A cascade nanozyme that targets and induces tumor pyroptosis, its preparation method and application

Abstract

This invention discloses a cascade nanozyme for targeted induction of tumor pyroptosis, its preparation method, and its application, belonging to the field of biomedical technology. The preparation method of the cascade nanozyme for targeted induction of tumor pyroptosis includes the following steps: synthesizing silver nanodots using glutathione as a ligand; reacting tetrachloroauric acid, manganese dioxide, and silver nanodots to obtain a gold / manganese nanozyme; activating the carboxyl groups of the gold / manganese nanozyme with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, followed by a directional covalent coupling reaction with GE11 peptide; after the reaction, purification yields the cascade nanozyme. The cascade nanozyme provided by this invention possesses dual enzyme activity, cascade catalytic ability, EGFR targeting ability, and fluorescence / CT dual-modal imaging capability, and can be used as a tumor-specific pyroptosis inducer to enhance immunotherapy for primary and metastatic OSCC.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a cascade nanozyme that targets and induces tumor pyroptosis, its preparation method, and its application. Background Technology

[0002] Head and neck squamous cell carcinoma is the sixth most common malignant tumor worldwide, with oral squamous cell carcinoma (OSCC) being the most common subtype. Current treatments for OSCC primarily include surgery, radiotherapy, and chemotherapy, but these methods face challenges such as maxillofacial defects, severe side effects, and a significant psychological and physical burden on patients. OSCC is characterized by high recurrence and metastasis rates. The high metastasis rate is a major cause of death in OSCC patients. These challenges highlight the urgent need to develop innovative strategies to address both primary and metastatic OSCC. Tumor immunotherapy, as one of the most transformative milestones in modern oncology, has broad application prospects. Unlike traditional local therapies, it eliminates tumors by activating a systemic immune response, especially targeting metastatic lesions. Although various immunotherapeutic drugs have been applied to OSCC, the low response rate and immune escape caused by the immunosuppressive tumor microenvironment pose challenges to immunotherapy. Therefore, transforming non-inflammatory tumors (“cold”) into inflammatory tumors (“hot”) and reshaping the immunosuppressive tumor microenvironment are crucial for improving the efficacy of immunotherapy.

[0003] Pyroptosis is a programmed cell death induced by inflammasome activation. It triggers a strong inflammatory response through gasdermin-dependent pore formation, cell swelling, cell membrane bubbling, and the release of pro-inflammatory cytokines, including interleukin-1β (IL-1β), interleukin-18 (IL-18), adenosine triphosphate (ATP), lactate dehydrogenase (LDH), and high mobility group box 1 (HMGB1). Due to its potent immunogenicity, pyroptosis not only induces immunogenic cell death but also recruits tumor-infiltrating lymphocytes, effectively reshaping the immunosuppressive tumor microenvironment and amplifying systemic anti-tumor immunity. Although chemotherapeutic drugs such as cisplatin and doxorubicin have been studied for inducing pyroptosis, their clinical translation remains limited due to insufficient tumor specificity leading to low pyroptosis induction efficiency, drug resistance, and off-target toxicity. Therefore, new strategies need to be developed to target and efficiently induce tumor pyroptosis.

[0004] ROS can induce pyroptosis through activation by classical inflammasomes, thus ROS-based therapies are considered a promising strategy. Compared to chemotherapy drugs, ROS-mediated pyroptosis offers a more direct, safer, and more efficient induction pathway. However, the limited production of ROS remains a bottleneck.

[0005] In summary, reversing the immunosuppressive tumor microenvironment remains a key challenge in OSCC immunotherapy. Pyroptosis offers a promising strategy for reshaping the "cold" tumor microenvironment, but highly effective pyroptosis inducers targeting OSCC are still relatively scarce. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing a cascade nanozyme that targets and induces tumor pyroptosis, in order to solve the problems mentioned in the background art.

[0007] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0008] A method for preparing a cascade nanozyme that targets and induces tumor pyroptosis, comprising the following steps:

[0009] Silver nanodots were synthesized using glutathione as a ligand.

[0010] The gold / manganese nanozyme was obtained by reacting tetrachloroauric acid, manganese dioxide and silver nanodots.

[0011] The gold / manganese nanozyme was mixed with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide for carboxyl activation, and then subjected to a directional covalent coupling reaction with GE11 peptide. After the reaction was completed, the cascaded nanozyme was obtained by purification.

[0012] Furthermore, the steps for synthesizing silver nanodots using glutathione as a ligand specifically include:

[0013] Silver nitrate and glutathione were dissolved in deionized water, and the pH was adjusted to 6-7. Then, hydrazine hydrate was added and stirred under light-protected conditions. Isopropanol was then added, and the supernatant was removed by centrifugation to obtain silver nanodots. The mass ratio of silver nitrate to glutathione was 1:(1.5-2); the mass-to-volume ratio of silver nitrate to deionized water was (7-10) mg:1 mL; and the volume ratio of hydrazine hydrate to deionized water was (0.08-0.12):1.

[0014] Further, the step of reacting tetrachloroauric acid, manganese dioxide, and silver nanodots to obtain gold / manganese nanozymes specifically includes:

[0015] Dissolve silver nanodots in deionized water to prepare a silver nanodot solution with a concentration of 8-12 mg / mL;

[0016] A mixture was obtained by sequentially adding 40-60 mM tetrachloroauric acid solution, manganese dioxide and silver nanoparticle solution to deionized water; the volume ratio of the silver nanoparticle solution to the tetrachloroauric acid solution was 1:(0.3-0.4); the mass-volume ratio of the manganese dioxide to the silver nanoparticle solution was (0.8-1.2) mg:1 mL.

[0017] The mixture was subjected to ultrasonic treatment, then the pH was adjusted to 7-8, and the mixture was heated to 70-90℃ for reaction. After the reaction was completed, the mixture was purified to obtain gold / manganese nanozyme.

[0018] Further, the mixture is sonicated, then the pH is adjusted to 7-8, and heated to 70-90℃ for reaction. After the reaction is completed, it is purified to obtain the gold / manganese nanozyme. The purification method is as follows:

[0019] The crude product solution containing the target product was centrifuged at 8500-9000 rpm, the supernatant was collected, and isopropanol was added. After thorough mixing, the mixture was centrifuged at 9500-10500 rpm. The supernatant was then completely discarded, and the precipitate was retained. The precipitate was then lyophilized to obtain the gold / manganese nanozyme.

[0020] Further, the gold / manganese nanozyme was mixed with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide for carboxyl activation, followed by a directional covalent coupling reaction with the GE11 peptide. After the reaction, the nanozyme was purified to obtain the cascade nanozyme. The specific steps included:

[0021] Gold / manganese nanozymes were added to deionized water for dispersion to prepare a gold / manganese nanozyme aqueous dispersion with a concentration of 4-6 mg / mL.

[0022] To the gold / manganese nanozyme aqueous dispersion, 60-70 mg / mL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride aqueous solution and 35-45 mg / mL of N-hydroxysuccinimide aqueous solution were added sequentially and the mixture was ultrasonically treated to activate the exposed carboxyl groups on the surface of the gold / manganese nanozyme, forming a highly reactive N-hydroxysuccinimide ester intermediate, thus obtaining the activated system.

[0023] A directional covalent coupling reaction was carried out by adding 0.5-1.5 mg / mL GE11 peptide solution to the activation system. After the reaction was completed, the cascaded nanozyme was obtained by purification. The volume ratio of the GE11 peptide solution, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride aqueous solution, N-hydroxysuccinimide aqueous solution and gold / manganese nanozyme aqueous dispersion was (0.5-1.5):(0.02-0.03):(0.03-0.04):1.

[0024] Furthermore, in the step of adding GE11 peptide solution to the activation system for a directional covalent coupling reaction, and then purifying the cascade nanozyme, the purification method is as follows:

[0025] After the reaction, the crude product was transferred to a dialysis bag, which was then placed in continuously stirred deionized water for dialysis purification. After dialysis, the solution containing the target product was removed from the dialysis bag and freeze-dried to obtain the cascade nanozyme.

[0026] Another objective of this invention is to provide a cascade nanozyme that targets and induces tumor pyroptosis, prepared using the above-described method.

[0027] Another objective of this invention is to provide the application of the above-mentioned targeted tumor pyroptosis-inducing cascade nanozyme in the preparation of anti-tumor drugs.

[0028] Furthermore, the tumor is oral squamous cell carcinoma.

[0029] Another objective of this invention is to provide the application of the above-mentioned targeted tumor pyroptosis-inducing cascade nanozyme as a tumor imaging contrast agent.

[0030] The cascaded nanozyme for targeted induction of tumor pyroptosis provided by this invention possesses dual enzyme-like activities (GOx-like and POD-like), cascade catalytic ability, EGFR targeting capability, and fluorescence / CT dual-modal imaging capability. It can serve as a tumor-specific pyroptosis inducer to enhance immunotherapy for primary and metastatic OSCC. The GOx-like activity of this cascaded nanozyme consumes glucose at the tumor site, continuously generating hydrogen peroxide in situ. This hydrogen peroxide is then catalyzed by the POD-like activity of the cascaded nanozyme to generate ROS, which is further accelerated by photothermal effects, leading to a local ROS storm that directly kills tumor cells. Importantly, the ROS storm induces Caspase-1 / GSDMD-dependent pyroptosis, resulting in the release of a large number of pro-inflammatory factors, effectively recruiting immune effector cells, and enhancing systemic anti-tumor immunity against primary and metastatic tumors. Furthermore, this cascaded nanozyme can also serve as an excellent nanoprobe, enabling fluorescence and CT dual-modal imaging to visualize the location of OSCC, facilitating precise image-guided therapy. In summary, these findings demonstrate that the cascaded nanozymes provided by this invention are a promising multifunctional therapeutic platform for targeting pyroptosis-induced and enhancing immunotherapy for primary and metastatic OSCC. Attached Figure Description

[0031] Figure 1Morphological characterization of AMNZ-GE. (a) Transmission electron microscopy image of AMNZ. (b) Size distribution histogram of AMNZ. (c) Transmission electron microscopy image of AMNZ-GE. (d) Size distribution histogram of AMNZ-GE.

[0032] Figure 2 Structural characterization of AMNZ-GE. (a) UV-Vis spectra of AMNZ, GE11, and AMNZ-GE. (b) Fourier transform infrared spectra of AMNZ, GE11, and AMNZ-GE. (c) Zeta potential determination of AMNZ, GE11, and AMNZ-GE. (d) Proton nuclear magnetic resonance spectra of AMNZ, GE11, and AMNZ-GE.

[0033] Figure 3 The chemical composition and elemental valence states of AMNZ-GE are shown in Figure 1. (a) X-ray photoelectron spectrum of Au 4f in AMNZ-GE. (b) X-ray photoelectron spectrum of Mn 2p in AMNZ-GE.

[0034] Figure 4 The dual-modal imaging performance of AMNZ-GE. (a) Fluorescence spectrum under 464 nm excitation, with insets showing photographs of AMNZ-GE aqueous solution under visible and ultraviolet light. (b) Fluorescence spectrum under high concentration (200 mM) of interfering ions (K+). + Ca 2+ Na + and Mg 2+ (c) Fluorescence intensity at different pH values. (d) CT intensity curves at different concentrations, with insets showing representative CT images.

[0035] Figure 5 Photothermal performance of AMNZ-GE. (a) Temperature rise over 10 minutes under laser irradiation at different power densities. (b) Photothermal stability during repeated heating-cooling cycles. (c) Temperature changes over time for the control group and AMNZ-GE aqueous solution under 808 nm laser irradiation. (d) Infrared thermal imaging of the control group and AMNZ-GE aqueous solution over time under 808 nm laser irradiation.

[0036] Figure 6 (a) GOx-like activity of AMNZ-GE with ABTS as the chromogenic substrate. (b) pH changes of different reaction solutions. (c) Glucose consumption by different concentrations of AMNZ-GE.

[0037] Figure 7POD-like activity of AMNZ-GE. (a) POD-like activity with ABTS as the chromogenic substrate. (b) Absorbance of methylene blue after adding AMNZ-GE at different hydrogen peroxide concentrations.

[0038] Figure 8 Photothermal enhanced nanocatalytic performance. (a) Absorbance of methylene blue under different reaction conditions. (b) Cascade catalytic activity with ABTS as the chromogenic substrate. (c) Cascade catalytic activity of AMNZ-GE with 3,3',5,5'-tetramethylbenzidine as the chromogenic substrate. (d) Electron spin resonance spectra under different reaction conditions.

[0039] Figure 9 The in vitro biocompatibility of AMNZ-GE. (a) Viability of SCC7, SCC25, and HaCaT cells (n = 3) after incubation with ANZ at different concentrations (0, 15, 30, 45, 60, and 75 μg / mL) for 24 hours without glucose. (b) Viability of SCC7, SCC25, and HaCaT cells (n = 3) after incubation with ANZ, (c) AMNZ, and (d) AMNZ-GE at different concentrations of glucose for 24 hours.

[0040] Figure 10 (a) Fluorescence and bright-field images of AMNZ or AMNZ-GE cell uptake (scale bar: 50 μm). (b) Quantitative percentage of BV650-positive cells in flow cytometry uptake (n = 3).

[0041] Figure 11 To demonstrate the in vitro antitumor activity of AMNZ-GE. (a) Representative images of SCC7 cell colony formation assays treated with different concentrations of AMNZ-GE (scale bar: 200 μm). (b) Quantitative analysis of colony-forming units per well (n = 3). (c) Representative images of scratch assays (scale bar: 200 μm). (d) Quantitative analysis of migration rate (n = 3).

[0042] Figure 12 The in vitro ROS generation performance of AMNZ-GE+NIR. (a) ROS fluorescence images in SCC7 cells under different treatment conditions. (b) Quantitative analysis images of ROS in SCC7 cells by flow cytometry (n = 3).

[0043] Figure 13 Observation of pyroptosis induced by AMNZ-GE+NIR. Live-cell imaging shows the morphology of pyroptotic cells in the AMNZ-GE+NIR group (arrows indicate membrane bubbling) (scale bar: 10 μm).

[0044] Figure 14To validate the mechanism of AMNZ-GE+NIR-induced pyroptosis. (a) Western blot analysis of pyroptosis-related proteins under different treatment conditions. (b) Semi-quantitative results of NLRP3. (c) Semi-quantitative results of ASC. (d) Semi-quantitative results of Cleaved Caspase-1. (e) Semi-quantitative results of GSDMD-N. (n = 3).

[0045] Figure 15 Release of cellular contents associated with pyroptosis induced by AMNZ-GE+NIR. (a) Quantitative results of IL-1β levels in cell culture supernatant under different treatment conditions. (b) Quantitative results of IL-18 levels. (c) Quantitative results of LDH levels. (d) Quantitative results of ATP levels. (n = 3). (e) Detection of HMGB1 transport (red) by laser confocal microscopy, with cell nuclei stained with DAPI (blue) (scale bar: 50 μm).

[0046] Figure 16 OSCC cells treated with AMNZ-GE+NIR were used to induce DC maturation. After co-culturing with SCC7 cells treated under different conditions, mature DCs (CD11c) were induced. + CD80 + CD86 + Quantitative analysis of the percentage of mature DCs in flow cytometry (n = 3).

[0047] Figure 17 The in vivo targeting and bimodal imaging performance of AMNZ-GE. (a) CT imaging of SCC7 tumor-bearing mice at different time points after tail vein injection of AMNZ or AMNZ-GE (red dashed circles indicate tumor location). (b) Quantitative analysis of relative CT intensity (n = 3). (c) Fluorescence imaging of SCC7 tumor-bearing mice (red dashed circles indicate tumor location). (d) Quantitative analysis of relative fluorescence intensity (n = 3). (e) Fluorescence imaging of tumors and major organs (heart, liver, spleen, lung, and kidney) in SCC7 tumor-bearing mice.

[0048] Figure 18 The in vivo photothermal properties of AMNZ-GE are shown. Infrared images reveal tumor temperature changes in tumor-bearing mice under 808 nm laser irradiation with different treatments.

[0049] Figure 19 To assess the in vivo biocompatibility of AMNZ-GE. (a) Representative hematoxylin-eosin histological images of major organs (heart, liver, spleen, lung, and kidney) of SCC7 tumor-bearing mice after different treatments (scale bar: 200 μm). (b) Changes in body weight of SCC7 tumor-bearing mice under different treatments (n=5).

[0050] Figure 20 The antitumor effects of AMNZ-GE+NIR on primary and metastatic OSCC. (a) Images of primary tumors after different treatments (tumors that have disappeared are marked with red dashed circles). (b) Quantitative analysis of primary tumor volume. (c) Images of metastatic tumors after different treatments. (d) Quantitative analysis of metastatic tumor volume. (n = 5).

[0051] Figure 21 Pyroptosis induced by AMNZ-GE+NIR in primary OSCC. (a) Immunofluorescence staining images of NLRP3 and GSDMD-N in primary OSCC under different treatment groups (scale bar: 50 μm). (b) Quantification of NLRP3 fluorescence intensity. (c) Quantification of GSDMD-N fluorescence intensity (n = 5, except for the AMNZ-GE group, n = 3 due to complete tumor eradication). (d) Detection of plasma pyroptosis-related secreted protein IL-1β levels by enzyme-linked immunosorbent assay (ELISA). (e) Plasma IL-18 levels (n = 3).

[0052] Figure 22 This demonstrates the dual immunomodulatory effects of AMNZ-GE+NIR on the spleen in vivo. (a) CD11c in the spleen + CD80 in cells (DCs) + CD86 + (a) Flow cytometry quantitative analysis of the percentage of mature dendritic cells (DCs). (b) CD3 count in the spleen. + CD8 + (CD8) + Quantitative analysis of T cell percentage by flow cytometry. (c) F4 / 80 of the spleen. + CD11b + CD86 in macrophages + Quantitative analysis of the percentage of cells (M1 macrophages) by flow cytometry. (d) F4 / 80 of the spleen. + CD11b + CD206 in macrophages + Flow cytometry quantitative analysis of the percentage of M2 macrophages (n = 5).

[0053] Figure 23 This demonstrates the dual immunomodulatory effects of AMNZ-GE+NIR on metastatic OSCC in vivo. (a) CD11c in metastatic OSCC + CD80 in cells (DCs) + CD86 + Quantitative analysis of the percentage of cells (mature DCs). (b) CD3 in metastatic OSCC. + CD8 + Cells (CD8)+ Quantitative analysis of the percentage of T cells. (c) F4 / 80 in metastatic OSCC. + CD11b + CD86 in macrophages + Quantitative analysis of the percentage of cells (M1 macrophages). (d) F4 / 80 of metastatic OSCC. + CD11b + CD206 in macrophages + Quantitative analysis of the percentage of cells (M2 macrophages). (n = 5, except for the AMNZ-GE group, n = 3 due to complete tumor regression).

[0054] In the figure, Control refers to the control group treated with physiological saline; ANZ refers to the experimental group treated with 10 mg / kg ANZ; AMNZ refers to the experimental group treated with 10 mg / kg AMNZ; and AMNZ+NIR refers to the experimental group treated with 10 mg / kg AMNZ and 2 W / cm². 2 The experimental group treated with near-infrared laser irradiation; the AMNZ-GE group refers to the experimental group treated with 10 mg / kg AMNZ-GE; AMNZ-GE+NIR refers to the experimental group treated with 10 mg / kg AMNZ-GE and 2 W / cm² laser. 2 The experimental group treated with near-infrared laser irradiation. Data in the figure are expressed as mean ± standard error. One-way ANOVA with Tukey correction was used to compare differences among multiple groups, and t-tests were used to compare differences between two groups. P <0.05 is considered statistically significant and is indicated by an asterisk (*). P <0.05;** P <0.01; *** P <0.001; **** P <0.0001). Detailed Implementation

[0055] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0056] To address the limited generation of reactive oxygen species (ROS) in existing technologies, nanocatalytic therapy utilizes nanomaterials with enzyme-like catalytic activity—nanozymes—to amplify intracellular ROS levels through reactions with specific substrates in the tumor microenvironment, thus overcoming this limitation. Through rational biomimetic design, nanozymes can be synthesized with tunable catalytic activity and multiple functions, such as mimicking the activities of POD, oxidases, and catalases, and have therefore attracted widespread attention. In particular, nanozymes containing multivalent metals such as Mn... 2 + / Mn 3+ Nanozymes possess POD-like activity, catalyzing the conversion of endogenous hydrogen peroxide (H₂O) in the tumor microenvironment into reactive oxygen species (ROS). However, a major challenge in nanocatalytic therapy is the low concentration of H₂O in tumors and the limited catalytic efficiency of nanozymes. GOx can overcome this limitation by oxidizing abundant tumor glucose (Glu) to gluconic acid and H₂O, providing substrates for the cascade catalytic reaction of POD-like nanozymes. However, natural GOx is unstable under physiological conditions. Ultrasmall gold nanoparticles (AuNPs), on the other hand, possess GOx-like activity, efficiently catalyzing the oxidation of intracellular glucose to H₂O while avoiding the instability of GOx, effectively overcoming the drawbacks of insufficient H₂O and low catalytic efficiency in nanocatalytic therapy. Furthermore, gold nanoparticles exhibit excellent optical, thermodynamic, and X-ray attenuation properties, making them suitable for photothermal therapy, fluorescence, and CT imaging of tumors. According to the Arrhenius equation, increased temperature accelerates the catalytic reaction of nanozymes, indicating that photothermal therapy can synergistically enhance the efficiency of nanocatalysis. Therefore, the design of biomimetic nanozymes with cascade catalysis and photothermal functions can amplify the in-situ generation of hydrogen peroxide and improve catalytic efficiency, which is a promising strategy for realizing enhanced nanocatalytic therapy to generate ROS.

[0057] Precisely inducing pyroptosis in tumor cells while protecting normal tissues is a key challenge in cancer treatment, requiring tumor-specific targeting strategies. In OSCC, EGFR is characteristically overexpressed, making it an ideal molecular target. The GE11 peptide (YHWYGYTPQNVI), identified through phage display peptide library screening, exhibits high affinity and specificity for EGFR. This dodecapeptide has been validated in in vitro cell models and in vivo tumor-bearing animal experiments, demonstrating its ability to specifically target EGFR-overexpressing tumor cells. Compared to clinically used anti-EGFR monoclonal antibodies such as cetuximab, the GE11 peptide offers several advantages: its smaller molecular size allows for deeper penetration into tumor tissues; its lower immunogenicity reduces the risk of adverse immune responses; and its simpler structure facilitates more economical and controllable conjugation with nanomaterials. Therefore, functionalizing nanomaterials through GE11 conjugation holds promise for achieving highly efficient EGFR-targeted delivery, improving the diagnosis and treatment of OSCC while minimizing off-target effects.

[0058] Specifically, this invention presents a biomimetic cascade nanozyme—gold / manganese nanozyme-GE11 (AMNZ-GE)—which is prepared as follows:

[0059] (1) Preparation of template / reducing agent AgNDs: Dissolve 80-100 mg of silver nitrate (AgNO3) and 120-200 mg of GSH in 10 mL of deionized water, stir for 30 minutes, and add 1 M sodium hydroxide dropwise to adjust the pH of the solution to 6-7. Then add 0.8-1.2 mL of hydrazine hydrate and stir under light-protected conditions. Next, add excess isopropanol, centrifuge to remove the supernatant, and redissolve the precipitate in 10 mL of water to obtain an AgNDs solution with a concentration of 8-12 mg / mL.

[0060] (2) Preparation of AMNZ: Add 300-400 μL of HAuCl4, 40-60 mM of MnO2 powder, and 1 mL of AgNDs solution sequentially to a round-bottom flask containing deionized water. Sonicate the mixture at room temperature using an ultrasonic cell disruptor, then add 1 M sodium hydroxide solution to adjust the pH to 7-8, and heat to 70-90℃ for 3-5 hours. After the reaction, purify the mixture to obtain dry, loose AMNZ powder. Dissolve the AMNZ powder in deionized water to prepare an AMNZ aqueous dispersion with a concentration of 4-6 mg / mL.

[0061] (3) Preparation of AMNZ-GE: Take 1 mL of the AMNZ aqueous dispersion prepared above and place it in a reaction flask. Accurately add 20-30 μL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride aqueous solution with a concentration of 60-70 mg / mL and 30-40 μL of N-hydroxysuccinimide aqueous solution with a concentration of 35-45 mg / mL to the mixture. Sonicate the mixture and then gently stir it to efficiently activate the exposed carboxyl groups on the surface of AMNZ to form a highly reactive N-hydroxysuccinimide ester intermediate, thus obtaining the activated system. Add 0.5-1.5 mL of 0.5-1.5 mg / mL GE11 peptide solution to the activated system to carry out a directional covalent coupling reaction. After the reaction is completed, the cascade nanozyme is obtained after purification.

[0062] In this embodiment of the invention, the cascaded nanozyme AMNZ-GE serves as a targeted pyroptosis inducer to enhance the immunotherapeutic effect on primary and metastatic OSCC. AMNZ-GE, functionalized with the GE11 peptide, actively targets OSCC cells overexpressing epidermal growth factor receptor (EGFR). This cascaded nanozyme possesses glucose oxidase-like (GOx) and peroxidase-like (POD) activities, enabling self-sufficiency in hydrogen peroxide and continuous generation of reactive oxygen species (ROS) through cascade catalysis. Furthermore, its inherent photothermal properties can be used for photothermal therapy, further enhancing its catalytic activity. This ROS storm activates the Caspase-1 / gasdermin D (GSDMD) pathway, inducing pyroptosis and releasing immunostimulatory molecules, thereby enhancing CD8+. + T-cell-mediated anti-tumor immunity. Therefore, AMNZ-GE, as a pyroptosis inducer, transforms "cold" tumors into highly immunogenic "hot" tumors by inducing ROS storms, triggering pyroptosis, and reversing immunosuppressive TME, significantly enhancing the immunotherapeutic effect of OSCC. Furthermore, AMNZ-GE possesses dual-modal imaging capabilities of fluorescence and computed tomography (CT), enabling precise tumor localization and image-guided therapy. In summary, this invention establishes a targeted cascade nanocatalytic therapy, providing a new direction for advancing OSCC immunotherapy.

[0063] Example 1: This example provides a cascade nanozyme that targets and induces tumor pyroptosis. Its preparation method includes the following steps:

[0064] (1) Preparation of template / reducing agent AgNDs: 84.5 mg of silver nitrate (AgNO3) and 153.5 mg of GSH were dissolved in 10 mL of deionized water and stirred for 30 minutes. The pH of the solution was adjusted to 6-7 by adding 1 M sodium hydroxide dropwise. Then 1 mL of hydrazine hydrate was added and stirred for 48 hours in the dark. Then excess isopropanol was added, and after centrifugation to remove the supernatant, the precipitate was redissolved in 10 mL of water to obtain an AgNDs solution with a concentration of 10 mg / mL.

[0065] (2) Preparation of AMNZ: 345 μL of 50 mM HAuCl4, 1 mg of MnO2 powder, and 1 mL of AgNDs solution were added sequentially to a round-bottom flask containing 10 mL of deionized water. The mixture was subjected to strong sonication for 5 minutes at room temperature using an ultrasonic cell disruptor to ensure that HAuCl4, MnO2, and AgNDs were highly uniformly dispersed in the solution, forming a stable initial mixture. Then, 1 M sodium hydroxide solution was added dropwise to adjust the pH to 7-8, and the mixture was heated to 80°C for 4 hours with gentle stirring. After the reaction was complete, the reaction system was allowed to cool naturally to room temperature to obtain a crude product solution containing the target product. The cooled crude product solution was transferred to a centrifuge tube and centrifuged at 8800 rpm for 15 minutes. The supernatant was collected, and isopropanol was added. After thorough vortexing or shaking to mix, the mixture was centrifuged at 10000 rpm for 15 minutes. The supernatant was then completely discarded, and the precipitate at the bottom of the centrifuge tube was retained. The precipitate was freeze-dried to obtain a dry, loose AMNZ powder. 10 mg of AMNZ powder was measured and dissolved in 2 mL of deionized water. The solution was gently vortexed or sonicated to ensure complete dissolution, yielding an AMNZ aqueous dispersion with a concentration of 5 mg / mL. This stock solution should be stored at 4°C protected from light for later use.

[0066] (3) Preparation of AMNZ-GE: Take 1 mL of the 5 mg / mL AMNZ aqueous dispersion prepared above and place it in a reaction flask. Add 24 μL of 64 mg / mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride aqueous solution and 36 μL of 42 mg / mL N-hydroxysuccinimide aqueous solution to the mixture in sequence. Sonicate the mixture at room temperature for 5 minutes, and then gently stir for 10 minutes to efficiently activate the exposed carboxyl groups on the AMNZ surface and form a highly reactive N-hydroxysuccinimide ester intermediate, which is prepared for the subsequent coupling reaction with the amino group. Add 1 mL of 1 mg / mL GE11 peptide solution to the activated AMNZ system. Place the mixture on a magnetic stirrer and gently stir for 1 hour at room temperature. During this process, the activated AMNZ carboxyl groups undergo an efficient amide bond condensation reaction with the primary amino group at the end of the GE11 peptide, realizing the directional covalent coupling of the GE11 peptide on the AMNZ surface. The mixture after the coupling reaction was completed was then transferred to a dialysis bag with a molecular weight cutoff of 3500 Da. The dialysis bag was placed in a large volume of continuously stirred deionized water for dialysis purification for a total of 12 hours. During this period, the external dialysis water was replaced every 3 hours. This step aims to completely remove unreacted 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, uncoupled free GE11 peptide, and possible small molecule byproducts and salt ions. After dialysis, the solution containing the target conjugate was removed from the dialysis bag. This solution was freeze-dried to obtain a dry solid product, denoted as AMNZ-GE. 10 mg of the freeze-dried AMNZ-GE solid was accurately weighed and dissolved in 5 mL of deionized water or physiological saline solution. The solution was gently vortexed or sonicated to completely reconstitute it, yielding an aqueous solution of the targeted pyroptosis inducer AMNZ-GE at a concentration of 2 mg / mL. This solution should be stored in a light-protected refrigerator at 4°C for later use.

[0067] Comparative Example 1: This comparative example provides a gold nanozyme (ANZ) which is prepared in the same way as AMNZ in Example 1, except that MnO2 is not added.

[0068] Example 2: The cascaded nanozyme AMNZ-GE prepared in Example 1 was characterized, its performance was tested, and related biological experiments were conducted, as follows:

[0069] (1) Characterization of materials: Transmission electron microscopy images were obtained using a JEM-2100F transmission electron microscope at an accelerating voltage of 200 kV. UV-Vis absorption spectra were acquired using a Lambad 800 UV-Vis spectrophotometer. Zeta potential measurements were performed using Zetasizer-nanozs dynamic light scattering. X-ray photoelectron spectroscopy was obtained using a Thermo Fisher Scientific ESCALAB 250Xi spectrometer. Fluorescence properties were measured using a Shimadzu RF-5301 PC fluorescence spectrophotometer.

[0070] (2) Catalytic performance testing of the material: The GOx-like activity of the nanomaterial was demonstrated by monitoring the generation of H2O2. 200 μg / mL of ANZ, AMNZ, or AMNZ-GE was reacted with 10 mM glucose for 30 minutes, followed by centrifugation at 12000 rpm for 5 minutes. The supernatant was mixed with 100 μL of 5 mM ABTS and 10 μL of 1 mg / mL horseradish peroxidase for 10 minutes. The absorbance at 420 nm was measured by UV-Vis absorption spectroscopy.

[0071] Further evaluation of the POD-like activity of the nanomaterials was conducted. 100 μL of 5 mM ABTS and 20 μL of 100 mM H2O2 were added to 200 μg / mL ANZ, AMNZ, or AMNZ-GE. After incubation for 10 minutes, the absorbance at 420 nm was recorded.

[0072] To assess the generation of functional hydroxyl radicals (ROS with the strongest oxidizing power), 2 mL of nanomaterial solution, 100 μg / mL methylene blue solution, and 10, 20, 40, 60, and 80 mM H₂O₂ solutions were added sequentially, and UV-Vis absorption spectra were measured at 600–700 nm. The generation of •OH was further confirmed by electron spin resonance spectroscopy using 5,5'-dimethylpyrrolidone-N-oxide as a spin trapping agent. 20 μL of DMPO, 2 mL of different samples (ANZ, AMNZ, or AMNZ-GE solution), and H₂O₂ solution were uniformly mixed, transferred to a quartz capillary, and measured on a Bruker E500 spectrometer.

[0073] To evaluate the cascade catalytic activity involving both GOx-like and POD-like activities, 200 μg / mL ANZ, AMNZ, or AMNZ-GE was incubated with 10 mM glucose at 37 °C for 30 min, followed by incubation with 100 μL of 5 mM ABTS for 10 min. The absorbance at 420 nm was measured.

[0074] (3) Cell Culture: Mouse OSCC cell line SCC7 (generously provided by Shanghai Ninth People's Hospital), human OSCC cell line SCC25 (purchased from Suzhou Haixing Biotechnology Co., Ltd., TCH-C479), and human keratinocyte cell line HaCaT (provided by the Second Hospital of Jilin University) were cultured in high-glucose DMEM complete medium under humidified conditions of 37°C and 5% carbon dioxide. Bone marrow-derived dendritic cells were isolated from the femur and tibia of 6-8 week old female C57BL / 6J mice (purchased from Changchun Yisi Experimental Animal Technology Co., Ltd.) and cultured in RPMI-1640 complete medium supplemented with 20 ng / mL granulocyte-macrophage colony-stimulating factor and 20 ng / mL interleukin-4. Half of the medium was replaced every two days. Cells collected on day 6 were considered immature DCs and used for subsequent experiments.

[0075] (4) In vitro biocompatibility assessment: SCC7, SCC25 and HaCaT cells in logarithmic growth phase were cultured at 1×10⁻⁶ cells per well. 4 Cells were seeded at a density of 1,000 cells / well in 96-well plates and incubated for 24 hours. Then, ANZ, AMNZ, or AMNZ-GE at concentrations of 0, 15, 30, 45, 60, and 75 μg / mL were added, and incubation continued for another 24 hours. For GOx activity assays, cells were cultured in glucose-free complete medium. Cell viability was determined using a cell counting kit-8, with absorbance measured at 450 nm using a multi-mode microplate reader (Synergy HT, BioTek). Relative cell viability was calculated using the following formula: (Sample absorbance - Blank absorbance) / (Control absorbance - Blank absorbance) × 100%.

[0076] (5) In vitro cell uptake experiment: SCC7, SCC25 and HaCaT cells were added at 4 × 10⁶ cells per well. 4 Cells were seeded at a density of [number] cells per well in 24-well plates. After incubation for 24 hours, cells were treated with 60 μg / mL AMNZ or AMNZ-GE for 8 hours. For confocal laser scanning microscopy, cells were fixed with 4% paraformaldehyde for 15 minutes and then mounted. Images were taken using a confocal laser microscope (Nikon AXR). For flow cytometry analysis, cells were digested, collected, and analyzed using a flow cytometer (MACSQuant Analyzer 16).

[0077] (6) Cell viability assay: SCC7 cells were seeded into 6-well plates at a density of 600 cells per well. After incubation for 24 hours, the cells were treated with AMNZ-GE at concentrations of 0, 15, 30, 45, 60, and 75 μg / mL, with the culture medium changed every two days. After 14 days, the cells were fixed with 4% paraformaldehyde for 15 minutes and then stained with crystal violet solution for 15 minutes. After rinsing, the plates were scanned using a scanner, and colony-forming units were counted using an inverted microscope (Olympus IX83).

[0078] (7) Cell migration assay: SCC7 cells were injected at a rate of 2 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 6-well plates. When cell confluence exceeded 95%, a vertical scratch was made on the bottom of the plate using a yellow pipette tip. Exfoliated cells were washed away. Cells were then treated with serum-free high-glucose medium at concentrations of AMNZ-GE (0, 15, 30, 45, 60, and 75 μg / mL). Images were taken using an inverted microscope at 0, 12, and 24 hours, and quantitative analysis was performed using ImageJ software. Cell migration was calculated as follows: Migration = (0-hour scratch area - 24-hour scratch area) / 0-hour scratch area × 100%.

[0079] (8) Intracellular ROS measurement: SCC7 cells were measured at 4 × 10⁻⁶ cells per well. 4 Cells were seeded at a density of [number] cells per well in 24-well plates and incubated for 24 hours. The following experimental groups were established: control group, ANZ group, AMNZ group, AMNZ + near-infrared (NIR) laser irradiation group, AMNZ-GE group, and AMNZ-GE + NIR group. After 24 hours of treatment, cells were incubated at 37°C in the dark with 10 μmol / L 2',7'-dichlorofluorescein diacetate for 20 minutes. After washing with serum-free high-glucose medium, ROS production was detected using an inverted fluorescence microscope and flow cytometry.

[0080] (9) Live-cell imaging of pyroptosis: SCC7 cells treated with AMNZ-GE+NIR were incubated with propidium iodide and transferred to a laser confocal scanning microscope live-cell imaging workstation maintained at 37°C, 5% carbon dioxide and high humidity. Images were captured every minute using bright field and fluorescence modes for time-lapse photography.

[0081] (10) Detection of pyroptosis contents release: To detect IL-1β and IL-18 released by SCC7 cells, cells were exposed to various experimental conditions, and cell culture supernatants were collected. To measure the levels of IL-1β and IL-18 in mouse serum, whole blood was collected via orbital sampling, coagulated at room temperature for 30 minutes, and then centrifuged at 1000 g for 15 minutes at 4 °C to collect the supernatant. The levels of IL-1β and IL-18 in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA). To detect ATP and LDH released by SCC7 cells, cells were exposed to various experimental conditions, and cell culture supernatants were collected. ATP concentration was measured using an ATP luminescent cell viability assay kit, and LDH concentration was measured using an LDH cytotoxicity assay kit. To detect the intracellular localization of HMGB1 by immunofluorescence staining, SCC7 cells were exposed to various treatment conditions and then fixed with 4% paraformaldehyde for 15 minutes. After permeabilization and cell blocking, the cells were incubated overnight at 4°C with rabbit anti-HMGB1 antibody, followed by incubation with CoraLite594-labeled goat anti-rabbit immunoglobulin G for 2 hours, and then stained with 4',6-diamidinyl-2-phenylindole (DAPI) for 5 minutes. The cells were mounted with mounting medium, and the localization of HMGB1 was detected using a laser confocal scanning microscope.

[0082] (11) Western blot analysis: To detect the expression of pyroptosis-related proteins, SCC7 cells were blotted at 2 × 10⁻⁶ cells per well. 5 Cells were seeded at a density of 1,000 cells per well in 6-well plates and treated for 24 hours under different experimental conditions. After cell lysis, proteins were fully released through repeated freeze-thaw cycles. Protein concentrations were collected and measured before Western blotting experiments. Primary antibodies included rabbit anti-NLRP3, rabbit anti-ASC, rabbit anti-Caspase-1, rabbit anti-GSDMD, and mouse anti-β-actin. Secondary antibodies included horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G or horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G. Finally, the chemiluminescent signals of the proteins were detected using a multifunctional gel imaging system (Tanon 5200, Tanon).

[0083] (12) In vitro activation and maturation of bone marrow-derived DCs: The isolation and culture methods for immature bone marrow-derived DCs are the same as those described above for cell culture. When co-culturing bone marrow-derived DCs with SCC7 cells, SCC7 cells were first cultured at a density of 2 × 10⁶ cells per well. 5Cells were seeded at a density of [number] cells per cell type in the lower chamber of a Transwell plate and incubated for 24 hours, followed by treatment under specified experimental conditions. Immature DCs were seeded at the same density in the upper chamber of the Transwell plate. After cell adhesion, both cell types were co-cultured for 24 hours. Bone marrow-derived DCs from the upper chamber were collected, blocked with mouse Fc receptor blocking agent for 10 minutes, and stained with FITC-labeled anti-mouse CD11c antibody, PE-labeled anti-mouse CD80 antibody, and APC-labeled anti-mouse CD86 antibody. The maturity of bone marrow-derived DCs was assessed by flow cytometry.

[0084] (13) In vivo fluorescence and CT imaging: Using 6-8 week old female C57BL / 6J mice (purchased from Changchun Yisi Experimental Animal Technology Co., Ltd.) and BALB / c nude mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.), 2×10⁻⁶ mice were subcutaneously injected into the left dorsal side. 6 A tumor-bearing model was established using SCC7 cells. For fluorescence imaging, BALB / c nude mice were intravenously injected with 10 mg / kg of AMNZ or AMNZ-GE. Fluorescence imaging was performed using an in vivo optical three-dimensional imaging system (IVIS Spectrum, Perkin Elmer) before and 2 hours after injection. After euthanasia, the heart, liver, spleen, lungs, and kidneys were collected for in vitro fluorescence imaging. Quantitative analysis of fluorescence intensity was then performed. For CT imaging, C57BL / 6J mice were intravenously injected with 10 mg / kg of AMNZ or AMNZ-GE. CT imaging was performed using a small animal CT system (μCT 760, United Imaging) before and 2 hours after injection. Quantitative analysis of CT values ​​was then performed.

[0085] (14) In vivo photothermal therapy: Tumor-bearing C57BL / 6J mice were intravenously injected with 10 mg / kg AMNZ-GE or saline as a control group. One hour after injection, the mice were treated with an 808 nm laser at 2 W / cm². 2 The tumor site was continuously irradiated with a high power density for 5 minutes. Temperature changes after laser irradiation were recorded using an infrared thermal imager.

[0086] (15) Establishment and treatment of primary and distant tumor models: To establish a primary OSCC model, 2×10⁻⁶ mcg of the tumor was subcutaneously injected into the left back of C57BL6 / J mice. 6 1.5 × 10⁶ SCC7 cells were injected subcutaneously into the right back of the mice on day 6. 6SCC7 cells were used to establish distant metastatic tumors. On day 7, tumor-bearing mice were randomly divided into 6 treatment groups: control group (saline), ANZ group (10 mg / kg), AMNZ group (10 mg / kg), AMNZ (10 mg / kg) + near-infrared laser (NIR) irradiation group (2 W / cm², 5 min), AMNZ-GE group (10 mg / kg), and AMNZ-GE (10 mg / kg) + NIR group (2 W / cm², 5 min). Treatment was administered once daily for 4 consecutive days. Mice were euthanized on day 8 after the first treatment. Mouse weight was monitored every 2 days. Tumor volume was measured every 2 days and calculated using the formula: Tumor volume = (length × width) / (length × width) 2 ) / 2. At the end of the experiment, the primary tumor and metastatic tumors were collected for imaging and volume measurement.

[0087] (16) In vivo biocompatibility assessment: After euthanasia of mice, major organs, including heart, liver, spleen, lung and kidney, were removed. The samples were fixed overnight with 4% paraformaldehyde, embedded in paraffin, sectioned, stained with hematoxylin-eosin, and histological changes were observed under an optical microscope.

[0088] (17) Detection of pyroptosis in vivo: After euthanasia of mice, serum was collected, and the levels of IL-1β and IL-18 were measured using enzyme-linked immunosorbent assay (ELISA). Primary tumor tissue was collected, fixed overnight at 4°C with 4% paraformaldehyde, and then frozen sections were prepared. The sections were incubated overnight at 4°C with rabbit anti-NLRP3 antibody or rabbit anti-GSDMD-N antibody. The sections were then incubated with FITC-labeled goat anti-rabbit secondary antibody for 30 minutes. Subsequently, the sections were stained with DAPI for 5 minutes, mounted, and images were taken using a laser confocal scanning microscope, and the fluorescence intensity was quantified.

[0089] (18) Detection of changes in in vivo immune status: After euthanasia of mice, spleens and distant metastases were collected, digested into single-cell suspensions, and analyzed by flow cytometry staining. To assess DC maturity, the following antibodies were used: FITC-labeled anti-mouse CD11c antibody, PE-labeled anti-mouse CD80 antibody, and APC-labeled anti-mouse CD86 antibody. To assess CD8... + T cells were analyzed using FITC-labeled anti-mouse CD3ε antibody and PE / Cy5-labeled anti-mouse CD8a antibody. To assess macrophage polarization, the following antibodies were used: FITC-labeled anti-mouse / human CD11b antibody, PE / Cy5-labeled anti-mouse F4 / 80 antibody, PE / Cy7-labeled anti-mouse CD206 antibody, and APC-labeled anti-mouse CD86 antibody. Finally, counterstaining with DAPI was performed, and analysis was performed using flow cytometry.

[0090] The results of the above characterization, performance testing, and related biological experiments are as follows: Figures 1-23 As shown.

[0091] The morphology and structure of AMNZ and AMNZ-GE were characterized using transmission electron microscopy. Figure 1 As shown in a, AMNZ is spherical; as Figure 1 As shown in b, the average size of the AMNZ is approximately 2.4 nm. Figure 1 As shown in c, after GE11 coupling, AMNZ-GE maintains a spherical morphology; as Figure 1 As shown in d, the average particle size of AMNZ-GE increases to 3.0 nm, exhibiting ultra-small size and uniform size distribution.

[0092] The structure of AMNZ-GE was further characterized. For example... Figure 2 As shown in Figure a, in the UV-Vis absorption spectrum, GE11 exhibits a distinct characteristic absorption peak at 274 nm, while AMNZ shows no characteristic peak across the entire measurement range. However, AMNZ-GE shows a peak at the corresponding wavelength, confirming the successful connection of GE11. Figure 2 As shown in b, in the Fourier transform infrared spectrum, AMNZ at 820 cm⁻¹ -1 No peak was observed at the GE11, but characteristic peaks of the benzene ring fingerprint signal appeared in both GE11 and AMNZ-GE, further supporting the successful connection between GE11 and AMNZ. Figure 2 As shown in Figure c, the Zeta potential values ​​are -25.2 mV for AMNZ and -28.6 mV for AMNZ-GE, which also proves the successful synthesis of AMNZ-GE. Figure 2 As shown in d, in the proton nuclear magnetic resonance spectrum, characteristic hydrogen signals from the GE11 peptide can be seen in AMNZ-GE, mainly including aromatic hydrogen on the tryptophan indole ring (shift of about 7.01 ppm) and ortho hydrogen on the tyrosine benzene ring (shift of about 6.75 ppm), which are not present in AMNZ, confirming that GE11 has been successfully attached to the surface of AMNZ nanozyme.

[0093] The chemical composition and elemental valence states of AMNZ-GE were assessed using X-ray photoelectron spectroscopy. Figure 3 As shown in Figure a, in the Au4f spectrum, the characteristic peaks at 88.3 eV and 84.5 eV correspond to Au(0) and Au(I), respectively. Au(0) facilitates the nucleation of nanodots and provides GOx-like activity, while Au(I) can react with thiol groups to form stable Au-S bonds, which not only improves the stability of nanodots but also endows them with bright and stable fluorescence properties. Figure 3 As shown in b, in the Mn 2p spectrum, the characteristic peaks at 641.2 eV and 653.3 eV correspond to Mn 2p 3 / 2 and Mn 2p 1 / 2, respectively, confirming that Mn 2+The presence of this substance is fundamental to the catalytic production of ROS from hydrogen peroxide. The above data confirms the successful synthesis of an AMNZ-GE nanozyme with an ultra-small size.

[0094] To address the limitations of existing OSCC detection methods, particularly the constraints of single-modal imaging, the designed AMNZ-GE also features fluorescence / CT dual-modal imaging capabilities. Dual-modal imaging provides more accurate localization and supplementary diagnostic information, which is crucial for guiding clinical decisions in tumor diagnosis and treatment. Figure 4 As shown in Figure a, the fluorescence spectrum reveals a strong excitation peak at 464 nm and a strong emission peak at 660 nm. Since fluorescence stability is crucial for in vivo applications, it is important to consider common physiological cations (Ca). 2+ K + Na + Mg 2+ The ionic stability of AMNZ-GE was examined in the presence of [a specific substance / factor]. For example... Figure 4 As shown in b, even in the presence of high concentrations of interfering ions, the fluorescence intensity of AMNZ-GE remained stable, demonstrating excellent ion stability. Furthermore, the fluorescence stability of AMNZ-GE within the physiologically relevant pH range (4–9) was also evaluated. Figure 4 As shown in c, the fluorescence intensity of AMNZ-GE remains stable within this range. Overall, these results indicate that the prepared AMNZ-GE can serve as a stable and durable fluorescent probe suitable for in vivo biomedical applications. To evaluate the multimodal imaging capabilities of AMNZ-GE, its CT imaging performance was further investigated. Based on the X-ray attenuation ability of gold, AMNZ-GE is expected to exhibit strong CT imaging performance. Figure 4 As shown in d, both CT signal intensity and image brightness increase proportionally with AMNZ-GE concentration, confirming its potential as a CT contrast agent. In summary, the synthesized AMNZ-GE nanosystem shows promise as a dual-modal fluorescence / CT imaging probe for accurate detection of OSCC.

[0095] Photothermal therapy is a newly developed cancer treatment that uses a photothermal agent to convert near-infrared light into heat energy, ultimately leading to the thermal ablation of the tumor. Figure 5 As shown in Figure a, at different power densities (1.0, 1.5, 2.0 and 2.5 W / cm²), 2 Under irradiation with an 808nm laser, the temperature increased by 7.3, 13.5, 19.8, and 27.9℃, respectively. For example... Figure 5 As shown in b, the heating efficiency of AMNZ-GE showed almost no loss after four laser-switching cycles, indicating its excellent photothermal stability and suitability for continuous, repetitive photothermal therapy applications. Figure 5 As shown in c, at 2.0 W / cm2 At the specified power density, after 10 minutes of irradiation, the temperature of AMNZ-GE increased significantly, while almost no temperature change was observed in phosphate-buffered saline (PBS). Figure 5 As shown in d, infrared thermal imaging visually reveals the temperature difference between the two groups. In conclusion, AMNZ-GE can serve as an excellent photothermal agent for photothermal therapy.

[0096] Thanks to the presence of gold and manganese, AMNZ-GE is expected to exhibit dual enzyme-like activities (GOx-like and POD-like activities) to catalyze cascade reactions. First, the gold-catalyzed decomposition of glucose to hydrogen peroxide was demonstrated. For example... Figure 6 As shown in Figure a, GOx-like activity was assessed using the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) colorimetric method. In the presence of glucose, the hydrogen peroxide concentration in the aqueous solutions of ANZ (gold nanozyme), AMNZ, and AMNZ-GE increased, indicating GOx-like activity. Furthermore, the incorporation of manganese and modification with GE11 did not affect the GOx-like activity of gold. Figure 6 As shown in b, the pH value in AMNZ-GE decreases, further confirming its GOx-like activity. Figure 6 As shown in Figure c, the decrease in glucose content is positively correlated with the content of AMNZ-GE. These experiments confirm that AMNZ-GE possesses good GOx-like activity.

[0097] like Figure 7 As shown in Figure a, the POD-like catalytic activity of AMNZ-GE was detected by ABTS colorimetric method. The results showed that the incorporation of manganese endowed it with POD-like activity, enabling it to catalyze the decomposition of hydrogen peroxide to produce ROS, while the connection of GE11 did not affect its catalytic effect. Figure 7 As shown in b, the POD-like activity of AMNZ-GE was further evaluated using methylene blue as an indicator. A concentration-dependent relationship was observed between ROS generation and hydrogen peroxide concentration, indicating high catalytic efficiency. These data demonstrate that AMNZ-GE possesses excellent GOx-like and POD-like catalytic activity, showing potential for application in nanocatalytic therapy.

[0098] Since the catalytic activity of POD-like enzymes increases with increasing temperature, photothermal activity promotes the POD-like activity of AMNZ. Figure 8 As shown in Figure a, the methylene blue absorbance was lowest in the AMNZ+NIR group, demonstrating that photothermal energy promotes the peroxidase-like activity of AMNZ. Subsequently, the cascade catalytic effect of AMNZ-GE was investigated. Figure 8As shown in b, under near-infrared irradiation, the AMNZ-GE+Glu+NIR group exhibits the highest absorbance at 420 nm, indicating that AMNZ-GE can perform photothermally promoted cascade catalysis, catalyzing the production of hydrogen peroxide from glucose, and further catalyzing the generation of ROS from hydrogen peroxide. Figure 8 As shown in Figure c, the cascade catalytic activity in the presence of glucose was further investigated using the 3,3',5,5'-tetramethylbenzidine colorimetric method. The results showed that the oxidized form of 3,3',5,5'-tetramethylbenzidine exhibited the strongest absorbance at 652 nm under both acidic and photothermal conditions. This is because, under photothermal and weakly acidic conditions, AMNZ-GE generates hydroxyl radicals (the most potent oxidizing ROS) through GOx-like and POD-like catalytic activities. To further evaluate the ability of AMNZ-GE to generate hydroxyl radicals, electron spin resonance and 5,5-dimethylpyrrolidone N-oxide were used as spin traps to monitor the formation of hydroxyl radicals. Figure 8 As shown in d, the AMNZ-GE+NIR group exhibits the strongest electron spin resonance signal for hydroxyl radicals, with a characteristic peak intensity ratio of 1:2:2:1. These results indicate that AMNZ-GE can effectively catalyze the decomposition of hydrogen peroxide into hydroxyl radicals, and that photothermal enhancement significantly improves the catalytic activity, achieving photothermally enhanced nanocatalytic activity.

[0099] To assess the biocompatibility of the synthesized nanomaterials, SCC7 (mouse OSCC cell line), SCC25 (human OSCC cell line), and HaCaT (human keratinocyte cell line) cells were treated with gradient concentrations (0, 15, 30, 45, 60, and 75 μg / mL) of ANZ, AMNZ, and AMNZ-GE, followed by cell viability assays. A glucose-free control group was established to assess Gox-like activity. Figure 9 As shown in figure a, cell viability remained above 90% in ANZ-treated cells. Figure 9 As shown in b, under glucose conditions, treatment with ANZ at concentrations of 60 and 75 μg / mL reduced cell viability to below 90%, confirming that ANZ-induced cytotoxicity is glucose-dependent. Figure 9 As shown in c, after treatment with 60 μg / mL AMNZ, HaCaT cells maintained >90% cell viability. Figure 9 As shown in Figure d, when treated with 60 μg / mL AMNZ-GE, HaCaT cells maintained good cell viability (>85%), while the viability of SCC7 cells decreased to 66.13 ± 3.39%, and the viability of SCC25 cells decreased to 69.61 ± 2.08%. This indicates that the nanomaterial exhibits excellent biocompatibility with normal cells while specifically killing tumor cells. Based on these results, a concentration of 60 μg / mL was selected for subsequent cell experiments.

[0100] To verify the cellular uptake and targeting capabilities of AMNZ and AMNZ-GE, the inherent fluorescence properties of the materials were used for detection. For example... Figure 10 As shown in Figure a, laser confocal microscopy results revealed that in SCC25 and SCC7 cells, at a concentration of 60 μg / mL, the AMNZ-GE group exhibited a stronger BV650 signal (red) than the AMNZ group, attributed to the interaction between GE11 and EGFR; while in HaCaT cells, both the AMNZ and AMNZ-GE groups showed similar and weak red fluorescence signals, which is related to the relatively low EGFR expression level in normal cells. Figure 10 Flow cytometry analysis of B showed that in SCC7 cells, the percentage of BV650-positive cells increased significantly from 50.17 ± 1.72% in the AMNZ group to 73.40 ± 3.54% in the AMNZ-GE group; in SCC25 cells, it increased from 51.13 ± 4.55% to 76.50 ± 4.56%, while there was no difference in HaCaT cells. These results indicate that AMNZ-GE significantly enhances cellular uptake in OSCC cells without affecting normal cells, demonstrating excellent tumor targeting.

[0101] like Figure 11 As shown in figure a, colony formation experiments revealed that AMNZ-GE inhibited the colony formation ability of SCC7 cells in a concentration-dependent manner. Figure 11 Quantitative analysis of b showed that the average colony-forming units per well decreased from 148.7 ± 13.45 in the control group to 4.33 ± 0.33 in the 60 μg / mL AMNZ-GE group, indicating its strong anti-proliferative effect. Figure 11 As shown in c, the scratch assay revealed that AMNZ-GE inhibited cell migration in a concentration-dependent manner. Figure 11 Quantitative analysis of the d-cell count showed that the cell migration rate in the 60 μg / mL group decreased to 6.00 ± 2.08% compared to the control group. These findings indicate that AMNZ-GE has antitumor activity, and 60 μg / mL AMNZ-GE significantly inhibited the proliferation and migration of OSCC in vitro. These findings lay the foundation for further research on AMNZ-GE combined with photothermal therapy for OSCC.

[0102] Given the excellent photothermal properties and POD-like catalytic activity of AMNZ-GE, photothermal therapy holds promise for synergistic effects with nanocatalytic therapy to generate excess ROS and establish an integrated photothermally enhanced nanocatalytic therapy. The ROS generation level of this therapy was first examined. For example... Figure 12As shown in Figure a, intracellular ROS levels were assessed using the oxidation-sensitive probe 2',7'-dichlorodihydrofluorescein diacetate. The results showed that the AMNZ-GE+NIR group exhibited the highest fluorescence intensity. Figure 12 Flow cytometry further confirmed these results. Quantitative analysis showed that the proportion of ROS-positive cells in the AMNZ-GE+NIR group reached 51.97 ± 1.61%, which was higher than that in the AMNZ+NIR group and the AMNZ-GE group. These results indicate that the AMNZ-GE+NIR-mediated photothermal enhanced nanocatalytic therapy has excellent ROS generation capacity.

[0103] Since ROS is crucial for initiating pyroptosis, this study further investigated whether AMNZ-GE+NIR-mediated photothermally enhanced nanocatalytic therapy induces pyroptotic cell death. Figure 13 As shown, confocal time-lapse microscopy revealed that cells in the AMNZ-GE+NIR group exhibited typical pyroptosis morphology. Initially, significant cell swelling was observed, followed by the rapid formation of large cell membrane bubbles (indicated by arrows), a clear hallmark of pyroptosis. Ultimately, all these pyroptotic cells showed strong propidium iodide-positive staining, confirming membrane rupture and cell death.

[0104] To further validate the pyroptosis induced by AMNZ-GE+NIR-mediated photothermal-enhanced nanocatalytic therapy, the expression of pyroptosis-related proteins in SCC7 cells was examined. Pyroptosis is a programmed cell death process requiring inflammasome activation. It typically involves the recruitment of apoptosis-associated speckled protein (ASC) and Pro Caspase-1 via ROS-mediated NOD-like receptor family pyrin domain proteins (3NLRP3), leading to their autocatalytic cleavage into Cleaved Caspase-1. Cleaved Caspase-1 cleaves GSDMD, releasing the N-terminal fragment of cleaved gasderminD (GSDMD-N). This fragment oligomerizes to form membrane pores, ultimately leading to cell membrane rupture and cell lysis. Figure 14 As shown in Figure a, Western blot analysis revealed the expression levels of the aforementioned pyroptosis-related proteins in different groups. Figure 14 As shown in b, NLRP3 expression was significantly increased in the AMNZ-GE+NIR group. Figure 14 As shown in c, ASC expression was significantly increased in the AMNZ-GE+NIR group, indicating enhanced inflammasome assembly. Figure 14As shown in d, in the AMNZ-GE+NIR group, the ratio of Cleaved Caspase-1 to Pro Caspase-1 was significantly increased by 12.50 ± 0.54 times compared to the control group, indicating that Cleaved Caspase-1 was effectively activated. Figure 14 As shown in Figure e, the ratio of GSDMD-N to GSDMD also increased significantly, consistent with pore formation and pyroptosis membrane rupture. These results indicate that AMNZ-GE+NIR-mediated photothermal-enhanced nanocatalytic therapy effectively induces tumor cell pyroptosis through inflammasome formation, cleaved caspase-1 activation, and subsequent GSDMD-N cascade.

[0105] like Figure 15 As shown in a, the release of the pyroptosis-related cytokine IL-1β was significantly increased in the AMNZ-GE+NIR group. Figure 15 As shown in b, the release of the pyroptosis-related cytokine IL-18 was significantly increased. Figure 15 As shown in c, LDH release is significantly enhanced. Figure 15 As shown in d, ATP release increased significantly. Figure 15 As shown in Figure e, the expression of HMGB1 in the cell nucleus was reduced or even disappeared, indicating that HMGB1 was released from the nucleus of pyroptotic cells. These results demonstrate that the release of pyroptosis-related cellular contents was enhanced in the AMNZ-GE+NIR group.

[0106] Given that the contents released by pyroptosis cells can effectively activate immune responses, the effect of pyroptosis on dendritic (DC) maturation was evaluated in vitro. Figure 16 As shown, after co-culturing treated SCC7 cells with immature primary bone marrow-derived dendritic cells (BMDCs), the expression of co-stimulatory molecules CD11c, CD80, and CD86 was analyzed by flow cytometry. Quantitative flow cytometry results showed that the proportion of mature DCs increased from 18.17 ± 1.50% in the control group to 55.87 ± 1.72% in the AMNZ-GE+NIR group. This indicates that the AMNZ-GE+NIR-mediated photothermal enhanced nanocatalytic therapy can not only induce pyroptosis in OSCC in vitro but also promote dendritic cell maturation, laying the foundation for enhancing T cell-mediated anti-tumor immune responses. This has significant implications for the treatment of OSCC, especially metastatic OSCC.

[0107] Precision imaging technologies play a crucial role in diagnosis, monitoring, and evaluation of treatment outcomes. CT and fluorescence imaging complement each other; CT provides high spatial resolution and detailed anatomical information, while fluorescence imaging enables real-time, safe tracking. Integrating CT and fluorescence in a dual-modal imaging approach leverages the advantages of both, providing precise guidance for targeted cancer therapy. Figure 17As shown in Figure a, after intravenous administration, AMNZ-GE exhibited a stronger CT signal at the tumor site compared to AMNZ. Figure 17 Quantitative results of b showed that the enhancement of the CT signal exhibited a time-dependent accumulation, with the CT signal of AMNZ-GE reaching its peak 1 hour after injection. Figure 17 As shown in Figure c, compared to AMNZ, AMNZ-GE exhibits higher fluorescence intensity and a more concentrated distribution in the tumor region. Figure 17 Quantitative results showed that the enhancement of the fluorescence signal also exhibited a time-dependent accumulation, with the fluorescence signal of AMNZ-GE reaching its peak 1 hour after injection. Figure 17 As shown in Figure e, AMNZ-GE, in addition to its excellent tumor accumulation, is primarily cleared through renal excretion. These results indicate that AMNZ-GE possesses excellent in vivo tumor targeting capabilities for OSCC, minimizing off-target effects, and exhibits superior fluorescence / CT dual-modal imaging capabilities, supporting its potential application in improving diagnostic accuracy and treatment outcomes.

[0108] Thanks to the superior dual-modal imaging capabilities of the AMNZ-GE, it can achieve precise tumor localization, thereby guiding precise tumor ablation mediated by photothermal therapy. Infrared thermal imaging technology was used to further verify its in vivo photothermal effects. Figure 18 As shown, one hour after intravenous injection of AMNZ-GE, 808 nm laser irradiation can raise the local tumor temperature to 50 °C, which is sufficient to induce irreversible tumor cell damage, indicating that AMNZ-GE has excellent in vivo photothermal properties and therapeutic potential.

[0109] Given the promising antitumor properties of AMNZ-GE-mediated photothermal enhanced nanocatalytic therapy in vitro, its therapeutic efficacy was further validated in SCC7 tumor-bearing mouse models of primary and metastatic OSCC. Tumor-bearing mice were randomly divided into six groups and treated with saline, ANZ, AMNZ, AMNZ+NIR, AMNZ-GE, or AMNZ-GE+NIR, respectively, for four consecutive days. On day 8, all mice were sacrificed, and primary tumors, metastatic tumors, serum, and vital organs (heart, liver, spleen, lung, and kidney) were collected for further analysis. First, the biocompatibility of the treatment regimen was assessed. Figure 19 As shown in a, the major organs in each group showed no obvious histological abnormalities. Figure 19 As shown in b, there was no significant decrease in body weight among the groups, confirming the excellent biocompatibility of AMNZ-GE.

[0110] like Figure 20 As shown in a, the efficacy of treatment for primary OSCC in each treatment group was evaluated. Figure 20As shown in b, the AMNZ, AMNZ+NIR, AMNZ-GE, and AMNZ-GE+NIR groups all inhibited the volume growth of primary OSCC, with the AMNZ-GE+NIR group exhibiting the strongest tumor suppression effect, in which the tumors of two mice completely regressed. Figure 20 As shown in c, the efficacy of each treatment group against metastatic OSCC was evaluated. Figure 20 As shown in Figure d, although no photothermal or other treatments were applied to metastatic OSCC, the AMNZ-GE+NIR group also exhibited the strongest tumor-suppressive ability, with no metastatic tumors detected in two of the mice. These results suggest that, in addition to targeting tumor accumulation to treat primary OSCC, AMNZ-GE-mediated photothermal-enhanced nanocatalytic therapy may have excellent therapeutic effects on metastatic OSCC by effectively activating a systemic immune response.

[0111] The therapeutic effects of AMNZ-GE-mediated photothermal enhanced nanocatalytic therapy were further elucidated to clarify its potential in vivo mechanisms. Figure 21 As shown in a, immunofluorescence staining was performed on each group of primary tumors to detect the expression of pyroptosis-related proteins. Figure 21 The quantitative results of b showed that, compared with the control group, the fluorescence intensity of NLRP3 in the AMNZ+NIR, AMNZ-GE, and AMNZ-GE+NIR groups was significantly enhanced, with the AMNZ-GE+NIR group showing the strongest signal. Figure 21 The quantitative results for c also show that the AMNZ-GE+NIR group has the strongest GSDMD-N signal. For example... Figure 21 As shown in d, the serum level of pyroptosis-related secreted cytokine IL-1β was highest in the AMNZ-GE+NIR group. Figure 21 As shown in Figure e, the AMNZ-GE+NIR group had the highest serum IL-18 levels. These results confirm that AMNZ-GE-mediated photothermal enhanced nanocatalytic therapy strongly induces pyroptosis in primary OSCC.

[0112] Pyroptosis can enhance immune activation and reverse the immunosuppressive microenvironment. This was further analyzed by examining changes in the immune cell population in the spleen of tumor-bearing mice. For example... Figure 22 Quantitative analysis of a showed that the proportion of mature DCs was significantly increased in the AMNZ-GE+NIR group, consistent with in vitro results. DC recruitment and maturation can significantly promote subsequent CD8... + Activation of T lymphocytes. For example... Figure 22 Quantitative analysis of b showed that in the AMNZ-GE+NIR group, CD3 + CD8 + The proportion of T lymphocytes was the highest. CD8 +T lymphocytes are core effector cells in cytotoxic anti-tumor immunity, capable of directly recognizing and eliminating tumor cells. These results indicate that AMNZ-GE+NIR-mediated photothermal enhanced nanocatalytic therapy can effectively induce pyroptosis, thereby promoting DC maturation and activating anti-tumor T cell immune responses in vivo. Figure 22 As shown in c, CD86 in the AMNZ-GE+NIR group + The proportion of M1 macrophages increased significantly. For example... Figure 22 As shown in d, CD206 + M2 macrophages were significantly reduced. While M1 macrophages have anti-tumor effects, M2 macrophages are typically associated with immunosuppression and tumor progression. These changes suggest that this treatment can effectively reverse the immunosuppressive tumor microenvironment. In summary, AMNZ-GE+NIR-mediated photothermal-enhanced nanocatalytic therapy not only effectively enhanced CD8... + T lymphocyte-mediated anti-tumor immune responses also increase the infiltration of M1 macrophages while reducing the infiltration of M2 macrophages, thereby exerting a dual immunomodulatory effect and enhancing the host's anti-tumor defense capabilities.

[0113] To gain a deeper understanding of the regulation of the immune microenvironment in metastatic tumors after treatment, a comprehensive analysis of immune cell subsets in metastatic OSCC was conducted. For example... Figure 23 Quantitative analysis of a showed that the proportion of mature DCs was significantly increased in the AMNZ-GE+NIR group. Figure 23 Quantitative analysis of b showed that CD3 in the AMNZ-GE+NIR group + CD8 + A significant increase in T lymphocytes was observed. These findings indicate that AMNZ-GE+NIR-mediated photothermal-enhanced nanocatalytic therapy effectively increased CD8+ T lymphocyte infiltration in metastatic lesions, which is key to achieving durable therapeutic effects in metastatic OSCC. Figure 23 As shown in c, the proportion of M1 macrophages in metastatic tumors was significantly increased after AMNZ-GE+NIR treatment. Figure 23 As shown in Figure d, the number of M2 macrophages was significantly reduced in the AMNZ-GE+NIR group. This indicates that the immunosuppressive state in metastatic OSCC was effectively reversed. Notably, the trends in these immune cell subsets were consistent with those observed in the spleen, further supporting the role of this treatment in modulating systemic immunity. In summary, these results demonstrate that AMNZ-GE+NIR-mediated photothermal-enhanced nanocatalytic therapy not only activates a robust anti-tumor immune response but also effectively reprograms the immunosuppressive tumor microenvironment (TME), transforming a “cold tumor” into a “hot tumor,” thereby enhancing the host’s immune response to the tumor.

[0114] In summary, this invention successfully developed a biomimetic cascade nanozyme, AMNZ-GE, which serves both as a targeted delivery platform for tumor cells and a potent pyroptosis inducer. Through a cascade catalytic reaction that generates excess reactive oxygen species, which are further amplified by photothermal therapy, pyroptosis is induced, effectively targeting both primary and metastatic OSCC by enhancing immunotherapy. Furthermore, AMNZ-GE, as a nanoprobe, exhibits satisfactory fluorescence / CT dual-modal imaging performance, demonstrating its potential applications in clinical tumor detection and precise image-guided therapy. Overall, this invention establishes a new therapeutic paradigm for advancing OSCC immunotherapy strategies.

[0115] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification.

Claims

1. A method for preparing a cascade nanoscale enzyme targeting induction of tumor pyroptosis, characterized in that, The method comprises the following steps: Synthesizing silver nanodots by taking glutathione as a ligand; Reacting tetrachloroauric acid, manganese dioxide and the silver nanodots to obtain a gold / manganese nanoscale enzyme; Mixing the gold / manganese nanoscale enzyme with 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide to activate carboxyl groups, and then performing a directional covalent coupling reaction with a GE11 peptide, and after the reaction is completed, purifying to obtain a cascade nanoscale enzyme.

2. The method of claim 1, wherein the preparation of the targeted tumor pyroptosis inducing cascade nanoscale enzyme is characterized by, The step of synthesizing silver nanodots by taking glutathione as a ligand comprises the following steps: Dissolving silver nitrate and glutathione in deionized water, adjusting the pH value to 6-7, then adding hydrazine hydrate, and stirring in the dark; then adding isopropanol, removing the supernatant after centrifugation to obtain silver nanodots; the mass ratio of the silver nitrate and the glutathione is 1:(1.5-2); the mass-volume ratio of the silver nitrate and the deionized water is (7-10) mg:1 mL; the volume ratio of the hydrazine hydrate and the deionized water is (0.08-0.12):

1.

3. The method of claim 1, wherein the preparation of the targeted tumor pyroptosis-inducing cascade nanoscale enzyme is characterized by, The step of reacting tetrachloroauric acid, manganese dioxide and the silver nanodots to obtain a gold / manganese nanoscale enzyme comprises the following steps: Dissolving the silver nanodots in deionized water to prepare a silver nanodot solution with a concentration of 8-12 mg / mL; Adding 40-60 mM tetrachloroauric acid solution, manganese dioxide and the silver nanodot solution into deionized water in sequence to obtain a mixture; the volume ratio of the silver nanodot solution and the tetrachloroauric acid solution is 1:(0.3-0.4); the mass-volume ratio of the manganese dioxide and the silver nanodot solution is (0.8-1.2) mg:1 mL; Ultrasonic treatment is performed on the mixture, then the pH value is adjusted to 7-8, and the mixture is heated to 70-90 DEG C for reaction, and after the reaction is completed, purification is performed to obtain the gold / manganese nanoscale enzyme.

4. The method of claim 3, wherein the preparation of the targeted tumor pyroptosis inducing cascade nanoszyme is characterized by, In the step of ultrasonic treatment, pH value adjustment and heating, the purification method is as follows: The crude product solution containing the target product is centrifuged at 8500-9000 rpm, the supernatant is collected, isopropanol is added, and after uniform mixing, centrifugation is performed at 9500-10500 rpm, then the supernatant is completely discarded, the precipitate is retained, and then freeze-drying treatment is performed to obtain the gold / manganese nanoscale enzyme.

5. The method of claim 1, wherein the preparation of the targeted tumor pyroptosis-inducing cascade nanoscale enzyme is characterized by, The step of mixing the gold / manganese nanoscale enzyme with 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide to activate carboxyl groups, and then performing a directional covalent coupling reaction with a GE11 peptide, and after the reaction is completed, purifying to obtain a cascade nanoscale enzyme comprises the following steps: The gold / manganese nanoscale enzyme is added into deionized water for dispersion to prepare a gold / manganese nanoscale enzyme aqueous dispersion with a concentration of 4-6 mg / mL. adding 60-70 mg / mL 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride aqueous solution and 35-45 mg / mL N-hydroxysuccinimide aqueous solution into the gold / manganese nanoscale enzyme water dispersion in sequence for ultrasonic treatment, to activate the carboxyl groups exposed on the surface of the gold / manganese nanoscale enzyme, form a highly reactive N-hydroxysuccinimide ester intermediate, and obtain an activation system; adding 0.5-1.5 mg / mL GE11 peptide solution into the activation system for directional covalent coupling reaction, and after the reaction is completed, purifying to obtain the cascade nanoscale enzyme; the volume ratio of the GE11 peptide solution, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride aqueous solution, N-hydroxysuccinimide aqueous solution, and gold / manganese nanoscale enzyme water dispersion is (0.5-1.5):(0.02-0.03):(0.03-0.04):

1.

6. The method of claim 5, wherein the preparation of the targeted tumor oncosis inducing cascade nanoszyme is characterized by, In the step of adding the GE11 peptide solution into the activation system for directional covalent coupling reaction, and after the reaction is completed, purifying to obtain the cascade nanoscale enzyme, the method for purifying is: transferring the crude product after the reaction into a dialysis bag, placing the dialysis bag in deionized water under continuous stirring for dialysis purification; after the dialysis is completed, taking out the solution containing the target product in the dialysis bag, and freeze-drying the solution to obtain the cascade nanoscale enzyme.

7. A cascade nanoscale enzyme for targeting and inducing pyroptosis of a tumor, which is prepared by the method of any one of claims 1-6.

8. Use of the cascade nanoscale enzyme for targeting and inducing pyroptosis of a tumor according to claim 7 in the preparation of a drug for treating oral squamous cell carcinoma.

9. Use of the cascade nanoscale enzyme for targeting and inducing pyroptosis of a tumor according to claim 7 in the preparation of an imaging contrast agent for oral squamous cell carcinoma.

Images