Preparation method and application of degradation type copper-tannic acid-chlorin e6 nanocomposite for inducing pyroptosis of cells

Abstract

The present application relates to a kind of degradation type copper-tannin acid-chlorin e6 nanocomposite preparation method and its application for inducing pyroptosis, belong to antitumor drug field.The nanocomposite preparation method is simple, low energy consumption, including the following steps: after the mixed solution of polyvinylpyrrolidone and copper salt is stirred for a fixed time, tannin acid solution is added and solution pH is adjusted, copper-tannin acid nanomaterial is obtained after reaction, 3-aminopropyl triethoxysilane is added, centrifugal dispersion is carried out, then carboxyl activated chlorin e6 is added, reaction is carried out, and copper-tannin acid-chlorin e6 nanocomposite is obtained.The nanocomposite can be biodegraded under simulated tumor microenvironment, it can be quickly metabolized and eliminated in vivo, has lower cytotoxicity and biological toxicity.The nanocomposite has multiple simulated enzyme activities, can occur cascade reaction in tumor microenvironment, cooperates with sonodynamic therapy to amplify the generation of reactive oxygen species and induces pyroptosis of tumor cells.

Description

Technical Field

[0001] This invention belongs to the field of antitumor drug technology, specifically relating to a method for preparing a degradable copper-tannic acid-dihydroporphyrin E6 nanocomposite that induces pyroptosis and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Nanozymes are nanomaterials that act as catalysts themselves and possess enzyme-mimicking activities similar to natural enzymes. Currently, nanozymes mimicking catalase (CAT), peroxidase (POD), oxidase (OXD), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) activities have been reported. Due to their ability to generate reactive oxygen species (ROS) within tumor cells through their enzyme-like properties, they have been widely used in tumor catalytic therapy.

[0004] Ideally, multifunctional nanozymes should, while ensuring biocompatibility, be able to reshape the immunosuppressive microenvironment (TME) through enzyme-driven cascade reactions, thereby achieving highly effective tumor therapy. However, currently reported nanozymes often use noble metals as catalytic active sites, which means the materials cannot be degraded and metabolized in vivo, posing certain biocompatibility issues. Furthermore, they often rely on programmed cell death (PCD) characterized by apoptosis for anti-tumor activity, failing to reverse the refractory immunosuppressive TME of tumor cells and achieve long-term effective immunotherapy.

[0005] Pyroptosis is a novel and unique proliferative cytodegenerative disorder (PCD) characterized by membrane pore formation, cell swelling and blistering, and the release of contents and inflammatory factors, driven by inflammasomes. Due to its ability to release high levels of damage-associated molecular patterns and pro-inflammatory factors, triggering a strong inflammatory response, it is increasingly considered a promising new avenue for activating tumor immunotherapy. However, current pyroptosis inducers often induce pyroptosis in an uncontrolled manner, causing adverse reactions in vivo and leading to potential biosafety issues. Ultrasound (US), with its precise controllability, non-invasiveness, and high tissue penetration, allows deep infiltration into tumor tissues with dense extracellular matrix for sonodynamic therapy (SDT), and is considered one of the most promising and versatile physical stimulation methods. However, the hypoxic environment of tumors and the high expression of reduced glutathione (GSH) significantly reduce the efficacy of sonosensitive agent-mediated SDT, limiting its further application in tumor treatment. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a method for preparing a degradable copper-tannic acid-dihydroporphyrin e6 nanocomposite that induces pyroptosis, and its application. The copper-tannic acid-dihydroporphyrin e6 nanocomposite provided by this invention catalyzes superoxide anion (O2) in TME under simulated SOD activity. ·- The formation of hydrogen peroxide (H₂O₂) and O₂ further enhances the ability to generate O₂ and hydroxyl radicals (·OH) in simulated CAT and POD activities. Under exogenous US stimulation, dihydroporphyrin e₆ attached to the CuTA surface further utilizes the O₂ accumulated through a dual pathway to generate a large amount of... 1 ROS storms, including O2 and ·OH, are also present. Simultaneously, under simulated GSH-Px activity, reduced reduced glutathione (GSH) is consumed, reducing off-target effects of ROS. The copper-tannic acid-dihydroporphyrin e6 nanocomposite with quadruple enzyme-mimicking activity can induce pyroptosis in tumor cells while undergoing degradation within the TME, disrupting the immunosuppressive microenvironment and achieving excellent therapeutic effects.

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

[0008] In a first aspect, the present invention provides a method for preparing a degradable copper-tannic acid-dihydroporphyrin E6 nanocomposite that induces pyroptosis, comprising:

[0009] The surfactant and copper salt were mixed evenly in the solution, and then tannic acid solution was added to allow them to react, thus obtaining copper-tannic acid nanomaterials.

[0010] The copper-tannic acid nanomaterials were added to the amino modifier dispersion and mixed evenly to obtain the first mixed solution;

[0011] An activator was added to the dispersion of dihydroporphyrin E6 and mixed thoroughly to obtain a second mixed solution.

[0012] The first mixed solution was added to the second mixed solution to carry out the reaction, and copper-tannic acid-dihydroporphyrin E6 nanocomposite was obtained.

[0013] In some embodiments, the surfactant is PVP, with a viscosity grade of K30 and a molecular weight of 44,000 to 54,000.

[0014] In some embodiments, the copper salt is copper chloride dihydrate.

[0015] In some embodiments, the amino modifier is APTES.

[0016] In some embodiments, the activator is EDC / NHS.

[0017] In some embodiments, the mass ratio of the copper salt to tannic acid is 12 to 22:1.

[0018] In some embodiments, the mass ratio of the dihydroporphyrin E6 to the copper salt is 1 to 5:85.

[0019] More specifically, it includes the following steps:

[0020] (1) Disperse the surfactant in an aqueous solution and add copper salt while stirring vigorously to obtain mixed solution A;

[0021] (2) After vigorously stirring the mixed solution A for a fixed time, add tannic acid aqueous solution to obtain mixed solution B;

[0022] (3) Add NaOH aqueous solution to the mixed solution B to adjust the pH value, and continue to stir the reaction to obtain a uniformly dispersed mixed solution C;

[0023] (4) The mixed solution C was centrifuged to collect the precipitate, and the precipitate was ultrasonically dispersed to obtain copper-tannic acid nanomaterials;

[0024] (5) Disperse the amino modifier in an ethanol solution, add copper-tannic acid solution and stir continuously, then centrifuge to obtain mixed solution D;

[0025] (6) After dispersing dihydroporphyrin E6 in methanol solution, an activator is added and stirred for a fixed time to obtain mixed solution E;

[0026] (7) Add mixed solution E to the mixed solution D and continue stirring to react, to obtain mixed solution F;

[0027] (8) The mixed solution F was centrifuged to collect the precipitate, and the precipitate was ultrasonically dispersed to obtain copper-tannic acid-dihydroporphyrin E6 nanocomposite.

[0028] In some embodiments, the stirring speed in step (1) is 400-800 rpm; the stirring time in step (2) is 60-120 min; the pH value is adjusted to 8-14 in step (3), and the stirring reaction time is 8-16 h; the stirring time in step (5) is 10-14 h; the stirring time in step (6) is 60-90 min; and the stirring time in step (7) is 10-14 h.

[0029] In some embodiments, the particle size of copper-tannic acid in step (4) is 62–97 nm.

[0030] In a second aspect, the present invention provides a degradable copper-tannic acid-dihydroporphyrin E6 nanocomposite that induces pyroptosis, prepared by the above method.

[0031] A third aspect of the present invention provides the use of the above-described pyroptosis-inducing degradable copper-tannic acid-dihydroporphyrin E6 nanocomposite in any one or more of the following:

[0032] (a1) Application in the preparation of products that induce pyroptosis, wherein the cells include: tumor cells;

[0033] (a2) Application in the preparation of ROS-generated products amplified under exogenous US.

[0034] A fourth aspect of the present invention provides the application of the copper-tannic acid nanomaterials prepared by the above method in the preparation of tumor microenvironment improvers or pyroptosis inducers.

[0035] The copper-tannic acid nanomaterials of the present invention can be degraded in a simulated tumor microenvironment. The simulated tumor microenvironment has a pH of 4.0–7.5, a glutathione concentration of 2–10 mM, and an H₂O₂ concentration of 0.01–0.1 mM.

[0036] The copper-tannic acid nanomaterials of the present invention also possess quadruple simulated activities of SOD, CAT, POD and GSH-Px.

[0037] Beneficial effects of the present invention

[0038] (1) The copper-tannic acid-dihydroporphyrin e6 nanocomposite of the present invention has four-fold mimicry activities of SOD, CAT, POD and GSH-Px, which can generate a large amount of ROS in the TME in conjunction with SDT, induce tumor cell pyroptosis, break the immunosuppressive microenvironment, and achieve long-term effective immunotherapy.

[0039] (2) In the preparation process of the copper-tannic acid-dihydroporphyrin e6 nanocomposite of the present invention, PVP, as a surfactant, can improve the water solubility, biocompatibility and control the size of the material; the copper-tannic acid-dihydroporphyrin e6 nanocomposite can be biodegraded in TME and has excellent safety. The analysis of biochemical indicators and blood routine results of material metabolism, liver function and kidney function in mice shows that the material has no long-term toxicity to mice.

[0040] (3) The needle-shaped copper-gallic acid nanozyme only exhibits catalase-like activity to produce O2, which will produce e6 from dihydroporphyrin. 1 Unlike other inventions that limit O2 production, the copper-tannic acid-dihydroporphyrin E6 of this invention possesses catalase-like and superoxide dismutase-like activities, enabling dual O2 generation and thus enhancing dihydroporphyrin E6 production. 1 The ability of O2.

[0041] (4) The preparation method of the present invention is simple, practical and easy to promote. Attached Figure Description

[0042] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. Exemplary embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0043] Figure 1 The image shows a transmission electron microscope (TEM) image of the copper-tannic acid nanomaterials prepared in step (4) of Example 1.

[0044] Figure 2 According to Figure 1 Particle size distribution obtained from the average size calculation of copper-tannic acid nanomaterials.

[0045] Figure 3 The simulated SOD activity diagram of the copper-tannic acid nanomaterials prepared in step (4) of Example 1 is shown.

[0046] Figure 4 The simulated CAT activity diagram of the copper-tannic acid nanomaterials prepared in step (4) of Example 1 is shown.

[0047] Figure 5 The simulated GSH-Px activity diagram of the copper-tannic acid nanomaterials prepared in step (4) of Example 1 is shown.

[0048] Figure 6 The simulated POD activity diagram of the copper-tannic acid nanomaterials prepared in step (4) of Example 1 is shown.

[0049] Figure 7 The electron spin resonance (ESR) diagram of the amount of ·OH generated by the copper-tannic acid nanomaterial prepared in step (4) of Example 1 under 0.1mM H2O2 conditions.

[0050] Figure 8 The image shows the degradation TEM image of the copper-tannic acid nanozyme prepared in step (4) of Example 1 under simulated TME.

[0051] Figure 9 This is a comparison of the emission intensity of SOSG at 525 nm for different materials under US stimulation.

[0052] Figure 10 The copper-tannic acid-dihydroporphyrin E6 nanocomposite prepared in Example 1 was generated under different conditions. 1 ESR diagram of O2 levels.

[0053] Figure 11 The image shows the cytotoxicity (MTT) curve of the copper-tannic acid-dihydroporphyrin e6 nanocomposite prepared in Example 1.

[0054] Figure 12The image shows the pyroptosis morphology of the copper-tannic acid-dihydroporphyrin E6 nanocomposite prepared in Example 1.

[0055] Figure 13 This is a metabolic diagram of the material after tumor-bearing mice were injected with the copper-tannic acid-dihydroporphyrin E6 nanocomposite prepared in Example 1.

[0056] Figure 14 The images show hematological and biochemical parameters of tumor-bearing mice after injection of the copper-tannic acid-dihydroporphyrin E6 nanocomposite prepared in Example 1. Detailed Implementation

[0057] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0058] Terminology Explanation:

[0059] APTES refers to 3-aminopropyltriethoxysilane.

[0060] EDC stands for 1-ethyl-(3-dimethylaminopropyl)carbodiimide.

[0061] NHS stands for N-hydroxysuccinimide.

[0062] The present invention will be further described in detail below with reference to specific embodiments. It should be noted that the specific embodiments are explanations of the present invention and not limitations thereof.

[0063] Example 1

[0064] The copper-tannic acid-dihydroporphyrin E6 nanocomposite was prepared using the following method:

[0065] (1) Disperse 0.25g PVP in 5mL of deionized water, and add 40mL of deionized water containing 0.085g CuCl2·2H2O while stirring at 700rpm to obtain mixed solution A;

[0066] (2) After vigorously stirring the mixed solution A for 90 minutes, add 5 mL of 1 mg / mL tannic acid aqueous solution to obtain mixed solution B;

[0067] (3) Add NaOH aqueous solution to mixed solution B to adjust the pH value to 12, and continue stirring for 12 hours to obtain a uniformly dispersed mixed solution C;

[0068] (4) The mixed solution C was centrifuged at 13500 rpm for 20 min to collect the precipitate. The precipitate was then ultrasonically dispersed to obtain copper-tannic acid nanomaterials.

[0069] (5) Disperse 300 μL of APTES in 3 mL of ethanol solution, add copper-tannic acid solution and stir continuously for 12 h, then centrifuge to obtain mixed solution D;

[0070] (6) Disperse 1 mg of dihydroporphyrin E6 in 1 mL of methanol solution, then add methanol solution containing 8 mg EDC and 12 mg NHS and stir for 60 min to obtain mixed solution E;

[0071] (7) Add mixed solution E to the mixed solution D and stir continuously for 12 hours to obtain mixed solution F;

[0072] (8) The mixed solution F was centrifuged at 10,000 rpm for 10 min to collect the precipitate. The precipitate was then ultrasonically dispersed to obtain a copper-tannic acid-dihydroporphyrin E6 nanocomposite.

[0073] Examples 2-4 describe the synthesis of copper-tannic acid with different mass ratios of copper salt and tannic acid.

[0074] Example 2:

[0075] (1) Disperse 0.25g PVP in 5mL of deionized water, and add 40mL of deionized water containing 0.085g CuCl2·2H2O while stirring at 700rpm to obtain mixed solution A;

[0076] (2) After vigorously stirring the mixed solution A for 90 minutes, add 5 mL of 0.5 mg / mL tannic acid aqueous solution to obtain mixed solution B;

[0077] (3) Add NaOH aqueous solution to mixed solution B to adjust the pH value to 12, and continue stirring for 12 hours to obtain a uniformly dispersed mixed solution C;

[0078] (4) Centrifuge the mixed solution C at 13500 rpm for 20 min to collect the precipitate, and then disperse the precipitate by ultrasonication.

[0079] Example 3

[0080] (1) Disperse 0.25g PVP in 5mL of deionized water, and add 40mL of deionized water containing 0.085g CuCl2·2H2O while stirring at 700rpm to obtain mixed solution A;

[0081] (2) After vigorously stirring the mixed solution A for 90 minutes, add 5 mL of 2 mg / mL tannic acid aqueous solution to obtain mixed solution B;

[0082] (3) Add NaOH aqueous solution to mixed solution B to adjust the pH value to 12, and continue stirring for 12 hours to obtain a uniformly dispersed mixed solution C;

[0083] (4) Centrifuge the mixed solution C at 13500 rpm for 20 min to collect the precipitate, and then disperse the precipitate by ultrasonication.

[0084] Example 4

[0085] (1) Disperse 0.25g PVP in 5mL of deionized water, and add 40mL of deionized water containing 0.085g CuCl2·2H2O while stirring at 700rpm to obtain mixed solution A;

[0086] (2) After vigorously stirring the mixed solution A for 90 minutes, add 5 mL of 10 mg / mL tannic acid aqueous solution to obtain mixed solution B;

[0087] (3) Add NaOH aqueous solution to mixed solution B to adjust the pH value to 12, and continue stirring for 12 hours to obtain a uniformly dispersed mixed solution C;

[0088] (4) Centrifuge the mixed solution C at 13500 rpm for 20 min to collect the precipitate, and then disperse the precipitate by ultrasonication.

[0089] Example 5: Characterization Analysis

[0090] TEM analysis was performed on the copper-tannic acid nanomaterials prepared in step (4) of Example 1, and the results were obtained. Figure 1 TEM images, by Figure 1 As shown, the copper-tannic acid nanomaterials are needle-shaped.

[0091] Record Analysis Figure 1 The size of the copper-tannic acid nanomaterials was obtained. Figure 2 The particle size distribution diagram shows that the copper-tannic acid nanomaterials have a particle size of 62–97 nm.

[0092] Example 6: Study on simulated superoxidase activity

[0093] Nitrotetrazole blue chloride (NBT) was used as an indicator for evaluating SOD enzyme activity, as it can be reacted with O2. ·- It is reduced to formazan, which has a characteristic absorption peak. O2 is generated through riboflavin and methionine under ultraviolet irradiation. ·- No O2 is produced under dark conditions. ·- As positive and negative controls, the simulated SOD activity of the copper-tannic acid nanomaterials prepared in step (4) of Example 1 was detected, and the results were as follows: Figure 3 The diagram shows the simulated SOD activity of copper-tannic acid nanomaterials at different concentrations.

[0094] Figure 3The concentrations of copper-tannic acid nanomaterials were 0, 1, 5, 10, and 20 μg / mL from low to high. It was found that the intensity of the characteristic absorption peak of formazan was negatively correlated with the concentration of copper-tannic acid nanomaterials, indicating that it has strong SOD-like activity.

[0095] Example 7: Study on simulated catalase activity

[0096] The oxygen production of the copper-tannic acid nanomaterials prepared in step (4) of Example 1 under different concentrations of H2O2 was detected using a dissolved oxygen analyzer, and the results were as follows: Figure 4 The simulated CAT activity diagram of copper-tannic acid nanomaterials under different concentrations of H2O2 is shown.

[0097] Figure 4 The concentrations of H2O2 were 10, 30, 60, 120, and 180 μg / mL from low to high. It was found that the ability of copper-tannic acid nanomaterials to catalyze the production of O2 from H2O2 increased with the increase of H2O2 concentration, indicating that it has strong SOD-like activity.

[0098] Example 8: Study on simulated glutathione peroxidase activity

[0099] 5,5-Dithio-bis-(2-nitrobenzoic acid) (DTNB) was used as an indicator for GSH-Px enzyme activity, generating a yellow product with a characteristic absorption peak at 412 nm under GSH. Simulated GSH-Px activity was detected by reacting GSH with the copper-tannic acid nanomaterials prepared in step (4) of Example 1 for different times, followed by the addition of DTNB. The results were as follows: Figure 5 The simulated GSH-Px activity diagram of the copper-tannic acid nanomaterial is shown.

[0100] Figure 5 The reaction times of copper-tannic acid nanomaterials with GSH were 0, 5, 10, 15 and 20 min, respectively. The absorption peak at 412 nm showed a time-dependent decreasing trend, indicating that copper-tannic acid nanomaterials have strong GSH-Px mimicking activity.

[0101] Example 9: Study on simulated peroxidase activity

[0102] 3,3',5,5'-Tetramethylbenzidine (TMB) was used as an indicator for POD activity detection. It can be oxidized to blue oxTMB with a characteristic absorption peak at 652 nm under ·OH conditions. The simulated POD activity of the copper-tannic acid nanomaterials prepared in step (4) of Example 1 was detected by H2O2 and TMB, yielding the following results: Figure 6 The diagram shows the simulated POD activity of copper-tannic acid nanomaterials at different concentrations.

[0103] Figure 6 The concentrations of copper-tannic acid nanomaterials, from low to high, were 2.5, 5.0, 7.5, 10.0, and 12.5 μg / mL, respectively, showing strong concentration-dependent simulated POD activity.

[0104] Using 5,5-dimethyl-1-pyrrolidone-N-oxide (DMPO) as an ·OH scavenger, the ·OH generation of the copper-tannic acid nanomaterials prepared in step (4) of Example 1 was evaluated by ESR method, and the results were obtained. Figure 7 .

[0105] like Figure 7 As shown, copper-tannic acid nanomaterials exhibit a characteristic ·OH peak with a peak area of ​​1:2:2:1 in the presence of 0.1 mM H2O2, indicating that copper-tannic acid nanomaterials can catalyze the generation of ·OH from H2O2.

[0106] Example 10: Study on the Degradation Behavior of Simulated Tumor Microenvironment

[0107] To investigate whether the copper-tannic acid nanomaterials prepared in step (4) of Example 1 are biodegradable, a TME was simulated in vitro under conditions of pH = 5.5, GSH = 10 mM, and H2O2 = 0.1 mM. The degradation process of the copper-tannic acid nanomaterials prepared in step (4) of Example 1 was visualized using TEM. Figure 8 As shown.

[0108] The needle-like morphology of copper-tannic acid nanomaterials was found to gradually decompose over time, eventually forming tiny nanodots, exhibiting a time-dependent degradation behavior. This indicates that CuTA nanomaterials are biodegradable under TME conditions, which is beneficial for subsequent metabolic clearance by the body and meets the requirements for biosafety.

[0109] Example 11: US Stimulation Generation 1 O2 capacity research

[0110] A singlet oxygen sensor (SOSG) fluorescent probe is used as the generator. 1 O2 indicator, in 1 It emits green fluorescence at 525 nm in the presence of O2. The copper-tannic acid-dihydroporphyrin E6 nanocomposite prepared in Example 1 under US stimulation was detected by exogenous US stimulation and SOSG. 1 The ability of O2 to obtain, as Figure 9 The figure shows a comparison of the emission intensity of SOSG at 525 nm for different materials under US stimulation.

[0111] Figure 9In Example 1, step (4) of the Chinese study showed that the SOSG emission intensity of the copper-tannic acid nanomaterial at 525 nm did not increase with increasing US time, while the SOSG emission intensity of the copper-tannic acid-dihydroporphyrin e6 nanocomposite at 525 nm gradually increased with increasing US time. Furthermore, the presence of H2O2 further enhanced the emission intensity of the copper-tannic acid-dihydroporphyrin e6 nanocomposite under US stimulation. 1 O2 capacity, exhibiting that the copper-tannic acid-dihydroporphyrin E6 nanocomposite can generate under US stimulation. 1 O2, and can be amplified in the presence of H2O2. 1 O2 generation capability.

[0112] Using 2,2,6,6-tetramethylpiperidine (TEMP) as 1 O2 scavengers were evaluated by ESR method for the production of copper-tannic acid-dihydroporphyrin e6 nanocomposite prepared in Example 1 under US stimulation. 1 The generation of O2 yields Figure 10 .

[0113] like Figure 10 As shown, the copper-tannic acid-dihydroporphyrin E6 nanocomposite prepared in Example 1 exhibits a peak area of ​​1:1:1 under US stimulation. 1 O2 characteristic peak, and in the presence of H2O2, 1 The increasing intensity of the characteristic peak of O2 indicates that the copper-tannic acid-dihydroporphyrin E6 nanocomposite can be generated under exogenous US. 1 O2, and further amplified in the presence of H2O2. 1 The ability to generate O2.

[0114] Example 12: In vitro antitumor activity study

[0115] 4T1 cells were seeded at a density of 8000 cells / well in 96-well plates and incubated for 24 h. After incubation, the cells were co-incubated for 6 h with copper-tannic acid-dihydroporphyrin E6 nanocomposite from Example 1 at concentrations of 0, 6, 12.5, 25, 50, and 100 μg / mL, respectively. Following US treatment, incubation continued for another 6 h. Cytotoxicity was assessed using the MTT assay, and absorbance at 490 nm was read using a microplate reader. Figure 11 As shown.

[0116] Figure 11 As the concentration of the copper-tannic acid-dihydroporphyrin E6 nanocomposite increases, its killing effect on tumor cells also increases, demonstrating its tumor-killing effect.

[0117] 4T1 cells were seeded at a concentration of 1.0 × 10⁻⁶. 54T1 cells were seeded at a density of 100 μg / well in 6-well plates and incubated for 24 h. After incubation, the cells were co-incubated for 6 h with 100 μg / mL of the copper-tannic acid-dihydroporphyrin E6 nanocomposite from Example 1. Following US treatment, incubation continued for 4 h. The morphology of the 4T1 cells was observed using an inverted fluorescence microscope, yielding the following results: Figure 12 As shown.

[0118] from Figure 12 The images show that 4T1 cells exhibit distinct blistering and pyroptosis morphological characteristics.

[0119] Example 13: Biosafety Study

[0120] To ensure safe biological applications, the copper-tannic acid-dihydroporphyrin E6 nanocomposite from Example 1 was used in mice to evaluate the material's metabolism and blood biochemistry and routine blood counts after tumor treatment.

[0121] The copper-tannic acid-dihydroporphyrin E6 nanocomposite prepared in Example 1 at a concentration of 15 mg / kg was subcutaneously injected into the tumor sites of Balb / c mice bearing 4T1 cells on days 1, 3, and 5. Material metabolism was then analyzed after 14 days. Figure 13 .

[0122] like Figure 13 As shown, increasing copper content was detected in mouse feces during the first 6 days, and copper content was also detected in mouse feces during the last 6 days, indicating that the copper-tannic acid-dihydroporphyrin E6 nanocomposite of Example 1 can be metabolized and excreted in mice through feces.

[0123] Healthy Balb / c mice and healthy Balb / c mice treated with the copper-tannic acid-dihydroporphyrin E6 nanocomposite prepared in Example 1 at a concentration of 15 mg / kg were subjected to blood biochemistry and routine blood tests. Figure 14 .

[0124] like Figure 14 As shown, compared with healthy mice, the mice treated with the drug showed no significant differences in any of their indicators, all of which were within the normal range, indicating good biocompatibility.

[0125] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a degradable copper-tannic acid-dihydroporphyrin E6 nanocomposite that induces pyroptosis, characterized in that, include: The surfactant and copper salt were mixed evenly in the solution, and then tannic acid solution was added to allow them to react, thus obtaining copper-tannic acid nanomaterials. The copper-tannic acid nanomaterials were added to the amino modifier dispersion and mixed evenly to obtain the first mixed solution; An activator was added to the dispersion of dihydroporphyrin E6 and mixed thoroughly to obtain a second mixed solution. The first mixed solution was added to the second mixed solution to carry out the reaction, and copper-tannic acid-dihydroporphyrin E6 nanocomposite was obtained. The mass ratio of the copper salt to tannic acid is 17~22:

1.

2. The method for preparing the degradable copper-tannic acid-dihydroporphyrin E6 nanocomposite that induces pyroptosis as described in claim 1, characterized in that, The surfactant is PVP, with a viscosity grade of K30 and a molecular weight of 44,000 to 54,000.

3. The method for preparing the degradable copper-tannic acid-dihydroporphyrin E6 nanocomposite that induces pyroptosis as described in claim 1, characterized in that, The copper salt is copper chloride dihydrate.

4. The method for preparing the pyroptosis-inducing degradable copper-tannic acid-dihydroporphyrin E6 nanocomposite as described in claim 1, characterized in that, The amino modifier is APTES.

5. The method for preparing the degradable copper-tannic acid-dihydroporphyrin E6 nanocomposite that induces pyroptosis as described in claim 1, characterized in that, The activator is EDC / NHS.

6. The method for preparing the pyroptosis-inducing degradable copper-tannic acid-dihydroporphyrin E6 nanocomposite as described in claim 1, characterized in that, The mass ratio of dihydroporphyrin E6 to copper salt is 1~5:

85.

7. The pyroptosis-inducing degradable copper-tannic acid-dihydroporphyrin E6 nanocomposite prepared by the method of any one of claims 1-6.

8. The use of the degradable copper-tannic acid-dihydroporphyrin E6 nanocomposite for inducing pyroptosis according to claim 7 in the preparation of products inducing pyroptosis, wherein the cells comprise: Tumor cells.

9. The use of the copper-tannic acid nanomaterials prepared by the method according to any one of claims 1-6 in the preparation of tumor microenvironment improvers or tumor cell pyroptosis inducers.

Images