Perylene imide derivative pdic-no and application thereof

Abstract

The application discloses a perylene diimide derivative PDIC-NO and an application thereof, and belongs to the field of medicinal chemistry, wherein the structural formula of the perylene diimide derivative PDIC-NO is as follows: The PDIC-NO is a novel hypoxia-activated prodrug, can efficiently kill tumor cells through a 'hypoxia reduction-lysosome capture-ROS explosion-LMP-pyroptosis' cascade reaction, can induce immunogenic cell death, activate adaptive immune response, and reverse hypoxia-mediated immune escape, and exhibits excellent antitumor effect and good safety in vivo and in vitro, and provides a new strategy for tumor immunotherapy based on the pyroptosis mechanism.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry, specifically relating to a perylene imide derivative PDIC-NO and its applications. Background Technology

[0002] One of the core challenges in cancer treatment is achieving selective killing of malignant cells while maximally protecting normal tissues. Solid tumors, during their rapid growth, are often accompanied by vascular structural abnormalities and oxygen deprivation, forming a unique hypoxic microenvironment. This characteristic not only drives tumor invasion, metastasis, and drug resistance but also provides molecular targets for targeted therapy. Hypoxia-activated prodrugs (HAPs) are strategic drugs developed by utilizing the hypoxic environment of tumors. They maintain a relatively inert state under normoxic conditions and are activated by reductases in tumor tissues with low oxygen partial pressure, thereby releasing their active ingredients to exert a killing effect. However, traditional HAPs still have some limitations, such as limited activation efficiency, susceptibility to drug resistance due to a single mechanism of action, and difficulty in effectively activating anti-tumor immune responses. In recent years, perylenediimide derivatives (PDIs) have received widespread attention in the biomedical field due to their excellent photochemical stability, ease of structural modification, and superior fluorescence properties. In particular, their electron-deficient structural characteristics provide a theoretical basis for the design of hypoxia-activated prodrugs. Meanwhile, lysosomes, as highly active acidic organelles within tumor cells, play a crucial role in maintaining cellular homeostasis, mediating autophagy, and participating in cell death regulation. Combining hypoxia-activated properties with lysosomal targeting capabilities holds promise for achieving more precise multi-stage drug delivery and effect amplification. Based on this, this application designs and synthesizes a hypoxia-activated perylene imide prodrug, PDIC-NO, aiming to trigger its structural transformation in the hypoxic environment of tumors, and then achieve prodrug enrichment and protonation through the acidic environment of lysosomes. The aim is to systematically explore its antitumor effects, its role in remodeling the tumor immune microenvironment, and its antitumor molecular mechanisms. Summary of the Invention

[0003] To overcome the above-mentioned technical defects, the present invention provides a perylene imide derivative PDIC-NO and its application.

[0004] A perylene imide derivative, PDIC-NO, has the following structural formula:

[0005] .

[0006] The preparation method of the above perylene imide derivative PDIC-NO is as follows:

[0007] , Compound PDIC-NN was added to ethanol, and hydrogen peroxide was added under a protective atmosphere. The temperature was raised from room temperature to 45-55°C, and the reaction was stirred for 10-15 hours. The reaction mixture was cooled to room temperature and post-treated to obtain the target compound PDIC-NO. The molar ratio of compound PDIC-NN to hydrogen peroxide was 1:(90-110).

[0008] Furthermore, the mass concentration of hydrogen peroxide is 25~35wt%.

[0009] Furthermore, the post-treatment refers to the addition of diethyl ether for recrystallization and drying to obtain the target compound PDIC-NO.

[0010] The above-mentioned perylene imide derivative PDIC-NO is used in the preparation of anti-lung cancer drugs. PDIC-NO induces ROS burst in tumor cells under hypoxia, triggers LMP in tumor cells, and causes a large amount of CTSB to leak into the cytoplasm of tumor cells. This, in turn, activates the NLRP3 / Caspase-1 / GSDMD pathway in tumor cells, induces pyroptosis of tumor cells, and releases a large amount of LDH, ATP, IL-1β and IL-18.

[0011] Furthermore, the tumor cells are LLC cells.

[0012] The present invention has the following beneficial effects:

[0013] PDIC-NO is reduced to PDIC-NN under hypoxic conditions and further protonated and enriched in acidic lysosomes, with a colocalization coefficient as high as 0.89. The half-maximal inhibitory concentration (IC50) of PDIC-NO in LLC cells under hypoxia is also shown. 50The fluorescence intensity (ROS) decreased to 3.05 μM, a reduction of 88.71% compared to normoxic conditions, and was superior to classic HAPs drugs tirapazamine (TPZ) and banoxantrone (AQ4N). Mechanistic studies showed that PDIC-NO induced a ROS burst under hypoxia (2.2 times that of the normoxic group), triggered LMP (a decrease in dextran fluorescence of 81.88%), leading to a large leakage of CTSB into the cytoplasm, which in turn activated the NLRP3 / Caspase-1 / GSDMD pathway, inducing pyroptosis and releasing large amounts of LDH, ATP, IL-1β, and IL-18. In in vivo experiments, PDIC-NO (2 mg / kg) treatment reduced tumor volume in LLC tumor-bearing mice to 30.40% of that in the PBS group and reduced the number of lung metastatic nodules by 76.92%, significantly outperforming AQ4N and oxaliplatin (OXA). Simultaneously, PDIC-NO promoted the maturation of dendritic cells (DCs) in lymph nodes (the maturation rate was 4.05 times that of the PBS group) and increased CD8 levels in the spleen and tumor tissue. + T cell infiltration and activation (activated CD8) + The proportion of T cells reached 45.9%, and the expression of the immune checkpoint galectin 9 was downregulated by inhibiting lysosomal autophagy. Biosafety evaluation showed that PDIC-NO had no hemolysis or organ toxicity at therapeutic doses, and blood routine and liver and kidney function indicators were normal. Attached Figure Description

[0014] Figure 1 PDIC-NO 1H NMR spectrum;

[0015] Figure 2 PDIC-NO carbon NMR spectrum;

[0016] Figure 3 High-resolution mass spectrometry for PDIC-NO;

[0017] Figure 4 The UV and fluorescence spectra of PDIC-NO under (a) normoxic and (b) hypoxic conditions are shown.

[0018] Figure 5 Characterization of PDIC-NO hypoxia activation: (a) High-resolution mass spectrometry detection results of PDIC-NO co-incubated with liver microsomes and NADPH for 2 h under normoxic and (b) hypoxia conditions [M+1]; (c) Color and state of PDIC-NN in PBS solutions at pH = 7.4 and pH = 5;

[0019] Figure 6This diagram illustrates the colocalization of lysosomal probes (blue fluorescence) and PDIC-NO (green fluorescence) in LLC cells treated with 1 μM PDIC-NO for 2 h under normoxic and hypoxic conditions. R represents the colocalization coefficient.

[0020] Figure 7 High-resolution mass spectrometry results of LLC cells treated with PDIC-NO for 6 h under (a) normoxic and (b) hypoxic conditions [M+1];

[0021] Figure 8 The half-maximal inhibitory concentration (IC50) of PDIC-NO on LLC (a), B16F10 (b), HepG2 (c), and L929 (d) cells under normoxic and hypoxic conditions. 50 The results of the measurement of the value (n = 3);

[0022] Figure 9 The effect of PDIC-NO on reactive oxygen species (ROS) levels: (a) confocal fluorescence images of ROS (red fluorescence) in LLC cells treated with 2.5 μM PDIC-NO for 6 h under normoxic and hypoxic conditions, and (b) quantitative results; (c) flow cytometry results of ROS in LLC cells treated with 2.5 μM PDIC-NO for 6 h under normoxic and hypoxic conditions, and (d) quantitative results. Data are expressed as mean ± standard deviation (n = 3). Statistical analysis was performed using the t-test: ***P < 0.001;

[0023] Figure 10 Fluorescence image (a) and quantification results (b) of LLC cells after 12 h of dextran pretreatment and 8 h of PDIC-NO treatment.

[0024] Figure 11 To detect the leakage of CTSB protein in LLC cells treated with 2.5 μM PDIC-NO for 12 h under normoxic and hypoxic conditions using Western blot.

[0025] Figure 12 Images taken with an inverted microscope after 24 h of PDIC-NO treatment of LLC cells under normoxic and hypoxic conditions;

[0026] Figure 13 The expression of pyroptosis-related proteins in LLC cells treated with 2.5 μM PDIC-NO under normoxic and hypoxic conditions for 24 h was detected by Western blot.

[0027] Figure 14To investigate the effects of PDIC-NO on the release of cellular contents and inflammatory factors from LLC cells: ELISA kits were used to detect the levels of (a) LDH, (b) IL-1β, (c) IL-18, and (d) ATP in the cell culture supernatant after 24 h of treatment with 2.5 μM PDIC-NO under normoxic and hypoxic conditions. Data are expressed as mean ± standard deviation (n = 3). Statistical analysis was performed using t-tests: no statistically significant difference was found in ns, **P < 0.01, ***P < 0.001;

[0028] Figure 15 The effect of PDIC-NO on immune escape in LLC cells. (a) Release of HMGB1 in LLC cells after treatment with 2.5 μM PDIC-NO for 24 h under normoxic and hypoxic conditions and (b) Expression of immune escape-related proteins;

[0029] Figure 16 Concentration-dependent hemolysis assay for PDIC-NO (negative control: PBS, positive control: H2O);

[0030] Figure 17 Safety evaluation of repeated administration of PDIC-NO: (a) Different doses of PDIC-NO (0, 2, 4, 8 mg / kg) -1 (a) Effect of tail vein injection every other day on the survival rate of C57BL / 6 mice; (b) Changes in mouse body weight; (c) Blood routine and blood biochemical indicators after the last administration. Data are expressed as mean ± standard deviation (n = 6);

[0031] Figure 18 (a) Schematic diagram of LLC tumor-bearing model establishment and experimental treatment protocol; (b) Gross anatomical morphological observation of the tumor at the treatment endpoint; (c) Tumor growth kinetic curve; (d) Results of ex vivo tumor requantification analysis. Data are expressed as mean ± standard deviation (n = 5). Statistical analysis was performed using one-way ANOVA: *P < 0.05, ***P < 0.001;

[0032] Figure 19 Detection of pyroptosis-related biomarkers in tumor tissues: (a) expression levels of Cl. Caspase1 and GSDMD-N proteins in tumor tissues and (b) quantitative results; (c) serum levels of IL-1β and IL-18 in mice. Data are expressed as mean ± standard deviation (n = 3). Statistical analysis was performed using one-way ANOVA: **P < 0.01, ***P < 0.001;

[0033] Figure 20 The results of H&E staining of major organs (heart, liver, spleen, lung and kidney) in mice of different groups in the LLC tumor-bearing model;

[0034] Figure 21 The results of routine blood tests and blood biochemical parameters in mice of different groups in the LLC tumor-bearing model are presented. Data are expressed as mean ± standard deviation (n = 3).

[0035] Figure 22 H&E staining results of major organs (heart, liver, spleen, lung and kidney) in mice of different groups in the pulmonary orthotopic tumor model. Detailed Implementation

[0036] The present invention will be further illustrated below with specific examples. These embodiments should be understood as illustrative only and not as limiting the scope of protection of the present invention. After reading the description of the present invention, those skilled in the art can make various alterations or modifications to the present invention, and these equivalent changes and modifications also fall within the scope defined by the claims of the present invention.

[0037] I. Synthesis and Characterization of PDIC-NO

[0038] (1) Synthesis of PDIC-NO

[0039]

[0040] 5,6,12,13-Tetrachloro-2,9-bis(2-(dimethylamino)ethyl)anthracene(2,1,9-def:6,5,10-d'e'f')diisoquinoline-1,3,8,10(2H,9H)-tetraone (PDIC-NN, 668.05 mg, 1.0 mmol) was added to 100 mL of ethanol, and after purging with argon for 30 minutes, 10.2 mL of 30% H2O2 (100 mmol) was added, followed by reaction at 50 °C for 12 h. The reaction mixture was cooled to room temperature, and then 100 mL of diethyl ether was added. The mixture was recrystallized and then reacted at 50 °C. o Vacuum drying at C yielded a black solid of 2,2'-(5,6,12,13-tetrachloro-1,3,8,10-tetraoxo-1,3,8,10-tetrahydroanthracene(2,1,9-def:6,5,10-d'e'f')diisoquinoline-2,9-diyl)bis(N,N-dimethylethane-1-oxamine) (PDIC-NO) (511 mg, 73% yield).

[0041] Proton NMR, carbon NMR, and high-resolution mass spectrometry, such as Figures 1 to 3 As shown. Figure 1 As shown, PDIC-NO: 1H NMR (CF3COOD, 500 MHz) δ (ppm): 11.9 (s, 12 H), 11.74~11.76 (m, 4 H), 12.76~12.78 (m, 4 H), 16.74 (s, 4H). like Figure 2 As shown, PDIC-NO: 13 C NMR (CF3COOD, 125 MHz) delta (ppm): 30.56, 52.25, 52.35, 61.62, 117.35, 118.95, 126.05, 126.91, 130.00, 132.40, 160.19. like Figure 3 As shown, PDIC-NO: 701.0510. The above results demonstrate the successful preparation of PDIC-NO.

[0042] II. Ultraviolet absorption and fluorescence emission spectra of PDIC-NO

[0043] Dissolve PDIC-NO solid in 3 mL of ultrapure water to prepare a solution of 10 -3 The mother liquor of M was then diluted to 2 × 10⁻⁶. -5 The solution of M was analyzed by measuring its ultraviolet absorption and fluorescence emission spectra. The results were obtained through ultraviolet and fluorescence spectroscopy. Figure 4 As shown in (a), PDIC-NO exhibits the strongest absorption peak at 525 nm under normal oxygen conditions. It demonstrates good fluorescence stability in the 550-750 nm range, with a maximum fluorescence intensity at 668 nm. Figure 4 As shown in (b), PDIC-NO exhibits the strongest absorption peak at 514 nm under hypoxic conditions. It demonstrates good fluorescence stability in the 510-700 nm range, with a maximum fluorescence intensity at 548 nm. PDIC-NO exhibits good water solubility, excellent fluorescence performance, and photostability. The determination of the UV and fluorescence spectra of PDIC-NO lays the foundation for subsequent cell imaging experiments.

[0044] III. Characterization of PDIC-NO hypoxia activation and PDIC-NN protonation using a liver microsomal model

[0045] Mix 1×PBS and physiological saline at a 1:1 ratio, stir well, and adjust the pH to 7.4. Then, prepare a 50 μM NADPH solution with water. Take 889 μL of the 1×PBS and physiological saline mixture, add 10 μL of liver microsomes (concentration: 200 μg / mL) and 1 μL of PDIC-NO (concentration: 1 mg / mL). -1After mixing thoroughly, the mixture was incubated at 37 °C for 10 min. Then, 100 μL of prepared NADPH aqueous solution was added, and the mixture was mixed thoroughly. The mixture was then placed in an anoxic chamber (oxygen content less than 1%) and incubated at 37 °C for 2 h. The solution was then removed, and an equal volume of acetonitrile was added to terminate the reaction. The mixture was vortexed for 2 min, centrifuged at 3000 rpm for 5 min, and the supernatant was sent for mass spectrometry analysis.

[0046] Take two EP tubes and prepare 1 mL of 10 μg / mL PDIC-NN in each tube using 1 × PBS at pH 7.4. Adjust the pH of one of the EP tubes to 5.0, and then take a picture to record the color and state of the solution.

[0047] Through liver microsomal models and mass spectrometry detection, such as Figure 5 As shown in (a), the high-resolution mass spectrometry of PDIC-NO under normoxic conditions shows a molecular weight of 701.4868, while under hypoxic conditions... Figure 5 (b) The high-resolution mass spectrometry of PDIC-NO shows a molecular weight of 671.2615. The difference in molecular weight indicates that the structure of PDIC-NO has changed to PDIC-NN. Furthermore, compared to pH 7.4, the solution of PDIC-NN changed from pink and turbid to clear and transparent orange at pH 5. Figure 5 (c) indicates that PDIC-NN has been protonated at pH 5.

[0048] IV. Study on the in vitro anti-non-small cell lung cancer effect of PDIC-NO

[0049] (1) Cell culture

[0050] Mouse epithelial cells (L929), mouse Lewis lung cancer cells (LLC), and human non-small cell lung cancer cells (A549) were cultured in high-glucose DMEM medium supplemented with 10% fetal bovine serum, 1% penicillin, and streptomycin at 37 °C in an incubator with 5% CO2. For hypoxia treatment, cells were placed in an anoxic chamber (oxygen content less than 1%) and cultured at 37 °C in an incubator with 5% CO2.

[0051] (2) Lysosomal colocalization experiment

[0052] LLC cells were seeded into confocal microscopy dishes. After complete cell adhesion, PDIC-NO solution (2 μM) was added and incubated for 3 h. 1 μL of lysosomal blue probe solution was added to the culture dish and the cells were incubated for 30 min. Observation and photography were performed using a confocal microscope. Colocalization was calculated using ImageJ software. The enrichment of PDIC-NO in lysosomes was investigated using lysosomal fluorescent probes, such as... Figure 6 As shown, PDIC-NO exhibits high co-localization coefficients with lysosomes under both normoxic and hypoxic conditions, with coefficients of 0.87 and 0.89 under normoxic and hypoxic conditions, respectively. These results indicate that PDIC-NO is localized in lysosomes.

[0053] (3) Cell model characterization of PDIC-NO hypoxia activation

[0054] LLC cells were seeded into 6 cm dishes and incubated overnight at 37 °C. When the cells were adherent, in good condition, and at a cell density of approximately 80%, PDIC-NO was added to a final concentration of 20 μg / mL. After culturing under hypoxic conditions for 6 h, the cells were removed, washed three times with PBS, and collected into EP tubes using a scraper. 100 μL of RIPA lysis buffer was added, and the cells were incubated for 30 min, vortexing for 30 s every 5 min. The cells were then centrifuged at 12000 rpm for 20 min, and the supernatant was sent for mass spectrometry analysis. The experimental results are as follows: Figure 7 (a) Electrospray ionization mass spectrometry of cell supernatant after PDIC-NO treatment under normoxic conditions showed a molecular weight of 701.4864, while under hypoxic conditions... Figure 7 (b) Electrospray ionization mass spectrometry of the cell supernatant after PDIC-NO treatment showed a molecular weight of 671.2609. The difference in molecular weight between the two indicates that the structure of PDIC-NO has indeed changed to PDIC-NN. + .

[0055] (4) Cell proliferation experiment

[0056] Arrange the cells at approximately 10 per well. 4Cells were seeded into 96-well plates and cultured for 24 h. Different concentrations of PDIC-NO, TPZ, and AQ4N were added to the cells under normoxic and hypoxic conditions, and the cells were cultured at 37 °C for another 24 h. The old culture medium was discarded, and the cells were washed three times with 1×PBS. Subsequently, the cells were incubated with MTT working solution for 4 h, and methyl methacrylate was dissolved in DMSO. Cell viability was evaluated by measuring the absorbance at 492 nm using a microplate reader. The IC50 of each cell was calculated using a nonlinear regression logarithm (inhibitor) versus response variable slope plot in GraphPad Prism 8. 50 Value. For example... Figure 8 As shown, PDIC-NO has an IC50 effect on three cancer cell types (LLC, B16-F10, and HepG2) under normoxic conditions. 50 The values ​​were 27.03, 13.6, and 21.81 μM, respectively. The IC50 values ​​for normal L929 cells were... 50 The value was 21.43 μM. The IC50 value of PDIC-NO against four cell types (LLC, B16-F10, HepG2, and L929) under hypoxic conditions was... 50 The values ​​were 3.05, 4.6, 9.89, and 17.17 μM, respectively. IC50 values ​​for the four types of cellular hypoxia were... 50 The percentage decreases compared to normoxic conditions were, in descending order: 88.71%, 66.18%, 54.65%, and 19.88%. The results indicate that PDIC-NO exhibits the highest sensitivity to hypoxia in LLC lung cancer cells, and that the IC50 under hypoxia... 50 The values ​​were the lowest. Based on this, LLC cells were selected for subsequent experiments in this application. In addition, two positive control drugs (the classic hypoxia-activating prodrug TPZ and AQ4N) were set up, and the IC50 values ​​of the three compounds were detected on human lung cancer cells (A549 cells). 50 See Table 1. Taking LLC as an example, the results show that the hypoxia IC50 of the three compounds (TPZ, AQ4N, and PDIC-NO) is... 50 The corresponding IC values ​​are 45.68, 8.02, and 3.05 μM. Compared to TPZ and AQ4N, the IC of PDIC-NO... 50 The levels decreased by 93.32% and 61.97% respectively, indicating that PDIC-NO significantly inhibited tumor cell proliferation under hypoxia.

[0057] Table 1. IC50 of different HAPs on A549 and LLC cells under normoxic and hypoxic conditions. 50 value

[0058]

[0059] V. Study on PDIC-NO-induced lysosomal permeability of tumor cells

[0060] (1) Detection of reactive oxygen species

[0061] Confocal laser scanning microscopy: A suitable amount of LLC lung cancer cells were seeded into confocal microscopy dishes and incubated at 37 ℃ for 12 h. Then, under normoxic and hypoxic conditions, PDIC-NO solution (final concentration 2.5 μM) was added and incubated for 6 h, followed by washing three times with 1×PBS. Cell ROXTm Deep Red was diluted 1:1000 with serum-free medium. 1 mL of the prepared Cell ROXTm Deep Red working solution was added to each well of the confocal microscopy dish and incubated in a 37 ℃ cell culture incubator in the dark for 20 min. The working solution was discarded, and the cells were slowly washed three times with 1×PBS. Observation was performed using a laser confocal microscope in the dark. ImageJ software was used for quantitative fluorescence analysis, and GraphPadPrism software was used for data analysis.

[0062] Flow cytometry analysis: PDIC-NO (2.5 μM) was added to logarithmic growth phase cells for 8 h under normoxic and hypoxic conditions, respectively. Cells were collected, centrifuged, and washed with 1×PBS to prepare single-cell suspensions, which were then stained with DCFH-DA. Cells were cultured at 37 °C (30 min) and examined using a CytoFLEX flow cytometer, and analyzed using Flow J software.

[0063] See results Figure 9 The results of laser confocal microscopy analysis of ROS production in LLC cells after 6 h of PDIC-NO treatment are shown in [Figure 1]. Figure 9 (a) Under normoxic conditions, compared with PBS, the total intracellular ROS increased only slightly after treatment with PDIC-NO; however, under hypoxic conditions, compared with PBS, the total intracellular ROS increased significantly after treatment with PDIC-NO. Fluorescence intensity quantification results showed that the total ROS produced by PDIC-NO under normoxic conditions was 1.2 times that of the PBS group, while the total ROS produced by PDIC-NO under hypoxic conditions was 2.2 times that of the PBS group. (See [reference needed]) Figure 9 (b). The above results were verified by flow cytometry, such as... Figure 9 The results (c and d) also show that PDIC-NO can produce a large amount of ROS under hypoxic conditions, while only a very small amount of ROS is generated under normoxic conditions.

[0064] (2) Detection of lysosomal membrane permeability

[0065] LLC cells were seeded into confocal microplates. After complete cell adhesion, 100 μg / mL Alexa Fluor 647-Dextran was added and incubated for 8 h. The probe solution was discarded, and PDIC-NO (4 μM) was added. Cells were then incubated for another 6 h under normoxic and hypoxic conditions, respectively. Cells were washed three times with 1×PBS and imaged using CLSM. Results are shown below. Figure 10 Dextran (Alexa Fluor 647-Dextran) is a dye that is located in lysosomes and exhibits red fluorescence in the acidic environment of lysosomes. When LMP occurs, dextran leaks from the lysosomes into the cytoplasm and no longer exhibits red fluorescence; therefore, the degree of weakening of red fluorescence represents the degree of LMP. Results showed that after 6 h of PDIC-NO treatment of LLC cells under hypoxia, the fluorescence of dextran in lysosomes was significantly reduced. Fluorescence quantification results showed that the fluorescence intensity of the PDIC-NO group under normoxic conditions decreased by 27.86% compared to the PBS group, while the fluorescence intensity of the PDIC-NO group under hypoxia decreased by 81.88% compared to the PBS group. Figure 10 (a and b). Therefore, PDIC-NO significantly induces lysosomal membrane permeability under hypoxia.

[0066] (3) Detection of cathepsin B leakage

[0067] Western blotting was used to analyze cathepsin B leakage. An appropriate amount of LLC cells were seeded into 15 cm dishes, and 2.5 μM PDIC-NO was added. In the rescue experiment, 10 μM NAC was also added to remove ROS. Cells were incubated for 24 h under both normoxic and hypoxic conditions. The culture medium was discarded, and the cells were washed three times with PBS. Cells were collected in EP tubes and centrifuged at 1000 rpm for 4 min at room temperature to collect the cell pellet. Lysosomal and cytoplasmic proteins were extracted according to the lysosome extraction kit instructions, and the supernatant was used for protein quantification. After balancing with Loading Buffer based on the quantification results, the cells were boiled in 100 °C for 11 min and cooled to room temperature before loading.

[0068] SDS-PAGE gel electrophoresis: Load samples as needed, set a constant voltage of 150 V for 1 h.

[0069] Electrophoretic imprinting transfer: PVDF membrane was placed in methanol and activated for 30 s to create a sandwich structure. The transfer device was assembled and the membrane was transferred at a constant current of 280 mA for 100 min.

[0070] Incubation and Development: After transfer, the PVDF membrane was placed in 5% skim milk powder and blocked at room temperature for 3 h. Then, it was washed with 1×TBST, and primary antibody diluted with 5% BSA was added, followed by incubation at 4 ℃ for 12 h. Afterward, the primary antibody was recovered, washed with 1×TBST, and the appropriate secondary antibody was incubated at room temperature for 2 h, depending on the source of the primary antibody. The secondary antibody was then recovered, washed with 1×TBST, and developed and images were acquired using the chemiluminescence imaging system according to the ECL chemiluminescence kit instructions. Grayscale values ​​of the images were calculated using ImageJ software.

[0071] See results Figure 11 Using LAMP1 as a reference for lysosomal components and β-actin as a reference for cytoplasmic components, the ratio of cytoplasmic CTSB to lysosomal CTSB (C / L) was used to characterize the degree of CTSB leakage. Under normoxic conditions, the CTSB leakage rate in the PDIC-NO group was 2.2 times that of the PBS group, while under hypoxic conditions, the CTSB leakage rate in the PDIC-NO group was 18.2 times that of the PBS group, an increase of 8 times, suggesting that PDIC-NO-induced LMP is more severe in hypoxic environments. Furthermore, to confirm that ROS is a cause of LMP, a rescue experiment was also conducted in this application. After using the ROS scavenger NAC, the CTSB leakage rate under normoxic conditions after PDIC-NO treatment was 1.7 times that of the PBS group, a reduction of 28.55% compared to the group without NAC. Under hypoxic conditions, the CTSB leakage rate after PDIC-NO treatment was 2.5 times that of the PBS group, a reduction of 86.25% compared to the group without NAC. This confirms that ROS is a key factor in PDIC-NO-induced LMP.

[0072] VI. Research on PDIC-NO-induced pyroptosis

[0073] (1) Detection of tumor cell death morphology under light microscopy

[0074] LLC cells were seeded into four 2 cm dishes and incubated overnight at 37 °C. When the cell density reached approximately 60%, the four dishes were divided into four groups: normoxic PBS group, normoxic PDIC-NO group, hypoxic PBS group, and hypoxic PDIC-NO group. After incubation for 24 h with 1× PBS or PDIC-NO (2.5 μM) under normoxic or hypoxic conditions, images were taken using an inverted microscope. Figure 12As shown, under hypoxia, cells in the PDIC-NO group could no longer maintain their normal morphology and exhibited obvious membrane blistering, i.e., pyroptosis bodies. In contrast, under normoxic conditions, most cells in the PDIC-NO group maintained their normal morphology, with only a very small number showing pyroptosis bodies (as indicated by the red arrows in the figure). These results confirm that PDIC-NO treatment of LLC cells can induce pyroptosis and further demonstrate its significant killing effect on tumor cells under hypoxic conditions.

[0075] (2) Detection of pyroptosis-related proteins

[0076] Mouse LLC cells were seeded into 6 cm dishes, and 2.5 μM PDIC-NO was added. In the rescue experiment, 10 μM CA-074Me was also added to inhibit cathepsin B. The cells were incubated for 24 h under normoxic and hypoxic conditions, respectively. The culture medium was discarded, and the cells were washed with PBS. 100 μL of RIPA lysis buffer containing protease and phosphatase inhibitors was added to each 6 cm dish. Cells were collected using a cell scraper and lysed on ice for 30 min. Afterward, the cells were centrifuged at 12000 rpm for 25 min at 4 ℃. A portion of the supernatant was collected for protein quantification. Subsequent experimental procedures were performed according to step six (3). Results are as follows: Figure 13As shown, under hypoxia, the levels of NLRP3, Cleaved Caspase-1, and GSDMD-N in the PDIC-NO group were 2.6, 2.5, and 3.4 times higher than those in the PBS group, respectively, while under normoxic conditions, they were 1.1, 1.1, and 1.3 times higher, respectively. Under hypoxic conditions, the expression levels of NLRP3, Cleaved Caspase-1, and GSDMD-N in the PDIC-NO group increased by 136.36%, 127.27%, and 209.09%, respectively, compared to the normoxic group. Therefore, it is believed that PDIC-NO induces more intense pyroptosis in a hypoxic environment. A rescue experiment was also used to further verify whether the PDIC-NO-induced pyroptosis was attributed to CTSB leakage. After the addition of the protease inhibitor CA-074Me, the expression levels of NLRP3, Cleaved Caspase-1, and GSDMD-N in the normoxic PDIC-NO group were 1.0, 1.0, and 1.1 times higher than those in the PBS group, respectively, representing decreases of 9.09%, 9.09%, and 15.38% compared to the level without CA-074Me. Under hypoxia, the expression levels of NLRP3, Cleaved Caspase-1, and GSDMD-N in the PDIC-NO group were 1.4, 2.0, and 1.8 times higher than those in the PBS group, respectively, representing decreases of 46.15%, 20%, and 47.06% compared to the level without CA-074Me. These results suggest that PDIC-NO can significantly induce pyroptosis in tumor cells under hypoxic conditions. Further analysis indicates that CTSB is a key molecule mediating PDIC-NO-induced pyroptosis.

[0077] (3) Detecting the released ATP

[0078] After cells had fully adhered to 2 cm culture dishes under normoxic and hypoxic conditions, PDIC-NO solution (2.5 μM) was added, and the cells were cultured for another 24 h. The culture medium was then collected for the following tests. 100 μL of ATP detection working solution and 20 μL of culture medium were added to each well, and fluorescence intensity was measured using a microplate spectrophotometer. An ATP standard curve was plotted with fluorescence intensity on the ordinate and ATP concentration on the abscissa. The fluorescence values ​​of each sample were substituted into the standard curve to calculate the ATP concentration.

[0079] (4) Detection of released LDH

[0080] After cells had fully adhered to 2 cm culture dishes under normoxic and hypoxic conditions, PDIC-NO solution (2.5 μM) was added, and the cells were cultured for another 24 h. The culture medium was then collected for the following tests. 100 μL of LDH detection working solution and 20 μL of culture medium were added to each well, and fluorescence intensity was measured using a microplate spectrophotometer. An LDH standard curve was plotted with fluorescence intensity on the ordinate and LDH concentration on the abscissa. The LDH concentration was calculated by substituting the fluorescence values ​​of each sample into the standard curve.

[0081] (5) Detection of cytokines in cell supernatant

[0082] LLC cells were seeded into six-well plates under normoxic and hypoxic conditions, respectively, and incubated at 37 °C for 12 h. PDIC-NO (2.5 μM) was added and incubated for 24 h. Cell supernatant was collected into EP tubes, and the contents of IL-1β and IL-18 in the cell culture supernatant were measured by enzyme-linked immunosorbent assay (ELISA).

[0083] (6) Detection of HMGB1 immunofluorescence

[0084] LLC cells were seeded in confocal microscopy dishes under normoxic and hypoxic conditions, and incubated at 37 °C for 12 h. After incubation with PDIC-NO for 24 h, the culture medium was discarded, and the cells were washed three times with 1×PBS. Cells were then fixed with 4% paraformaldehyde and incubated at room temperature for 10 min. After washing twice with PBS, cells were permeabilized with 0.1% Triton X-100 and incubated at room temperature for 10 min. After washing twice with 1×PBS, primary antibody against high mobility group box 1 protein (HMGB1) was added, and the cells were incubated overnight at 4 °C. After washing three times with 1×PBS, fluorescently labeled secondary antibody was added, and the cells were incubated at room temperature in the dark for 2 h. After washing three times with 1×PBS, cell nuclei were stained with DAPI and incubated at room temperature in the dark for 5 min. After washing three times with 1×PBS, the cells were observed and photographed using a confocal microscope to analyze the expression and localization of HMGB1.

[0085] (7) Detection of NFX1 and Galectin 9 proteins

[0086] Mouse LLC cells in the exponential growth phase were plated in 6 cm dishes, and 2.5 μM PDIC-NO was added. The cells were incubated under hypoxic conditions for 24 h. The culture medium was discarded, and the cells were washed three times with PBS. 100 μL of RIPA lysis buffer containing 1% protease inhibitor and phosphatase inhibitor was added to each 6 cm dish. Cells were scraped off and placed on ice for lysis for 30 min. The cells were vortexed every 5 min for 30 s each time. The cells were centrifuged at 12000 rpm for 20 min at 4 ℃. The supernatant containing intracellular proteins was aspirated, and the protein concentration of each group was determined using a BCA protein concentration quantification kit. After balancing with loading buffer, the cells were boiled in 100 ℃ boiling water for 11 min. After cooling to room temperature, the cells were stored in a -20 ℃ refrigerator. The expression of NFX1 and Galectin 9 in LLC cells was analyzed using Western blot technology. The specific experimental steps are described in step six (3).

[0087] For details of the above results, please see Figure 14 and Figure 15 Given that GSDMD-N ultimately forms pore structures on the cell membrane, intracellular LDH, ATP, and mature inflammatory factors are released extracellularly, subsequently triggering a strong inflammatory response; furthermore, the release of DAMPs and mature inflammatory factors also indirectly confirms the occurrence of pyroptosis. Therefore, the levels of lactate dehydrogenase (LDH), IL-1β, IL-18, and adenosine triphosphate (ATP) in the supernatant of cells cultured after PDIC-NO treatment were measured under normoxic and hypoxic conditions, respectively. (See...) Figure 14 (ad) The results showed that under normoxic conditions, the levels of LDH, IL-1β, IL-18, and ATP in the PDIC-NO group were 1.12, 1.13, 1.22, and 1.37 times higher than those in the PBS group, respectively, while under hypoxia, they were 1.86, 1.82, 3.11, and 2.08 times higher, respectively. Under hypoxia, the levels of LDH, IL-1β, IL-18, and ATP in the supernatant of cultured cells in the PDIC-NO group increased by 66.07%, 61.06%, 154.92%, and 51.82%, respectively, compared to normoxic conditions. These results indicate that PDIC-NO can induce significant pyroptosis under hypoxic conditions, accompanied by the release of large amounts of cellular contents and inflammatory factors. Figure 15 (a) Blue fluorescence represents the cell nucleus, and red fluorescence represents HMGB1. The experimental results show that PDIC-NO induces tumor cells to release large amounts of HMGB1 under hypoxia. This indicates that PDIC-NO can inhibit tumor immune escape by promoting the release of DAMPs.

[0088] VII. Evaluation of the in vivo antitumor effect of PDIC-NO

[0089] Five-week-old SPF-grade female C57BL / 6 mice were purchased from Beijing Vital River Company and were housed in accordance with the regulations of the Animal Management and Ethics Committee of Henan University (Ethics Code: HUSOM-2021-001).

[0090] (1) Hemolysis test

[0091] Blood samples obtained from the retro-orbital region of mice were placed in heparin sodium collection tubes. The blood samples were centrifuged at 2000 rpm for 5 minutes to collect red blood cells. 0.5 mL of red blood cells was diluted with 10 mL of 1×PBS. Simultaneously, different concentrations of 0.5 mL PDIC-NO solution were prepared. After preparation, 0.5 mL of diluted red blood cells was added to each tube to ensure a final drug concentration of 10, 25, 50, 100, and 200 μg / mL. The negative control used only 0.5 mL of 1×PBS, and the positive control used 0.5 mL of H2O. The mixture was incubated at 37 °C for 4 hours, then centrifuged at 2000 rpm for 10 minutes, and photographs were taken to observe red blood cell hemolysis. Figure 16 As shown, after erythrocytes were incubated with different concentrations of PDIC-NO for 4 h, the concentration of PDIC-NO increased to 200 µg / mL. -1 No hemolysis was observed in mouse erythrocytes, indicating that PDIC-NO at 200 µg / mL... -1 Within a certain range, it exhibits good biocompatibility in blood.

[0092] (2) Pharmacokinetic experiments

[0093] Three five-week-old C57BL / 6 mice were injected with 2 mg / kg via tail vein. -1 PDIC-NO was extracted. 100 μL of tail vein blood was collected from mice at 0, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 6, 9, 12, 24, 36, and 48 h. 100 μL of 1% Triton X-100 was added, and the mixture was stirred. Then, 400 μL of DMSO was added, and the mixture was sonicated. The mixture was then extracted at 4 ℃ for 12 h, followed by centrifugation at 4000 rpm for 15 min. The supernatant was collected, and the OD value was measured. Pharmacokinetic data were processed using a non-compartmental model in Phoenix WinNonlin software.

[0094] To investigate the metabolism of PDIC-NO in vivo, a pharmacokinetic experiment was conducted in mice. As shown in Table 2, a single intravenous injection of 2 mg / kg... -1 After PDIC-NO, the half-life (T) of PDIC-NO 1 / 2 The peak concentration (C) was 9.92 h. max ) is 2 µg mL -1 Total content (AUC) last The value was 117.84 h µg mL -1 Mean residence time of PDIC-NO in mice (MRT) 0-inf The blood circulation time was 12.89 h. These results indicate that PDIC-NO possesses moderate blood drug concentrations and a relatively long blood circulation time, laying the foundation for its application in tumor treatment.

[0095] Table 2 Pharmacokinetic parameters of PDIC-NO

[0096]

[0097] (3) Preliminary investigation on biosafety

[0098] Eligible C57BL / 6 mice were randomly divided into 4 groups, with 0 mg / kg of each group. -1 Group, 2 mg kg -1 Group, 4 mg kg -1 Group, 8 mg kg -1 During administration, the groups were injected with 1 × PBS and 2 mg / kg, respectively. -1 PDIC-NO, 4 mg kg -1 PDIC-NO, 8 mg / kg -1 PDIC-NO was administered once every two days for a period of 14 days. During treatment, the mice's body weight and survival status were monitored daily. After treatment, blood samples were collected for complete blood count and blood biochemistry analysis. Figure 17 As shown, the experimental results indicate that all mice survived during the 14-day observation period. Figure 17 (a). With 0 mg kg -1 Compared to the three dosages (2, 4, 8 mg kg), the three dosages were... -1 The mice all showed stable weight gain and were at 0 mg / kg. -1 There were no significant differences between the groups, see Figure 17(b) Further hematological parameters of the mice were examined, including routine blood counts such as white blood cell count (WBC), red blood cell count (RBC), hemoglobin (HGB), and platelet count (PLT), as well as liver and kidney function indicators such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), and blood urea nitrogen (BUN). The results showed that all groups were within the normal physiological range. Figure 17 (c).

[0099] (4) Construction of animal models

[0100] Constructing a tumor-bearing model: An appropriate amount of LLC cells were used to prepare a cell concentration of 6 × 10⁻⁶. 6 mL -1 The cell suspension was injected at a rate of 100 μL per mouse into the back of the right hind limb. The tumor volume reached approximately 100 mm. 3 Five animals were randomly assigned to four groups of four. The PBS group received 1 × PBS, while the AQ4N, PDIC-NO, and OXA groups received 2 mg / kg PBS. -1 AQ4N, PDIC-NO, and OXA were administered every two days for a total of six doses. During treatment, mouse body weight, tumor volume, and survival status were recorded every two days. After treatment, the model animals were euthanized, and the tumor, heart, liver, spleen, lungs, kidneys, and tumor drainage lymph nodes were dissected for later use. The tumor was also photographed and weighed. The recorded data were then processed using GraphPad Prism software.

[0101] Lung in situ tumor model: Intraperitoneal injection of ethyl carbamate (800 mg / kg) twice a week. -1 A stable C57BL / 6 mouse lung adenocarcinoma model was established for 5 consecutive weeks and allowed to grow for 90 days. The mice were then randomly divided into 4 groups of 5 mice each. The PBS group received 1 × PBS before administration, while the AQ4N, PDIC-NO, and OXA groups received 2 mg / kg of PBS before administration. -1AQ4N, PDIC-NO, and OXA were administered every two days for a total of six doses. The mice's weight and survival status were recorded every two days during treatment. After treatment, the model animals were euthanized, and the heart, liver, spleen, lungs, and kidneys were dissected for later use. The lungs were also photographed, weighed, and the number of pulmonary nodules was recorded. The recorded data were then processed using GraphPad Prism software.

[0102] (5) Immune activation status in tumor draining lymph nodes

[0103] Mouse tumor-draining lymph nodes were placed in 1×PBS. The lymph nodes were ground using a 100 μm cell sieve, washed with physiological saline, and the filtrate was collected. The cells were centrifuged at 2000 rpm for 10 min at room temperature, the supernatant was discarded, and the cell pellet was resuspended in PBS for subsequent analysis. Staining was performed as follows: each group was divided into three EP tubes, and each EP tube contained FITC anti-mouse CD86, APC anti-mouse CD80, and PE anti-mouse CD11c. Single-staining tubes of FITC anti-mouse CD86, APC anti-mouse CD80, and PE anti-mouse CD11c were also prepared. Flow cytometry analysis was then performed.

[0104] (6) Immune activation in tumors

[0105] Tumor tissue was soaked in PBS, surrounding connective tissue was removed, washed with 1×PBS, and cut into pieces approximately 1 mm in size. 3 Small pieces, add collagenase IV (200 U / mL) -1 ) and DNase I (40 U mL) -1 Cells were digested in serum-free DMEM medium at 37 °C for 50 min using a shaker. The digest was filtered through a 100 μm cell sieve and digestion was terminated with DMEM containing 2 mM EDTA. The cells were centrifuged at 2000 rpm for 5 min, the supernatant was discarded, and the cell pellet was resuspended in PBS for subsequent antibody fluorescence detection. The staining method was as follows: each group was divided into 3 EP tubes, and FITC anti-mouse CD3 and APC anti-mouse CD4 were added to each EP tube for CD3 detection. + CD4 + T cells. FITC anti-mouse CD3 and APC anti-mouse CD8 were added to each EP tube for CD3 detection. + CD8 +T cells. Each EP tube contained FITC anti-mouse CD3, APC anti-mouse CD8, and PE anti-mouse CD38 for CD3 detection. + CD8 + CD38 + T cells. Four single-staining tubes were prepared: FITC anti-mouse CD3, APC anti-mouse CD4, APC anti-mouse CD8, and PE anti-mouse CD38. Detection was performed using flow cytometry.

[0106] (7) Detect the proportion of immune cells in the spleen

[0107] Mouse spleens were placed in PBS. The spleens were homogenized using a 100 μm fine cell sieve, washed with 10% FBS-1640 medium, and the filtrate was collected. The cells were centrifuged at 2000 rpm for 10 min, and the supernatant was discarded. The cells were resuspended in erythrocyte lysis buffer, allowed to stand for 5 min, and then centrifuged (2000 rpm, 10 min), repeated once. The cells were resuspended in 1640 medium and centrifuged at 2000 rpm for 15 min, repeated once. The cell pellet was resuspended in serum-free 1640 medium, filtered, and the cell concentration adjusted to 2.5 × 10⁶ cells / mL. 6 Cells / mL, for later use. Resuspend in 1 × PBS for subsequent antibody fluorescence detection. Staining method is the same as in step (6).

[0108] (8) Detect the proportion of immune cells in lung tissue

[0109] Mouse lung tissue was placed in PBS. The lung tissue was milled through a 100 μm cell sieve, washed with 10% FBS-1640 medium, and the filtrate was collected. The cells were centrifuged at 2000 rpm for 10 min, and the supernatant was discarded. The cells were resuspended in erythrocyte lysis buffer, allowed to stand for 5 min, and then centrifuged (2000 rpm, 10 min). The cells were resuspended in 1640 medium, centrifuged at 2000 rpm for 15 min, and the cell pellet was resuspended in serum-free 1640 medium. The cells were filtered, and the cell concentration was adjusted to 2.5 × 10⁶ cells / mL. 6 Cells / mL, ready for use. Resuspend in PBS for subsequent antibody fluorescence detection. Staining method is the same as in step (6).

[0110] (9) H&E staining of tissue sections

[0111] Mice were dissected, and the heart, liver, spleen, lungs, and kidneys were harvested. Connective tissue was removed, and the tissues were washed with PBS, then fixed in 4% paraformaldehyde for at least 24 hours. After fixation, the tissues were washed three times with PBS, placed in embedding cassettes, rinsed with running water for 30 min, and then dehydrated sequentially using an automated dehydrator (70%-100% ethanol, xylene). Finally, the tissues were embedded in paraffin. After trimming, the tissue blocks were sectioned (5 μm), flattened in 46 ℃ warm water, and dried at 63 ℃. The dewaxing procedure included xylene and graded ethanol treatment, followed by hematoxylin-eosin (H&E) staining, differentiation, washing with water, and finally mounting with neutral resin. Images were taken using an automated scanner, and the data were analyzed using Motic DSAssistant Lite software. Figure 20 As shown, the results of each experimental group were as follows: the myocardial fibers in all groups were neatly arranged and no inflammatory cell infiltration was observed; the ratio of white pulp to red pulp in the spleen tissue was normal and the tissue structure was intact; the lung tissue showed clear alveolar structure and no abnormal pathological phenomena such as hemorrhage, edema or inflammatory exudation were observed; the glomeruli in the kidney tissue were normal in morphology and no abnormal casts were observed in the renal tubules.

[0112] (10) Immunohistochemical staining of tissue sections

[0113] Dissected mice, tumor tissue was fixed in 4% paraformaldehyde for at least 24 hours. After washing with PBS, the tissue was cut into blocks, placed in an embedding cassette, rinsed with running water for 30 min, and then dehydrated sequentially using an automated dehydrator (70%-100% ethanol, xylene), and finally embedded in paraffin. The tissue blocks were trimmed and sectioned (5 μm), flattened in 45 ℃ warm water, and then dried at 63 ℃. The dewaxing procedure included xylene and graded ethanol treatment, followed by hydrogen peroxide blocking, permeabilization, and antigen retrieval. Blocking was performed with 5% BSA for 30 min, followed by the addition of Cl. Caspase 1 and GSDMD-N primary antibodies, and incubation at 4 ℃ for 12 h. After washing with PBS, HRP-labeled secondary antibody was added, and incubation was performed at room temperature for 1 h. DAB staining was performed for 5 min, hematoxylin staining of cell nuclei for 3 min, and finally, the slides were mounted with neutral resin, photographed and recorded using an automated scanner, and analyzed using Motic DSAssistant Lite software. Figure 21 As shown,

[0114] (11) Blood index analysis

[0115] Mouse ocular blood was collected in an anticoagulant tube, mixed immediately, and then analyzed using a fully automated veterinary blood cell analyzer. Alternatively, mouse ocular blood was collected in an EP tube without anticoagulant, centrifuged, and the supernatant was analyzed using a fully automated veterinary biochemical analyzer.

[0116] The treatment process is as follows Figure 18 As shown in (a), mice were euthanized on day 24, the tumor mass was completely removed and weighed, and important organs such as the heart, liver, spleen, lungs, kidneys, and lymph nodes were collected for subsequent histopathological and immune cell analysis. Figure 18 As shown in (b) and (c), after 10 days of treatment, the tumor volume in PDIC-NO-treated mice was significantly suppressed (467 mm). 3 ), is the PBS group (1536 mm) 3 The percentage of 30.40% in the AQ4N group (1041 mm) was lower than that in the AQ4N group (1041 mm). 3 ) and OXA group (673 mm) 3 The tumor inhibition rates in the PDIC-NO group were 67.77% and 43.82% respectively, while those in the AQ4N and OXA groups were 2.16 times and 1.24 times higher, respectively. Furthermore, the tumor quality in the PDIC-NO group was significantly reduced, such as... Figure 3 .20 (d). The above results fully demonstrate that PDIC-NO has excellent anti-tumor activity.

[0117] like Figure 19 As shown in (a) and (b), compared with the PBS group, the fluorescence intensity of Cl.Caspas1 and GSDMD-N in the PDIC-NO group was significantly enhanced, being 1.61 times and 2.40 times that of the PBS group, respectively. However, there were no significant differences between the AQ4N group and the OXA group and the PBS group. Furthermore, compared with the PBS group, the serum IL-1β and IL-18 levels in the PDIC-NO group mice were also significantly increased, being 1.75 times and 2.15 times that of the PBS group, respectively. Figure 19 (c). The above results indicate that PDIC-NO can induce significant tumor cell pyroptosis and inhibit tumor growth in solid tumors.

[0118] like Figure 20 As shown, the results of each experimental group were as follows: the myocardial fibers in all groups were neatly arranged and no inflammatory cell infiltration was observed; the ratio of white pulp to red pulp in the spleen tissue was normal and the tissue structure was intact; the lung tissue showed clear alveolar structure and no abnormal pathological phenomena such as hemorrhage, edema or inflammatory exudation were observed; the glomeruli in the kidney tissue were normal in morphology and no abnormal casts were observed in the renal tubules.

[0119] like Figure 21As shown, the results of relevant detection indicators of blood routine (RBC, WBC, HGB, PLT, etc.) and blood biochemistry (ALT, AST, BUN, etc.) in mice of each experimental group were all within the normal range, further indicating that AQ4N, OXA, and PDIC-NO have good biosafety at therapeutic doses. Finally, further biosafety assessments were performed on the pulmonary orthotopic tumor model, such as... Figure 22 As shown, after treatment with AQ4N, OXA, and PDIC-NO, histological analysis of the major organs of mice in each group revealed no significant histopathological changes, indicating that AQ4N, OXA, and PDIC-NO all have relatively good biocompatibility in the treatment of in situ tumors.

[0120] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A perylene imide derivative PDIC-NO, characterized in that, The structure is as follows: 。 2. The method for preparing the perylene imide derivative PDIC-NO according to claim 1, characterized in that, The process is as follows: ， Compound PDIC-NN was added to ethanol, and hydrogen peroxide was added under a protective atmosphere. The temperature was raised from room temperature to 45-55°C, and the reaction was stirred for 10-15 hours. The reaction mixture was cooled to room temperature and post-treated to obtain the target compound PDIC-NO. The molar ratio of compound PDIC-NN to hydrogen peroxide was 1:(90-110).

3. The method for preparing the perylene imide derivative PDIC-NO according to claim 2, characterized in that, The mass concentration of hydrogen peroxide is 25~35wt%.

4. The method for preparing the perylene imide derivative PDIC-NO according to claim 2, characterized in that, The post-treatment refers to the addition of diethyl ether for recrystallization and drying to obtain the target compound PDIC-NO.

5. The use of the perylene imide derivative PDIC-NO according to claim 1 in the preparation of anti-lung cancer drugs, characterized in that, The PDIC-NO induces ROS burst in tumor cells under hypoxia, triggers LMP in tumor cells, and causes a large amount of CTSB to leak into the cytoplasm of tumor cells, thereby activating the NLRP3 / Caspase-1 / GSDMD pathway in tumor cells, inducing pyroptosis of tumor cells, and releasing large amounts of LDH, ATP, IL-1β and IL-18.

6. The application according to claim 5, characterized in that, The tumor cells were LLC cells.

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