Cysteine-mediated Hf / Cu bimetallic synergistic self-assembled nano-particles as well as preparation method and application thereof

Abstract

The invention discloses cysteine mediated Hf / Cu bimetal synergistic self-assembled nano-particles as well as a preparation method and application of the cysteine mediated Hf / Cu bimetal synergistic self-assembled nano-particles. According to the method, cysteine is used as a multifunctional ligand, carboxyl and sulfydryl of cysteine are in bridge connection with metal through coordination, ligand chelation and assembly guiding effects are synchronously exerted, bimetallic ions are induced to be cooperatively self-assembled in an alkaline system (pH is 9-10), and metal cysteine self-assembled nanoparticles with uniform structures are formed through in-situ nucleation and multistage aggregation. According to the method, the ligand function and the bimetallic synergistic effect are accurately matched, the synthesis process is mild (room temperature), the operation is simple and convenient, and the product is excellent in dispersity and stability. Meanwhile, a close coordination structure constructed by cysteine provides a key channel for energy / electron transfer from sensitizing metal to Cu under X-ray irradiation, and lays a structural foundation for subsequent catalytic effect enhancement.

Description

Cysteine-mediated Hf / Cu bimetallic co-assembled nanoparticles, their preparation method and applications Technical Field

[0001] This invention belongs to the field of biomaterials, specifically, it relates to a cysteine-mediated Hf / Cu bimetallic synergistic self-assembly nanoparticle, its preparation method and application. Background Technology

[0002] Malignant tumors are a major disease threatening human life and health worldwide. Radiotherapy, as an indispensable anti-tumor treatment in clinical practice, plays a key role in local tumor control. However, radiotherapy alone has many technical bottlenecks: the inherent radioresistance of tumor cells limits the therapeutic effect; increased radiation dose easily causes damage to normal tissues; and it lacks the ability to specifically respond to the tumor microenvironment. Nano-hafnium oxide, as a commonly used radiosensitizing material in clinical practice, has shown a certain sensitizing effect in clinical practice due to its strong X-ray attenuation ability brought by its high Z atomic number (72). However, it still has significant limitations: relying solely on a single radiosensitizing mechanism, it lacks synergistic therapeutic function support, making it difficult to overcome the bottleneck of tumor radioresistance, and the overall anti-tumor effect is limited; the synthesis process often requires complex processes such as high-temperature calcination and organic phase dispersion, which is not only cumbersome and costly, but may also lead to poor product dispersibility, thereby affecting biocompatibility and clinical application safety; at the same time, the material does not have the ability to target the tumor microenvironment, and its selectivity for normal tissues during treatment is insufficient, which can easily cause side effects.

[0003] As tumor treatment technologies advance towards "precision and synergy," traditional single-function nano-hafnium oxide radiosensitizers are no longer sufficient to meet the current clinical demand for highly effective and low-toxicity anti-tumor therapy. Developing novel radiosensitizing materials that combine multi-mechanism synergistic therapeutic functions with excellent fabrication properties has become an urgent research need. To overcome these challenges, multi-mechanism synergistic anti-tumor strategies have become a core development direction in the field of nanobiomedicine. Bimetallic / multimetallic nanocomposites, through the functional complementarity and synergistic effects between components, integrate multiple therapeutic mechanisms such as "physical sensitization, chemical catalysis, and biological regulation," providing a new approach to solving the therapeutic bottlenecks of traditional single-function materials.

[0004] However, the development of existing bimetallic radiosensitizing materials still faces core technological challenges: on the one hand, the synthesis systems often rely on high temperature and pressure, toxic organic solvents, or complex template agents, making it difficult to achieve precise preparation under mild conditions, and it is also difficult to simultaneously ensure product size uniformity, colloidal stability, and biocompatibility; on the other hand, the functional synergy between metal components lacks precise regulation, and is mostly achieved through simple physical mixing or surface modification, failing to achieve a synergistic effect of "1+1>2", and the responsive design to the tumor microenvironment is insufficient, making it impossible to specifically activate the synergistic therapeutic effect at the tumor site. These problems make it difficult for existing bimetallic nanomaterials to move from the laboratory to clinical application, and there is an urgent need to establish a new nanomaterial design and synthesis system that is "easy to prepare, functionally synergistic, and precisely responsive".

[0005] It is worth noting that the Fenton-like catalytic activity of copper-based nanomaterials is closely related to their valence state. +1 valent copper exhibits superior tumor microenvironment-responsive catalytic performance compared to +2 valent copper and can trigger an emerging copper death effect—killing tumor cells by interfering with intracellular copper ion homeostasis and metabolic processes, providing a new functional dimension for synergistic radiosensitization. However, how to stably retain +1 valent copper in bimetallic nanomaterials and achieve its functional synergy with high-Z elements (such as Hf), while simplifying the synthesis process and improving product performance, remains a key technical challenge in current research.

[0006] In contrast, the Hf / Cu bimetallic synergistic self-assembled nanoparticle material (HC) proposed in this application not only continues the high Z-radiosensitization advantage of nano hafnium oxide, but also innovatively integrates Fenton-like catalysis and copper death dual synergistic mechanism through cysteine-mediated bimetallic synergistic design, solving the core problems of traditional nano hafnium oxide "single function, limited effect, and complex synthesis". Crucially, experiments confirmed that the Fenton-like catalytic effect of HC was significantly enhanced after X-ray irradiation. The core mechanism lies in the fact that Hf, as a high-z element (atomic number 72), can efficiently absorb energy and convert it into secondary electrons (such as photoelectrons and Auger electrons) under X-ray irradiation. Simultaneously, because Hf and Cu form a tight coordination structure through the carboxyl and thiol groups of cysteine ​​(providing a close-range contact channel for energy / electron transfer between the bimetals), the energy absorbed by Hf and the secondary electrons generated can be efficiently transferred to Cu sites. This can reduce any residual +2 valence copper during assembly to +1 valence copper (increasing the concentration of Fenton-like active centers) and lower the activation energy of the Fenton-like reaction (H2O2→·OH), thereby significantly enhancing the Fenton-like catalytic efficiency of Cu-based HC nanocatalysts. The synthesis process of this material is mild (room temperature), simple to operate, and requires no complex processes. The product exhibits superior dispersibility and stability and can achieve specific synergistic killing through tumor microenvironment response, significantly outperforming commonly used clinical nano-hafnium oxide radiosensitizing materials. Therefore, developing bimetallic nanomaterials with a synergistic three-in-one function of "radiotherapy sensitization-Fenton-like catalysis-copper death" and a superior preparation process is of great significance and application value for promoting the clinical upgrading of radiotherapy sensitization materials and the development of multimodal anti-tumor therapy. Summary of the Invention

[0007] The purpose of this invention is to provide a cysteine-mediated Hf / Cu bimetallic synergistic self-assembled nanoparticle material, and to provide a method for preparing the material, which exhibits good reactivity, selectivity and higher biosafety for cancer treatment.

[0008] The above-mentioned objectives of the present invention are achieved through the following technical solutions:

[0009] A method for preparing cysteine-mediated Hf / Cu bimetallic co-assembled nanoparticles includes the following steps:

[0010] S1. Dissolve cysteine ​​in deionized water, mix with alkaline solution to deprotonate cysteine, and obtain ligand solution;

[0011] S2. Add HfCl4 aqueous solution dropwise to the ligand solution and stir at room temperature to form Hf-cysteine ​​primary complex;

[0012] S3. Adjust the pH of the reaction mixture obtained in step S2 to 9-10;

[0013] S4. Add the CuCl2 aqueous solution to the alkaline reaction mixture obtained in step S3, and stir at room temperature to allow the CuCl2 solution to react. 2+ Hf / Cu bimetallic synergistic self-assembly nanoparticles were obtained by synergistic self-assembly with Hf-cysteine ​​primary complex, followed by centrifugation and washing.

[0014] Further, the alkaline solution mentioned in step S1 is a NaOH solution, and the molar ratio of cysteine ​​to NaOH is 1:1.

[0015] Furthermore, the molar ratio of cysteine, HfCl4, and CuCl2 is 2:1:1.

[0016] Furthermore, the pH in step S3 is 9.5.

[0017] Furthermore, the centrifugation conditions are 8000 rpm for 5 min.

[0018] The Hf / Cu bimetallic synergistic self-assembled nanoparticles prepared by this invention are spherical with a particle size of 30-40 nm; the Cu in the nanoparticles is predominantly in the +1 valence state. 2+ The proportion is ≤25%; under the carboxyl-thiol bridging of cysteine, Hf in the nanoparticles forms a molecular-level close coordination structure with Cu.

[0019] The Hf / Cu bimetallic synergistic self-assembled nanoparticles prepared by this invention can be used as X-ray radiosensitizers, Fenton-like catalytic-copper death synergistic therapeutic agents, or in the preparation of antibacterial or environmental remediation drugs. Under X-ray irradiation, the Hf / Cu bimetallic synergistic self-assembled nanoparticles enhance Cu energy transfer through Hf→Cu energy / electron transfer. + It reduces the content and lowers the activation energy of the Fenton-like reaction, thereby achieving tumor microenvironment-specific ·OH burst and activation of the copper death pathway.

[0020] The present invention also provides an antitumor composition comprising Hf / Cu bimetallic synergistic self-assembled nanoparticles prepared by the method thereof; the antitumor composition is an injection, a lyophilized powder, or a topical implant.

[0021] The present invention also provides a tumor treatment device, comprising: an X-ray emitting unit; and Hf / Cu bimetallic synergistic self-assembled nanoparticles prepared by the method and placed in the target area, for generating a triple synergistic killing effect of radiosensitization-Fenton-like catalysis-copper death under X-ray irradiation.

[0022] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects:

[0023] (1) Innovative Synthesis Method: This invention provides a novel strategy for the synergistic self-assembly synthesis of Hf / Cu bimetallic compounds using cysteine ​​ligands. Cysteine ​​is used as a multifunctional ligand, and its carboxyl and thiol groups are bridged to Hf via specific coordination interactions. 4+ With Cu 2+ This method simultaneously leverages the dual functions of ligand chelation and assembly guidance. By adjusting the system pH to 9-10, the deprotonation of the cysteine ​​functional group is promoted, providing a thermodynamic driving force for the synergistic coordination and orderly aggregation of bimetallic ions. This induces spontaneous in-situ nucleation and multi-level self-assembly at room temperature, ultimately forming small Hf-Cu bimetallic synergistic self-assembled nanoparticles. This method achieves a precise match between ligand function and bimetallic synergistic effect, and possesses technical advantages such as mild synthesis conditions (room temperature), simple operation process, and excellent product dispersibility and stability. Furthermore, the tight bimetallic coordination structure constructed by cysteine ​​provides the necessary spatial basis for subsequent energy / electron transfer from Hf to Cu under X-ray irradiation, which is a key structural guarantee for achieving enhanced catalytic effects.

[0024] (2) Functional Synergistic Innovation: Construct a three-in-one synergistic anti-tumor system of "radiotherapy sensitization-Fenton-like catalysis-copper death" to achieve efficient superposition of anti-tumor effects. Moreover, X-ray irradiation can significantly enhance the Fenton-like catalytic effect through energy / electron transfer between Hf and Cu, achieving efficient superposition and dynamic enhancement of anti-tumor effects. On the one hand, the high Z atomic number of Hf endows the material with strong radiotherapy sensitization properties, which can significantly improve the killing efficiency of radiotherapy on tumor cells; on the other hand, the +1 valent copper, which is mainly present in the Cu-cysteine ​​component, exhibits superior tumor microenvironment-responsive Fenton-like catalytic activity compared with +2 valent copper. Under X-ray irradiation, the secondary electrons generated by Hf after absorbing energy can further increase the concentration of +1 valent copper and reduce the activation energy of the catalytic reaction, efficiently catalyzing the conversion of highly expressed hydrogen peroxide in the tumor into highly toxic hydroxyl radicals, achieving dynamic enhancement of tumor microenvironment-specific chemical kinetic therapy; more importantly, +1 valent copper can simultaneously trigger the copper death effect. Through the multi-level synergistic effect of "radiotherapy sensitization - dynamic catalytic enhancement - copper death", the overall anti-tumor effect is further amplified, ultimately achieving highly efficient synergistic killing of tumor cells. Attached Figure Description

[0025] Figure 1 shows the characterization of Hf / Cu-cysteine ​​bimetallic synergistic self-assembled nanoparticles (HC) prepared in Example 1; (a) is the SEM image of HC, (b) is the TEM image of HC, (c) is the energy spectrum of HC material, and (d) is the HAADF and elemental mapping diagram of HC material.

[0026] Figure 2 shows the XPS spectrum of the HC material prepared in Example 1; (a)-(f) in the figure are the X-ray photoelectron spectra of Cu, Hf, C, S, O and N elements in HC, respectively.

[0027] Figure 3 shows the performance of the HC material prepared in Example 1; in the figure, (a) is the TMB fluorescence colorimetric reaction of HC at different pH, (b) is the TMB fluorescence colorimetric reaction of HC at different temperatures, (c) is the detection diagram of monovalent copper ions in HC material, (d) is the detection reaction diagram of free radicals of H2O2 catalyzed by HC with and without X-rays, and (e) is a comparison diagram of free radical generation catalyzed by HC and Cu-Cys assembly.

[0028] Figures 4-7 show the cytotoxicity of the HC material prepared in Example 1:

[0029] Figure 4 shows the cell viability after 24 h of incubation of 50 μg / mL HC material with 4T1 mouse breast cancer cells and HUVEC human normal umbilical vein endothelial cells.

[0030] Figure 5 shows the ROS fluorescence images of cells with and without X-rays for HC and HfO2.

[0031] Figure 6 shows cell viability experiments with HC and HfO2 in the presence and absence of X-rays.

[0032] Figure 7 shows the fluorescence images of DLAT protein aggregation in cells under X-ray conditions with and without HC and HfO2. Detailed Implementation

[0033] The present invention will be further described below with reference to the embodiments, but the scope of protection of the present invention is not limited to the scope shown in the embodiments.

[0034] Example 1

[0035] This embodiment provides a method for preparing cysteine-mediated Hf / Cu bimetallic co-assembled nanoparticles, the specific steps of which are as follows:

[0036] (1) Prepare 2 mmol NaOH (99% purity) particles (0.08 g) and 2 mmol cysteine ​​(0.2424 g). Cysteine ​​is the ligand required for the material. The carboxyl and thiol groups in its molecule are bridged to Hf through specific coordination. 4+ With Cu 2 + It simultaneously performs the dual functions of ligand chelation and assembly guidance.

[0037] (2) Dissolve NaOH and cysteine ​​in 10 ml of deionized water respectively. After they are completely dissolved, mix them to obtain solution 1.

[0038] (3) Dissolve 1 mmol HfCl4 (0.32 g) in 10 mL of deionized water and stir until clear and transparent to obtain solution 2.

[0039] (4) Continue stirring solution 1 and slowly add solution 2 to solution 1. As solution 2 is slowly added, solution 1 gradually turns into a white turbid liquid. After stirring at room temperature for 30 minutes, the turbidity is reduced, and mixed solution 3 is obtained.

[0040] (5) Weigh 1 millimole of CuCl2·2H2O (0.17 g) and dissolve it in 10 mL of deionized water to obtain solution 4.

[0041] (6) Continue stirring the mixture 3 and slowly add 5000 μL of 0.5 mol / L NaOH solution to the mixture 3 dropwise, with the goal of adjusting the pH of the mixture 3 to 9.5.

[0042] (7) Stir the pH-adjusted mixture 3 continuously, and add solution 4 dropwise at room temperature. The solution does not change color at first, but after solution 4 is added continuously, the solution turns blackish-gray, and the mixture 5 is obtained.

[0043] (8) The resulting mixture 5 was then centrifuged (8000 rpm, 5 min) to collect the reaction product and washed three times with deionized water.

[0044] The characterization of the Hf / Cu-cysteine ​​bimetallic synergistic self-assembled nanoparticles (HC) prepared in Example 1 is shown in Figure 1. Scanning electron microscopy (Figure 1a) and transmission electron microscopy (Figure 1b) reveal that the obtained HC nanoparticles are spherical with a size of approximately 30 nm, and the synthesized material exhibits a certain degree of stability. Energy dispersive spectroscopy (EDS) mapping data (Figure 1d) visually demonstrates the uniform distribution of Hf and Cu elements within the nanoparticles, confirming that Hf and Cu are not simply physically mixed, but rather form a "molecular-level tight coordination structure" through cysteine ​​bridging. This tight spatial arrangement provides a "close-range contact channel" for energy / electron transfer from Hf to Cu under X-ray irradiation, structurally eliminating the possibility of "transfer failure due to excessive distance between the bimetals," and forming the core structural basis for subsequent functional synergy.

[0045] The XPS pattern of the HC material prepared in Example 1 is shown in Figure 2. High-resolution XPS spectrum analysis of Hf, Cu, S, and O elements in HC shows that Cu appears near 932.6 eV in the Cu 2p orbital spectrum (Figure 2a). + The characteristic main peak of Cu near 934.5 eV. 2+ The characteristic peak intensity is only Cu +The 25% peak confirms that Cu in HC is predominantly in the +1 valence state, which aligns with the functional design of "highly active Fenton-like catalytic centers and copper death effect triggering". In the Hf 4f orbital spectrum (Figure 2b), the characteristic peaks of Hf 4f7 / 2 (≈16.8 eV) and Hf4f5 / 2 (≈18.5 eV) correspond to stable Hf 4+ The valence state ensures the radiosensitizing properties of its high Z element. A characteristic peak of 161.8 eV appears in the S 2p orbital spectrum (Figure 2d), corresponding to a metal-sulfur bond (Cu-S), confirming the interaction between the sulfhydryl group of cysteine ​​and Cu. + Specific coordination is formed; in the O 1s orbital spectrum (Figure 2e), the characteristic peak near 529.8 eV corresponds to a metal-oxygen bond (Hf-O), indicating that the carboxyl group of cysteine ​​interacts with Hf. 4+ To achieve effective coordination.

[0046] The performance of the HC material prepared in Example 1 is shown in Figure 3. By detecting the efficiency of HC in catalyzing the generation of free radicals from H2O2 (e.g., using TMB fluorescence colorimetry (Figure 3a, b) and TAOH colorimetry (Figure 3d, e)), it can be demonstrated that HC itself has Fenton-like activity in response to the tumor microenvironment (e.g., the ·OH production in an acidic, high-H2O2 tumor simulation environment is significantly higher than that in a neutral normal environment), laying the foundation for "Fenton-like catalysis participating in synergistic therapy";

[0047] The monovalent copper content in HC and single-metal Cu-cysteine ​​materials was directly detected using the new copper reagent (Figure 3c), which intuitively demonstrated that X-ray irradiation can significantly increase the content of monovalent copper in HC through energy / electron transfer via Hf.

[0048] The study using X-ray amplification effect data (Figure 3d) revealed that, comparing the ·OH production of the "no X-ray irradiation group" and the "X-ray irradiation group", the ·OH production of the irradiation group was significantly higher than that of the no-irradiation group, and the increase was far greater than the superposition effect of "simple X-rays + simple Cu-based materials". This directly confirms that X-ray irradiation can significantly enhance the Fenton-like catalytic efficiency through the energy / electron transfer of Hf.

[0049] Meanwhile, by setting up a control group of "single metal Cu-cysteine ​​+ X-ray" (Figure 3e), it was further demonstrated that "the amplification of the Fenton-like effect is most significant only when bimetallic synergy exists", eliminating the interference of single metal and clarifying the necessity of Hf-Cu synergy.

[0050] The cytotoxicity of the HC material prepared in Example 1 is shown in Figures 4-7. Using normal HUVEC cells and 4T1 tumor cells as models, multiple control groups were set up, and the experimental results are as follows (Figure 4):

[0051] In the CCK-8 assay for normal cells, the survival rate of HUVEC cells remained at 86.3% even when the HC concentration reached 200 μg / mL, confirming that HC has excellent biocompatibility and low toxicity to normal cells.

[0052] Cellular ROS experiment (Figure 5): DCFH-DA probe detection showed that the ROS fluorescence intensity of the HC+RT group was much higher than that of the HfO2+RT group, confirming that X-rays can enhance the Fenton-like catalytic production of ·OH by HC.

[0053] In the cell viability assay, Hoechst / PI double staining fluorescence (Figure 6) showed that the proportion of dead cells in the HC+RT group was 72.3%, which was significantly higher than that in the HfO2+RT group (28.7%), verifying the synergistic killing effect of "radiotherapy-Fenton-like-copper death".

[0054] Cellular DLAT assay (Figure 7) showed that the DLAT protein lipoacylation level in the HC+RT group was significantly increased, much higher than that in other groups, proving that HC+RT can specifically activate the copper death pathway.

[0055] In summary, cellular level experiments clearly demonstrate that HC possesses both excellent biocompatibility and synergistic killing activity of "radiotherapy sensitization-Fenton-like catalysis-copper death," with bimetallic synergy being the core of the effect amplification, providing direct cellular level evidence for the overall anti-tumor mechanism.

Claims

1. A method for preparing cysteine-mediated Hf / Cu bimetallic co-assembled nanoparticles, characterized in that, Includes the following steps: S1. Dissolve cysteine ​​in deionized water, mix with alkaline solution to deprotonate cysteine, and obtain ligand solution; S2. Add HfCl4 aqueous solution dropwise to the ligand solution and stir at room temperature to form Hf-cysteine ​​primary complex; S3. Adjust the pH of the reaction mixture obtained in step S2 to 9-10; S4. Add the CuCl2 aqueous solution to the alkaline reaction mixture obtained in step S3, and stir at room temperature to allow the CuCl2 solution to react. 2+ Hf / Cu bimetallic synergistic self-assembly nanoparticles were obtained by synergistic self-assembly with Hf-cysteine ​​primary complex, followed by centrifugation and washing.

2. The method according to claim 1, characterized in that, The alkaline solution mentioned in step S1 is a NaOH solution, and the molar ratio of cysteine ​​to NaOH is 1:

1.

3. The method according to claim 1, characterized in that, The molar ratio of cysteine, HfCl4, and CuCl2 is 2:1:

1.

4. The method according to claim 1, characterized in that, The pH in step S3 is 9.

5.

5. The method according to claim 1, characterized in that, The centrifugation conditions were 8000 rpm for 5 min.

6. An Hf / Cu bimetallic synergistic self-assembled nanoparticle prepared by the method according to any one of claims 1-5, characterized in that: The nanoparticles are spherical with a diameter of 30-40 nm; the Cu in the nanoparticles is predominantly in the +1 valence state. 2+ The proportion is ≤25%; under the carboxyl-thiol bridging of cysteine, Hf in the nanoparticles forms a molecular-level close coordination structure with Cu.

7. The use of the Hf / Cu bimetallic synergistic self-assembled nanoparticles prepared by the method of any one of claims 1-5 as an X-ray radiotherapy sensitizer, a Fenton-like catalytic-copper death synergistic therapeutic agent; or in the preparation of antibacterial or environmental remediation drugs.

8. The use according to claim 7, characterized in that: The Hf / Cu bimetallic synergistic self-assembled nanoparticles, under X-ray irradiation, enhance Cu energy / electron transfer via Hf→Cu. + It reduces the content and lowers the activation energy of the Fenton-like reaction, thereby achieving tumor microenvironment-specific ·OH burst and activation of the copper death pathway.

9. An antitumor composition comprising Hf / Cu bimetallic synergistic self-assembled nanoparticles prepared by the method of any one of claims 1-5; wherein the antitumor composition is an injection, a lyophilized powder, or a topical implant.

10. A tumor treatment device, comprising: X-ray emitting unit; And Hf / Cu bimetallic synergistic self-assembled nanoparticles prepared by the method according to any one of claims 1-5 and placed in the target area, for generating a triple synergistic killing effect of radiosensitization-Fenton-like catalysis-copper death under X-ray irradiation.

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