Composite nano vaccine and application thereof

Abstract

The invention relates to the technical field of medicines, in particular to a composite nano vaccine and application thereof. The composite nano vaccine is prepared by coupling a natural compound with endoplasmic reticulum stress induction capability on the surface of Ga-MOF nano particles. The composite nano vaccine can significantly enhance the anti-tumor effect through a ferroptosis mechanism, an endoplasmic reticulum stress and immunogenic cell death mechanism and a cryoablation technology synergistic effect mechanism, reverses an immunosuppressed tumor microenvironment, inhibits the growth of primary tumors and distant metastatic tumors, and has a good anti-tumor effect. A brand new strategy is provided for comprehensive treatment of tumors, and the application prospect is wide.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicines, in particular to a composite nano-vaccine and application thereof. BACKGROUND

[0002] In the field of tumor immunotherapy, nanoscale cancer vaccines have become a research hotspot due to their advantages of precise targeting, controllable drug release and multi-modal immune activation. Metal-organic framework (MOF) nano-vaccines have attracted extensive attention due to their high drug loading capacity, controllable degradation characteristics and excellent immune regulation effect. At present, cryoablation technology, as a minimally invasive treatment method, effectively inactivates tumor cells through rapid cooling and heating cycles, has the advantages of low treatment pain and good patient tolerance, and can also significantly activate the patient's own anti-tumor immune activity. However, the distant anti-tumor effect of cryoablation technology is uncertain, and the effect decays after short-term immune activation, which limits its widespread application in clinical practice. In addition, existing tumor vaccines also face many challenges, such as insufficient specificity, limited efficacy, difficulty in triggering strong and long-lasting immune responses, especially under the immunosuppressive effect of the tumor microenvironment, their effect is often greatly discounted, and they cannot achieve efficient tumor killing and immune activation, which cannot meet the expectations of clinical tumor treatment. SUMMARY

[0003] In order to solve the above technical problems, the present application provides a composite nano-vaccine, which is a natural compound with endoplasmic reticulum stress induction ability coupled on the surface of Ga-MOF nanoparticles.

[0004] In some embodiments, the natural compound with endoplasmic reticulum stress induction ability includes baicalein, curcumin or resveratrol. And / or, the surface of the Ga-MOF nanoparticles is also modified with a ligand or functional group with targeting function.

[0005] Among them, curcumin has various biological activities such as anti-inflammatory and antioxidant, its mechanism of inducing endoplasmic reticulum stress is similar to that of baicalein, which can enhance the sensitivity of tumor cells to immunotherapy by regulating related signaling pathways. Therefore, the above-mentioned natural compounds can also trigger endoplasmic reticulum stress response of tumor cells, induce immunogenic cell death, and thus realize the synergistic anti-tumor effect with Ga-MOF.

[0006] In some embodiments, the method for preparing the composite nano-vaccine comprises: mixing and reacting gallium salt and organic ligand in an organic solvent to prepare Ga-MOF nanoparticles; introducing functional groups to the Ga-MOF nanoparticles by amidation reaction (so that the Ga-MOF nanoparticles have active sites for binding with the natural compound), to prepare Ga-MOF nanoparticles modified with functional groups; mixing and reacting the Ga-MOF nanoparticles modified with functional groups with a natural compound having the ability to induce endoplasmic reticulum stress, to prepare the composite nano-vaccine.

[0007] In some embodiments, the gallium salt comprises gallium nitrate; and / or, the organic ligand comprises bis(3,5-dicarboxyphenyl) azo; and / or, the organic solvent is a mixture of acetonitrile and dimethyl sulfoxide (preferably, acetonitrile and dimethyl sulfoxide are mixed at a volume ratio of 1:1).

[0008] Preferably, the mass ratio of gallium salt to organic ligand is (1-3): 1.

[0009] In some embodiments, the temperature for mixing and reacting in the organic solvent is 110-130°C; preferably, the mixing and reacting in the organic solvent is carried out under the condition of continuous stirring by a magnetic stirrer, preferably for more than 3h.

[0010] In some embodiments, after mixing and reacting gallium salt and organic ligand in an organic solvent, the precipitate is washed with deionized water for three times (each washing condition: centrifugation at 8000 rpm for 10 minutes), to obtain light yellow Ga-MOF nanoparticles.

[0011] In some embodiments, the step of introducing functional groups to the Ga-MOF nanoparticles by amidation reaction comprises: mixing the Ga-MOF nanoparticles with EDC and NHS for amidation reaction, to prepare Ga-MOF nanoparticles modified with functional groups.

[0012] In some embodiments, the natural compound having the ability to induce endoplasmic reticulum stress and p-toluenesulfonamide are dissolved in DMSO to prepare a mixture, and the mixture is mixed and reacted with the Ga-MOF nanoparticles modified with functional groups, to prepare the composite nano-vaccine.

[0013] Preferably, in the mixture, the concentration of the natural compound having the ability to induce endoplasmic reticulum stress is 1-10 mg / mL; and / or, the concentration of p-toluenesulfonamide is 0.1-0.5 mg / mL.

[0014] In some embodiments, the particle size of the composite nano-vaccine is in the range of 100-200 nm.

[0015] In the implementation process, the synthesis process of Ga-MOF can be adjusted to optimize its particle size and surface properties to improve the targeting efficiency and drug release efficiency. For example, by changing the proportion of solvents, reaction temperature and time and other parameters in the synthesis process, the particle size and distribution of Ga-MOF can be controlled to better meet the permeability and retention effect (EPR effect) of tumor tissues. At the same time, the surface of Ga-MOF is modified to introduce specific targeting ligands or functional groups, which can further improve the targeting recognition ability of tumor cells and enhance the accuracy of drug release. The above optimization schemes are within the scope of protection of the present application.

[0016] Further, the present application provides a composition comprising the composite nano-vaccine and an immune modulator. Preferably, the immune modulator includes but is not limited to PD-1 inhibitors.

[0017] By combining the composite nano-vaccine with other immune modulators, the immunotherapy effect can be further enhanced. For example, PD-1 inhibitors can block the immune checkpoint signal pathway between tumor cells and immune cells, and relieve immune suppression. Combined with the composite nano-vaccine, it is expected to achieve a stronger immune synergistic effect. In combination therapy, PD-1 inhibitors can enhance the function of T cells and improve their killing ability to tumor cells, while cooperating with the immunogenic cell death induced by the composite nano-vaccine to further amplify the immune response, providing a more powerful means for the treatment of tumors.

[0018] Further, the present application provides the use of the composite nano-vaccine or the composition in the preparation of a pharmaceutical product; preferably, the pharmaceutical product is used in combination with cryoablation technology to treat cancer or tumors; preferably, the cancer is lung cancer.

[0019] Compared with the prior art, the present application has the following advantages: The present application provides a composite nano-vaccine based on gallium-based metal organic framework and natural compounds with endoplasmic reticulum stress induction ability (such as Baicalein), which can significantly enhance the anti-tumor effect through multiple mechanisms and has broad application prospects.

[0020] The specific mechanism is as follows: 1. Ferroptosis mechanism: Gallium ions (Ga 3+ ) in Ga-MOF can selectively interfere with the iron metabolism pathway of tumor cells. Iron metabolism plays a key role in the growth and survival of tumor cells, and Ga 3+Interventions that disrupt this metabolic balance can precisely induce ferroptosis in tumor cells. This process not only directly kills tumor cells, but also releases a large number of tumor-associated antigens, providing a material basis for subsequent immune activation.

[0021] 2. Endoplasmic reticulum stress and immunogenic cell death mechanism: Natural compounds with endoplasmic reticulum stress inducing ability such as Baicalein can effectively trigger endoplasmic reticulum stress (ER Stress) in tumor cells. Endoplasmic reticulum stress can further induce immunogenic cell death (ICD), in which process tumor cells release a series of key damage-associated molecular patterns (DAMPs) such as cell surface calreticulin (CRT) and extracellular release of high mobility group protein B1 (HMGB1). These molecules can be recognized by the immune system, activate the innate immune system, and initiate an anti-tumor immune response.

[0022] 3. The composite nano-vaccine can significantly activate immunogenic cell death (ICD) in cooperation with cryoablation technology, thereby prolonging and enhancing the anti-tumor immune response. In vitro and in vivo experimental results confirm that the combined treatment of the composite nano-vaccine and cryoablation can significantly promote the maturation of dendritic cells (DC) and increase T cell infiltration. Specifically, the combined treatment significantly increases the expression level of co-stimulatory molecules (such as CD80 / 86) on the surface of DC cells, enhances their antigen-presenting ability, and activates more specific T cells. At the same time, the number of T cells infiltrating the tumor tissue increases significantly, which can more effectively recognize and kill tumor cells. In addition, this combined treatment can also reverse the immunosuppressive tumor microenvironment, inhibit the growth of primary tumors and distant metastatic tumors, and provide a new strategy for comprehensive tumor treatment. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1are the preparation and characterization results of Ba@Ga-MOF nanoparticles; (A) synthesis route and coordination mechanism of Ga-MOF and Ba@Ga-MOF; (B) scanning electron microscope (SEM) images showing the surface morphology; (C) transmission electron microscope (TEM) images revealing the internal structure; (D) macroscopic morphology photographs of nanoparticles; (E) particle size distribution histogram measured by dynamic light scattering (DLS); (F) Zeta potential analysis results; (G) energy dispersive X-ray spectroscopy (EDS) element mapping; (H) Fourier transform infrared spectroscopy (FT-IR) for chemical bond characterization; (I) drug loading stability curves of nanoparticles under different pH conditions; (J) drug release kinetics curves of nanoparticles in different pH environments; (K) X-ray photoelectron spectroscopy (XPS) analysis of the chemical state of surface elements; (L) nitrogen adsorption-desorption isotherms and BET specific surface area determination; (M) element mapping analysis of Ba@Ga-MOF; results are expressed as mean ± standard deviation (n = 3); statistical analysis used one-way analysis of variance (ANOVA) p < 0.05, p < 0.01, p < 0.001).

[0024] Figure 2 are the in vitro anti-tumor activity evaluation results of baicalein and nanoparticles; (A) cell proliferation activity (CCK-8 assay) after baicalein treatment (24, 48 and 72 hours); (B) cell proliferation activity (CCK-8 assay) after Ba@Ga-MOF nanoparticle treatment (24, 48 and 72 hours); (C) representative images of colony formation test in each treatment group (control group, baicalein, Ga-MOF and Ba@Ga-MOF); (D) quantitative analysis of colony formation (**p < 0.01 vs control group); (E) and (F) flow cytometry detection of apoptosis rate (Annexin V-FITC / PI double staining) in different treatment groups (control group, baicalein, Ga-MOF and Ba@Ga-MOF); results are expressed as mean ± standard deviation (n = 3); statistical analysis used one-way analysis of variance (ANOVA) p < 0.05, p < 0.01, p < 0.001).

[0025] Figure 3are the effects of different treatments on tumor cell migration, invasion, and viability; (A) Transwell assay images (top: migration; bottom: invasion, using Matrigel matrix simulation); (B) Quantitative analysis of cell migration (BaGa-MOF vs control); (C) Quantitative analysis of cell invasion (BaGa-MOF vs control); (D) Scratch wound healing assay images (0 h and 24 h, scale bar = 200 pm); (E) Quantitative analysis of scratch closure rate (percentage of migration area); (F) Live-dead cell fluorescence staining images (green: Calcein-AM labeled live cells; red: PI labeled dead cells); (G) Statistical results of cell viability (percentage of live cells, n = 3); results are expressed as mean ± standard deviation (n = 3); statistical analysis by one-way ANOVA p < 0.05, p < 0.01, p < 0.001).

[0026] Figure 4 are the subcellular localization and in vivo biodistribution kinetics results of the nanoparticles; (A) Confocal microscopy images of nanoparticle-endoplasmic reticulum (ER) colocalization (4 h / 8 h / 16 h; green: nanoparticles; red: ER tracer; blue: nucleus); (B) Quantitative analysis of nanoparticle-ER colocalization (Pearson correlation coefficient, mean ± standard deviation, **p < 0.01 vs 4 h group); (C) In vivo fluorescence imaging results (top: only nanoparticle group; bottom: nanoparticle + cryoablation combination group; time points: 0 h / 8 h / 16 h / 24 h); (D) Statistical distribution of nanoparticle fluorescence intensity in major organs (heart, liver, spleen, lung, kidney; fluorescence intensity (unit / pg tissue)); results are expressed as mean ± standard deviation (n = 3); statistical analysis by one-way ANOVA p < 0.05, p < 0.01, p < 0.001).

[0027] Figure 5Molecular mechanism results of nanoparticle-induced ICD, ferroptosis and ER stress; (A) Schematic diagram of nanoparticle-induced ICD, ferroptosis and ER stress; (B) Effects of different treatments on the expression of proteins related to ferroptosis, ER stress and ICD (Western blot results: ferroptosis markers: ACSL4 (pro-ferroptosis), SLC7A11 (anti-ferroptosis); ER stress markers: GRP78 (chaperone), CHOP (apoptosis-inducing factor); ICD markers: CRT (cell surface exposure), HMGB1 (extracellular release)); (C-I) Quantitative statistical analysis of protein expression levels; the column chart represents the gray value of the protein band in panel B, normalized to the control group; error bars indicate SEM; results are expressed as mean ± standard deviation (n = 3); statistical analysis by one-way ANOVA p<0.05, p<0.01, p<0.001).

[0028] Figure 6 Multidimensional evaluation results of nanoparticle-induced oxidative stress and ICD; (A) Comparison of DCFH-DA fluorescence intensity between different treatment groups; (B) Comparison of Fluo-4 fluorescence intensity between different treatment groups; (C) Fluorescence expression of calreticulin (CRT) in different treatment groups; (D) Fluorescence expression of high mobility group protein 1 (HMGB1) in different treatment groups; (E) Quantitative statistical analysis of fluorescence experiments (column chart summarizes the average fluorescence intensity of groups A-D; *p<0.001); (F) Representative flow cytometry detection of in vitro LLC cell-induced dendritic cell (DC) maturation under different treatments (CD80+CD86+); results are expressed as mean ± standard deviation (n = 3); statistical analysis by one-way ANOVA p<0.05, p<0.01, p<0.001).

[0029] Figure 7are the results of the evaluation of the inhibitory effect of nanoparticles combined with cryoablation on primary and distant tumors in mice; (A) flow chart of the experimental design of mice, showing the experimental grouping, drug administration scheme, cryoablation time point, observation period and key time nodes; (B) trend graph (line graph) of the change of the volume of primary tumors in mice in each treatment group over time (from day 0 to day 18); (C) comparison of the weight of primary tumors in each group at the end of the experiment; (D) change of the body weight of mice over time during treatment; (E) direct representation of the change of tumors after cryoablation treatment; (F) representative images of gross specimens of primary tumors; (G-M) trend of the change of the volume of distant tumors in each group over time (from day 0 to day 18) (a series of line graphs); the experimental groups include: saline group, Ga-MOF group (5 mg / kg), baicalein group (5 mg / kg), Ba@Ga-MOF group (5 mg / kg in terms of Ga-MOF content), cryoablation group (-168°C, 3 minutes), and Ba@Ga-MOF + Cry group (5 mg / kg in terms of Ga-MOF content; -168°C, 3 minutes); all data are expressed as mean ± standard deviation (n = 3); statistical analysis uses one-way ANOVA p<0.05, p<0.01, p<0.001).

[0030] Figure 8Fig. 7 is the results of the influence of nanoparticle combination therapy on the whole body and tumor microenvironment (TME) of mice; (A) Kaplan-Meier survival curves from day 0 to day 60 were compared between the following groups: saline group, Ga-MOF group (5 mg / kg), baicalein group (5 mg / kg), Ba@Ga-MOF group (5 mg / kg based on Ga-MOF), cryoablation group (-168°C, 3 minutes), and Ba@Ga-MOF+Cry group (5 mg / kg based on Ga-MOF; -168°C, 3 minutes); (B-F) ELISA analysis further evaluated the levels of cytokines in the serum of mice, including interferon-γ (IFN-γ), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and monocyte chemoattractant protein-1 (MCP-1); (G) Multiplex immunofluorescence analysis of tumor tissues revealed molecular stress responses and apoptosis characteristics, including TUNEL detection (green: apoptotic cells; blue: nuclei), CRT surface exposure (red staining), HMGB1 nuclear translocation (red staining), and CHOP protein expression (green staining); data are expressed as mean ± standard deviation (n = 5); differences between groups were analyzed by one-way ANOVA p < 0.05, p < 0.01, p < 0.001.

[0031] Figure 9 Fig. 8 is the results of the regulation of tumor immune microenvironment and systemic immune response by nanoparticle therapy; (A) Dynamic analysis of regulatory T cells (Tregs) in the spleen (quantified by CD4+FoxP3+ double positive cells, reflecting Treg-mediated immune suppression function); (B) Myeloid cell reprogramming in the spleen (MDSC suppression: reduction of CD11b+Gr-1+ myeloid-derived suppressor cell infiltration); (C) T cell effector function in the spleen (quantified by CD4+or CD8+T cell number, assessing T cell activation status); (D-F) Flow cytometry analysis of related cells in the tumor microenvironment; (G) Representative flow cytometry data graph; results are expressed as mean ± standard deviation (n = 3); statistical analysis was performed by one-way ANOVA p < 0.05, p < 0.01, p < 0.001.

[0032] Figure 10is the results of transcriptome sequencing and differential gene analysis; (A) volcano plot of differentially expressed genes (DEGs) (significantly up-regulated genes are marked in red, and down-regulated genes are marked in blue); (B) protein-protein interaction (PPI) network of DEGs; (C) KEGG pathway enrichment analysis of protein processing in endoplasmic reticulum; (D) gene ontology (GO) biological process classification; (E) gene set enrichment analysis (GSEA).

[0033] Figure 11 is the results of distant lung metastasis inhibition effect and biological safety evaluation; (A) distant lung metastasis inhibition effect; (B) biological safety evaluation results. DETAILED DESCRIPTION

[0034] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions in the present application will be described clearly and completely below. Obviously, the described embodiments are some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application. In the embodiments provided in the present specification, the specific techniques or conditions not noted are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The reagents or instruments not noted by the manufacturer are conventional products that can be purchased through a regular channel.

[0035] Example 1 Preparation of composite nano vaccine The present embodiment provides a composite nano vaccine (Ba@Ga-MOF nanoparticle), which is baicalein coupled on the surface of Ga-MOF nanoparticle, and the preparation method is as follows: First, 60 milligrams of gallium nitrate (Ga(NO3)3) and 40 milligrams of bis(3,5-dicarboxyphenyl) azo ligand were dissolved in a mixed solvent of acetonitrile and dimethyl sulfoxide (DMSO, volume ratio 1:1), and then ultrasonic treatment was performed for 10 minutes to ensure complete dissolution. The mixed solution was transferred to a beaker and placed on a magnetic stirrer in an oil bath, and reacted at 120°C for 3 hours with constant magnetic stirring at a speed of 500 rpm to ensure uniform reaction. During this process, the solution color gradually changed from colorless to cream yellow.

[0036] After the reaction was completed, the system was naturally cooled to room temperature, and then the reaction solution was centrifuged at 8000 rpm for 10 minutes to collect the precipitated product. In order to remove unreacted substances and residual solvents, the precipitate was washed with deionized water three times (each washing condition: 8000 rpm centrifugation for 10 minutes). Finally, the obtained product was freeze-dried to obtain a light yellow Ga-MOF powder.

[0037] The specific steps for preparing the endoplasmic reticulum-targeted and baicalein-loaded Ba@Ga-MOF composite material through an amidation reaction are as follows: First, 50 mg of the above Ga-MOF powder material is dispersed in 5 mL of anhydrous DMSO, and then 10 mg of EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) and 6 mg of NHS (N-hydroxysuccinimide) are sequentially added, and the carboxyl groups on the surface of the Ga-MOF are activated by stirring at room temperature for 1 hour.

[0038] Next, 5 mg of baicalein and 1 mg of p-toluenesulfonamide (endoplasmic reticulum targeting agent) are added to the system, and the reaction is continuously stirred (300 rpm) for 24 hours in the dark to complete the drug loading process. After the reaction is completed, the mixture is centrifuged at 9000 rpm for 10 minutes to collect the precipitated product, which is washed with deionized water three times (each washing condition is the same). Finally, the precipitate is treated by freeze-drying to obtain Ba@Ga-MOF nanoparticles.

[0039] The concentration of free baicalein in the supernatant is determined by ultraviolet-visible spectrophotometry, and the drug encapsulation efficiency is calculated by the following formula: encapsulation efficiency (%) = (1 - C 上清液 / C 初始 ) x 100%.

[0040] The morphology, particle size, and surface charge of the nanoparticles are analyzed by various characterization methods. Transmission electron microscopy (TEM) is used to observe the internal structure and morphology of the nanoparticles, scanning electron microscopy (SEM) is used to show their surface morphology, dynamic light scattering (DLS) is used to measure the particle size distribution, and Zeta potential analysis is used to determine the surface charge characteristics of the nanoparticles. In addition, energy dispersive X-ray spectroscopy (EDS) is used for elemental analysis, Fourier transform infrared spectroscopy (FT-IR) is used for chemical bond characterization, X-ray photoelectron spectroscopy (XPS) is used for analysis of the chemical state of surface elements, and N2 adsorption-desorption isotherms and specific surface area determination (BET) are used to evaluate the porous structure characteristics.

[0041] The preparation and characterization results of Ba@Ga-MOF nanoparticles are shown in Figure 1 .

[0042] In this embodiment, a nanoparticle system was constructed using a green self-assembly strategy. Gallium nitrate and an azo-based organic ligand were used as building blocks to successfully synthesize a gallium-based metal-organic framework (Ga-MOF). Based on this, baicalin was effectively loaded using a supramolecular self-assembly method. Furthermore, p-toluenesulfonamide was covalently grafted onto the material surface, ultimately constructing a novel intelligent drug delivery system with targeted delivery capabilities. Figure 1 A). When Ba@Ga-MOF nanoparticles are dispersed in an aqueous solution, a uniform yellow solution is formed ( Figure 1 (D) indicates that the nanoparticles possess excellent colloidal stability, which is crucial for their potential biomedical applications as it ensures their uniform distribution in solution. Transmission electron microscopy (TEM) analysis ( Figure 1 C) shows that the Ba@Ga-MOF nanoparticles are regularly spherical and well dispersed with almost no aggregation. This regular morphology and good dispersion are conducive to their consistent interaction with biological systems such as cells and tissues. Scanning electron microscopy (SEM) further revealed the presence of nanoprotrusion structures on their surface. Figure 1 B). These surface nanostructures are thought to enhance cell adhesion, potentially facilitating the targeted delivery of nanoparticles to specific cells or tissues. Dynamic light scattering (DLS) measurements ( Figure 1 E) confirmed that the Ba@Ga-MOF nanoparticles have a narrow particle size distribution, ranging from 100 to 200 nm. This size range is ideally suited for utilizing the enhanced penetration and retention effect (EPR effect) in tumor tissue. The EPR effect allows the nanoparticles to preferentially accumulate in tumor tissue due to the poor permeability of tumor vessels and lymphatic drainage. Therefore, Ba@Ga-MOF nanoparticles hold promise as a candidate material for tumor-targeted drug delivery. Zeta potential analysis ( Figure 1 F) showed a significant charge change: the zeta potential of baicalin was -40 mV, while that of Ga-MOF was +18 mV, indicating that Ba@Ga-MOF exhibited a net negative charge. This change in zeta potential validated the successful synthesis of Ba@Ga-MOF nanoparticles, demonstrating the effective binding between baicalin and Ga-MOF, leading to novel surface charge properties. Energy-dispersive X-ray spectroscopy (EDS) Figure 1 G) confirmed the presence of nitrogen (N) and gallium (Ga) elements in the Ba@Ga-MOF nanoparticles. The strong Ga signal indicates that Ga-MOF constitutes the main framework structure of the nanoparticles, consistent with the design strategy. Fourier transform infrared spectroscopy (FT-IR) ( Figure 1H) shows some peak shifts, such as the C-O stretching vibration peak at 1041.55 cm-1, and intensity changes, such as the O-H / N-H peak at 3259.33 cm-1. These changes indicate that hydrogen bonding or coordination occurs between baicalein and Ga-MOF, indicating a strong interaction between the drug (baicalein) and the carrier (Ga-MOF). Ba@Ga-MOF nanoparticles exhibit significant pH-responsive drug release behavior and structural stability under different pH conditions. In a physiological environment (pH 7.4), the nanoparticles maintain a stable particle size of about 200 nm, and no significant changes are observed within 72 hours. However, in a simulated tumor acidic microenvironment (pH 5.4), the nanoparticles undergo a dynamic dissociation process: the particle size decreases significantly within 24 hours, and further decomposes to below 100 nm after 72 hours. This acid-dependent dissociation behavior confirms its potential as a controllable release mechanism - taking advantage of the pH gradient difference between tumors and normal tissues to achieve targeted drug release Figure 1 I). Ba@Ga-MOF nanoparticles exhibit pH-dependent behavior. At physiological pH (7.4), they maintain structural integrity within 72 hours, with only a small amount of drug leakage (only 25% within 48 hours). However, in an acidic tumor microenvironment (pH 5.4), the nanoparticles rapidly dissociate, leading to nearly complete drug release (100% within 8 hours) Figure 1 J). This tumor-specific responsiveness makes Ba@Ga-MOF nanoparticles very suitable for targeted drug delivery, as they can selectively release drugs at tumor sites while reducing side effects on normal tissues. X-ray photoelectron spectroscopy (XPS) Figure 1 K) confirms the successful loading of baicalein into the Ga-MOF framework. Brunauer-Emmett-Teller (BET) analysis Figure 1 L) shows that it has a mesoporous structure with a pore size of 12.3 nm. This mesoporous structure enhances the drug loading capacity of the nanoparticles and also helps with lysosomal escape, which is very important for efficiently delivering drugs to the cytoplasm of target cells. Elemental mapping analysis Figure 2 M) further verifies that Ga is the main component of Ba@Ga-MOF nanoparticles, which is consistent with the design strategy and EDS analysis results. This consistency in elemental composition analysis deepens the understanding of the structure of the nanoparticles and their potential in biomedical applications.

[0043] In summary, the comprehensive morphology and physicochemical characterization of Ba@Ga-MOF nanoparticles show that they have excellent colloidal stability, clear morphology, suitable particle size distribution, and pH-dependent drug release behavior, demonstrating their great potential as a tumor-targeted drug delivery system.

[0044] Example 2 Evaluation of baicalein and nanoparticles for in vitro anti-tumor activity This example evaluates baicalein and nanoparticles for in vitro anti-tumor activity, following the steps: 1. CCK-8 cytotoxicity assay: LLC cells were seeded in 96-well plates at a density of 3000 cells per well and incubated overnight (about 8 hours) to adhere. The cytotoxicity of free baicalein was first determined at a concentration range of 0.5 to 100 pg / mL: different concentrations of free baicalein were added and incubated for 48 hours. Then, in another 96-well plate, LLC cells were seeded at a density of 3000 cells per well and incubated overnight to adhere. The cytotoxicity of Ba@Ga-MOF and Ga-MOF was compared at a concentration range of 0 to 200 pg / mL (calculated based on Ga-MOF content): samples were added and incubated for 48 hours. After incubation, 10 pL of CCK-8 reagent was added to each well containing 100 pL of culture medium and incubated for 0.5-1 hour (or until color development was sufficient), then the absorbance at 450 nm was measured.

[0045] 2. Flow cytometry analysis of cell apoptosis: LLC cells were seeded in plates at a density of 1 x 10 5 cells per well and incubated for 8 hours to adhere. Then, Ba@Ga-MOF was used to treat the cells at a concentration equivalent to 100 pg / mL of Ga-MOF for 12 hours. After treatment, the cells were collected and washed with PBS, then stained with 5 pL of Annexin-V FITC and 10 pL of PI for 15 minutes at room temperature in the dark. The proportion of early apoptotic (Annexin V+ / PI ) and late apoptotic / necrotic (Annexin V+ / PI ) cells was immediately analyzed by flow cytometry.

[0046] The results of the evaluation of in vitro anti-tumor activity are shown in Figure 2 .

[0047] In the CCK-8 test ( Figure 2 A), the half-maximal inhibitory concentration (IC 50 ) of baicalein on LLC cells was about 20.77 pg / mL. These data provide a basis for subsequent studies on the anti-tumor activity of baicalein. Based on Figure 2 B, a detailed comparative analysis of Ga-MOF and Ba@Ga-MOF was performed. It was found that the proliferation inhibition efficiency was significantly improved after loading baicalein. Specifically, the IC 50The concentration decreased from 100 μg / mL for Ga-MOF to 80 μg / mL for Ba@Ga-MOF. This significant change indicates that the drug loading significantly enhanced the inhibitory effect, suggesting a strategy for optimizing the performance of antitumor nanomedicines. To more comprehensively evaluate the antitumor activity of Ba@Ga-MOF, we conducted a colony formation assay (CFA). Figure 3 D). The experimental results clearly show that Ba@Ga-MOF exhibits a strong ability to inhibit tumor cell colony formation compared to the unmodified material. The relative colony formation number in the Ba@Ga-MOF group was significantly reduced to 9%, while it was 74% in the Ga-MOF group, 44% in the baicalein group, and 91% in the control group. This significant difference strongly demonstrates the superior performance of Ba@Ga-MOF in inhibiting tumor cell proliferation. To further verify the ability of Ba@Ga-MOF to induce apoptosis in LLC tumor cells, we used flow cytometry for precise detection. The results of flow cytometry detection of apoptosis showed significant differences among the treatment groups. When baicalein was used alone, the apoptosis induction rate was 14.2%; the apoptosis induction rate of Ga-MOF was 17.49%; while the apoptosis induction rate of Ba@Ga-MOF was as high as 29.43%. The gradual increase in the apoptosis rate strongly indicates that this nanocomposite can synergistically activate cell death pathways, providing a new mechanism of action for anti-tumor therapy.

[0048] Example 3: Effects of different treatments on tumor cell migration, invasion, and viability This embodiment tests the effects of different treatments on tumor cell migration, invasion, and viability, and the steps are as follows: 1. Calcein-AM / PI live / dead cell staining: LLC cells were spaced at 1 × 10⁶ cells per well. 5 Cells were seeded at a density of 100 μg / mL in 6-well plates and cultured overnight to allow adhesion. Then, the cells were treated with Ba@Ga-MOF or Ga-MOF at a concentration of 100 μg / mL for 8 hours. After treatment, staining working solution containing 2.0 μM Calcein-AM and 4.5 μM PI was added, and the cells were incubated in the dark for 20 minutes. The cells were then washed 1-2 times with PBS to remove excess stain. Afterward, the cells were observed and photographed under a confocal laser scanning microscope (CLSM). The untreated group served as a control.

[0049] 2. Cloning experiment: LLC cells were seeded at low density of 400-1000 cells per well in 6-well plates and cultured at 37°C, 5% CO2 for 14 days (avoid moving the plates and change the medium regularly during the culture period). At the end of the culture, discard the medium, gently wash the cells with PBS for 1-2 times, add appropriate amount of fixation solution (4% paraformaldehyde) to fix the cells at room temperature for 15-30 minutes, discard the fixation solution and wash with PBS or deionized water for 1-2 times, then add appropriate amount of staining solution (such as 0.1% crystal violet solution or Giemsa staining solution) to stain the cells at room temperature for 15-30 minutes, discard the staining solution and gently rinse with running water until the background is clear, then dry at room temperature, finally observe under a microscope and count the number of colonies (usually defined as cell clusters with >50 cells) formed in each well using counting software or manual counting.

[0050] 3. Wound healing (wound closure) experiment: LLC cells were seeded into culture plates (such as 6-well plates or 24-well plates) and cultured to 90-100% confluence, then use a 200 μL sterile pipette tip to draw a straight line scratch on the monolayer cells perpendicular to the plate bottom (2-3 parallel lines per well are recommended), after gently washing with PBS for 2-3 times to remove the cell debris, replace with serum-free medium containing 1% FBS (or lower) (BaGa-MOF is added to the experimental group, and no addition to the control group). At 0 hours (immediately after scratching) and after 24 or 48 hours of culture, take pictures of the scratch in the same fixed field, and use image analysis software to measure the scratch width and calculate the wound healing rate (healing rate (%) = [(0 hour width - T hour width) / 0 hour width] x 100%).

[0051] 4. Migration experiment (Transwell): Transwell chamber (8 pm pore polycarbonate membrane) with uncoated Matrigel was used. LLC cells were prepared into serum-free medium suspension, Ba@Ga-MOF was added into the suspension in the upper chamber of the experimental group at a concentration of 100 pg / mL (based on the Ga-MOF content), and no drug was added into the suspension in the upper chamber of the control group. The medium containing 10% FBS was added into the lower chamber as a chemical attractant. The culture was carried out at 37°C and 5% CO2 for 24 hours. After the culture, the medium and un-migrated cells in the upper chamber were carefully removed, the cells on the upper surface of the membrane in the upper chamber were gently wiped off with a cotton swab, the cells migrated to the lower surface of the membrane were fixed with 4% paraformaldehyde for 10-15 minutes, then dyed with 0.1% crystal violet solution for 15-20 minutes, and then gently washed with PBS or deionized water to remove excess dye, and then dried at room temperature. Finally, the number of cells migrated to the lower surface of the membrane was counted under an optical microscope by randomly selecting multiple fields and taking pictures.

[0052] 5. Invasion experiment (Transwell): An invasion chamber (8 pm pore polycarbonate membrane) was used, in which the upper surface of the membrane in the upper chamber was coated with diluted Matrigel (1:8 diluted in serum-free cold culture medium) in advance. The coated chamber was placed in a 37°C incubator for 1-2 hours to allow the Matrigel to solidify. LLC cells were prepared into serum-free medium suspension, Ba@Ga-MOF was added into the suspension in the upper chamber of the experimental group at a concentration of 100 pg / mL (based on the Ga-MOF content), and no drug was added into the suspension in the upper chamber of the control group. The medium containing 10% FBS was added into the lower chamber as a chemical attractant. The culture was carried out at 37°C and 5% CO2 for 48 hours (usually longer than the migration experiment). After the culture, the medium and un-invasive cells in the upper chamber were carefully removed, the cells on the upper surface of the membrane in the upper chamber were gently wiped off with a cotton swab, the cells invaded to the lower surface of the membrane through the Matrigel matrix and the membrane were fixed with 4% paraformaldehyde for 10-15 minutes, then dyed with 0.1% crystal violet solution for 15-20 minutes (the steps were consistent with the migration experiment), and then gently washed with PBS or deionized water to remove excess dye, and then dried at room temperature. Finally, the number of cells invaded to the lower surface of the membrane was counted under an optical microscope by randomly selecting multiple fields and taking pictures.

[0053] The results of the effects of different treatments on tumor cell migration, invasion and viability are shown in Table 1. Figure 3

[0054] Migration and invasion experiments Figure 3 ​A, 3B-C) revealed the strong potential of Ba@Ga-MOF in inhibiting tumor metastasis. Ba@Ga-MOF treatment significantly reduced the relative number of migrated cells to about 22.2% ( Figure 3 B) and invaded cells to about 36.4% ( Figure 3 C), compared to the control group set at 100%. This significant change fully demonstrated that Ba@Ga-MOF could effectively inhibit the migration and invasion ability of tumor cells, providing strong experimental evidence for blocking tumor metastasis. Scratch healing test ( Figure 3 D-E) verified the anti-tumor effect of Ba@Ga-MOF from another perspective. After 24 hours, the relative wound closure area of the Ba@Ga-MOF group was significantly reduced to about 10%, while the control group was 95% ( Figure 3 E). This result intuitively showed the inhibitory effect of Ba@Ga-MOF on tumor cell migration and repair ability. Live and dead cell fluorescence imaging ( Figure 3 F) provided intuitive visual evidence to verify the enhanced anti-tumor killing activity of Ba@Ga-MOF. By observing the weakening of green fluorescence (live cells) and the enhancement of red signal (dead cells), we can clearly see the death of tumor cells. The proportion of PI positive (dead cells) was significantly increased from about 5% in the control group to about 55% in the Ba@Ga-MOF group ( Figure 4 G). Further confirmed the significant effect of Ba@Ga-MOF in killing tumor cells.

[0055] In summary, through a series of systematic experiments, we comprehensively and deeply studied the in vitro anti-tumor effect of Baikal skullcap root and Ba@Ga-MOF composite nanovaccine. These rich and powerful experimental results collectively demonstrate that Ba@Ga-MOF is a highly promising nanotherapeutic agent with multi-mechanism anti-tumor effect, including significant proliferation inhibition, efficient apoptosis induction, and strong metastasis blocking ability, bringing new hope and possibility to the field of tumor treatment.

[0056] Example 4 Subcellular localization and in vivo biodistribution kinetics of nanoparticles To verify the endoplasmic reticulum (ER) targeting ability of Ba@Ga-MOF, this study adopted multi-fluorescence labeling combined with laser confocal microscopy technology. Specifically, Hoechst 33342 was used to dye the cell nucleus blue, ER-Tracker Green was used to dye the ER structure green, and Rhodamine B was used to fluorescently label Ba@Ga-MOF red. Observations at 4, 8, and 16 hour time points ( Figure 4A) shows time-dependent difference in distribution: almost no nanoparticles enter the ER at 4 hours, significant accumulation at 8 hours, and strong red-green fluorescence overlap (indicating ER colocalization) at 16 hours. Quantitative analysis (Pearson correlation coefficient (r) and overlap coefficient) confirms the time-dependent ER targeting efficiency. Figure 4 B) shows that the colocalization indicator gradually increases: the Pearson correlation coefficient (r) increases from 0.65 (4h) to 0.83 (16h), and the overlap coefficient increases from 0.77 to 0.90, confirming the time-dependent ER targeting efficiency. To investigate the specific accumulation of nanoparticles in tumors, a cryoablation intervention experiment was designed. The experimental procedure is as follows: After establishing a C57 mouse subcutaneous tumor model, the tumor-bearing mice were randomly divided into a cryoablation group and an untreated control group; a commercial cryoablation system was used to subject the unilateral tumor of the cryoablation group of mice to -168°C ultralow-temperature freezing (for 3 minutes), and the control group did not receive any intervention; immediately after the ablation treatment, rhodamine B-labeled Ba@Ga-MOF nanoparticles were injected into the tumor tissue of all mice by intratumoral injection; at 0, 8, 16, and 24 hours after injection, the whole-body biodistribution was dynamically monitored using a PerkinElmer IVIS Spectrum in vivo imaging system (USA), and the fluorescence intensity data of the bilateral tumor regions were collected and the time course was recorded; at the 24-hour end time, all mice were sacrificed, and the heart, liver, spleen, lung, kidney, and bilateral tumor tissues were systematically removed for ex vivo fluorescence imaging, and the fluorescence intensity ratio of the tumor to the organs was quantitatively analyzed to verify the enhanced effect of cryoablation on the tumor-targeted accumulation of nanoparticles.

[0057] Comparison of nanoparticle distribution before and after cryoablation Figure 4 C) shows that tumor accumulation is significantly enhanced after ablation. Fluorescence intensity quantification Figure 5 D) shows that nanoparticles preferentially accumulate in tumor tissue and the lung, and tumor-related fluorescence significantly increases after cryoablation.

[0058] These results collectively indicate that Ba@Ga-MOF not only exhibits inherent ER targeting properties, but also achieves enhanced tumor-selective accumulation through cryoablation-assisted delivery, highlighting its potential in ER-targeted and tumor-selective therapeutic applications.

[0059] Example 5 Molecular mechanisms of nanoparticle-induced ICD, ferroptosis, and ER stress After confirming the efficient cellular uptake and tumor-targeted accumulation of Ba@Ga-MOF, we further investigated the molecular mechanisms of its synergistic anti-tumor effect with cryoablation. To comprehensively explore whether Ba@Ga-MOF combined with cryoablation can produce a synergistic anti-tumor effect, we performed a Western blot experiment. As shown in Figure 5As shown in Figure A, the combined treatment of Ba@Ga-MOF and cryoablation significantly enhanced the antitumor effect through three pathways—ferroptosis, endoplasmic reticulum (ER) stress, and immunogenic cell death (ICD). Figure 5 As shown in Figure B, in the ferroptosis pathway, the expression level of the pro-ferroptosis marker ACSL4 was significantly upregulated compared to the control group; while the expression of the key anti-ferroptosis proteins SLC7A11 and GPX4 was significantly downregulated. This indicates that nanoparticles induce iron-dependent cell death through enhanced lipid peroxidation mediated by ACSL4 and redox homeostasis disorder caused by the imbalance between SLC7A11 and GPX4. In the ER stress pathway, the expression level of the molecular chaperone GRP78 was increased, and the expression of the pro-apoptotic transcription factor CHOP was also significantly increased. This confirms that the combination therapy can activate the unfolded protein response (UPR) and trigger downstream apoptosis signaling pathways. In the ICD pathway, the exposure rate of cell surface calreticulin (CRT) was 4.3 times that of the control group, and the release of extracellular high-mobility group box 1 (HMGB1) increased by 3.7 times. This indicates that nanoparticles can effectively induce immunogenic tumor cell death, laying the foundation for subsequent recruitment of immune cells. Quantitative statistical analysis ( Figure 6 C-5I further showed that the expression changes of all key proteins in these three pathways were statistically significant. In summary, this combination therapy achieves a multidimensional synergistic anti-tumor effect through ferroptosis-mediated lipid metabolism disorders, ER stress-triggered mitochondrial apoptosis, and ICD-activated immune responses, providing a new strategy for comprehensive cancer treatment through multi-mechanism synergistic action.

[0060] Example 6: Multidimensional assessment of nanoparticle-induced oxidative stress and ICD To systematically elucidate the synergistic antitumor mechanism of Ba@Ga-MOF combined with cryoablation, this study constructed a multidimensional experimental validation system. The experimental steps are as follows: LLC cells were spaced at 1 × 10⁻⁶ cells per well. 5 Cells were seeded at a density of 100 μg / mL in 6-well plates and cultured at 37°C and 5% CO2 for 8 hours. After adhesion, the cells were divided into two groups: the experimental group was treated with Ba@Ga-MOF to a final concentration of 100 μg / mL (based on Ga-MOF content), and the control group consisted of a blank group (no treatment) and a solvent control group (equal volume of dissolved solvent). Each group had 3 replicates. After treatment, the following parameters were measured: To detect the changes of intracellular reactive oxygen species (ROS) levels in different treatment groups, DCFH-DA staining method was used: After BaGa-MOF treatment, the culture medium in the 6-well plate was carefully aspirated, and the cells were gently washed with preheated phosphate buffer solution (PBS) at 37°C for 3 times, 1-2 minutes each time, to completely remove the residual culture medium. Then 10 μM DCFH-DA was added to each well of serum-free medium, and incubated at 37°C, 5% CO2, avoiding light for 20 minutes. After incubation, the staining solution was aspirated, and preheated serum-free medium was used to wash 3 times to remove DCFH-DA that did not enter the cells. The samples were placed on the confocal microscope stage, and observed using the FITC channel, 488 nm excitation light. At least 10 different fields were selected under low magnification, and images were taken under high magnification, and the fluorescence intensity was quantitatively analyzed using ImageJ software to reflect the production of intracellular ROS.

[0061] To detect changes in intracellular calcium homeostasis, Fluo-4 probe (excitation wavelength 494 nm) was used, and the operation steps were the same as those for ROS detection. The intracellular free calcium ion concentration and calcium homeostasis were evaluated by changes in fluorescence signal. For the detection of calreticulin (CRT, excitation wavelength 495 nm) and high mobility group protein B1 (HMGB1, excitation wavelength 590 nm), the staining steps were consistent with those for ROS detection. The protein expression level was quantitatively analyzed by fluorescence intensity at a specific excitation wavelength, and the subcellular distribution characteristics were observed by fluorescence localization.

[0062] As shown in Figure 6 A-B, DCFH-DA probe detection results showed that the intracellular ROS level of the combined treatment group was significantly higher than that of the single treatment group. Fluo-4 calcium imaging further confirmed that this synergistic effect could induce calcium homeostasis imbalance related to ER stress. Immunofluorescence co-localization analysis showed that this treatment significantly up-regulated the membrane localization expression of damage-associated molecular pattern (DAMP) marker CRT ( Figure 6 C) and HMGB1 ( Figure 6 D). Quantitative analysis showed that the fluorescence intensity of CRT and HMGB1 in the combined treatment group was 3.2 times and 2.8 times that of the control group ( Figure 6 E). Based on the above findings, we established an in vitro dendritic cell (DC) maturation model to verify the ICD effect. The experimental steps are as follows: First, Lewis lung cancer cells (LLC) were cultured to the logarithmic growth phase and divided into four treatment groups: control group (only fresh medium was replaced), simulated freeze-thaw group (repeated freeze-thaw cycles in liquid nitrogen, controlled to a certain extent), Ba@Ga-MOF group (final concentration 100 μg / mL, based on Ga-MOF content), and Ba@Ga-MOF+Cry group. After treatment, the treatment medium was removed, and fresh low-serum medium was replaced for 24-48 hours of further culture. The supernatant was collected, centrifuged, and filtered to obtain cell-free conditioned medium (CM). Simultaneously, hematopoietic stem cells were isolated from mouse bone marrow and induced to differentiate for 6-8 days in a medium containing GM-CSF (20 ng / mL) and IL-4 (10 ng / mL) to obtain immature dendritic cells (BMDCs). Subsequently, BMDCs were seeded in 24-well plates (1×10⁻⁶). 6 Cells / mL were added to 50% (v / v) LLC conditioned medium for each group (control group CM, cryoablated CM, Ba@Ga-MOF CM, and combined CM) and stimulated for 24-48 hours. After stimulation, BMDCs were collected, Fc receptors were blocked with anti-CD16 / 32, and surface staining was performed with anti-CD11c-APC, anti-CD80-FITC, and anti-CD86-PE antibodies, respectively. Unstained, single-stained, and isotype controls were also included. Finally, the proportion of CD80+CD86+ double-positive mature DCs in CD11c+ cells was analyzed by flow cytometry, and the significance of differences between groups was statistically analyzed using FlowJo software to verify whether the combined treatment promotes DC maturation by enhancing DAMPs release.

[0063] Flow cytometry analysis showed ( Figure 7 (F) Compared to Ba@Ga-MOF alone or cryoablation, the combination therapy group significantly promoted the expression of mature DC markers (CD80+CD86+). The DC maturation efficiency reached 25.4±3.1%, 7.8 times that of the control group. This enhanced DC maturation efficiency showed a strong positive correlation, confirming that the synergistic therapy effectively activated innate immunity through enhanced DAMP release. In summary, this experimental evidence constitutes a coherent framework of action: the synergistic effect of Ba@Ga-MOF and cryoablation induces the classic ICD phenotype in tumor cells through dual regulation of oxidative stress (ROS burst) and ER stress (calcium overload), thereby activating dendritic cell-mediated adaptive immune responses. This multi-target synergistic mechanism provides a theoretical basis for developing novel tumor immunotherapy strategies.

[0064] Example 7: Evaluation of the inhibitory effect of nanoparticles combined with cryoablation on primary and distant tumors in mice. To systematically evaluate the anti-tumor effect of nanoparticle combined with cryoablation therapy in a mouse model, we focused on its effect on primary tumors and distant metastatic tumors. Experimental design Figure 7 A) included six treatment groups: saline group, Ga-MOF group, Baatin group, Ba@Ga-MOF group, cryoablation group, and Ba@Ga-MOF+Cry group, with a standardized dosing regimen and cryoablation intervention on day 6 Figure 7 E shows the visual operation process and temperature change, which dropped from about 16 °C to -168 °C). Tumor volume and mouse weight were monitored daily Figure 7 B, D) The results showed that the Ba@Ga-MOF+Cry group exhibited the most superior tumor inhibition effect. In contrast, the Ga-MOF group, Baatin group, Ba@Ga-MOF group, and cryoablation group only showed limited inhibition, with tumor inhibition rates of 19.47% ± 7.1%, 33.48% ± 8.35%, 55.52% ± 5.48%, and 83.77% ± 1.61%, respectively. These effects can be attributed to Ga-MOF-mediated ferroptosis, Baatin-induced endoplasmic reticulum stress, and direct cell rupture caused by cryoablation. While Ba@Ga-MOF+Cry combination therapy exhibited significant synergistic anti-tumor effects and immune responses, with an average inhibition rate as high as 96.68%. Tumors harvested and weighed at the end of treatment Figure 7 D, F) further confirmed the superior efficacy of this combination therapy. Although the Ba@Ga-MOF+Cry group had a primary tumor volume close to zero due to extensive necrosis, the remaining necrotic tissue still had a measurable weight of 0.13 ± 0.14 g. Compared with the saline group (1.18 ± 0.18 g), Ga-MOF group (0.90 ± 0.13 g), Baatin group (0.71 ± 0.11 g), Ba@Ga-MOF group (0.58 ± 0.09 g), and cryoablation group (0.39 ± 0.11 g), the tumor in the Ba@Ga-MOF+Cry group was almost completely eliminated. This significant regression was attributed to the combined effects of ferroptosis, endoplasmic reticulum stress, and nanoparticle-enhanced cryoablation immunotherapy. There were no significant changes in the body weight of mice in all treatment groups Figure 7 D), indicating that the treatment had good biological safety. Notably, this combination therapy also induced a clear "abscopal effect," causing distant tumor growth to be inhibited by 75% ± 6.8% (E) Figure 9 G-M), indicating that systemic anti-tumor immunity was effectively activated.

[0065] These comprehensive findings indicate that nanoparticle-enhanced cryoablation synergistically improves local tumor control capacity through immediate cryodestruction and nanoparticle-mediated ICD. In addition, this therapy successfully activated systemic antitumor immunity, effectively suppressing metastatic progression, thus providing solid preclinical evidence for clinical translation of this combined cancer treatment modality.

[0066] Example 8 Impact of nanoparticle combination therapy on mouse systemic and tumor microenvironment (TME) To systematically evaluate the synergistic anti-tumor effect of Ba@Ga-MOF combined with cryoablation (Cry) and its immunomodulatory role, a comprehensive in vivo evaluation was performed, including survival analysis, serum cytokine profiling, and tumor microenvironment (TME) characterization. Kaplan-Meier survival analysis showed that the Ba@Ga-MOF + Cry combination group exhibited the most significant survival advantage, with a median survival of over 60 days, significantly longer than the saline control group (46 days), baicalein group (50 days), Ga-MOF group (52 days), Ba@Ga-MOF group (56 days), and cryoablation alone group (59 days). This survival advantage was closely related to the strong systemic immune activation observed. Multiple cytokine detection confirmed this systemic immune activation phenomenon. Ba@Ga-MOF + Cry combination therapy significantly upregulated key pro-inflammatory cytokines in serum: IFN-γ (4-fold increase over the control group, a core effector cytokine in anti-tumor immunity, which can activate macrophages and enhance antigen presentation); IL-6 (2.5-fold increase, involved in acute phase response and T cell regulation); TNF-α (3-fold increase, with direct cytotoxicity to tumor cells and pro-inflammatory effects); IL-1β (1.5-fold increase, mediates inflammatory response and neutrophil recruitment); MCP-1 (3.5-fold increase, mainly chemoattracting monocytes / macrophages to inflammatory sites). The significant synergistic upregulation of these cytokines collectively indicates a strong systemic anti-tumor immune response. Further immunofluorescence analysis of tumor tissue revealed key mechanistic features: extensive TUNEL+ apoptosis areas co-localized with calreticulin (CRT) surface exposure spaces, with cytoplasmic translocation of high mobility group box 1 (HMGB1), providing strong evidence for immunogenic cell death (ICD). At the same time, CHOP protein expression was significantly increased, indicating that the combination therapy triggered sustained endoplasmic reticulum (ER) stress. Combining the significant survival advantage, strong systemic immune activation (manifested as 1.5 to 4-fold upregulation of key pro-inflammatory cytokines), and CRT / HMGB1 co-localization in the tumor microenvironment (as an ICD marker) and enhanced CHOP expression (as an ER stress marker), this study established a triple synergistic anti-tumor cascade of the Ba@Ga-MOF + Cry strategy: 1. Direct cytotoxicity provided by cryoablation, inducing rapid tumor cell apoptosis / necrosis; 2. Nanoparticle-mediated ER stress driving ICD through CRT exposure and HMGB1 release, enhancing the immunogenicity of tumor antigens; 3. Cytokine-mediated systemic immune activation (such as IFN-γ, IL-6, MCP-1 pathways), triggering a systemic anti-tumor immune response.

[0067] In summary, based on the remarkable 60-day survival improvement and multi-parameter immunological evidence (significant serum cytokine upregulation, ICD marker co-localization in TME, enhanced ER stress markers), this study clearly demonstrates that the Ba@Ga-MOF + Cry strategy combines local physical ablation with systemic immunotherapy—cryoablation provides initial tumor killing and antigen release, while nanoparticles amplify ICD via ER stress—highlighting the great potential of this strategy as a transformative combination therapy.

[0068] Example 9 Regulation of tumor immune microenvironment and systemic immune response by nanoparticle therapy Flow cytometry analysis results Figure 9 A-F) showed that Ba@Ga-MOF combined with cryoablation (Ba@Ga-MOF + Cry) exhibited a remarkable synergistic effect in systemically regulating the immunosuppressive tumor immune microenvironment (TIME). Compared with the saline group, the Ba@Ga-MOF + Cry group significantly reduced the proportion of CD25⁺FoxP3⁺ regulatory T cells (Tregs) in the spleen (by about 16.98%) and in tumor tissue (by 9.93%). This inhibitory effect on Tregs was significantly better than that of the Ba@Ga-MOF monotherapy group (14.19% reduction in the spleen and 6.06% reduction in tumor tissue) and the cryoablation alone group (Cry group) (15.28% reduction in the spleen and 8.69% reduction in tumor tissue). At the same time, this combined therapy significantly inhibited the infiltration of CD11b⁺Gr-1⁺ myeloid-derived suppressor cells (MDSCs), reducing them by 6.8% in the spleen and 1.93% in tumor tissue. This inhibitory effect was also better than that of the monotherapy group (3.3% reduction in the spleen and 0.77% reduction in tumor) and the cryoablation group (4.2% reduction in the spleen and 1.1% reduction in tumor), especially in the inhibition of spleen MDSCs. In terms of enhancing adaptive immune response, compared with the saline group, the proportion of CD4⁺ and CD8⁺ T cells in the spleen of the combined therapy group increased significantly by 25.42%, while the infiltration rate of CD4⁺ and CD8⁺ T cells in tumor tissue increased from 8.99% to 12.98% (an increase of 3.99%). In summary, these results collectively confirmed that Figure 10 G): Ba@Ga-MOF combined with cryoablation therapy reverses the immunosuppressive tumor immune microenvironment (TIME) by dual inhibition of Tregs and MDSCs, and effectively promotes T cell activation and infiltration, providing a solid experimental basis for the optimization of immunomodulatory-based anti-tumor combination therapy strategies.

[0069] Example 10 Transcriptome sequencing and differential gene analysis results This study systematically elucidated the molecular mechanism of Ba@Ga-MOF combined with cryoablation therapy using transcriptome sequencing technology, focusing on revealing the biological basis of the synergistic effect between nanomaterials and physical therapy. Figure 10 The volcano plot visualization analysis in Figure A showed significant differences in gene expression between the combined treatment group and the control group, with 658 genes significantly upregulated (|log2FC|>1, p<0.05) and 450 genes significantly downregulated. This provides important clues for revealing the molecular regulatory network behind the synergistic therapy. This was achieved by constructing a protein-protein interaction network (…). Figure 10 B), differentially expressed genes form a highly interconnected network, with key nodes corresponding to multiple key signaling pathways. KEGG pathway analysis ( Figure 10 C) Further analysis revealed significant enrichment of pathways such as glutathione metabolism, endoplasmic reticulum protein processing, and antigen presentation (p<0.01), suggesting that the combination therapy may exert its synergistic effect by activating the ER stress response and ICD pathway. GO functional enrichment analysis ( Figure 10 D) This validated that ferroptosis, ER stress, and immune regulation were significantly enriched across three dimensions: molecular function, biological process, and cellular component (FDR < 0.05). Gene set enrichment analysis (GSEA) results ( Figure 11 E) Further confirmation that pathways such as glutathione metabolism, ER stress, and antigen presentation are significantly positively correlated with treatment efficacy indicates that these pathways play a key role in the synergistic effect of combination therapy.

[0070] In summary, this study reveals the molecular mechanism by which Ba@Ga-MOF combined with cryoablation achieves its anti-tumor effects through multi-pathway synergistic regulation. This treatment activates the glutathione metabolic pathway, thereby inducing oxidative stress, which in turn triggers programmed cell death via the ER stress response. Furthermore, this therapy enhances antigen presentation efficiency, thereby activating adaptive immune responses. These findings provide an important theoretical foundation and technical pathway for developing novel tumor combination therapy strategies based on metal-organic framework (MOF) nanovaccines.

[0071] Example 11: Evaluation of the inhibitory effect and biosafety of distant lung metastases Ba@Ga-MOF exhibits significant advantages in inhibiting tumor metastasis and in terms of biosafety. For example... Figure 11A, HE staining pathological results showed that the lung of blank control group mice appeared extensive and obvious metastatic foci, indicating that the tumor metastasis process was highly active without intervention. The baicalein treatment group alone could effectively reduce the volume of metastatic foci, indicating that it had certain independent anti-metastatic ability. The inhibition effect of the cryoablation group and the Ba@Ga-MOF group was similar, and it was speculated that cryoablation achieved therapeutic effect by destroying tumor cell structure at low temperature, while Ba@Ga-MOF may inhibit the metastasis process by synergistic effect.

[0072] Most importantly, the combination therapy group of cryoablation and Ba@Ga-MOF completely eliminated the lung metastatic foci. This synergistic effect is due to the fact that cryoablation destroys the tumor microenvironment (such as blood vessel blockage, cell freeze-thaw injury) through a physical method, creating more efficient targeting conditions for nanoparticles (such as enhanced drug penetration and retention), while Ba@Ga-MOF further blocks the continuous process of tumor metastasis at the molecular level, forming a multiple mechanism of “physical destruction + nano immunity”. For example, ​ B, histological analysis showed that the main organs (heart, liver, spleen, kidney) of mice treated with Ba@Ga-MOF did not show pathological changes, and the tissue structure was normal. The cell morphology of all organs was normal, and no toxic reactions such as cell swelling, lipid vacuolization or nuclear fragmentation were found. There were no inflammatory cell infiltration, hemorrhage or tissue necrosis in the parenchymal and interstitial areas. The liver structure was complete, the liver lobules showed clear plate-like structure, and the liver cell cords were arranged in order without fatty degeneration or fibrosis. The glomerular filtration barrier structure in the kidney was complete, and the renal tubular epithelial cells were not atrophic or exfoliated, and there was no sign of interstitial fibrosis. The heart and spleen also showed no pathological changes such as inflammation or fibrosis, and the myocardial fibers were arranged in an orderly manner, and the boundary between red pulp and white pulp of the spleen was clear.

[0073] Overall, Ba@Ga-MOF achieved complete elimination of lung metastatic foci by synergistic cryoablation, and its combination therapy strategy successfully integrated the immediate effect of physical ablation and the molecular regulation advantage of nano-drugs. At the same time, the nanoparticle showed high biocompatibility in vivo and did not cause toxic reactions to important organs, which may be closely related to the good biocompatibility of the material itself (such as the stable properties of metal organic framework structure). This study not only provides an innovative combination therapy for the precise treatment of tumor metastasis, but also verifies the application potential of Ba@Ga-MOF as a safe and efficient drug carrier, especially in the clinical transformation of solid tumor metastasis, which has broad prospects.

[0074] In summary, Ba@Ga-MOF composite nanoparticle vaccine has the following advantages: (1) It has high drug loading capacity and controllable release characteristics, and can accurately target tumor cells. Its porous organic framework structure can effectively accommodate and load a large number of drug molecules, and achieve controllable release of drugs under the trigger of tumor microenvironment, improve the concentration of drugs in tumor sites, enhance the treatment effect, and reduce the side effects on normal tissues.

[0075] (2) Through multiple mechanisms of gallium ions and baicalin, the anti-tumor effect is significantly enhanced, and the tumor growth and metastasis are inhibited. The iron death induced by gallium ions and the endoplasmic reticulum stress and immunogenic cell death triggered by baicalin synergize with each other, killing tumor cells from multiple pathways, not only directly leading to tumor cell death, but also releasing tumor antigens, activating the immune system, and forming a persistent immune memory, effectively inhibiting the recurrence and metastasis of tumors.

[0076] (3) Cooperate with cryoablation technology to activate immunogenic cell death (ICD) and prolong and enhance anti-tumor immune response. Cryoablation technology can quickly destroy tumor cells and release tumor antigens, while Ba@Ga-MOF composite nanovaccine can further enhance the ICD effect, promote the release of antigens and the activation of the immune system, making the anti-tumor immune response more persistent and strong, providing long-term immune protection for the body.

[0077] (4) Reverse the immunosuppressive tumor microenvironment, promote the maturation of dendritic cells and T cell infiltration, and form a persistent anti-tumor immune memory. Combination therapy can significantly change the immune status of the tumor microenvironment, reduce the number of immunosuppressive cells (such as regulatory T cells and myeloid-derived suppressor cells), while increasing the infiltration of DC cells and T cells, promoting the interaction between immune cells, enhancing immune response, and ultimately forming a persistent anti-tumor immune memory, providing long-term anti-tumor ability for the body.

[0078] (5) It has good biological safety and no obvious toxic side effects. In mouse experiments, detailed histopathological examination and blood biochemical index detection found that Ba@Ga-MOF nanoparticles did not cause significant damage to major organs, indicating that its metabolism and clearance process in the body is safe and reliable, and has good biocompatibility, providing a theoretical basis for clinical application.

[0079] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and are not limited thereto; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can modify the technical solutions described in the foregoing examples, or make equivalent substitutions for some of the technical features; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A composite nanovaccine, characterized in that, It is a natural compound with endoplasmic reticulum stress induction ability coupled on the surface of Ga-MOF nanoparticles.

2. The composite nanovaccine of claim 1, wherein, The natural compound with endoplasmic reticulum stress induction ability includes baicalein, curcumin or resveratrol. And / or, the surface of the Ga-MOF nanoparticles is also modified with a ligand or a functional group with targeting function.

3. The composite nanovaccine according to claim 1 or 2, characterized in that, The preparation method includes: mixing and reacting gallium salt and organic ligand in an organic solvent to prepare Ga-MOF nanoparticles; introducing a functional group into the Ga-MOF nanoparticles through amidation reaction (so that the surface of the Ga-MOF nanoparticles has active sites for binding with the natural compound) to prepare Ga-MOF nanoparticles modified with the functional group; and mixing and reacting the Ga-MOF nanoparticles modified with the functional group with the natural compound with endoplasmic reticulum stress induction ability to prepare the composite nano-vaccine.

4. The composite nanovaccine of claim 3, wherein, The gallium salt includes gallium nitrate; and / or, the organic ligand includes bis(3,5-dicarboxyphenyl)azo; and / or, the organic solvent is a mixture of acetonitrile and dimethyl sulfoxide.

5. The composite nanovaccine of claim 4, wherein, The temperature for mixing and reacting in the organic solvent is 110-130°C; preferably, the mixing and reacting in the organic solvent is carried out under the condition that the magnetic stirrer continuously stirs, preferably for more than 3h.

6. The composite nanovaccine of claim 3, wherein, The step of introducing the functional group into the Ga-MOF nanoparticles through amidation reaction includes: mixing and reacting the Ga-MOF nanoparticles with EDC and NHS to prepare Ga-MOF nanoparticles modified with the functional group.

7. The composite nanovaccine of claim 6, wherein, The natural compound with endoplasmic reticulum stress induction ability and p-toluenesulfonamide are dissolved in DMSO to prepare a mixture, and the mixture is mixed and reacted with the Ga-MOF nanoparticles modified with the functional group to prepare the composite nano-vaccine.

8. The composite nanovaccine according to any one of claims 1 to 7, wherein, The particle size thereof is in the range of 100-200 nm.

9. A composition characterized in that, It includes the composite nano-vaccine according to any one of claims 1-8 and an immunomodulator. Preferably, the immunomodulator includes a PD-1 inhibitor.

10. Use of the composite nano-vaccine according to any one of claims 1-8 or the composition according to claim 9 in the preparation of a pharmaceutical product; preferably, the pharmaceutical product is used for treating cancer or tumor by combined cryoablation technology; preferably, the cancer is lung cancer.

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