Tandem amplification pyroptosis nano inducer, preparation method thereof and application of tandem amplification pyroptosis nano inducer in preparation of cancer treatment drugs

Abstract

The invention belongs to the technical field of biological medicine, and relates to a tandem amplification pyroptosis nano inducer, a preparation method thereof and application of the tandem amplification pyroptosis nano inducer in preparation of cancer treatment drugs. The tandem amplification pyroptosis nano inducer is of a nano-particle structure and comprises crystalline dendritic Gd2O3 NPs and a shell packaged on the surface of the crystalline dendritic Gd2O3 NPs, crystalline dendritic Gd2O3 nano-particles load CPPO and Ce6, and the shell is an inorganic layer formed by calcium carbonate nano-particles. The tandem amplification pyroptosis nano inducer can promote NF-kappa B pre-activation through pH response and replace a phosphate group on a lysosome membrane so as to cause lysosome rupture; in addition, the released CPPO and Ce6 generate singlet oxygen, so that the limitation of light penetration and tumor hypoxia is overcome, strong anti-tumor immune response can be initiated in vitro and in vivo through the synergistic effect with pyroptosis, and the tumor immune curative effect is further improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and relates to a tandem amplified pyroptosis nanoinducer, a preparation method thereof and an application thereof in the preparation of a drug for treating cancer. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Hepatocellular carcinoma (HCC) is often diagnosed in the middle or late stages, leaving most patients without surgical options. Tumor immunotherapy, which enhances the host immune response to attack tumors, holds great promise. Pyroptosis, a type of programmed cell death mediated by gasdermin (GSDM), is characterized by rapid cell swelling, membrane rupture, and the release of proinflammatory cytokines (such as IL-1β and IL-18). This process reshapes the tumor microenvironment (TME) and stimulates anti-tumor immunity, making it a promising immunotherapy target.

[0004] However, current strategies based on reactive oxygen species (ROS)-induced pyroptosis have significant limitations. 1. Insufficient NF-κB pre-activation: Effective pyroptosis usually requires pre-activation of the NF-κB pathway to initiate downstream processes, and existing methods often fail to address this critical step. 2. Weak single-signal inflammasome activation: Relying on a single signal (such as ROS) to activate inflammasomes has limited effect and is difficult to effectively trigger GSDM activation. 3. Limited physical penetration: For deep tumors, methods that rely on external light irradiation, such as photodynamic therapy, are limited by insufficient light penetration depth and the hypoxic environment inside the tumor.

[0005] Therefore, developing efficient pyroptosis-inducing strategies that can synergistically overcome NF-κB pre-activation and insufficient activation of single-signal inflammasomes and break through the limitations of light / oxygen penetration is a key need to improve the efficacy of tumor immunotherapy. Summary of the Invention

[0006] In order to address the deficiencies of the prior art, the present invention aims to provide a tandemly amplified pyroptosis nanoinducer, a preparation method thereof, and its use in the preparation of cancer treatment drugs. The tandemly amplified pyroptosis nanoinducer can promote the pre-activation of NF-κB through pH response and replace the phosphate groups on the lysosomal membrane, thereby causing lysosomal rupture; in addition, the released bis(3,4,6-trichloro-2-(pentyloxycarbonyl)phenyl)oxalate (CPPO) can react with hydrogen peroxide (H2O2) to form a high-energy intermediate, emit light and stimulate hematoporphyrin (Ce6) to produce singlet oxygen, thereby overcoming the limitations of light penetration and tumor hypoxia, synergizing with cell pyroptosis, and inducing a strong anti-tumor immune response both in vitro and in vivo, thereby enhancing the efficacy of tumor immunotherapy.

[0007] In order to achieve the above object, the present invention provides the following technical solutions: In the first aspect, a tandem amplified pyroptosis nanoinducer is provided, which is a nanoparticle structure, including crystalline dendritic Gd2O3 nanoparticles (crystalline dendritic Gd2O3NPs, denoted as DM-Gd2O3) and a shell encapsulated on the surface of the crystalline dendritic Gd2O3NPs, wherein the crystalline dendritic Gd2O3NPs contain a plurality of dendritic radial mesoporous channels, and the crystalline dendritic Gd2O3 nanoparticles are loaded with CPPO and Ce6, and the shell is an inorganic layer formed by calcium carbonate (CaCO3) nanoparticles.

[0008] The tandem amplified pyroptosis nanoinducer provided by the present invention can induce CaCO3 degradation and release Ca at low pH in the tumor area. 2+ , triggering NF-κB activation. Subsequently, Gd 3+ The pH-dependent release of DM-Gd2O3 can strip the lysosomal membrane of phosphate groups, causing the morphology to transform into a sea urchin-like structure, thereby destroying the lysosome and triggering the release of cathepsin B. At the same time, the decomposition and degradation of DM-Gd2O3 leads to the release of CPPO and Ce6. CPPO reacts with H2O2 to form high-energy intermediates, emits light and excites Ce6, thereby generating a large amount of 1 O2, thereby overcoming the limitations of low light transmittance and tumor hypoxia. This process triggers the production of mitochondrial ROS, which in turn enhances the activation of the NLRP3 inflammasome and amplifies pyroptosis.

[0009] In vivo experimental results in an H22 tumor-bearing mouse model showed that the tandemly amplified pyroptosis nanoinducer can effectively inhibit tumor growth by promoting dendritic cell (DC) maturation and T cell infiltration, synergistically promoting pyroptosis and triggering immune activation.

[0010] On the other hand, a method for preparing the above-mentioned tandem amplified pyroptosis nanoinducer comprises the following steps: Providing dendritic mesoporous gadolinium hydroxide (DM-Gd(OH) x ), and the dendritic mesoporous gadolinium hydroxide was calcined and pyrolyzed under an inert atmosphere to obtain crystalline dendritic Gd2O3NPs; DM-GCCNPs were obtained by encapsulating (or loading) CPPO and Ce6 in crystalline dendritic Gd2O3NPs via electrostatic adsorption; The CaCO3 inorganic shell was coated on DM-GCC NPs by co-precipitation method.

[0011] In the third aspect, a use of the above-mentioned tandem amplified pyroptosis nanoinducer in the preparation of cancer treatment drugs.

[0012] The beneficial effects of the present invention are: The tandem amplified pyroptosis nanoinducer provided by the present invention not only has a nanometer-scale particle size, but also has a positive charge on the surface of the nanoparticles after being coated with a CaCO3 inorganic layer, which is conducive to its efficient accumulation in the tumor area as a drug. In vitro studies have shown that the tandem amplified pyroptosis nanoinducer can consume lysosomal membrane phosphate groups and bioconvert into sea urchin-like structures, thereby inducing lysosomal rupture; pH-responsive Ca 2+ The release of CPPO and Ce6 promotes the activation of NF-κB and the production of large amounts of ROS, which synergizes with pyroptosis to trigger a strong anti-tumor immune response. In addition, in vivo experiments in H22 tumor-bearing mice showed that the tandemly amplified pyroptosis nanoinducer significantly inhibited tumor progression, while the toxicity to normal cells was negligible. This shows that the tandemly amplified pyroptosis nanoinducer provided by the present invention not only has a good cancer treatment effect, but also has good safety, providing a new strategy for tumor immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0014] Figure 1 Schematic diagram of DM-GCCA NPs for tumor immunotherapy in an embodiment of the present invention; (A) DM-GCCA NPs were synthesized by loading CPPO and Ce6 on DM-Gd2O3 NPs and then encapsulating them with a CaCO3 shell; (B) Tumors at low pH, CaCO3 degrade and release Ca 2+ , triggering NF-κB activation; at the same time, pH-dependent Gd 3+ Release of phosphate groups on the stripe lysosomal membrane induces morphological transformation into a sea urchin-like structure, destroys the lysosome, and triggers the release of cathepsin B; degradation of DM-Gd2O3 releases CPPO and Ce6, producing a large amount of 1 O2, independent of light penetration or tumor hypoxia; this process induces NF-κB activation and triggers pyroptosis, while mitochondrial ROS generation and NLRP3 inflammasome assembly and lysosomal rupture lead to pyroptosis activation.

[0015] Figure 2 The characterization results of DM-GCCA NPs in the embodiment of the present invention are shown in FIG; (A) prepared DM-Gd(OH) xTEM images of (A) DM-Gd2O3NPs, (B) DM-GCCNPs, and (C) DM-GCCANPs; scale bar, 100 nm; (D) N2 adsorption-desorption isotherms and pore size distribution of DM-Gd2O3NPs; (E) Evaluation of Ce6 loading efficiency using UV-visible spectroscopy calibration curve; (F) DM-Gd(OH) x (G) XRD spectra of Ce6, CPPO, DM-Gd2O3NPs, DM-GCC NPs and DM-GCCA NPs; (H) Hydrodynamic particle size distribution of DM-Gd2O3NPs, DM-GCC NPs and DM-GCCA NPs; (I) Zeta potential of Ce6, CPPO, DM-Gd2O3NPs, DM-Gd2O3@PEI NPs, DM-GCC NPs or DM-GCCA NPs.

[0016] Figure 3 The results of the PSF morphological changes and ROS generation ability of DM-GCCA NPs in the examples of the present invention are shown in Figure 2; (A) Schematic diagram of the morphological changes and lysosomal rupture induced by DM-GCCA NPs; (B) Ca2+-induced DM-GCCA NPs in PBS buffer (pH = 4.5 and 7.4) 2+(C) Morphological analysis of DM-GCCA NPs after PSF / H2O exposure; (D) The molecular basis of lysosomal membrane disruption includes phospholipid dephosphorylation and subsequent formation of GdPO4 nanocrystals on the nanoparticle surface; (E) CPPO release from DM-GCCA NPs in PBS buffer (pH = 4.5 and 7.4) at 6 h; (F) Ce6 release from DM-GCCA NPs in PBS buffer (pH = 4.5 and 7.4) at 6 h; (G) UV-visible spectra of ABDA in the presence of Ce6, CPPO, DM-Gd2O3 NPs, DM-GCC NPs, or DM-GCCANPs; (H) ABDA in different groups at 380 Absorbance change at nm (A / A0); A and A0 are the absorbance of the sample and control group to the test solution ABDA, respectively; asterisks indicate statistical significance compared with the untreated control group by one-way ANOVA and Tukey's post hoc test; ****p<0.0001; (1) UV-visible spectra of DPBF degradation after incubation with Ce6, CPPO, DM-Gd2O3 NPs, DM-GCC NPs, or DM-GCCA NPs; (J) UV-visible spectra of DPBF degradation after incubation with different concentrations of DM-GCCA NPs.

[0017] Figure 4Figure 1 shows the results of in vitro cell tests of the embodiments of the present invention; (A) Confocal images of H22 cells cultured with DM-GCCA NPs for 0, 2, 4, and 6 hours; (B) Flow cytometry histogram of H22 cells cultured with DM-GCCA NPs; (C) Effect of different concentrations of DM-Gd2O3NPs, DM-GCC NPs, or DM-GCCA NPs (DM-Gd2O3NPs concentration) on the cell viability of H22 cells for 24 hours; (D) Effect of different concentrations of DM-Gd2O3NPs, DM-GCC NPs, or DM-GCCA Effect of NPs on cell viability of NIH / 3T3 cells for 24 h; (E) Colony formation assay using H22 cells according to the established protocol; (F) Colony-forming ability was quantified by counting colonies of ≥50 cells; asterisks indicate statistical significance compared with the untreated control group by one-way ANOVA and Tukey's post hoc test; ****p<0.0001; (G) Live and dead cell staining of H22 cell populations; (H) Apoptosis detection by flow cytometry of H22 cells stained with PI and Annexin-FITC after 24 h of treatment with PBS, DM-Gd2O3NPs, DM-GCCNPs, or DM-GCCANPs; (I) Quantitative assessment of apoptotic cell death in H22 cells after 24 h of incubation with different formulations using Annexin V-FITC / PI double staining and flow cytometric analysis; Q1-Q4 regions represent necrotic cells, late apoptotic cells, early apoptotic cells, and live cells, respectively.

[0018] Figure 5 Figure 2 shows the experimental results of DM-GCCA NPs activating NF-κB pathway and inducing pyroptosis in vitro in the embodiment of the present invention; (A) After incubation for 24 h, flow cytometry was used to observe the intracellular Ca 2+Levels; (B) Fluorescence microscopy analysis and nuclear staining of NF-κB translocation p65 subunit in cells treated with PBS, DM-Gd2O3NPs, DM-GCC NPs and DM-GCCA NPs; (C) Quantification of nuclear fluorescence intensity of p65 staining; (D) The release of cathepsin B from lysosomes to the cytoplasm in H22 cells was observed using commercial Magic Red MR-(RR)2; the scale bar represents 10 μm; (E) The corresponding fluorescence quantitative intensity of MitoSOX release; (F) Confocal images of intracellular ROS levels under treatment with PBS, DM-Gd2O3NPs, DM-GCC NPs or DM-GCCA NPs; the scale bar represents 10 μm; (G) Flow cytometry was used to observe the levels of JC-1 aggregates and JC-1 monomers in cells after 24 h of culture in each group; (H) The bar graph shows the percentage of JC-1 monomer-positive cells; MMP: mitochondrial membrane potential; (1) Western Blot analysis of NLRP3 and GSDMD-N in H22 cells after different treatments; (J) Extracellular lactate dehydrogenase levels in H22 cells after different treatments; (K) Extracellular IL-1β levels in H22 cells after different treatments; Asterisks indicate statistical significance compared with the control group by one-way ANOVA and Tukey's post hoc test; ***p<0.001, ****p<0.0001.

[0019] Figure 6 Figure 1 shows the in vivo study results of the anti-tumor and immune activation effects of DM-GCCA NPs in the H22 tumor-bearing mouse model according to the embodiments of the present invention; (A) Schematic timeline of the treatment process of PBS, DM-Gd2O3, DM-GCC NPs, or DM-GCCA NPs; (B) Digital photos of the DM-GCCA NPs hemolysis test and quantification of the hemolysis rate (HR%); asterisks indicate statistical significance compared with the untreated control group by one-way analysis of variance and Tukey's post hoc test; ****p<0.0001; (C) Representative fluorescence images of H22 tumor-bearing mice after intravenous injection of DM-GCCA@ICG NPs or free ICG; (D) Fluorescence images of major organs and tumors 24 hours after DM-GCCA@ICG NPs injection; (E) Quantification of Gd concentrations in mouse tissues and tumors 24 hours after intravenous injection of DM-GCCA NPs by ICP-MS; data are expressed as percentage of injected dose per gram of tissue (%) ID / g); (F) Body weight of mice in each group after 14 days of treatment; (G) Tumor volume of mice sacrificed 14 days after treatment; (H) Tumor images of mice sacrificed in each group on day 14 (n = 5, mean ± SD).

[0020] Figure 7The experimental results of the anti-tumor immune response of DM-GCCA NPs driven by pyrolysis in the embodiment of the present invention are shown in FIG; (A) Lymph node CD4 + and CD8 + Flow cytometric analysis of T cells; Quantitative determination of (B) CD4 in lymph nodes of different treatment groups by flow cytometry + T cells and (C) CD8 + The proportion of T cell populations; Quantitative determination of spleen CD4 in different treatment groups by flow cytometry (D) + T cells and (E) CD8 + The proportion of T cell population; (F) CD80 + and CD86 + The expression frequency of DC in tumor tissue; (G) Quantitative determination of the proportion of DC population in lymph nodes in different treatment groups by flow cytometry; (H) Lymph node CD80 + and CD86 + Frequency of DCs; Fluorescence staining of (I) NLRP3 and (J) Caspase-1 targets in tumor tissues 14 days after treatment; Quantitative fluorescence intensity of (K) NLRP3 and (L) Caspase-1; 1: PBS; 2: DM-Gd2O3; 3: DM-GCC; 4: DM-GCCA; Data are expressed as mean ± SD; Student's t-test or one-way ANOVA was used, *p < 0.05, **p < 0.01, ****p < 0.0001. DETAILED DESCRIPTION

[0021] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0022] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0023] Given that the effectiveness of cell pyroptosis in tumor immunotherapy is hampered by key factors such as the inability to resolve NF-κB pre-activation, insufficient activation of single-signal inflammasomes, and limited light and oxygen penetration, the present invention proposes a tandemly amplified pyroptosis nanoinducer, a preparation method thereof, and its application in the preparation of cancer treatment drugs.

[0024] A typical embodiment of the present invention provides a tandem amplified pyroptosis nanoinducer, which is a nanoparticle structure, including crystalline dendritic Gd2O3NPs and a shell encapsulated on the surface of the crystalline dendritic Gd2O3NPs, the crystalline dendritic Gd2O3NPs containing a number of dendritic radial mesoporous channels, and the crystalline dendritic Gd2O3 nanoparticles loaded with CPPO and Ce6, and the shell is an inorganic layer formed by CaCO3 nanoparticles.

[0025] In some embodiments, the hydrodynamic average particle size of the tandemly amplified pyroptosis nanoinducer is 215.0-225.0 nm. Under such conditions, the tandemly amplified pyroptosis nanoinducer is more conducive to the efficient accumulation of the drug in the tumor area.

[0026] In some embodiments, the zeta potential of the tandemly amplified pyroptosis nanoinducer is 7.0-9.0 mV. Under such conditions, the tandemly amplified pyroptosis nanoinducer is more conducive to the efficient accumulation of the drug in the tumor area.

[0027] In some embodiments, the weight of CPPO and Ce6 loaded per 100 μg of crystalline dendritic Gd2O3 nanoparticles is 0.01-4.92 μg and 0.01-42.65 μg, respectively. Specifically, the weight of CPPO and Ce6 loaded per 100 μg of crystalline dendritic Gd2O3 nanoparticles is 4.88-4.92 μg and 42.55-42.65 μg, respectively.

[0028] Another embodiment of the present invention provides a method for preparing the above-mentioned tandem amplified pyroptosis nanoinducer, comprising the following steps: Providing dendritic mesoporous gadolinium hydroxide (DM-Gd(OH) x ), and the dendritic mesoporous gadolinium hydroxide was calcined and pyrolyzed under an inert atmosphere to obtain crystalline dendritic Gd2O3NPs; CPPO and Ce6 were loaded on crystalline dendritic Gd2O3NPs via electrostatic adsorption to obtain DM-GCC NPs; The CaCO3 inorganic shell was coated on DM-GCC NPs by co-precipitation method.

[0029] In some embodiments, the calcination temperature under an inert atmosphere is 550-650° C. and the calcination time is 5-7 h.

[0030] In some embodiments, after calcining in an inert atmosphere, the sintered product is then calcined in an air atmosphere to remove residual carbon. Specifically, the calcination temperature in an air atmosphere is 550-650°C for 1-3 hours.

[0031] In some embodiments, polyethyleneimine is used to modify crystalline dendritic Gd2O3NPs so that the surface of the crystalline dendritic Gd2O3NPs is positively charged, and then mixed with CPPO and Ce6 to achieve electrostatic adsorption of CPPO and Ce6 by the crystalline dendritic Gd2O3NPs. Since the surface of the crystalline dendritic Gd2O3NPs is negatively charged and the polyethyleneimine is positively charged, the polyethyleneimine can be modified on the surface of the crystalline dendritic Gd2O3NPs through electrostatic interaction and cause the surface to be positively charged, thereby facilitating the electrostatic adsorption of CPPO and Ce6. Specifically, the mass ratio of polyethyleneimine to crystalline dendritic Gd2O3NPs is 1:3.5~4.5.

[0032] In some embodiments, the coprecipitation method involves mixing DM-GCC NPs with a calcium salt and a carbonate in an aqueous solution. The calcium salt herein refers to a compound whose cation is calcium ion, such as calcium chloride, calcium nitrate, and calcium acetate. The carbonate herein refers to a water-soluble compound whose anion is carbonate ion, such as sodium carbonate, potassium carbonate, and ammonium carbonate. Specifically, the mass ratio of DM-GCC NPs to calcium salt and carbonate is 1:0.9-1.1:1.5-2.0.

[0033] In some embodiments, the preparation method of dendritic mesoporous gadolinium hydroxide is as follows: citric acid, hexadecyltrimethylammonium bromide, sodium salicylate, hexamethylenetetramine and gadolinium salt are added to water and mixed evenly, and then cyclohexane is added and mixed, and heated to react. The gadolinium salt described in the present invention refers to a compound whose cation is a gadolinium ion, such as gadolinium chloride, gadolinium nitrate, etc. Specifically, the mass ratio of citric acid, hexadecyltrimethylammonium bromide, sodium salicylate, hexamethylenetetramine and gadolinium salt is 1.8~2.2:18~22:11~13:2.7~3.3:13.5~16.5. Specifically, the volume ratio of water to cyclohexane is 4~6:1. Specifically, the temperature of the heating reaction is 65~75°C, and the reaction time is 6.5~7.5 h.

[0034] In the third aspect, a use of the above-mentioned tandem amplified pyroptosis nanoinducer in the preparation of cancer treatment drugs.

[0035] In some embodiments, the cancer includes but is not limited to lung cancer, intestinal cancer, gastric cancer, breast cancer, cervical cancer, etc. Specifically, the cancer is liver cancer.

[0036] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples.

[0037] Example DM-Gd(OH) x Synthesis of NPs 2.0 mg of citric acid, 20.0 mg of cetyltrimethylammonium bromide (CTAB), 12.0 mg of sodium salicylate, 3.0 mg of hexamethylenetetramine (HMTA), and 15.0 mg of gadolinium chloride hexahydrate (GdCl3·6H2O) were sequentially added to 10.0 mL of distilled water. 2.0 mL of cyclohexane was added, and the mixture was vigorously stirred for 1 h. The reaction was continued at 70°C for 7 h. After the reaction was complete, the product was centrifuged and washed with ethanol three times. Finally, the product was dispersed in 100 mL of ethanol and refluxed at 60°C for 4 h. This was repeated four times to effectively remove the CTAB surfactant, yielding DM-Gd(OH) x NPs.

[0038] Synthesis of DM-Gd2O3NPs DM-Gd(OH) x NPs were calcined in a tubular furnace under nitrogen atmosphere at a heating rate of 2 °C·min -1 , until it reaches 600 ℃ and is maintained for 6 h. Subsequently, the sample is further calcined in air at a heating rate of 3 ℃·min -1 to 600 °C for 2 h to eliminate residual carbon. Then, 5 mL of polyethyleneimine (PEI, 0.5 mg mL -1 ) was added with 10 mL of DM-Gd2O3 (1 mg mL -1 ) and stirred continuously for 2 h to obtain a uniform suspension. After thorough washing with DI water, the DM-Gd2O3@PEINPs were collected, centrifuged, and freeze-dried for later use. After PEI modification, DM-Gd2O3@PEI acquired a large amount of positive charge.

[0039] Synthesis of DM-GCC NPs 4.0 mg of CPPO was dissolved in 2 mL of tetrahydrofuran (THF), and 4.0 mg of Ce6 was dissolved in 2 mL of DMSO. These solutions were then mixed with 8.0 mg of DM-Gd2O3@PEI NPs and incubated at 37°C overnight. After incubation, the mixture was centrifuged at 6000 rpm. The resulting particles were then washed three times with deionized water to remove any unreacted material, yielding DM-GCCNPs.

[0040] Synthesis of DM-GCCA NPs 3.0 mg of the prepared DM-GCC NPs were lightly suspended in 10 mL of ddH2O containing 3.0 mg of calcium chloride (CaCl2) and stirred for 6 h. Then, excess Na2CO3 (10 mL, 500 μg mL -1) was quickly added to the mixture and stirred at high speed overnight in the dark. Finally, the DM-GCCA NPs were collected and centrifuged at 12,000 g for 10 minutes. The NPs were washed three times with ethanol and ddH2O, and then resuspended in ddH2O to obtain DM-GCCA NPs.

[0041] In the embodiment of the present invention, dendritic mesoporous gadolinium oxide (DM-Gd2O3NPs) is constructed, loaded with CPPO and Ce6 wrapped by calcium carbonate (CaCO3), and Figure 1 DM-GCCA NPs are shown in Figure A. Low pH in the tumor area induces CaCO3 degradation to release Ca 2+ , triggering NF-κB activation. Subsequently, Gd 3+ The pH-dependent release of DM-Gd2O3 can strip lysosomal membrane phosphate groups, leading to a morphological transformation into a sea urchin-like structure, thereby destroying the lysosome and triggering the release of cathepsin B. In addition, the decomposition and degradation of DM-Gd2O3 leads to the release of CPPO and Ce6, which can produce a large amount of 1 O2, without being limited by low light transmittance and tumor hypoxia. This process triggers the production of mitochondrial ROS, which in turn enhances the activation of NLRP3 inflammasome and amplifies pyroptosis, such as Figure 1 As shown in B. In the H22 tumor-bearing mouse model, in vivo experimental results also showed that DM-GCCA NPs can effectively inhibit tumor growth by promoting dendritic cell (DC) maturation and T cell infiltration, synergistically promoting pyroptosis and triggering immune activation.

[0042] Results and Discussion 1. Synthesis and characterization of DM-GCCA NPs The synthesis process of DM-GCCA NPs is as follows Figure 1 As shown in A. Dendritic mesoporous gadolinium hydroxide (DM-Gd(OH)x) has a uniform spherical morphology and was first synthesized by viscosity-mediated assembly in an oil-water two-phase system, as shown in Figure 2 A. Then, the obtained DM-Gd(OH)x was calcined at high temperature in a nitrogen atmosphere to form crystalline dendritic Gd2O3NPs (DM-Gd2O3) with well-defined dendritic radial mesoporous channels, as shown in Figure 2 As shown in A. Subsequently, CPPO and Ce6 were encapsulated in DM-Gd2O3 by electrostatic adsorption to form DM-GCC NPs. Finally, a mild co-precipitation method was used to coat the surface of DM-GCC NPs with a CaCO3 inorganic layer shell. Transmission electron microscopy (TEM) ( Figure 2 A) and scanning electron microscopy (SEM) images ( Figure 2B) Confirms the formation of monodisperse spherical DM-GCCA nanoparticles with an average size of ~200 nm. In addition, scanning transmission electron microscopy (STEM) and elemental mapping of the corresponding DM-GCCA nanoparticles clearly show that the Gd, O, and Ca elements are uniformly distributed, as shown in Figure 2. Figure 2 As shown in Figure C, it was preliminarily confirmed that Gd2O3 and CaCO3 coexisted in the nanoparticles (NPs). In addition, the Ca 2p and Gd 3d5 / 2 peaks in the X-ray photoelectron spectroscopy (XPS) further confirmed the presence of Ca and Gd elements in DM-GCCA NPs. Figure 2 It can be seen that the BET specific surface area and average pore size of dendritic mesoporous DM-Gd2O3 nanoparticles are 9.04 m 2 / g and 10.5 nm. These results indicate that DM-Gd2O3NPs are beneficial for drug encapsulation in tumor therapy. Subsequently, the chimera of CPPO and Ce6 with DM-Gd2O3NPs was verified by high-performance liquid chromatography (HPLC) and UV-visible absorption spectroscopy. The loading capacities of CPPO and Ce6 were calculated to be 4.9±0.02µg and 42.6±0.05µg per 100µg DM-Gd2O3NPs, respectively (n = 3). Figure 2 E. These results confirm the successful loading of Ce6 and CPPO onto DM-Gd2O3 NPs. To elucidate the crystal structure of DM-GCCA NPs, their crystallinity and orientation characteristics were investigated using X-ray powder diffraction (XRD). Preliminary XRD analysis revealed that the synthesized DM-Gd(OH)x NPs were amorphous. After calcination, these NPs underwent a phase transformation to form crystalline DM-Gd2O3 NPs, which closely matched the diffraction peaks of previously reported Gd2O3 (PDF#00-012-0797). During drug loading, the synthesized DM-GCC NPs exhibited identical XRD patterns to those of DM-Gd2O3 NPs, demonstrating the high stability of the nanoparticle framework throughout the synthesis process. Furthermore, a diffraction peak at 2θ = 29.4° was observed in the XRD patterns of DM-GCCA NPs, corresponding to the characteristic peak of calcite (PDF#05-0586). This observation confirms that CaCO3 was successfully incorporated into the nanoparticles in the form of calcite, e.g. Figure 2 As shown in F.

[0043] To further demonstrate the successful loading of Ce6 and coating of CaCO3 shell, the UV-visible absorption spectra of various samples (including free Ce6, DM-Gd2O3NPs, DM-GCC NPs and DM-GCCA NPs) were analyzed. Figure 2As shown in Figure G, DM-Gd2O3 lacks a specific UV-visible absorption peak. In contrast, DM-GCC NPs show characteristic absorption peaks of Ce6 at 400 nm and 655 nm, confirming the successful loading of Ce6. It is worth noting that the characteristic absorbance peak of Ce6 at 400 nm in the UV-visible spectrum of DM-GCCA NPs is weakened. This attenuation may be due to the formation of the CaCO3 shell, as shown in Figure 3. Figure 2 G. In addition, the hydrodynamic size of DM-Gd2O3, DM-GCC and DM-GCCA NPs was evaluated by dynamic light scattering (DLS). The measurement results showed that the hydrodynamic diameters of DM-Gd2O3, DM-GCC and DM-GCCA NPs were 183.6±11.8 nm, 187.7±3.5 nm and 219.8±4.6 nm, respectively. Figure 2 H. In addition, the measurement of zeta potential further confirmed the successful preparation of NPs at each stage of the synthesis process. The zeta potential of DM-Gd2O3NPs was -9.0±1.5 mV. The zeta potentials of Ce6 and CPPO were -5.6±2.2 mV and -28±3.3 mV, respectively. After modification with cationic PEI, the zeta potential of DM-Gd2O3NPs increased significantly to 5.6±2.0 mV, which was consistent with expectations. The zeta potential of DM-GCC NPs was -11.9±1.2 mV, indicating that it effectively encapsulated the negatively charged CPPO and Ce6. The zeta potential value of DM-GCCA NPs was 8.0±1.0 mV, indicating that the positively charged CaCO3 shell was successfully formed, as shown in Figure 5. Figure 2 As shown in I. Overall, these results demonstrated the successful synthesis of DM-GCCA NPs.

[0044] 2. In vitro pH-responsive biotransformation and ROS generation like Figure 3 As shown in A, pH responsiveness triggers the rupture of the CaCO3 outer layer, thereby releasing Ca 2+ The results reveal that DM-GCC NPs undergo morphological changes within the lysosomes, forming a sea urchin-like structure. Phosphate groups on the lysosomal membrane participate in this biotransformation process, triggering lysosomal rupture and the release of CPPO and Ce6. Afterwards, the presence of H2O2 within the tumor cells induces the production of a large amount of ROS without the need for light or ultrasound activation. Based on this, this example uses inductively coupled plasma optical emission spectroscopy (ICP-OES) to analyze the Ca in DM-GCCA NPs. 2+ The content was quantitatively analyzed and determined to be about 1.6%. Subsequently, this example studied the Ca content in the supernatant after centrifugation at a predetermined time point.2+ Specifically, in PBS at pH 4.5 and 7.4, 6 h Ca 2+ The release rates of α and β were 81.6% and 33.9%, respectively. Figure 3 As shown in Figure 2B, exposure of DM-GCCA NPs to acidic conditions revealed the pH-dependent dissociation properties of the CaCO3 coating. Subsequently, considering the biotransformation of ReO in lysosomes, we evaluated the abiotic transformation of DM-GCCA NPs in a synthetic fluid that simulates the phagolysosomal environment (PSF). This example compares the behavior of DM-GCCA NPs in PSF with their behavior in H2O. The transformation of DM-GCCA NPs in PSF resulted in the formation of urchin-like structures displaying needle-like protrusions, as shown in Figure 2B. Figure 3 C. Lysosomal processing of DM-GCCA NPs results in enhanced pH-dependent particle dissolution. The released Gd(III) ions are rapidly incorporated by bystander phosphates, leading to the deposition and crystallization of GdPO4 on the particle surface. This results in a morphological transformation to a sea urchin-shaped structure, as shown in Figure 3 As shown in D.

[0045] The pH-responsive biotransformation of DM-GCCA NPs forces the responsive release of Ce6 and CPPO. This example studies the in vitro release profiles of DM-GCCA NPs under different pH environments. At pH 7.4, only 18.2% of CPPO was released in 6 hours. Figure 3 E. In contrast, in PBS at pH 4.5, the release of CPPO increased significantly, with the drug release reaching 83.8% at 6 h. Compared with the release curve of CPPO, Ce6 also showed similar pH-dependent drug release behavior, as shown in Figure 5. Figure 3 F. At pH 7.4, the release of Ce6 within 6 h was only 21.3%, while at pH 4.5, the release of Ce6 increased significantly, reaching 85.2% within 6 h.

[0046] In summary, DM-GCCA NPs are easily degraded and shed under acidic stimulation, leading to the complexation of biological phosphates. This enables them to release Ca in the acidified lysosomal environment. 2+ ions, CPPO and Ce6. We then evaluated the production of DM-GCCA NPs. 1 O2's capabilities. First, adopt 1 The efficiency was evaluated using the O2 indicator [9,10-anthracenediyl-bis(methylene)malonic acid, ABDA]. The decrease in ABDA absorbance at 380 nm was used as the basis for evaluation. Figure 3As shown in G and 3H, at pH 4.5 and in the presence of H2O2, the absorbance of ABDA in the DM-GCC and DM-GCCA NPs groups decreased by 1.9 and 2.0 times compared with the PBS group. These results indicate that DM-GCC and DM-GCCA NPs can produce Ce6 through CPPO activation without external excitation light. 1 O2, thus overcoming the limitation of traditional PDT penetration depth.

[0047] In addition, this example also uses the common ROS probe 1,3-diphenylisobenzofuran (DPBF) to evaluate the ROS generation efficiency. 1 When O2 binds, DPBF undergoes irreversible oxidation, resulting in a rapid decrease in the absorption intensity of the UV-visible spectrometer. When DPBF is incubated with DM-GCC NPs and DM-GCCA NPs, the absorbance of DPBF at 410 nm decreases significantly, as shown in Figure 2. Figure 3 As shown in Figure 1, it shows that Ce6 can be activated to efficiently generate ROS in the presence of CPPO and H2O2. At the same time, with the increase of DM-GCCA NPs concentration, the absorption of DPBF at 410 nm also decreases accordingly, indicating that the ability of DM-GCCA NPs to catalyze the conversion of H2O2 into ROS is enhanced with the increase of DM-GCCA NPs concentration. Figure 3 J. In summary, the intermediates produced by the reaction of CPPO with H2O2 can excite Ce6, indicating that ROS can be generated without external laser irradiation.

[0048] 3. Enhanced anti-tumor efficacy and immune response activation in vitro After synthesizing and characterizing DM-GCCA NPs, this example investigated their performance at the cellular level. Cellular uptake was initially assessed by incubating DM-GCCA NPs with mouse hepatoma cell lines (H22 cells) for varying periods of time, followed by observation using confocal laser scanning microscopy (CLSM). Figure 4 As shown in A, the red fluorescence signal of DM-GCCA NPs increased over time, indicating that the cellular uptake was enhanced with the extension of incubation time. Flow cytometry was also used to evaluate the cellular uptake of DM-GCCA NPs at different incubation times, as shown in Figure 4 As shown in B. Similarly, the fluorescence intensity of DM-GCCA NPs increased with time, with durations of 0, 2, 4, and 6 h. Subsequently, the cytotoxicity of H22 cells and mouse embryonic fibroblast 3T3 cells (NIH / 3T3 cells) was further studied using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT method). Figure 4 C, 100 μg·mL -1DM-Gd2O3NPs, DM-GCCNPs, and DM-GCCANPs caused 17.3%, 22.3%, and 42.9% mortality in H22 cells, respectively. This significant increase may be attributed to the intracellular Ca 2+ The combined effects of increased lysosomal levels, lysosomal destruction, and ROS generation, which together activate NLRP3 inflammasome-mediated pyroptosis. In contrast, when NIH / 3T3 cells were incubated with DM-Gd2O3NPs, DM-GCC NPs, and DM-GCCA NPs for 24 h, their activity remained above 90%, indicating the safety of DM-GCCA NPs for normal cells, such as Figure 4 Then, the effects of various treatments on cell proliferation were detected by colony formation assay. Figure 4 As shown in Figures E and 4F, the colony-forming ability of cancer cells treated with DM-Gd2O3 NPs, DM-GCC NPs, and DM-GCCA NPs was significantly inhibited by 78.8%, 17.1%, and 7.1% of the control group, respectively. In contrast, the colony-forming rate of cells treated with DM-GCCA NPs was 7.1% compared to the untreated PBS group. These findings indicate that treatment with DM-GCCA NPs significantly inhibits the formation of cancer cell colonies.

[0049] To further evaluate the cytotoxic effects of these NPs on H22 cells, this example used fluorescein-annexin V and propidium iodide (PI) staining to determine their lethal effects. Figure 4 The results of G and Figure 4 The results of MTT assay were consistent with those of C. In addition, flow cytometry with Annexin V-FITC / PI staining was performed to quantify the apoptotic cell population after treatment with PBS, DM-Gd2O3 NPs, DM-GCC NPs, and DM-GCCA NPs, as shown in Figure 3. Figure 4 H and 4I. The cell apoptosis rates were 1.5%, 12.4%, 27.2% and 42.8%, respectively, indicating that the degree of cell apoptosis induced by DM-GCCA NPs was higher than that of other treatments.

[0050] Subsequently, this example investigated the activation of NF-κB, as well as the induction of lysosomal disruption and mitochondrial ROS production. First, the colocalization of DM-GCCA NPs with endocytic organelles in H22 cells was analyzed. DM-GCCA NPs were labeled with Ce6 (red), lysosomes with Lysotracker (green), and nuclei with Hoechst 33342 (blue). After treatment of H22 cells with DM-GCCA NPs, the red fluorescence signal from the DM-GCCA NPs significantly overlapped with the green fluorescence signal from the lysosomes, confirming that the DM-GCCA NPs could be internalized and subsequently enter the lysosomes.

[0051] The destruction of the CaCO3 shell can lead to the 2+ Therefore, this example uses Rhod-2 AM probe to detect intracellular Ca 2+ level, which is a cell-permeable Ca 2+ Fluorescent probes are detected by flow cytometry and fluorescence microscopy. Figure 5 As shown in A, the Rhod-2 fluorescence intensity in the DM-GCCA NPs group was higher than that in the PBS, DM-Gd2O3NPs, and DM-GCC NPs groups. Immunofluorescence staining was used to further evaluate the intracellular Ca 2+ CLSM images showed that cells treated with DM-GCCANPs exhibited obvious intracellular Ca 2+ accumulation, indicating a strong Ca 2+ internal flow. Ca 2+ Flux and Ca 2+ The NF-κB-dependent signaling pathway has been shown to be crucial for the activation of NF-κB and subsequently triggers the expression of immune factors, thereby regulating the activity of immune cells and immune responses. Therefore, the present example detected the potential induction of NF-κB activation and nuclear translocation. The nuclei of H22 cells in the PBS, DM-Gd2O3NPs and DM-GCC NPs groups showed blue fluorescence, and the green fluorescence corresponding to the NF-κB p65 subunit was mainly confined to the cytoplasm, indicating that NF-κB was not activated. It is worth noting that after treatment with DM-GCCA NPs, the most intense green fluorescence indicating the p65 subunit of NF-κ bloalization in the nucleus was observed, which means that DM-GCCA NPs may have the strongest effect on NF-κB activation and nuclear translocation, which may be attributed to the degradation of the external CaCO3 coating, as shown in Figure 5. Figure 5 B and 5C.

[0052] Furthermore, considering the biotransformation of ReO within lysosomes, the translocation of phosphates in the lysosomal lipid bilayer leads to biophosphate complexation and subsequent lysosomal damage. Cathepsin B is a lysosomal enzyme that can cleave Magic Red-labeled substrates, such as Figure 5As shown in D. In untreated cells, Magic Red appeared as dot-like structures, indicating that the enzyme was confined to intact lysosomes. However, when the lysosomes were damaged, the fluorescent marker was released and diffused throughout the cytoplasm. Notably, DM-Gd2O3NPs, DM-GCC NPs, and DM-GCCA NPs were able to trigger the release and dispersion of red fluorescence, while the PBS group showed bright dot-like red fluorescence. This indicates that DM-Gd2O3NPs, DM-GCC NPs, and DM-GCCA NPs were able to cause lysosomal rupture and subsequent release of cathepsin B, which is consistent with the Gd 3+ The release is closely related to the damage of lysosomal membrane.

[0053] The biotransformation process of DM-GCCA NPs can trigger the release of CPPO and Ce6. CPPO can react with H2O2 to generate high-energy intermediates, which activate Ce6 to produce a large amount of 1 O2, leading to a surge in exogenous ROS. Therefore, this example uses CLSM staining to detect changes in mitochondrial ROS levels. After treatment with DM-GCC NPs and DM-GCCA NPs, the red fluorescence intensity increased significantly, which is due to the presence of CPPO and Ce6, as shown in Figure 2. Figure 5 E. From Figure 5 F As can be seen, after treatment with DM-GCC NPs and DM-GCCA NPs, the fluorescence intensity (green) of the 2'-7' dichlorofluorescein diacetate (DCFH-DA) fluorescent probe was significantly enhanced, which also confirmed that treatment with DM-GCCNPs and DM-GCCA NPs can enhance the production of ROS. Excessive production of ROS can lead to a decrease in mitochondrial transmembrane potential (ΔΨm), thereby inducing mitochondrial dysfunction. Subsequently, this example used the JC-1 probe to measure changes in mitochondrial membrane potential (MMP). The JC-1 dye forms polymers at high membrane potentials, emitting red fluorescence (JC-1 Aggregate), and exists as monomers at low membrane potentials, emitting green fluorescence (JC-1 monomer). Figure 5 G and 5H show the changes after treatment with PBS, DM-Gd2O3NPs, DM-GCC NPs, and DM-GCCA NPs. The decreasing trend of MMP became increasingly obvious, indicating severe mitochondrial damage, which may be related to the generation of ROS and the increase of Ca2+ levels.

[0054] Inspired by the above phenomenon, this example explored the mechanism of DM-GCCANPs-induced pyroptosis by detecting the expression of pyroptosis-related proteins. Figure 5As shown in Figure 1, the expression level of NLRP3 showed the highest amplitude in the DM-GCCA NPs group, indicating a large recruitment of inflammasomes. At the same time, unlike the other groups, GSDMD-N was also highly expressed in the DM-GCCA group, and the NLRP3 / β-actin and GSDMD-N / β-actin ratios were the highest. In this case, this example further studied the release of cell contents and cytokines such as lactate dehydrogenase (LDH) and IL-1β. The LDH content in the culture supernatant of H22 cells after different treatments was then detected, and it was found that the LDH content in the DM-GCCA group was the highest, as shown in Figure 2. Figure 5 J. IL-1β is a typical marker of NLRP3 inflammasome activation. The levels of IL-1β and IL-18 in the culture supernatant of H22 cells after different treatments were also detected. Similarly, the levels of IL-1β and IL-18 were significantly increased at 100 μg mL -1 The highest in the DM-GCCA NPs group, e.g. Figure 5 As shown in K.

[0055] The NLRP3 inflammasome is typically activated through three mechanisms, including potassium (K + ) efflux, ROS generation, and lysosomal disruption. This example first pretreated H22 cells with N-acetylcysteine ​​(NAC), a potent ROS inhibitor, and CA-074 ME, a cathepsin B inhibitor. The study showed that NAC pretreatment significantly reduced IL-1β release in cells exposed to various materials. Furthermore, lysosomal disruption was associated with the release of cathepsin B, which provides a signal for the assembly of the NLRP3 inflammasome. Pretreatment with CA-074 ME also resulted in a significant decrease in IL-1β levels. This suggests that DM-Gd2O3 NPs, DM-GCC NPs, and DM-GCCA NPs induce NLRP3 inflammasome activation, in part, through lysosomal disruption and ROS release. Upon stimulation of cells by external particles, NLRP3 recruits the adaptor protein ASC and pro-caspase-1 to form the NLRP3 inflammasome, regulating caspase-1 release. To investigate whether caspase-1 is involved in IL-1β production, H22 cells were pretreated with Z-YVAD-FMK, a potent caspase-1 inhibitor, and then treated with DM-Gd2O3 NPs, DM-GCC NPs, and DM-GCCANPs. In the presence of Z-YVAD-FMK, the nanomaterial-induced IL-1β levels were significantly lower than in its absence, suggesting that Z-YVAD-FMK can block IL-1β secretion by inhibiting caspase-1 activity.

[0056] The above results confirmed that biodegradable DM-GCCA NPs could induce lysosomal rupture and ROS production, leading to pyroptosis of cancer cells and the release of a large amount of cytokines and cellular contents.

[0057] 4. In vivo evaluation of the therapeutic effect of DM-GCCA NPs DM-GCCA NPs have good immune activation effects, which prompted this example to study its anti-tumor therapeutic effect in vivo. Mice were intravenously injected with PBS, DM-Gd2O3NPs, DM-GCC NPs and DM-GCCA NPs on days 1, 3 and 5. Figure 6 As shown in A. Prior to this, a systematic toxicological study was conducted on DM-GCCA NPs to ensure their safe biological applications. Figure 6 As shown in Figure B, the hemolysis rates of DM-GCCA NPs at various concentrations were all below 5%, demonstrating the safety of the DM-GCCA NPs injection. Blood biochemistry and hematology analyses in all treatment groups were within normal reference ranges. These results demonstrate the good biocompatibility of the tested NPs. Hematoxylin and eosin (H&E) staining of major organs after intravenous injection in all groups showed no significant toxicity, further supporting the good in vivo biocompatibility of DM-GCCA NPs. Figure 6 C shows the biodistribution of DM-GCCA@ICG NPs in H22 tumor-bearing mice by in vivo imaging. After intravenous injection, DM-GCCA NPs can effectively accumulate in the tumor area and reach a maximum value 24 hours after injection. The free ICG group has a faster clearance rate in vivo. In the tumor area, the fluorescence signal of ICG is relatively weak and almost invisible after 24 hours, indicating that the circulation time of DM-GCCA@ICG NPs is longer. The corresponding ICP-MS elemental analysis ( Figure 6 D) shows that the contents of Gd in tumor, heart, liver, spleen, lung and kidney are 1.0%, 0.8%, 2.5%, 2.3%, 0.4% and 1.3% ID g-1, respectively. DM-GCCA NPs have obvious accumulation in tumor tissue. Figure 6 As shown in E, intravenous injection of DM-GCCA NPs had no significant effect on the body weight of mice during treatment, while the anti-tumor effect of the DM-GCCA NPs group was the most significant after treatment. Figure 6 F. This is consistent with the in vitro antitumor results, confirming the successful tumor inhibition effect of DM-GCCA NPs.

[0058] To further verify the anti-tumor effect of DM-GCCA NPs, mice were euthanized after treatment, and their major organs were collected. A series of experiments were performed to comprehensively evaluate the therapeutic effect. Compared with the other groups, H&E staining showed that the cell nuclei in the DM-GCCA NPs group were significantly condensed and the structure was obviously damaged. Figure 6 G. Activation of pyroptosis not only triggers an inflammatory response but also induces a large release of inflammatory cytokines. ELISA results further confirmed this observation, showing that the secretion level of the key pro-inflammatory cytokine IL-1β was significantly increased in the DM-GCCA NPs-treated group compared with other groups. Cancer cell pyroptosis is beneficial for inducing anti-tumor immune responses and enhancing tumor immunogenicity in vivo by promoting DC maturation. In addition, we used immunofluorescence staining to evaluate the expression of cytotoxic T cells (CD8 + ) and helper T cells (CD4 + ) infiltration in the tumor. Figure 6 As shown in H, compared with the PBS group, the number of CD8 + T cells (green) and CD4 + The proportion of T cells (red) was the highest, significantly enhancing T cell-mediated immunotherapy.

[0059] To further investigate the systemic remodeling of the mouse immune response, this example examined the proportion of T cells in lymph nodes and spleen. + and CD4 + The infiltration rates of immune cells were 1.2%, 4.6%, 7.1% and 12.9% respectively, and CD4 + The infiltration rates of T cells were 4.0%, 15.2%, 23.6% and 37.2% respectively. + and CD4 + The infiltration rates of T lymphocytes were 10.8 and 9.3 times higher than those in the PBS group, respectively. Figure 7 As shown in A, 7B and 7C, CD8 + and CD4 + Immunofluorescence staining showed that the proportion of CD8 T cells in the DM-GCCANPs treatment group + and CD4 + T cell infiltration increased significantly, indicating a strong activation of adaptive immunity in vivo. Flow cytometry analysis of spleen CD8 + The proportions of T cells were 5.3%, 14.6%, 20.4% and 20.8% respectively, and CD4 + The proportions of T cells were 13.6%, 22.0%, 24.2% and 25.3%, respectively. Figure 7D and 7E. These data together emphasize the potent immunostimulatory capacity of DM-GCCA NPs. Notably, dendritic cells play a key role in antigen presentation to initiate adaptive antitumor immunity. Flow cytometry analysis showed that CD80 + and CD86 + The infiltration rate of mature DC is the highest, e.g. Figure 7 F. Tumor immunofluorescence staining was used to detect CD80 + and CD86 + Infiltration of DCs. DM-GCCA NPs group CD80 + and CD86 + The distribution of DCs increased significantly, which was consistent with the results of flow cytometry analysis. Flow cytometry analysis showed that CD80+ and CD86 + DC has the highest infiltration rate, such as Figure 7 GH shown.

[0060] To clarify the role of NLRP3 inflammasome activation in inducing pyroptosis, this example used immunofluorescence staining to assess the activation status of NLRP3 inflammasome. Figure 7 As shown in IL, the expression levels of NLRP3 and caspase-1 in tumors treated with DM-GCCA NPs were significantly higher than those in the PBS group, providing evidence for the successful activation of NLRP3 inflammasome.

[0061] In summary, DM-GCCA NPs have good biocompatibility and tumor-targeting accumulation, and have strong in vivo antitumor activity. Pyroptosis-induced nanotherapy promotes the release of proinflammatory cytokines and enhances CD8 + / CD4 + These findings highlight the potential of DM-GCCA NPs as a new strategy for cancer immunotherapy.

[0062] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A tandem amplification pyroptosis nanoinducer, characterized in that: It is a nanoparticle structure, including crystalline dendritic Gd2O3NPs and a shell encapsulated on the surface of the crystalline dendritic Gd2O3NPs, the crystalline dendritic Gd2O3NPs contain a number of dendritic radial mesoporous channels, and the crystalline dendritic Gd2O3 nanoparticles are loaded with CPPO and Ce6, and the shell is an inorganic layer formed by CaCO3 nanoparticles.

2. The tandem amplified pyroptosis nanoinducer according to claim 1, wherein the tandem The hydrodynamic average particle size of the amplified pyroptosis nanoinducers was 215.0–225.0 nm; Or, it is characterized in that the zeta potential of the tandem amplified pyroptosis nanoinducer is 7.0~9.0 mV.

3. The tandem amplified pyroptosis nanoinducer according to claim 1, wherein: The masses of CPPO and Ce6 loaded per 100 μg of crystalline dendritic Gd2O3 nanoparticles are 0.01~4.92 μg and 0.01~42.65 μg, respectively.

4. The tandem amplified pyroptosis nanoinducer according to claim 1, characterized in that The masses of CPPO and Ce6 loaded per 100 μg of crystalline dendritic Gd2O3 nanoparticles are 4.88~4.92 μg and 42.55~42.65 μg, respectively.

5. A method for preparing the tandem amplified pyroptosis nanoinducer according to claim 1, characterized in that: The steps include: Providing dendritic mesoporous gadolinium hydroxide, and calcining and pyrolyzing the dendritic mesoporous gadolinium hydroxide under an inert atmosphere to obtain crystalline dendritic Gd2O3NPs; CPPO and Ce6 were loaded on crystalline dendritic Gd2O3NPs via electrostatic adsorption to obtain DM-GCC NPs; The CaCO3 inorganic shell was coated on DM-GCC NPs by co-precipitation method.

6. The preparation method according to claim 5, characterized in that: The calcination temperature under inert atmosphere is 550~650℃, and the calcination time is 5~7 hours; Alternatively, the sintered product may be calcined in an inert atmosphere and then calcined in an air atmosphere.

7. The preparation method according to claim 5, characterized in that: The crystalline dendritic Gd2O3NPs were modified with polyethyleneimine so that the surface of the crystalline dendritic Gd2O3NPs had a positive charge, and then mixed with CPPO and Ce6 to achieve electrostatic adsorption of CPPO and Ce6 by the crystalline dendritic Gd2O3NPs.

8. The preparation method according to claim 5, characterized in that: The co-precipitation process is as follows: DM-GCC NPs, calcium salt and carbonate are mixed and stirred in an aqueous solution.

9. Use of the tandemly amplified pyroptosis nanoinducer according to any one of claims 1 to 4 in the preparation of a drug for treating cancer.

10. The use according to claim 9, characterized in that: The cancer is lung cancer, intestinal cancer, stomach cancer, breast cancer or cervical cancer.

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