Application of a cerium monatomic functional material in preparation of a tumor radiotherapy sensitizer

Abstract

The application discloses application of a cerium monatomic functional material in preparation of a tumor radiotherapy sensitizer. SA In the material, cerium is dispersed on a nitrogen-doped carbon carrier in a monatomic form and forms a stable coordination structure with N / O. The application determines the structure-activity relationship of the unique physical and chemical structure and multiple enzyme activities, verifies the radiotherapy sensitization effect and biological safety in and out of the body. The material has multiple enzyme activities such as simulating peroxidase and glutathione oxidase, can efficiently catalyze ROS and consume GSH, and enhances DNA damage and apoptosis induced by ionizing radiation. In vitro clone formation experiments show that the radiotherapy sensitization ratio reaches 1.38, which is better than that of CeO2 nano clusters (1.17). In vivo experiments on tumor-bearing mice prove that the material combined with radiotherapy can significantly inhibit tumor growth, prolong survival time, and has good biocompatibility. The application provides a new strategy of high efficiency and low toxicity for tumor radiotherapy sensitization, and has a good clinical application prospect.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and nanocatalysis, specifically to the application of a cerium single-atom functional material in the preparation of tumor radiosensitizers, and a method for preparing the cerium single-atom functional material. Background Technology

[0002] Cancer, a highly prevalent and deadly malignant disease worldwide, has become a major public health challenge posing a serious threat to human life and health. Data from the World Health Organization shows that the number of new cancer cases and deaths globally remains high, and both incidence and mortality rates continue to rise, making prevention and treatment extremely challenging. Currently, surgical resection, chemotherapy, and radiotherapy are the three core methods for combating cancer in clinical practice. Among them, radiotherapy, as a crucial local radical and adjuvant therapy, is widely used clinically. Over 70% of cancer patients require radiotherapy during their disease course, and nearly 40% of malignant tumors can be cured with radiotherapy, occupying an irreplaceable core position in the comprehensive cancer treatment system. The anti-tumor mechanisms of radiotherapy are mainly divided into two categories: first, ionizing radiation directly destroys the double-stranded DNA of tumor cells, inducing cell death; second, it generates a large amount of reactive oxygen species by radiating water molecules in the body, thereby killing tumor cells through oxidative stress damage. However, in clinical practice, traditional radiotherapy still has many inherent shortcomings, which seriously restrict the treatment effect and long-term application: On the one hand, ionizing radiation lacks tumor targeting, and while killing tumor cells, it is very easy to damage the surrounding normal tissues, causing a series of adverse reactions; on the other hand, the hypoxic state unique to the tumor microenvironment and the high expression of antioxidants in tumor cells (such as the content of reduced glutathione is significantly higher than that in normal cells) will clear a large amount of ROS generated by radiotherapy, thereby inducing radiotherapy resistance and significantly reducing the efficacy of radiotherapy.

[0003] Addressing the pain points of traditional radiotherapy, such as radioresistance, collateral damage to normal tissues, and limited clinical efficacy, the development of highly efficient and adaptable radiosensitizers has become a key direction for overcoming the bottlenecks of traditional radiotherapy, possessing significant clinical practical value. The core of radiosensitization lies in using chemical, pharmacological, or physical agents to specifically enhance the sensitivity of tumor cells to ionizing radiation, strengthen the targeted killing efficiency of radiation on tumor cells, optimize tumor eradication rates, and simultaneously minimize collateral damage to normal tissues. This fundamentally compensates for the inherent defects of traditional radiotherapy, providing a new pathway for upgrading the efficacy of tumor radiotherapy. Based on this, in-depth exploration has been conducted on novel functional materials for radiosensitization, with the rapid development of nanomaterials and nanotechnology injecting new vitality into the field of tumor radiosensitization and gradually becoming a research hotspot for radiosensitizers.

[0004] In recent years, enzyme therapy has shown broad application prospects in the clinical treatment of various diseases. However, as biocatalysts, natural enzymes promote various biochemical reactions in organisms under physiological conditions. Their well-defined and uniform active site structure and coordination environment give enzyme catalysis high efficiency and specificity. However, natural enzymes are prone to denaturation and inactivation under harsh environments, are difficult to store, and require cumbersome preparation and refining processes, severely limiting their widespread application. Therefore, there is an urgent need to develop new materials to solve these problems.

[0005] In 2007, Fe3O4 nanoparticles were discovered to possess peroxidase-like activity, indicating that nanomaterials are not inert in biological systems but possess catalytic activity. Compared to natural enzymes, nanozymes offer advantages such as low production cost, high stability, and ease of surface functionalization, combining the "powers of natural and artificial catalysis" and demonstrating great potential to replace natural enzymes. Nanozymes exhibit a variety of outstanding characteristics, including synergistic effects, cascade reaction activity, and environmental responsiveness, and have been widely applied in fields such as biosensing, catalytic medicine, environment, and energy.

[0006] To date, researchers have sought to improve the catalytic efficiency of traditional nanozymes by adjusting their size, morphology, composition, and surface modification. While nanozymes overcome many drawbacks of natural enzymes, challenges remain in their application. Due to various defects, steps, and dislocations in nanomaterials, the active sites on the surface exist in different local environments, leading to decreased catalytic activity and selectivity. Furthermore, the heterogeneous active sites and their diverse surface coordination environments hinder the study of the structure-activity relationship between structure and catalytic activity. Therefore, improving the catalytic activity and selectivity of nanozymes remains a significant challenge.

[0007] With the rapid development of synthetic methods, characterization techniques, and theoretical calculations, the field of catalysis science has achieved remarkable success. A groundbreaking study published in 2019 reported a single-atom nanozyme (SAzymes) with an Fe-N4 active center. This material exhibits activity similar to natural antioxidant enzymes, effectively scavenging ROS and thus protecting cells. This exciting discovery sparked great interest in the exploration of single-atom nanozymes in the following years. In particular, the well-defined and uniform active sites provide models for determining catalytic reaction mechanisms, which is crucial for exploring structure-activity relationships and studying catalytic mechanisms. Furthermore, the high metal atom utilization and low metal loading can solve the problem of high cost in large-scale catalytic applications of traditional nanozymes. Compared with natural enzymes, the difficulty in separating feedstocks and products has also been effectively solved. Understanding the structure-activity relationship of enzyme catalysts is one of the important means to study reaction mechanisms and construct optimal catalytic systems. Single-atom nanozymes provide models and possibilities for understanding enzyme catalysis mechanisms at the atomic and molecular level, and provide a theoretical basis for designing more efficient nanozymes. Therefore, single-atom nanozymes are considered an effective bridge connecting natural enzymes and traditional nanozyme catalysis.

[0008] To date, researchers have designed single-atom nanozymes with multiple metal active centers, capable of mimicking the activities of natural enzymes such as peroxidase, oxidase, catalase, and superoxide dismutase. Due to their superior performance and high catalytic activity, various single-atom nanozymes have been developed and applied in cancer treatment research. Notably, these single-atom nanozymes often exhibit multiple enzyme-mimicking activities, a characteristic that may bring unique advantages—such as synergistic effects, cascade reactions, and selective responses to the tumor microenvironment—thereby improving the utilization efficiency of overexpressed substances (such as H2O2) in the tumor microenvironment, thereby enhancing the efficacy of tumor therapy. This effectively solves multiple bottlenecks inherent in traditional nanozymes, such as lattice defect dislocations, non-uniform surface active sites, and low catalytic activity and selectivity.

[0009] These studies demonstrate that the catalytic activity of single-atom nanozymes can be optimized through multiple strategies, including atomic coordination regulation, bimetallic synergy, and biomimetic structural design, providing new insights for achieving highly efficient and low-toxicity radiosensitization therapy. Cerium, as the most abundant element among the lanthanides, also possesses the well-known redox pair (Ce...). 3+ / Ce 4+ ), Ce 3+ and Ce 4+The interconversion between valence states is of great value in heterogeneous catalysis, making it one of the most attractive rare earth metals. CeO2 nanoparticles and cerium-based materials exhibit excellent oxygen activation ability and stability, enabling them to adapt to the complex reaction conditions in heterogeneous catalysis and demonstrating superior performance in applications such as fuel cells, water treatment, automotive exhaust purification, and disease treatment. Summary of the Invention

[0010] Radiotherapy (RT), a crucial treatment for malignant tumors, induces DNA damage and reactive oxygen species (ROS) bursts in tumor cells through high-dose, high-energy ionizing radiation (IR) to achieve its killing effect. However, tumor radioresistance, high expression of reduced glutathione (GSH) in the tumor microenvironment (TME), and hypoxia significantly weaken the efficacy of radiotherapy. Furthermore, high-dose ionizing radiation can damage normal lung tissue and even lead to pulmonary fibrosis, severely impacting patient prognosis. Therefore, developing radiosensitizers with both high catalytic activity and good biocompatibility is crucial for overcoming the bottlenecks in radiotherapy efficacy. Single-atom nanozymes, due to their well-defined and uniform active sites, high metal atom utilization, and excellent ROS regulation capabilities, show great potential in the field of tumor catalytic therapy. Cerium possesses reversible Ce... 3+ / Ce 4+ The redox cycle and excellent oxygen activation ability provide an ideal foundation for constructing a highly efficient multi-enzyme catalytic system.

[0011] Based on this, this study employs a host-guest synthesis strategy to construct a highly efficient and stable cerium single-atom nanozyme (Ce). SA -N / O), including the following steps:

[0012] Step 1: Zinc source, cerium acetylacetone, and organic ligand are mixed in a solvent and Ce(acac)3@ZIF-8 composite material is prepared by host-guest synthesis; wherein the molar ratio of zinc source to cerium acetylacetone is greater than 3:1.

[0013] Step 2: Place the Ce(acac)3@ZIF-8 composite material obtained in Step 1 in a protective atmosphere, heat it to 800-1000 ℃ at a heating rate of 1-10℃ / min, hold it at that temperature for 1-5 hours, and then cool it to room temperature to obtain the cerium single-atom functional material Ce. SA -N / O.

[0014] The molar ratio of the zinc source to cerium acetylacetone is 4:1 to 10:1; further, the molar ratio of the zinc source to cerium acetylacetone is preferably 5:1.

[0015] Based on the above technical solution, in step one, the zinc source is zinc nitrate or its hydrate, the organic ligand is 2-methylimidazole, and the solvent is methanol or ethanol; in step two, the protective atmosphere is one or more of nitrogen, argon, or helium; the heating rate is 5 ℃ / min, the heating temperature is 900 ℃, and the holding time is 3 hours.

[0016] This invention, through a series of physicochemical structural characterizations (aberration-corrected scanning transmission electron microscopy, X-ray photoelectron spectroscopy, etc.), confirms that Ce is uniformly dispersed in atomic form on the surface of a nitrogen-doped carbon support, forming a stable coordination structure with N / O (Ce). SA (-N / O), this unique structure not only effectively avoids the aggregation of Ce single atoms, but also endows it with excellent structural stability and electron transport properties. This Ce SA -N / O exhibits synergistically coupled catalase, peroxidase, and oxidase-like activities, efficiently catalyzing the generation of highly reactive ROS such as hydroxyl radicals (•OH) from H₂O₂. More importantly, Ce... SA -N / O can efficiently deplete reduced glutathione (GSH) within tumor cells. As a major antioxidant in tumor cells, GSH depletion can directly disrupt the tumor cells' own antioxidant defense system. In in vitro experiments, Ce... SA -N / O significantly enhances ionizing radiation-induced ROS levels and DNA damage, and significantly reduces tumor cell survival. In vivo experimental results show that Ce SA -N / O combined radiotherapy significantly inhibited tumor growth, demonstrating superior anti-tumor effects compared to the radiotherapy-only group, and no significant systemic toxicity or damage to vital organs was observed, further confirming the efficacy of Ce2-O combined radiotherapy. SA -N / O has good biocompatibility and can effectively protect normal tissues and organs while exerting a highly effective radiosensitizing effect. Attached Figure Description

[0017] Figure 1 For Ce SA Physical and chemical characterization of -N / O. (a) AC-STEM image; (b) intensity surface profile and intensity line profile; (c) EDS elemental mapping; (df) EELS spectrum.

[0018] Figure 2 For Ce SA BET specific surface area and pore size distribution of -N / O.

[0019] Figure 3 For N / C, CeO2 NCs and Ce SA DLS curves for -N / O.

[0020] Figure 4 For N / C, CeO2 NCs and Ce SA Zeta potential of -N / O.

[0021] Figure 5 For CeO2 NCs and Ce SA -N / O POD-mimic kinetic analysis. (ab) Michaelis-Menten kinetic analysis and Lineweaver-Burk plotting with H2O2 as substrate; (cd) Steady-state kinetic determination with TMB as substrate.

[0022] Figure 6 For CeO2 NCs and Ce SA -N / O GSHox-mimic kinetic analysis. (ab) Steady-state kinetic determination using GSH as substrate.

[0023] Figure 7 For Ce SA Stability assessment of -N / O. (a) Long-term storage stability; (b) Recovery and reusability; (c) HRTEM, SAED and AC-STEM images after multiple catalytic reactions.

[0024] Figure 8 For Ce SA -N / O and CeO2 NCs cytotoxicity to HUVECs and L929 cells.

[0025] Figure 9 For Ce SA Effects of -N / O combined RT on LLC cells. (a) LLC cell viability after treatment with different concentrations of materials; (b) Colony formation photographs; (c) Corresponding survival fractions.

[0026] Figure 10 To assess DNA damage using immunofluorescence staining for γ-H2AX. (a) γ-H2AX (red fluorescence) expression; (b) number of γ-H2AX focal points.

[0027] Figure 11 To detect the levels of GSH, NADPH, and MDA in cells.

[0028] Figure 12 Photographic curves showing the in vivo antitumor effect. (a) Digital image of the removed tumor; (b) Tumor growth curve; (c) Tumor weight; (d) Survival curve.

[0029] Figure 13 Representative images of tumor tissue stained with H&E, Ki67, and TUNEL. Detailed Implementation

[0030] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and do not limit the scope of the invention. The experimental materials, reagents, etc. used in the following embodiments can be obtained commercially or by known experimental methods.

[0031] Example 1: Ce SA Preparation of -N / O

[0032] Weigh 5.58 g of Zn(NO3)2·6H2O and 1.55 g of Ce(acac)2 (Zn:Ce molar ratio = 5:1) and disperse them in 150 mL of methanol solution, stirring until completely dissolved. Slowly add 150 mL of methanol solution containing 6.16 g of 2-methylimidazole to this solution. After the addition is complete, immediately place the mixture in an ice-water bath and sonicate for 5 minutes, then allow it to age overnight at 37 °C. After the reaction is complete, wash the resulting white precipitate three times with methanol by repeated centrifugation, and dry it under vacuum at 65 °C to obtain the Ce(acac)3@ZIF-8 precursor.

[0033] The dried precursor powder was placed in a tube furnace and heated to 900 °C at a rate of 5 °C / min under a flowing N2 atmosphere, and held at this temperature for 3 hours. After the holding period, the tube furnace was quickly opened, and the sample was allowed to cool completely to room temperature before Ce was collected. SA -N / O.

[0034] Comparative Example 1: Preparation of CeO2 NCs

[0035] The preparation method is the same as in Example 1, except that the amount of Ce(acac)3 added is 2.65 g (Zn:Ce molar ratio = 3:1), and the product obtained is CeO2 NCs.

[0036] Comparative Example 2: Preparation of N / C Support

[0037] The preparation method is the same as in Example 1, except that Ce(acac)3 is not added, and only pure ZIF-8 is used for calcination to obtain nitrogen-doped carbon support N / C.

[0038] Ce prepared in Example 1 SA -N / O was the experimental group, and CeO2NCs prepared in Comparative Example 1 and N / C prepared in Comparative Example 2 were used as control groups. Structural characterization, stability assessment, enzyme-mimicking activity assay, and radiosensitization effects were performed, as detailed below:

[0039] 1. Characterization and Technical Effects of Cerium Single-Atom Functional Materials

[0040] 1.1 Morphological and structural characterization

[0041] Figure 1 Ce was showcased SA The physical and chemical structural characterization results of -N / O. For example... Figure 1 As shown in (a), the aberration-corrected scanning transmission electron microscope (AC-STEM) image reveals Ce SA In the -N / O matrix, Ce is uniformly dispersed on the N / C support as isolated single-atom bright spots (marked by red circles). No Ce-C aggregates or lattice fringes were observed, confirming that Ce exists in single-atom form. Figure 1 (b) The intensity surface profile and intensity line profile further confirm the isolated dispersion characteristics of single atoms.

[0042] Figure 1 (c) Energy dispersive X-ray spectroscopy (EDS) elemental mapping shows that Ce, C, N and O elements are uniformly distributed on the N / C structure, indicating that Ce atoms are successfully anchored on the nitrogen-doped carbon support surface.

[0043] Figure 1 The electron energy loss spectrum (EELS) of (df) showed that spot scanning of the white bright spots (green circles) produced signals of Ce, O and N elements, confirming that O and N form a stable coordination structure (Ce-N / O) with Ce atoms.

[0044] The above results demonstrate that the present invention successfully prepared cerium single-atom functional materials with atomic-level dispersion, and their unique coordination structure provides a structural basis for subsequent excellent catalytic performance.

[0045] 1.2 Specific surface area and pore structure analysis

[0046] Figure 2 Ce was showcased SA BET specific surface area and pore size distribution of -N / O. CeO2 NCs and CeO2 were calculated using nitrogen adsorption-desorption isotherms. SA The BET specific surface areas of -N / O are 1019.87 m². 2 / g and 1095.22 m 2 / g. Pore size distribution analysis showed that the pores in both materials were predominantly micropores. The high specific surface area and abundant porous structure significantly increased the contact area between the material and the enzyme substrate for catalytic reaction, which helped to fully expose the Ce atom active sites and enhance the catalytic activity of the mimic enzyme.

[0047] 1.3 Particle size distribution and stability analysis

[0048] Figure 3 Dynamic light scattering (DLS) analysis showed that Ce SA -N / O has a narrower particle size distribution and a lower polydispersity index (PDI) than CeO2 NCs, indicating that CeSA -N / O exhibits good dimensional uniformity.

[0049] Figure 4 The zeta potential shows that Ce SA The -N / O surface carries a negative charge and has a higher absolute potential value, indicating better colloidal stability. This result is favorable for Ce. SA -N / O achieves long-term circulation in the blood, making it more suitable for tumor treatment.

[0050] 2. Simulated enzyme activity analysis

[0051] 2.1 Analysis of POD-mimic activity

[0052] Figure 5 CeO2NCs and Ce were showcased. SA -N / O POD-mimic kinetic analysis results. To quantitatively analyze Ce... SA The POD-mimic catalytic activity of -N / O and CeO2 NCs was investigated through steady-state kinetic experiments at different H2O2 or TMB concentrations. Key kinetic parameters were obtained by fitting the Michaelis-Menten equation curves and Lineweaver-Burk double reciprocal plots.

[0053] The results showed that Ce SA -N / O versus the Michaelis constant (K) for H2O2 and TMB double substrates m The values ​​are all lower than those of CeO2NCs, indicating that Ce... SA -N / O exhibits a stronger affinity for dual substrates. This indicates that when Ce is dispersed in single-atom form, its catalytic active centers are fully exposed, significantly improving substrate accessibility and thus demonstrating superior POD-mimic activity compared to CeO2 nanoclusters.

[0054] 2.2 Analysis of Glutathione Oxidase (GSHOx-mimic) Activity

[0055] Figure 6 CeO2NCs and Ce were showcased. SA GSHOx-mimic kinetic analysis results for -N / O. Although Ce SA -N / O exhibits excellent performance in catalyzing ROS generation, but the tumor microenvironment (TME) contains high concentrations of reduced glutathione (GSH), which can continuously consume newly generated ROS. This invention further evaluates Ce... SA -N / O mimics glutathione peroxidase (GSHO) x The activity of γ-mimic can convert GSH into oxidized glutathione (GSSG).

[0056] Steady-state kinetics were determined using DTNB as a probe and GSH as a substrate. The results showed that Ce... SA -N / O exhibits a significantly higher affinity (Km) for GSH substrates than CeO2NCs, enabling more efficient GSH consumption. This characteristic makes CeO2NCs more efficient at consuming GSH. SA -N / O can effectively weaken the antioxidant defense capabilities of tumor cells and synergistically enhance the effects of radiotherapy.

[0057] 2.3 Summary of Multi-Mimetic Enzyme Activities

[0058] comprehensive Figure 5 and Figure 6 Multi-mimetic enzyme activity analysis, Ce SA The catalytic activity of -N / O (including POD and GSHOx activities) is superior to that of CeO2NCs. This result indicates that Ce is uniformly dispersed atomically on the surface of the nitrogen-doped carbon support, forming a stable coordination structure with N / O, which significantly improves the material's enzyme-mimicking catalytic performance. Furthermore, Ce... SA -N / O also exhibits a concentration-dependent enhancement of ROS generation and effectively consumes reducing substances.

[0059] 3. Stability Assessment

[0060] Figure 7 Ce was showcased SA -N / O stability assessment results. For example... Figure 7 As shown in (a), Ce SA -N / O maintained high catalytic activity after long-term storage, demonstrating excellent long-term storage stability. Figure 7 (b) shows that after multiple recycling and reuse, Ce SA No significant attenuation was observed in the catalytic activity of -N / O. Figure 7 (c) The HRTEM, SAED, and AC-STEM images show that after multiple simulated enzyme-catalyzed reactions, Ce SA -N / O showed no obvious morphological and structural changes, and Ce single atoms remained well dispersed.

[0061] The above results indicate that the Ce prepared in this invention... SA -N / O exhibits excellent catalytic stability, laying the foundation for its practical application in living organisms.

[0062] 4. In vitro cell experiments

[0063] 4.1 Cytotoxicity assessment

[0064] Figure 8 Ce was showcased SACytotoxicity assessment results of -N / O and CeO2NCs on human umbilical vein endothelial cells (HUVECs) and mouse fibroblasts (L929).

[0065] The results, obtained using a CCK-8 assay kit, showed that different concentrations of Ce... SA After treatment with -N / O and CeO2NCs, HUVECs and L929 cells still maintained high cell viability (>85%), indicating that both materials have good biocompatibility. This provides a basis for Ce... SA -N / O provides a safety basis as a radiosensitizer for in vivo application.

[0066] 4.2 Radiosensitizing effect - CCK-8 and combination index

[0067] Figure 9 (a) Demonstrates the effects of different concentrations of CeO2NCs or CeO2 with or without radiotherapy (RT). SA LLC cell viability after -N / O treatment. Ce SA -N / O and CeO2NCs were co-incubated with Lewis lung cancer (LLC) cells, and ionizing radiation (IR, 4 Gy) was introduced as an exogenous stimulus. The results showed:

[0068] Ce SA -N / O itself exerted a concentration-dependent inhibitory effect on LLC cell viability; calculation of Ce SA The combination index (Q) of -N / O combined with RT therapy revealed CeO2NCs and Ce SA -N / O both enhanced the radiosensitivity of LLC cells after 4 Gy irradiation, with Ce... SA -N / O has a greater effect on improving radiosensitivity (Q > 1.15, indicating a synergistic therapeutic effect).

[0069] 4.3 Clonogenesis Experiment and Radiosensitization Ratio (SER)

[0070] Figure 9 (b) and (c) show the clonogenic experiments and the corresponding survival scores. To further quantify Ce... SA The radiosensitizing effect of -N / O was demonstrated in a clonogenic experiment under different ionizing radiation doses (2, 4, and 6 Gy). The results showed that Ce... SA -N / O exhibited the strongest inhibitory effect on colony formation; the calculated radiosensitization ratio (SER) was: Ce SA The -N / O group has a value of 1.38, while the CeO2 NCs group has a value of 1.17. Ce SA The SER value of -N / O is significantly higher than that of CeO2NCs, indicating that Ce has a single-atom structure. SA-N / O exhibits superior radiosensitization effects. This result verifies the significant advantages of the material of this invention compared to traditional cerium-based nanomaterials.

[0071] 4.4 DNA damage assessment - γ-H2AX immunofluorescence staining

[0072] Figure 10 This study demonstrates the expression and number of γ-H2AX foci in LLC cells treated with different materials. Ionizing radiation irradiation induces DNA damage in the cell nucleus, and changes in the number and protein levels of phosphorylated histone H2AX (γ-H2AX) foci can reflect the degree of DNA damage. The results show that Ce... SA The number and expression level of γ-H2AX foci in LLC cells of the -N / O + RT group were significantly increased, indicating that Ce SA -N / O combined radiotherapy significantly enhanced the degree of DNA damage in tumor cells. This explains Ce at the molecular level. SA -N / O radiosensitization mechanism.

[0073] 4.5 Redox State Regulation - Detection of GSH, NADPH and MDA

[0074] Figure 11 This study demonstrates the intracellular GSH, NADPH, and MDA levels in LLC cells treated with different materials, regardless of whether they received RT. The results show that Ce... SA LLC cells treated with -N / O showed more significant GSH and NADPH depletion after irradiation with 4 Gy compared to the CeO2NCs + RT group and the RT group.

[0075] Meanwhile, the content of malondialdehyde (MDA, which reflects the degree of lipid peroxidation) was significantly increased, indicating that the oxidative damage to the cell membrane was aggravated.

[0076] The above results indicate that Ce SA -N / O weakens the antioxidant defense capacity by consuming GSH and NADPH in tumor cells, while enhancing ROS production, which together lead to redox imbalance and cell damage, thereby enhancing the effect of radiotherapy.

[0077] 5. In vivo animal experiments

[0078] 5.1 Tumor Models and Grouping

[0079] Establish a subcutaneous LLC tumor-bearing C57BL / 6J mouse model (tumor volume approximately 100 mm). 3 ), randomly divided into 6 groups (n=5): (1) PBS control group; (2) CeO2NCs group; (3) Ce SA(3) -N / O group; (4) RT group (6 Gy × 3F); (5) CeO2NCs +RT group; (6) Ce SA -N / O + RT group.

[0080] The material (10 mg / kg) was injected via the tail vein, followed by IR irradiation (6 Gy, dose rate 600 cGy / min) 6 hours later, with irradiation every other day for a total of 3 times.

[0081] 5.2 Tumor growth inhibition

[0082] Figure 12 (a) Digital photographs of tumors removed two weeks after treatment in different groups are shown; (b) Tumor growth curves are shown; (c) Tumor weight is shown. Results showed that compared to the PBS group, the CeO2NCs group had no significant inhibitory effect on tumor growth; Ce... SA The -N / O group showed moderate inhibition of tumor growth, similar to the inhibitory effect of the RT group alone; Ce SA The -N / O + RT group exhibited the most significant tumor suppression effect: the smallest tumor volume and lightest tumor weight, and the suppression effect was significantly better than that of the CeO2NCs + RT group. This result is attributed to the radiation-induced damage and CeO2 induced tumor growth. SA Synergistic effects between -N / O mediated biocatalysis.

[0083] 5.3 Survival Analysis

[0084] Figure 12 (d) shows the survival curves of LLC tumor-bearing mice after treatment. The results show that Ce SA The synergistic effect between -N / O and RT significantly improved the overall survival rate of mice, Ce SA The median survival of mice in the -N / O + RT group was significantly longer than that of other groups.

[0085] 5.4 Histological Analysis

[0086] Figure 13 Representative images of tumor sections from each treatment group, stained with H&E, Ki67, and TUNEL, are presented. Results show: Ce... SA -N / O + RT group effectively inhibited tumor cell proliferation (lowest Ki67 positivity rate); Ce SA The -N / O + RT group showed the most significant tumor cell necrosis and apoptosis (highest TUNEL positivity rate, H&E showed obvious necrotic areas).

[0087] 5.5 Biosafety Assessment

[0088] No significant systemic toxicity or damage to vital organs (heart, liver, spleen, lungs, and kidneys) was observed in mice in any of the treatment groups, further confirming the efficacy of Ce. SA -N / O exhibits good biocompatibility and in vivo safety.

[0089] 6. Experiment Summary

[0090] Based on the above experimental data, the present invention has the following beneficial effects:

[0091] (1) First application of cerium single-atom functional materials for tumor radiosensitization: This invention creatively applies cerium single-atom functional materials to tumor radiotherapy. SA -N / O is used for radiosensitization, and by utilizing its highly efficient catalytic activity due to its single-atom structure and well-defined and uniform active sites, it achieves excellent radiosensitization effects.

[0092] (2) Significant radiosensitization effect (SER=1.38): Clonogenic experiments confirmed that Ce SA The radiosensitization ratio of -N / O (1.38) was significantly higher than that of CeO2NCs (1.17), demonstrating the superiority of the single-atom structure.

[0093] (3) Synergistic catalysis by multiple mimic enzymes: Ce SA -N / O simultaneously possesses multiple mimicry enzyme activities, including POD and GSHox. Figure 5 , Figure 6 It can efficiently catalyze the production of ROS, and also consume GSH to weaken the tumor's antioxidant defense, thus synergistically enhancing the radiotherapy effect.

[0094] (4) Excellent DNA damage induction ability: γ-H2AX immunofluorescence staining ( Figure 10 ) Confirm Ce SA -N / O combined radiotherapy significantly enhances DNA damage in tumor cells.

[0095] (5) Good biocompatibility: in vitro cytotoxicity test ( Figure 8 Both in vivo animal experiments and other studies have confirmed that Ce SA -N / O has no significant toxicity to normal cells and vital organs.

[0096] (6) Controllable preparation method: This invention reveals for the first time that a Zn / Ce molar ratio greater than 3:1 is the key critical condition for obtaining Ce single atoms rather than CeO2 clusters, thus realizing the controllable preparation of cerium single-atom functional materials.

[0097] (7) Excellent stability: structural characterization after long-term storage, repeated use, and multiple catalytic reactions ( Figure 7 ) Confirm Ce SA -N / O exhibits excellent catalytic stability.

[0098] In summary, this invention, by constructing CeSA-N / O single-atom nanozymes, clarified their unique physicochemical structure and structure-activity relationship with multiple enzyme-mimicking activities, and systematically verified their tumor radiosensitization effects and biosafety in vitro and in vivo. This not only provides new ideas for the structural design and functional optimization of single-atom nanozymes, but also provides a solid theoretical basis and feasible technical strategy for the translational application of single-atom nanozymes in tumor radiotherapy, showing good prospects for clinical application.

[0099] The applicant declares that, based on the above embodiments, those skilled in the art can combine the specific content values ​​of a certain component in the above embodiments with the technical solutions in the invention content section to generate new numerical ranges, which are also within the scope of this invention. To keep the specification concise, this application will not list these numerical ranges.

Claims

1. Application of a cerium single-atom functional material in the preparation of tumor radiosensitizers.

2. The application according to claim 1, characterized in that, In the cerium single-atom functional material, cerium is dispersed in single-atom form on a nitrogen-doped carbon support and forms a coordination structure with nitrogen and / or oxygen.

3. The application according to claim 1 or 2, characterized in that, The cerium single-atom functional material has catalytic activity that mimics peroxidase, catalase, oxidase and / or glutathione oxidase.

4. The application according to any one of claims 1-3, characterized in that, The cerium single-atom functional material is obtained by a preparation method including the following steps: Step 1: Zinc source, cerium acetylacetone, and organic ligand are mixed in a solvent and Ce(acac)3@ZIF-8 composite material is prepared by host-guest synthesis; wherein the molar ratio of zinc source to cerium acetylacetone is greater than 3:

1. Step 2: Place the Ce(acac)3@ZIF-8 composite material obtained in Step 1 in a protective atmosphere, heat it to 800-1000 ℃ at a heating rate of 1-10 ℃ / min, hold it at that temperature for 1-5 hours, and then cool it to room temperature to obtain the cerium single-atom functional material Ce. SA -N / O.

5. The application according to claim 4, characterized in that, The molar ratio of the zinc source to cerium acetylacetone is 4:1 to 10:

1.

6. The application according to claim 5, characterized in that, The molar ratio of the zinc source to cerium acetylacetone is 5:

1.

7. The application according to claim 4, characterized in that, In step one, the zinc source is zinc nitrate or its hydrate, the organic ligand is 2-methylimidazole, and the solvent is methanol or ethanol.

8. The application according to claim 4, characterized in that, In step two, the protective atmosphere is one or more of nitrogen, argon, or helium.

9. The application according to claim 4, characterized in that, In step two, the heating rate is 5 ℃ / min, the heating temperature is 900 ℃, and the holding time is 3 hours.

Images