A composite nanomaterial capable of activating calcium overload and tumor cell pyroptosis, its preparation method and application

Abstract

This invention relates to the field of biomedical nanomaterial preparation, and particularly to a novel composite nanomaterial capable of activating calcium overload and pyroptosis in tumor cells, its preparation method, and its applications. The composite nanomaterial of this invention comprises calcium peroxide, a zeolite imidazole ester framework, curcumin, and hyaluronic acid. The calcium peroxide is embedded within the zeolite imidazole ester framework, forming a core-shell structure. The curcumin is loaded within the shell layer of this core-shell structure, and the hyaluronic acid is attached to the particle surface. The composite material of this invention releases a large amount of calcium ions in response to the acidic environment of tumor cell lysosomes, inducing calcium overload in tumor cells and activating pyroptosis. The preparation method of this invention is simple and reliable, and the resulting composite nanomaterial, by inducing calcium overload and pyroptosis, shows significant application potential in combating tumor immunity.

Description

Technical Field

[0001] This invention relates to the field of biomedical nanomaterial preparation, and in particular to a novel composite nanomaterial capable of activating calcium overload and tumor cell pyroptosis, its preparation method, and its application. Background Technology

[0002] Pyroptosis is a unique form of programmed cell death characterized by the formation of pores in the cell membrane, causing the cell to swell, produce large gas bubbles, and release its contents. The classical pyroptosis pathway activates caspase-1, a caspase-containing protease, which cleaves Gasdermin D protein, promoting pore formation in the cell membrane and inducing pyroptosis. Studies have shown that caspase-3, an apoptosis-associated caspase, can cleave Gasdermin E (GSDME) protein, similarly promoting cell membrane pore formation and pyroptosis. Pyroptosis in tumor cells releases large amounts of damage-associated pattern molecules (DAMPs) and inflammatory factors, such as adenine triphosphate (ATP) and high-mobility group box 1 (HMGB1), promoting tumor inflammation. Therefore, activating tumor cell pyroptosis is a powerful cancer immunotherapy strategy that can stimulate tumor immunity and inhibit tumor growth. Related studies have confirmed that some small-molecule chemotherapeutic drugs can activate tumor cell pyroptosis, achieving the effect of inhibiting tumor growth. However, small molecule chemotherapy drugs have poor tumor-suppressing effects due to their systemic circulation and non-targeted nature, and are prone to toxic side effects on normal tissues and organs.

[0003] Nanomaterials, with their enhanced permeability and high retention properties in blood vessels and tumor tissues, have been widely used in cancer treatment in recent years. Calcium ions (Ca...) 2+ CaO is one of the most important metal ions in the human body, playing a crucial role in maintaining cellular function. Nano-CaO2 and its analogues have a strong ability to disrupt the redox balance within tumor cells, thereby inducing calcium overload. CUR, as a natural compound, is a good source of intracellular calcium. 2+ Outflow inhibitor. However, CaO2 is sensitive to acidic environments and readily reacts with hydrogen ions to form CaO in acidic conditions. 2+ And hydrogen peroxide (H2O2), this property is not conducive to CaO2 being enriched in the tumor site through blood circulation to promote apoptosis in tumor cells.

[0004] In 2020, Qianhua Feng (patent number: CN112274495B, article doi:10.1016 / j.ijpharm.2021.120937) modified the surface of CaO2 with a layer of transferrin to protect CaO2. However, due to the protection of transferrin, CaO2 is not easily degraded and burst to release a large amount of Ca after reaching the tumor site.2+ H2O2 induces calcium overload in tumor cells. In 2022, Xiaoming Ma (article doi:10.1016 / j.jcis.2022.08.125) grew a layer of ZIF-8 on the surface of CaO2, but additional steps were required to load tetraphenylporphyrin drugs, and the overall synthesis steps were cumbersome and the reaction time was long.

[0005] Therefore, exploring a composite nanomaterial and its synthesis method that can replace small molecule chemotherapy drugs to activate tumor cell pyroptosis, while having a short reaction time, few steps, good stability, and explosive release in the required environment to enhance the tumor-suppressing effect, is of great value for the practical application of such compounds. Summary of the Invention

[0006] The purpose of this invention is to address the problems in existing technologies, such as the toxic side effects of small molecule pyroptosis inducers in tumor treatment, the long reaction time, cumbersome steps, poor stability, and inability to release CaO2 nanomaterials explosively. The invention proposes a novel composite nanomaterial that can activate calcium overload and tumor cell pyroptosis, along with its preparation method and applications.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In a first aspect of the invention, a composite nanomaterial capable of activating calcium overload and tumor cell pyroptosis is provided, the composite nanomaterial comprising CaO2, ZIF-8, CUR and HA synthesized in the ethanol phase, wherein the CaO2 is embedded in ZIF-8 to form a core-shell structure, the CUR is loaded in the shell layer of the core-shell structure, and the HA is attached to the particle surface.

[0009] Preferably, the electron microscope size of the composite nanomaterial is 100-150 nm. More preferably, it is 120 nm.

[0010] In a second aspect of the present invention, a method for preparing composite nanomaterials as described in the first aspect is provided, comprising the following steps:

[0011] 1) Prepare CaO2 methanol solution with a concentration of 0.6 mg / mL, Zn(NO3)2·6H2O methanol solution with a concentration of 5 mg / mL, CUR methanol solution with a concentration of 2 mg / mL and 2-MI methanol solution with a concentration of 1 mg / mL respectively;

[0012] 2) In the CaO2 methanol solution prepared in step 1), the Zn(NO3)2·6H2O methanol solution, CUR methanol solution and 2-MI methanol solution prepared in step 1) are added sequentially, and the mass ratio of CaO2, Zn(NO3)2·6H2O, 2-MI and CUR is 0.3:5:1:0.5-2. The reaction is carried out under low temperature stirring, and the synthesized CaZC nanoparticles are obtained by centrifugation.

[0013] 3) CaZC nanoparticles were dispersed in an aqueous HA solution and stirred for modification, and then centrifuged to obtain CaZCH nanoparticles.

[0014] Preferably, in step 2), the mass ratio of CaO2, Zn(NO3)2·6H2O, 2-MI and CUR is 0.3:5:1:1.

[0015] Preferably, in step 2), the reaction temperature is 5±0.5℃, the stirring speed is 1600rpm, the stirring time is 0.5-2h, and the centrifugation speed is 11000rpm.

[0016] Preferably, in step 3), the mass ratio of CaZC, ultrapure water, and HA is 1.2:8:20.

[0017] Preferably, in step 3), the reaction temperature is 20-25℃, the stirring speed is 1400-1600 rpm, the stirring reaction time is 12h, and the centrifugation speed is 7000-8000 rpm.

[0018] In this invention, the method for preparing CaO2 synthesized from ethanol is existing technology. The specific process is as follows: A 35 mg / mL polyvinylpyrrolidone ethanol solution is added to a 20 mg / mL calcium chloride dihydrate ethanol solution. The pH is adjusted by adding a 0.8 M ammonium hydroxide aqueous solution, followed by the addition of a 1 M H2O2 aqueous solution. The mixture is stirred for 30 min, and then centrifuged to obtain CaO2 nanoparticles. The mass ratio of calcium chloride dihydrate, polyvinylpyrrolidone, ammonium hydroxide, and H2O2 is 20:70:5.6:4.1. The prepared CaO2 nanoparticles can be dispersed in methanol to form a 0.6 mg / mL CaO2 methanol solution.

[0019] In a third aspect of the invention, the use of composite nanomaterials as described in the first aspect or prepared by the preparation method as described in the second aspect in the preparation of anti-tumor drugs for colorectal cancer, breast cancer, cervical cancer, and melanoma is provided.

[0020] Compared with the prior art, the advantages of this invention are:

[0021] (1) In this invention, ZIF-8 successfully encapsulates CaO2 nanoparticles to form a core-shell structure through low-temperature stirring growth, thus protecting the unstable CaO2 nanoparticles. CUR, on the other hand, is grown in one step within the shell of the core-shell structure via ZIF-8 growth, solving the problems of cumbersome synthesis steps and long reaction times in existing technologies. ZIF-8 is a responsive material that can degrade in acidic environments. A layer of HA is then modified and attached to the material surface to protect the composite nanomaterial and improve its colloidal stability.

[0022] (2) When the nanoparticles are dispersed in an acidic environment, their ZIF-8 shell will rapidly degrade within 30 minutes, exposing CaO2 and then explosively releasing a large amount of Ca. 2+ This solves the problem of Ca2+ being affected by transferrin modification in existing technologies. 2+ The explosive release can more effectively activate calcium overload and tumor cell pyroptosis.

[0023] (3) When nanoparticles are internalized by tumor cells, they degrade and release a large amount of free Ca2+ by utilizing the acidic intracellular environment. 2+ And CUR, leading to mitochondrial Ca 2+ Overload, and also because of O2 2- The presence of this substance generates a large amount of reactive oxygen species, which activates caspase-3 to cleave GSDME protein, activates tumor cell pyroptosis, releases DAMPs to enhance immunogenicity, and at the same time solves the problem of toxic side effects on normal cells caused by small molecule chemotherapy drugs inducing tumor cell pyroptosis.

[0024] (4) A novel composite nanomaterial prepared by the above structural design, CUR inhibits the extracellular Ca2+ excretion. 2+ ZIF-8 and HA protect the internal CaO2 nanoparticles and CUR, thereby maximizing the therapeutic effect of tumor treatment by activating calcium overload and pyroptosis.

[0025] (5) In this invention, the problems of toxic side effects caused by small molecule pyroptosis inducers, long reaction time, complicated steps, poor stability and inability to release CaO2 nanomaterials in the prior art of tumor treatment are solved. This invention provides new theoretical support for the development of new pyroptosis inducers and anticancer drugs, and has important scientific significance, application value and economic value. Attached Figure Description

[0026] Figure 1 In the image, a and b are scanning electron microscope images of CaO2 nanoparticles and CaZC nanoparticles in this invention.

[0027] Figure 2 This is a transmission electron microscope (TEM) image of the CaZCH nanoparticles in this invention.

[0028] Figure 3 In the figures, a represents the UV-Vis absorption spectrum of the CaZC nanoparticles in this invention; b represents the zeta potential measurement result of the CaZCH nanoparticles; and c represents the concentration of H2O2 generated in the solution after the CaZCH nanoparticles in this invention are incubated with phosphate buffered saline (PBS) solutions of different pH values.

[0029] Figure 4 The zinc ions and Ca2+ ions in CaZCH nanoparticles after incubation with buffer solutions of different pH values ​​in this invention are... 2+ The relationship between cumulative release and time.

[0030] Figure 5 The cell survival rate of CT26 cells after incubation with CUR, CaZH and CaZCH nanoparticles in this invention.

[0031] Figure 6 The results of laser confocal microscopy observation of reactive oxygen species production after incubation of PBS, CUR, CaZH and CaZCH nanoparticles with CT26 cells in this invention.

[0032] Figure 7 To observe the free Ca2+ after incubation of PBS, CUR, CaZH, and CaZCH nanoparticles with CT26 cells using laser confocal microscopy in this invention. 2+ The result.

[0033] Figure 8 The results of laser confocal microscopy observation of changes in mitochondrial membrane potential in CT26 cells after incubation of PBS, CUR, CaZH and CaZCH nanoparticles with the cells in this invention.

[0034] Figure 9 In the figures, a represents the results of microscopic observation of cell morphology after incubation of CT26 cells with PBS, CUR, CaZH and CaZCH nanoparticles in this invention, with the white arrows indicating pyroptotic cells; b represents the cell survival rate after incubation of CaZCH nanoparticles with normal cell lines L-02 and NIH / 3T3 in this invention.

[0035] Figure 10 In the figures, a represents the result of detecting the ATP concentration in the cell culture supernatant after incubation of PBS, CUR, CaZH and CaZCH nanoparticles with CT26 cells in this invention; b represents the result of detecting intracellular HMGB1 expression in CT26 cells using laser confocal microscopy after incubation of PBS, CUR, CaZH and CaZCH nanoparticles with CT26 cells in this invention.

[0036] Figure 11 This presents the tumor growth inhibition results of different treatments in the subcutaneous tumor model in this invention.

[0037] Figure 12 This represents the maturation level of dendritic cells (DCs) in the axillary lymph nodes on the tumor side of mice in subcutaneous tumor models treated with different methods in this invention.

[0038] Figure 13 CD8 at the tumor sites of mice in different treatment models of subcutaneous tumors in this invention. + Degree of T cell infiltration. Detailed Implementation

[0039] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the invention, and not all of them.

[0040] Methanol, ethanol, ammonium hydroxide, H2O2, and Zn(NO3)2·6H2O mentioned in the following examples of this invention were purchased from Sinopharm Chemical Reagent Co., Ltd.; calcium chloride dihydrate and polyvinylpyrrolidone were purchased from Sigma-Aldrich Reagents Ltd.; CUR and 2-MI were purchased from Beijing Bailingwei Technology Co., Ltd.; HA was purchased from Shanghai Maclean Biotechnology Co., Ltd.; PBS, CCK-8 kit, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), calcium ion fluorescent probe (Fluo-4 AM), and mitochondrial red fluorescent probe (Mito-Tracker Red CMXRos) were purchased from Shanghai Beyotime Biotechnology Co., Ltd.; the mouse colon cancer cell line CT26 was purchased from Shanghai Fuheng Cell Biotechnology Co., Ltd.; the mouse embryonic fibroblast cell line NIH / 3T3 and the human normal liver cell line L-02 were purchased from Jiangsu Kaiji Biotechnology Co., Ltd.; and the experimental mice were Balb / c mice purchased from the Comparative Medicine Center of Yangzhou University.

[0041] Example 1

[0042] The composite nanomaterial in this embodiment, capable of activating calcium overload and pyroptosis, includes CaO2, ZIF-8, CUR, and HA. The CaO2 is embedded in ZIF-8, forming a core-shell structure. The CUR is loaded within the shell of the core-shell structure, and the HA is modified on the particle surface. Figure 2 As shown, arrow ① indicates the core-shell structure, i.e., ZIF-8. The CUR is loaded inside arrow ①, i.e., inside the shell. Arrow ② points to the particle surface, and HA is attached to the outer surface of this particle. Here, the particle refers to a particle with a core-shell structure. The electron microscope size of the composite nanomaterial is 120 nm.

[0043] (1) The synthesis of CaZCH in this invention includes the following steps:

[0044] a. Preparation of CaO2 nanoparticles

[0045] At room temperature, 10 mL of calcium chloride dihydrate ethanol solution (20 mg / mL) and 20 mL of polyvinylpyrrolidone ethanol solution (35 mg / mL) were added sequentially to a 50 mL Erlenmeyer flask. After stirring until homogeneous, 2 mL of ammonium hydroxide aqueous solution (0.8 M) was added, followed by 1.2 mL of H₂O₂ aqueous solution (1 M). The mixture was stirred continuously at 1400 rpm for 30 min. After stirring, the CaO₂ nanoparticles were collected by centrifugation at 11000 rpm for 15 min, dried, and stored for later use. The obtained CaO₂ nanoparticles can be dispersed in methanol to form a CaO₂ methanol solution.

[0046] b. Preparation of CaZC nanoparticles

[0047] First, prepare CaO2 methanol solution with a concentration of 0.6 mg / mL, Zn(NO3)2·6H2O methanol solution with a concentration of 5 mg / mL, CUR methanol solution with a concentration of 2 mg / mL and 2-methylimidazolium methanol solution with a concentration of 1 mg / mL, respectively.

[0048] Then, at 5℃, 10 mL of CaO2 methanol solution (0.6 mg / mL), 20 mL of Zn(NO3)2·6H2O methanol solution (5 mg / mL), and 10 mL of CUR methanol solution (2 mg / mL) were added sequentially to a 50 mL Erlenmeyer flask, and the mixture was stirred until homogeneous. Under stirring at 1600 rpm, 20 mL of pre-cooled 2-methylimidazolium methanol solution (1 mg / mL) was added to the mixture, and stirring was continued for 30 min. After stirring, the CaZC nanoparticles were collected by centrifugation at 11000 rpm for 15 min, dried, and stored for later use. The synthesis steps show that, compared to the existing method of first synthesizing ZIF-8 and then loading the drug, this method for synthesizing CaZC nanoparticles solves the problems of cumbersome synthesis steps and long reaction times, making it more conducive to the widespread application of the material.

[0049] c. Preparation of CaZCH nanoparticles

[0050] CaZC, ultrapure water, and HA were mixed thoroughly at a mass ratio of 1.2:8:20 at 20-25℃ and stirred continuously at 1600 rpm for 12 h. After stirring, the CaZC nanoparticles were collected by centrifugation at 8000 rpm for 15 min and dispersed in PBS for later use.

[0051] (2) Characterization of CaO2, CaZC and CaZCH nanomaterials

[0052] like Figure 1As shown in Figure a, the CaO2 nanoparticles synthesized in ethanol exhibit uniform size and morphology, with a size of approximately 90 nm. Using these CaO2 nanoparticles, [the following was successfully synthesized]... Figure 1 The CaZC nanoparticles with a size of 120 nm shown in figure b. Among them, as... Figure 2 As shown, after HA connection, the size of CaZCH nanoparticles did not change significantly. TEM clearly shows that CaZCH nanoparticles have a core-shell structure, and the thickness of their ZIF-8 shell is about 15 nm.

[0053] The CaZC nanoparticles prepared in this embodiment were analyzed using a UV-Vis spectrophotometer, and the results were as follows: Figure 3 The UV-Vis absorption spectrum shown in Figure a reveals that neither CaO2 nor ZIF-8 exhibits a distinct UV absorption peak; CUR shows a UV absorption peak around 425 nm; and CaZC nanoparticles also show a characteristic absorption peak at around 425 nm, attributed to CUR. Figure 3 As shown in Figure b, the zeta potential of the nanoparticles was measured and compared, revealing that CaZC nanoparticles have a lower zeta potential. Figure 3 As shown in Figure c, CaZCH nanoparticles were dispersed in PBS at different pH values, and more H2O2 was generated under low pH conditions.

[0054] (3)Ca 2+ Explosive release test

[0055] After being internalized by tumor cells, CaZCH nanomaterials enter lysosomes and degrade due to their acidic environment (pH 4.5–6.0), releasing large amounts of Ca. 2+ Induced mitochondrial dysfunction leads to calcium overload in tumor cells. Therefore, the inventors simulated the acidic environment of lysosomes to monitor the degradation and ion release of CaZCH nanoparticles. Figure 4 As shown, when CaZCH nanoparticles were dispersed in a buffer solution with a pH of 5.5, it was found that more than 90% of zinc ions were released cumulatively within 30 minutes. 2+ The cumulative release exceeded 80% within 4 hours, demonstrating that the CaZCH material achieved explosive release, thus solving the problem of Ca release being affected by transferrin modification in existing technologies. 2+ The explosive release can more effectively activate calcium overload and tumor cell pyroptosis.

[0056] Comparative Example 1: Synthesis of (CaO2@ZIF-8-HA)CaZH nanoparticles

[0057] The synthesis of CaZH includes the following steps:

[0058] a. Preparation of CaO2@ZIF-8-HA(CaZ) nanoparticles

[0059] CaZ nanoparticles were prepared by replacing the CUR methanol solution with an equal volume of methanol, following the method and proportions for preparing CaZC nanoparticles in Example 1.

[0060] b. Preparation of CaZH nanoparticles

[0061] By replacing CaZC with CaZ, and following the method and proportions for preparing CaZCH nanoparticles in Example 1, CaZH nanoparticles were prepared.

[0062] Example 2

[0063] The following experiments were conducted using the CaZCH and CaZH nanoparticles prepared in Example 1 and Comparative Example 1, respectively:

[0064] (1) Cytotoxicity studies

[0065] The cytotoxic effect of the CaZCH nanoparticles prepared in Example 1 on CT26 cells was determined using the CCK-8 assay. The experimental results are as follows: Figure 5 As shown, when the calcium concentration was 25 μg / mL, the survival rate of CT26 cells after incubation was 36.5%, indicating that CaZCH nanoparticles have a significant killing effect on CT26 tumor cells.

[0066] (2) Research on intracellular reactive oxygen species production

[0067] DCFH-DA was used as a fluorescent dye to verify the generation of reactive oxygen species (ROS). DCFH-DA reacts with intracellular ROS to generate DCFH, which emits a distinct green fluorescence. Fluorescence imaging was then performed using laser scanning confocal fluorescence microscopy. Figure 6 As shown, the intracellular reactive oxygen species (ROS) generation of the CaZCH nanoparticles prepared in Example 1 was investigated. The figure shows that CT26 cells treated with CaZCH nanoparticles exhibited strong green fluorescence after DCFH-DA staining, indicating that the CaZCH nanoparticles prepared in Example 1 possess a strong ROS generation capacity and can effectively induce tumor cell death.

[0068] (3) Intracellular free Ca 2+ Genesis Research

[0069] Validation of intracellular free Ca using Fluo-4 AM 2+ The production of Fluo-4 AM. After entering the cell, Fluo-4 AM can be cleaved by intracellular esterases to form Fluo-4, which is then retained within the cell. Fluo-4 can react with Ca... 2+ Combination, combination of Ca 2+This produces strong green fluorescence, which is then imaged using a laser scanning confocal fluorescence microscope. For example... Figure 7 As shown, intracellular Ca2+ nanoparticles prepared in Example 1 were subjected to intracellular Ca2+ processing. 2+ Release study. After CaZCH nanoparticles were internalized by CT26 cells, they were degraded in the acidic liquid environment within lysosomes, releasing large amounts of Ca. 2+ The image shows that CT26 cells treated with CaZCH nanoparticles and stained with Fluo-4 AM exhibit a strong green Ca2+ staining. 2+ Fluorescence indicates that the nanoparticles release a large amount of free Ca after being internalized by cells. 2+ This also demonstrates that the CaZCH nanoparticles prepared in Example 1, after being internalized, can explosively release a large amount of Ca into cells. 2+ .

[0070] (4) Study on changes in intracellular mitochondrial membrane potential

[0071] Changes in mitochondrial membrane potential were detected using Mito-Tracker Red CMXRos. Mito-Tracker Red CMXRos is a small molecule dye with cell permeability that specifically labels biologically active mitochondria in cells, allowing for the detection of mitochondrial membrane potential. When mitochondrial activity is normal, Mito-Tracker Red CMXRos-labeled mitochondria initially emit red fluorescence. When mitochondria are damaged, the membrane potential changes, and the red fluorescence weakens. Fluorescence imaging can be performed using laser scanning confocal fluorescence microscopy. Figure 8 As shown, the ability of CaZCH nanoparticles prepared in Example 1 to induce mitochondrial dysfunction was investigated. After internalization by CT26 cells, the CaZCH nanoparticles were degraded in the acidic liquid environment within lysosomes, releasing a large amount of Ca. 2+ The CaZCH nanoparticles accumulate in mitochondria, inducing mitochondrial dysfunction and causing changes in mitochondrial membrane potential. The figure shows that after CT26 cells were treated with CaZCH nanoparticles, the mitochondrial membrane potential changed significantly, and the red fluorescence was significantly weakened, indicating that the CaZCH nanoparticles prepared in Example 1 induced mitochondrial dysfunction and calcium overload.

[0072] Example 3

[0073] The CaZCH nanoparticles prepared in Example 1 were incubated with CT26 cells in four groups (Control, CUR, CaZH, and CaZCH). The Control group was incubated with an equal volume of PBS, the CUR group was incubated with a concentration of 8.2 μg / mL (calculated as CUR), and the CaZH and CaZCH groups were incubated with concentrations calculated as Ca... 2+The concentration was calculated to be 25 μg / mL. Subsequent pyroptosis observation, biocompatibility verification, ATP release detection, and HMGB1 production detection were performed.

[0074] (1) Observation of pyroptosis morphology and verification of biocompatibility

[0075] Changes in cell morphology were observed using an inverted biological microscope. For example... Figure 9 As shown in Figure a, the CaZCH nanoparticles prepared in Example 1 were incubated with CT26 cells for 10 hours in a cell culture incubator. This verified that the nanoparticles, after internalization by CT26 cells, could activate pyroptosis in CT26 cells. The figure shows that after 10 hours of incubation with CaZCH, CT26 cells exhibited a large number of pyroptotic cell morphologies under a microscope (the white arrows indicate pyroptotic cells), indicating that the CaZCH nanoparticles prepared in Example 1 have a good ability to induce pyroptosis in tumor cells. Incubation of the CaZCH nanoparticles prepared in Example 1 with normal cell lines NIH / 3T3 and L-02 further demonstrated the following effects: Figure 9 As shown in b, even if Ca 2+ Even at a concentration as high as 25 μg / mL, the cell survival rate remained above 95%, indicating that CaZCH nanoparticles have high biocompatibility with normal cells, thus solving the problem of small molecule chemotherapy drugs causing toxic side effects on normal cells while inducing pyroptosis.

[0076] (2) ATP release detection

[0077] The release of ATP from the cells was verified by detecting the ATP concentration in the supernatant of the cell culture medium after incubating CT26 cells with CaZCH nanoparticles prepared in Example 1 for 24 hours. Figure 10 As shown in Figure a, CT26 cells were incubated with the CaZCH nanoparticles prepared in Example 1 for 24 hours in a cell culture incubator. This verified that after the nanoparticles were internalized by CT26 cells, they activated pyroptosis and released intracellular ATP. The figure shows that after CT26 cells were incubated with CaZCH, a significantly enhanced ATP release was detected in the cell culture medium, indicating that the CaZCH nanoparticles prepared in Example 1 induced pyroptosis and promoted ATP release, thereby enhancing the immunogenicity of the cells.

[0078] (3) HMGB1 generation detection

[0079] The expression level of HMGB1 in CT26 cells after incubation of CaZCH nanoparticles prepared in Example 1 for 4 hours was detected by immunofluorescence assay. Figure 10As shown in Figure b, after incubating CT26 cells with the CaZCH nanoparticles prepared in Example 1 for 4 hours, the cells underwent fixation, permeabilization, blocking, primary antibody incubation, fluorescent secondary antibody incubation, and nuclear fluorescent dye incubation. Fluorescence imaging was then performed using a laser scanning confocal fluorescence microscope. This verified that internalization of the nanoparticles induced a large expression of HMGB1 in CT26 cells. The figure shows that after incubation with CaZCH for a period of time, CT26 cells exhibited stronger intracellular HMGB1 fluorescence, indicating that the CaZCH nanoparticles prepared in Example 1 can induce immunogenicity in cells after incubation.

[0080] Example 4

[0081] The CaZCH and CaZH nanoparticles prepared in Example 1 and Comparative Example 1, respectively, were used for in vivo subcutaneous treatment of CT26 tumor-bearing mice. The tumor volume of the mice was approximately 80 mm². 3 They were randomly divided into four groups (PBS group, CUR group, CaZH group and CaZCH group), with 5 animals in each group.

[0082] Experimental methods: Intratumoral injections were performed every two days for a total of four injections over one week. Mice were then observed for one week, with tumor volume and body weight recorded every two days. The PBS group served as a control group, the CUR group received CUR at a dose of 1.6 mg / kg, the CaZH group received CaZH at a dose of 5.0 mg / kg, and the CaZCH group received CaZCH at a dose of 5.0 mg / kg.

[0083] The results are as follows Figure 11 As shown, after a week of treatment and a week of observation, the average tumor volume of mice in the CaZCH treatment group was 5 times that before treatment, which was much lower than 17.7 times that of the PBS group, 15.3 times that of the CUR group, and 9.5 times that of the CaZCH group, indicating that CaZCH nanoparticles can effectively inhibit tumor growth.

[0084] In addition, such as Figure 12 and Figure 13 As shown, compared to the other three groups, the CaZCH treatment group had the highest proportion of mature dendritic cells (DCs) in the axillary lymph nodes on the tumor side of the mice, and stronger CD8 activity at the tumor site. + T cell infiltration demonstrates the significant advantages of the CaZCH nanoparticles prepared in Example 1 in tumor immunotherapy.

[0085] In summary, the novel composite nanomaterial of this invention, CUR, inhibits the extracellular Ca2+ excretion. 2+ ZIF-8 and HA protect the internal CaO2 nanoparticles and CUR, thereby maximizing the therapeutic effect of tumor treatment by activating calcium overload and pyroptosis.

[0086] This invention solves the problems of toxic side effects caused by small molecule pyroptosis inducers, long reaction time, cumbersome steps, poor stability, and inability to release pyroptosis in existing tumor treatment technologies. It provides new theoretical support for the development of novel pyroptosis inducers and anticancer drugs, and has important scientific, practical, and economic value.

[0087] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitution or modification made by those skilled in the art within the scope of the technology disclosed in the invention based on the technical solution and inventive concept of the present invention, such as CaO2@ZIF-8 / KAE-HA for pyroptosis-induced tumor therapy, CaO2@ZIF-90 / CUR-HA or CaO2@ZIF-8 / CUR-PVP, or the combined use of the above two nanoparticles for the treatment of tumors such as breast cancer and cervical cancer, should all be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a composite nanomaterial capable of activating calcium overload and pyroptosis in tumor cells, characterized in that, The composite nanomaterials include calcium peroxide (CaO2) synthesized from ethanol, zeolite imidazole ester framework material (ZIF-8), curcumin (CUR), and hyaluronic acid (HA). The CaO2 is embedded in ZIF-8 to form a core-shell structure, the CUR is loaded in the shell of the core-shell structure, and the HA is attached to the particle surface. The electron microscope size of the composite nanomaterial is 100-150 nm. The preparation method includes the following steps: 1) Prepare CaO2 methanol solution with a concentration of 0.6 mg / mL, zinc nitrate hexahydrate (Zn(NO3)2·6H2O) methanol solution with a concentration of 5 mg / mL, CUR methanol solution with a concentration of 2 mg / mL and 2-methylimidazole (2-MI) methanol solution with a concentration of 1 mg / mL respectively; 2) In the CaO2 methanol solution prepared in step 1), the Zn(NO3)2·6H2O methanol solution, CUR methanol solution and 2-MI methanol solution prepared in step 1) are added sequentially, and the mass ratio of CaO2, Zn(NO3)2·6H2O, 2-MI and CUR is 0.3:5:1:0.5-2.

0. The reaction is carried out under low temperature stirring, and the synthesized CaO2@ZIF-8 / CUR(CaZC) nanoparticles are obtained by centrifugation. 3) CaZC nanoparticles were dispersed in an aqueous HA solution and stirred for modification. The mixture was then centrifuged to obtain CaO2@ZIF-8 / CUR-HA(CaZCH) nanoparticles. In step 2), the mass ratio of CaO2, Zn(NO3)2·6H2O, 2-MI and CUR is 0.3:5:1:1; In step 2), the reaction temperature is 5±0.5℃, the stirring speed is 1600rpm, the stirring time is 0.5-2.0h, and the centrifugation speed is 11000rpm.

2. The method for preparing the composite nanomaterial capable of activating calcium overload and tumor cell pyroptosis according to claim 1, characterized in that, In step 3), the mass ratio of CaZC, ultrapure water and HA is 1.2:8:

20.

3. The method for preparing the composite nanomaterial capable of activating calcium overload and tumor cell pyroptosis according to claim 1, characterized in that, In step 3), the reaction temperature is 20-25℃, the stirring speed is 1400-1600 rpm, the stirring reaction time is 12h, and the centrifugation speed is 7000-8000 rpm.

4. The application of the composite nanomaterial prepared by the method described in claim 1 in the preparation of tumor therapeutic drugs.

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