumor treatment device, characterized in that: The invention comprises an X-ray laser and a bimetallic nanoassembly prepared by the bimetallic nanoassembly preparation method according to any one of claims 1 to 8 or the bimetallic nanoassembly according to claim 9 or 10.

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