Anti-tumor nano vaccine, preparation method and application thereof

Abstract

This invention relates to the field of tumor immunotherapy technology, specifically to an anti-tumor nanovaccine, its preparation method, and its application. The anti-tumor nanovaccine comprises a metal-organic framework nanosheet formed by a tetra(4-carboxyphenyl)porphyrin ligand centrally chelated with divalent manganese ions and coordinated with manganese ions, and a ROCK inhibitor and tumor antigen loaded on the nanosheet. The nanovaccine provided by this invention can promote precise recognition of antigens and antigen-presenting cells, effectively initiate innate and adaptive immune responses, thereby enhancing and maintaining the positive feedback of the cancer-immune cycle, enhancing the aggregation and maturation of antigen-presenting cells in lymph nodes, precisely maintaining the synergistic effect between antigens and immune cells in the cancer-immune cycle, thus enhancing the therapeutic effect of the tumor vaccine, and exhibiting high biosafety.

Description

Technical Field

[0001] This invention relates to the field of tumor immunotherapy technology, specifically to an anti-tumor nanovaccine, its preparation method, and its application. Background Technology

[0002] In recent years, cancer vaccines have attracted much attention due to their ability to stimulate active immune responses, effectively inhibit tumor growth, and reduce the risk of tumor metastasis and recurrence. These vaccines exert cytotoxic effects by utilizing the cancer-immune cycle. In the tumor microenvironment, dendritic cells (DCs) first capture antigens released by the tumor. These antigens are then taken up and processed by antigen-presenting cells (APCs) to generate antigen peptide-MHC complexes. The initiation of the T-cell immune response is achieved through the binding of T-cell receptors (TCRs) to the antigen peptide-MHC complexes on APCs, as well as the interaction between co-stimulatory molecules. In the later stages of the cancer-immune cycle, activated cytotoxic T lymphocytes (CTLs) migrate to and infiltrate the tumor site via the bloodstream, leading to the clearance of cancer cells and the production of tumor-associated antigens. These antigens are further recognized by APCs, thereby achieving a positive cycle of the immune system. However, the effectiveness of the antitumor immune cycle depends on repeated and precise cooperation and maintenance among immune cells (especially antigen-producing cells, cellular progenitor cells, and tumor antigens). When antigen presentation, phagocytosis, and recognition by APCs are inhibited, tumor cells can evade immune system-mediated destruction by downregulating major histocompatibility complex class I (MHC-I) molecules, and may even promote pro-tumor inflammatory progression. Therefore, precisely and efficiently maintaining the antigen processing, presentation, and phagocytosis of APCs is crucial for the successful execution of the cancer-immune cycle.

[0003] One approach to developing tumor vaccines currently utilizes TLR receptor agonists, such as the TLR3 agonist PolyI:C, the TLR4 agonist bacterial lipopolysaccharide (LPS), the TLR7 / 8 agonists R848 / R837, and the TLR9 receptor agonist CpG oligodeoxynucleotides. These agonists mimic pathogen invasion and activate APCs, thereby triggering an innate immune response and promoting the differentiation of various helper T cell (Th) subsets by releasing related cytokines. In this way, stimulation enhances the adaptive immune response, ultimately improving anti-tumor efficacy. Another approach involves studying tumor vaccine delivery systems composed of liposomal nanoparticles and TLR agonists to enhance vaccine responsiveness by promoting APC activation and antigen uptake. However, these strategies still require further refinement because they only focus on one aspect of the tumor-immune cycle, neglecting the re-recognition of neoantigens by circulating immune surveillance cells.

[0004] Therefore, building a multifunctional platform that can synergistically improve the killing efficiency of the entire cancer-immune cycle, including improving the activation, cross-presentation, and repeated uptake and recognition of neoantigens of APCs, remains crucial and challenging. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides an anti-tumor nanovaccine, its preparation method, and its applications. The nanovaccine provided by this invention can promote precise recognition of antigens and antigen-presenting cells, effectively initiate innate and adaptive immune responses, thereby enhancing and maintaining the positive feedback loop of the cancer-immune cycle, and enhancing the aggregation and maturation of antigen-presenting cells in lymph nodes. This precisely maintains the synergistic effect between antigens and immune cells in the cancer-immune cycle, thus enhancing the therapeutic effect of the tumor vaccine, and exhibiting high biosafety.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides an anti-tumor nanovaccine comprising a metal-organic framework nanosheet formed by a tetra(4-carboxyphenyl)porphyrin ligand centrally chelating divalent manganese ions and coordinating with manganese ions, and a ROCK inhibitor and a tumor antigen loaded on the nanosheet.

[0007] The tetra(4-carboxyphenyl)porphyrin ligand with a centrally chelated divalent manganese ion, namely tetra(4-carboxyphenyl)manganese porphyrin (abbreviated as TCPP(Mn) in the embodiments of this invention), is a porphyrin compound containing manganese metal ions. It consists of four carboxyphenyl porphyrin rings and a central divalent manganese ion. In this invention, the TCPP(Mn) is coordinated with manganese ions to form metal-organic framework (MOF) nanosheets (abbreviated as Mn-TCPP(Mn) nanosheets, or TM-NSs in the embodiments of this invention). The TM-NSs has a binuclear manganese structure, that is, while Mn is chelated at the center of the porphyrin ring, another Mn is also chelated. 2+ As a metal node in a MOF, it coordinates with the carboxyl group of tetrakis(4-carboxyphenyl)manganese porphyrin to form a MOF framework. This framework possesses a uniform morphology, good crystallinity, even elemental distribution, and a negatively charged two-dimensional surface, which is beneficial for uptake by antigen-presenting cells. Its negatively charged surface can efficiently load tumor antigens and ROCK inhibitors through multiple interactions, and can achieve Mn in an acidic lysosomal environment. 2+ The controlled release of reactive oxygen species effectively activates antigen-presenting cells, initiates the cGAS-STING pathway, promotes antigen cross-presentation, and ultimately enhances the anti-tumor immune response.

[0008] The nanovaccine uses TM-NSs as a substrate and further synergistically self-assembles TM-NSs with ROCK inhibitors and tumor antigens through electrostatic interactions, hydrogen bonds, and π-π stacking. Even after loading antigens and drugs, the nanovaccine maintains a complete two-dimensional sheet structure, good crystallinity, and stable coordination structure with uniform elemental distribution, ensuring efficient uptake by antigen-presenting cells and stable ROS generation and Mn production in an acidic endosome / lysosome environment. 2+ It releases the function and simultaneously achieves the synergistic delivery of antigens and inhibitors and immune activation, providing a reliable structural basis and functional guarantee for initiating and amplifying the cancer-immune cycle and enhancing the anti-tumor immune response.

[0009] Preferably, the ROCK inhibitor is Y27632 or a pharmaceutically acceptable salt thereof. The ROCK inhibitor of this application can not only act as an immune adjuvant, but also effectively regulate the function of antigen-presenting cells (APCs). By inhibiting the activation of the ROCK pathway in APCs, it promotes the phagocytosis of neoantigens generated by direct killing of tumor cells, thereby effectively amplifying the vaccine-mediated cancer-immune cycle.

[0010] Preferably, the tumor antigen is ovalbumin (OVA).

[0011] Preferably, the mass ratio of the metal-organic framework nanosheets, ROCK inhibitor, and tumor antigen is (43~44):(6~7):(24~26). More preferably, the mass ratio of the metal-organic framework nanosheets, ROCK inhibitor, and tumor antigen is 43.137:6.863:25.

[0012] Preferably, the nanosheets have a two-dimensional crystalline structure.

[0013] Preferably, the average particle size of the nanosheets is 200-300 nanometers.

[0014] Preferably, the nanosheets have a negatively charged surface.

[0015] Preferably, the nanosheets are formed by coordination of manganese salt, tetra(4-carboxyphenyl)manganese porphyrin, and organic amine ligands under the regulation of surfactant.

[0016] Preferably, the nanosheets are capable of generating reactive oxygen species (ROS) and releasing Mn in acidic endosomes or lysosomes. 2+ This activates the cGAS-STING pathway.

[0017] Preferably, the average particle size of the anti-tumor nanovaccine is 200-300 nanometers.

[0018] Preferably, the antitumor nanovaccine has a two-dimensional crystalline structure.

[0019] Secondly, the present invention provides a method for preparing the anti-tumor nanovaccine, comprising: preparing the metal-organic framework nanosheet, and then loading ROCK inhibitor and tumor antigen onto the nanosheet through self-assembly to obtain the nanovaccine.

[0020] Preferably, the preparation method includes the following steps: (1) Preparation of metal-organic framework nanosheets: After dispersing manganese salt, organic amine ligand and surfactant in solvent, tetrakis(4-carboxyphenyl)manganese porphyrin solution is added to obtain the reaction system; (2) Loaded ROCK inhibitor: The solution containing ROCK inhibitor is added to the reaction system of step (1), dispersed, and then dried to remove impurities, thus obtaining metal-organic framework nanosheets loaded with ROCK inhibitor. (3) Loaded with tumor antigens: The solution containing tumor antigens is mixed with a solution of metal-organic framework nanosheets containing the ROCK inhibitor loaded in step (2) and incubated. The resulting product is obtained after removing impurities.

[0021] Preferably, in step (1), the mass ratio of manganese salt, organic amine ligand, and surfactant is 1:(0.2~0.3):(4~5). More preferably, in step (1), the mass ratio of manganese salt, organic amine ligand, and surfactant is 1:0.23:4.18.

[0022] Preferably, in step (1), the mass ratio of manganese salt to tetra(4-carboxyphenyl)manganese porphyrin is (19~20):(20~22). More preferably, in step (1), the mass ratio of manganese salt to tetra(4-carboxyphenyl)manganese porphyrin is 19.16:21.5.

[0023] Preferably, step (1) of preparing metal-organic framework nanosheets further includes: subjecting the obtained reaction system to a light-protected heating reaction, and removing impurities and drying the reaction product. Preferably, the heating reaction conditions are 70~90°C for 20~28h.

[0024] Preferably, in step (2), the mass ratio of the ROCK inhibitor to the metal-organic framework nanosheets of step (1) is 1:(6~7). More preferably, in step (2), the mass ratio of the ROCK inhibitor to the metal-organic framework nanosheets of step (1) is 1:6.3.

[0025] Preferably, in step (3), the mass ratio of the tumor antigen to the metal-organic framework nanosheet loaded with the ROCK inhibitor in step (2) is 1:(1.5~3). More preferably, in step (3), the mass ratio of the tumor antigen to the metal-organic framework nanosheet loaded with the ROCK inhibitor in step (2) is 1:2.

[0026] Thirdly, the present invention provides the application of the aforementioned anti-tumor nanovaccine or the aforementioned preparation method in the preparation of tumor therapeutic drugs.

[0027] Preferably, the tumor is melanoma.

[0028] Preferably, the tumor treatment drug is a drug that promotes the antigen uptake and antigen cross-presentation capabilities of APC cells.

[0029] Preferably, the tumor treatment drug is a drug that enhances the anti-tumor immune response.

[0030] Preferably, the tumor treatment drug is a drug that activates the cGAS-STING pathway.

[0031] Preferably, the tumor treatment drug is a drug that enhances the accumulation and maturation of APCs in lymph nodes.

[0032] Fourthly, the present invention provides a tumor treatment drug, including the aforementioned anti-tumor nanovaccine.

[0033] Preferably, the tumor treatment drug further includes any one or a combination of at least two of pharmaceutically acceptable carriers, diluents, or excipients.

[0034] The mechanism of action of the nano-vaccine in this application: like Figure 1 As shown, after vaccination, the antigen pool formed at the injection site initially attracts antigen-presenting cells (APCs), including dendritic cells (DCs) and macrophages. Due to the properties of nanoparticles, which facilitate easy uptake, this antigen pool is internalized. Within the weakly acidic lysosomes of APCs, the nanoadjuvant TM-NSs induces the release of reactive oxygen species (ROS) and Mn through the Russell mechanism. 2+ The generation of ROS, this process, shows a synergistic effect in enhancing cGAS-STING pathway stimulation, thereby promoting APC activation. At the same time, the presence of ROS helps intracellular antigens escape from lysosomes and promotes APCs to cross-present them to subsequent cells, thus strongly initiating the cancer-immune cycle. Subsequently, the ROCK inhibitor contained in the nanovaccine effectively modulates APCs. By inhibiting the activation of the ROCK pathway in APCs, the ROCK inhibitor promotes the phagocytosis of tumor neoantigens generated by direct killing of tumor cells, thereby effectively amplifying the vaccine-mediated cancer-immune cycle.

[0035] The beneficial effects of this invention are: The antitumor nanovaccine of this invention uses metal-organic framework nanosheets (TM-NSs) as a multifunctional nanoadjuvant and delivery carrier, which can efficiently generate reactive oxygen species (ROS) and release manganese (Mn) in the weakly acidic endosomes / lysosomes of antigen-presenting cells via the Russell mechanism. 2+ The material synergistically activates the cGAS-STING innate immune pathway, promoting antigen escape from lysosomes and cross-presentation, effectively initiating an anti-tumor immune response. ROCK inhibitors suppress the ROCK signaling pathway in antigen-presenting cells, significantly improving the phagocytosis and processing efficiency of tumor antigens, improving the tumor immunosuppressive microenvironment, and further amplifying and maintaining the cancer-immune cycle. Tumor antigens provide precise immune recognition targets, endowing the vaccine with specific anti-tumor effects. The three components work together to achieve a unified multi-functional approach, including efficient antigen delivery, potent activation of innate immunity, precise initiation of adaptive immunity, and regulation of the immunosuppressive microenvironment, significantly enhancing the overall anti-tumor immune effect. Simultaneously, the material exhibits good structural stability and biosafety, providing a reliable solution for safe and efficient tumor immunotherapy. Attached Figure Description

[0036] Figure 1 This is a diagram illustrating the mechanism of action of the nano-vaccine of this invention.

[0037] Figure 2 A simplified diagram of the process for preparing TM-NSs.

[0038] Figure 3 A simplified diagram of the process for preparing OVA-Y27632@TM-NSs.

[0039] Figure 4 Partial characterization images of TM-NSs material ((a) TEM image of TM-NSs (scale bar = 200 nm); (b) EDS spectrum of TM-NSs; (c) Zeta potential spectrum of TM-NSs; (d) XRD spectrum of TM-NSs).

[0040] Figure 5Summary diagrams of partial characterization of TM-NSs materials, evaluation of ROS generation, in vitro cytotoxicity detection, and activation of the cGAS-STING pathway in APCs ((a) Energy scattering spectral (EDS) mapping of TM-NSs (scale bar = 5 μm); (b) Statistical graphs and representative flow cytometry histograms of ROS generation in DC 2.4 cells after treatment with different formulations; (c) Statistical graphs and representative flow cytometry histograms of ROS generation in Raw264.7 cells after treatment with different formulations; (d, e) MTT assay results 24 hours after adding different concentrations of TM-NSs to DC2.4 cells (d) and Raw264.7 cells (e); (f) Western blot analysis of the activation of the cGAS-STING signaling pathway in Raw264.7 cells 2 hours after adding different formulations; (gh) RNA sequencing analysis of Raw264.7 cells treated with saline or TM-NSs, showing volcano plot (g) and KEGG enrichment analysis (h)).

[0041] Figure 6 The image shows the MTT assay of BMDC after treatment with different concentrations of TM-NSs.

[0042] Figure 7 The diagram shows the activation of the cGAS-STING pathway in APCs by TM-NSs ((a) Western blot (WB) analysis of cGAS-STING pathway activation in Raw264.7 cells 4 hours after the addition of different preparations; (b) RNA sequencing analysis of Raw264.7s treated with saline or TM-NSs, with the image showing gene ontology (GO) enrichment analysis).

[0043] Figure 8 Evaluation diagram of TM-NSs promoting antigen-presenting cell (APC) maturation and activation ((af) BMDCs via TM-NSs, Mn) 2+ After 24 hours of cGAMP treatment, the expression of maturation markers CD40(a), CD80(b), CD83(c), CD86(d), major histocompatibility complex I (MHC-I)(e), and major histocompatibility complex II (MHC-II)(f) (n=3-4); (gh) the secretion of TNF-α and IL-1β by BMDCs after incubation with each formulation; (in) the expression of maturation markers CD40(i), CD80(j), CD83(k), CD86(l), MHC-I(m), and MHC-II(n) by BMDMs after treatment with each formulation; (o) a schematic diagram of how TM-NSs promote APC maturation and related cytokine secretion by activating the cGAS-STING signaling pathway.

[0044] Figure 9 For BMDCs via TM-NSs, Mn 2+ Representative flow cytometry histograms of maturation markers (CD40(a), CD80(b), CD83(c), CD86(d), MHC-I(e), and MHC-II(f)) after 24 hours of cGAMP treatment.

[0045] Figure 10 The graph shows the IL-1β produced after BMDC is incubated with various formulations.

[0046] Figure 11 For BMDMs via TM-NSs, Mn 2+ Representative flow cytometry histograms of maturation markers (CD40(a), CD80(b), CD83(c), CD86(d), MHC-I(e), and MHC-II(f)) after 24 hours of cGAMP treatment.

[0047] Figure 12 Partial characterization images of OVA-Y27632@TM-NSs ((a) TEM image of OVA-Y27632@TM-NSs (scale bar = 200 nm); (b) EDS image of OVA-Y27632@TM-NSs; (c) XRD image of OVA-Y27632@TM-NSs; (d) Infrared images of TM-NSs and OVA-Y27632@TM-NSs).

[0048] Figure 13 Release behavior curves of OVA and Y27632 for OVA-Y27632@TM-NSs nanovaccine at different pH values.

[0049] Figure 14 Summary of material characterization and evaluation of the ability of OVA-Y27632@TM-NSs nanovaccine to promote antigen uptake and cross-presentation in APC cells ((a) EDS spectrum of OVA-Y27632@TM-NSs (scale bar = 5 μm); (b) EDS spectrum of OVA-Y27632@TM-NSs or OVA-FITC-Mn 2+ Laser microscopy images of DC 2.4s after 24 hours of treatment (scale bar = 20 μm); (cf) Statistical plots and representative flow cytometry histograms of OVA-FITC uptake in DC 2.4s (c, d) and Raw 264.7 (e, f) after different treatments; (gj) Flow cytometry density plots and statistical charts of OVA antigen cross-presentation efficiency in BMDC (g, h) and BMDM (i, j) after different treatments.

[0050] Figure 15 For use with OVA-FITC, OVA-FITC@TM-NSs, OVA-FITC-Y27632, OVA-Y27632@TM-NSs or OVA-FITC-Mn 2+ Laser microscope image (scale bar = 20 μm) after 24 hours of processing, with a raw time of 264.7 seconds.

[0051] Figure 16 Statistical graphs and representative flow cytometry histograms of OVA-FITC uptake in BMDCs after treatment with free OVA or OVA-Y27632.

[0052] Figure 17 Flow cytometry density plots and statistical charts of OVA antigen cross-presentation efficiency in BMDCs (a, c) and BMDMs (b, d) after treatment with OVA-Y27632@TM-NSs or OVA-cGAMP.

[0053] Figure 18 The following are graphs for evaluating the cellular immune response of the OVA-Y27632@TM-NSs nanovaccine: (a) Schematic diagram of the vaccination regimen for C57 / BL-6 mice; (be) Ten days after the first vaccination, the production of OVA-specific antibodies in mouse serum was detected, including anti-OVA IgG titers (b), IgG1 titers (c), IgG2b titers (d), and IgG2c titers (e); (fi) On day 21, the titers of secondary OVA-specific antibodies were analyzed by enzyme-linked immunosorbent assay (ELISA) (n=4); (jk) After OVA restimulation, the secretion of IL-5 and IFN-γ in spleen lymphocytes was measured using an ELISA kit; (l) CCK8 detection in different spleen lymphocytes after OVA restimulation (n=3); (mp) Representative flow cytometry scatter plots and statistical graphs of CD4+IFN-γ+ T cells (mn) and CD8+IFN-γ+ T cells (op) in spleen lymphocytes after treatment with different formulations (n=4).

[0054] Figure 19 Representative flow cytometry scatter plots and statistical graphs (n=4) of CD4+CD69+ T cells (a, b) and CD8+CD69+ T cells (c, d) in spleen lymphocytes after treatment with different preparations.

[0055] Figure 20Figures show how the OVA-Y27632@TM-NSs nanovaccine precisely maintains the interaction cycle between cancer and the immune system, enhances anti-tumor immune response, and inhibits tumor growth in B16F16-OVA tumor-bearing mice. (a) Schematic diagram of mouse inoculation and vaccination protocols; (bc) Average tumor volume growth curves of mice after treatment with different formulations; (b) Tumor growth curves of each mouse in each group; (dg) Flow cytometry scatter plot and statistical graph of dendritic cell (DC) subtypes in lymph nodes; (h,i) CD4 infiltrating cells in tumors after treatment with different formulations. + T cells and CD8 + Flow cytometry density maps and statistical plots of T cells; (j)CD4 + T cells and CD8 + Statistical plots of T cell proportions. (k,l) Statistical plots of regulatory T cells (Treg) (k) and dendritic cells (DC) (l) infiltrating tumors after different treatments; (mo) Representative histograms and statistical plots of M1 tumor-associated macrophages (TAMs) and M2 TAMs in tumors by flow cytometry; (p) Quantitative analysis of the proportions of myeloid-derived suppressor cells (m-MDSCs), granulocytic myeloid-derived suppressor cells (g-MDSCs), and neutrophils in the tumor microenvironment by flow cytometry.

[0056] Figure 21 This is a graph showing the biochemical analysis of mouse serum after treatment with different formulations.

[0057] Figure 22 Images of H&E staining of major organs after different treatments (scale bar = 100 μm).

[0058] Figure 23 Representative flow cytometry plots and statistical graphs of dendritic cells (DCs) in lymph nodes (n = 4-5 / group).

[0059] Figure 24 Flow cytometry plots of DC cell maturation in lymph nodes (n=4-5 per group).

[0060] Figure 25 For Tregs (CD4+) in the tumor microenvironment + CD25 + Foxp3 + A representative flow cytometry scatter plot.

[0061] Figure 26 For DCs (CD11c) in the tumor microenvironment + A representative flow cytometry scatter plot.

[0062] Figure 27Representative flow cytometry scatter plots of m-MDSCs (a), g-MDSCs (b), and viable neutrophils (b) in the tumor microenvironment. Detailed Implementation

[0063] To enable those skilled in the art to better understand the technical solution of the invention, the invention will be further described in detail below with reference to specific embodiments.

[0064] Example 1 Synthesis of Mn-TCPP (Mn) nanosheets (TM-NSs), OVA-Y27632@TM-NSs and OVA@TM-NSs (1) Synthesis of TM-NSs: Manganese nitrate hydrate Mn(NO3)2∙4H2O (19.16 mg, 0.0762 mM), piperazine (4.38 mg, 0.051 mM), and polyvinylpyrrolidone (PVP 400, 80 mg) were dispersed in a mixed solvent of ethanol (5 mL) and N,N-dimethylformamide (DMF, 15 mL) to obtain a mixed solution. Then, under ultrasonic conditions, TCPP(Mn) (21.5 mg, 0.0254 mM) was dissolved in a mixed solvent of ethanol (1.5 mL) and N,N-dimethylformamide (DMF, 4.5 mL) and added dropwise to the above mixed solution to obtain a reaction system. The reaction system was then protected from light and stirred at 80°C for 24 hours. The product was centrifuged at 10,000 rpm for 15 minutes and washed three times with ethanol. Finally, the residual ethanol was completely evaporated to obtain TM-NSs. A simplified diagram of the preparation process is shown below. Figure 2 As shown.

[0065] (2) Synthesis of OVA-Y27632@TM-NSs: Y27632 (3.85 mg, 0.012 mM) was dispersed in a mixed solvent of ethanol (2 mL) and N,N-dimethylformamide (6 mL) and then added to the reaction system described in (1). The mixture was then sonicated for 10 minutes and stirred for 24 hours. The resulting solution was centrifuged at 10,000 rpm for 15 minutes, washed three times with ethanol, and evaporated for 12 hours to obtain Y27632@TM-NSs (TM-NSs to Y27632 mass ratio 6.3:1). Then, 50 μL of OVA solution (0.5 μg / μL in phosphate-buffered saline PBS, i.e., 25 μg) and 50 μL of Y27632@TM-NSs solution (1 mL in PBS) were added. Mix 50 μg (mg / mL) of the solution and incubate for 30 minutes to obtain OVA-Y27632@TM-NSs (the mass ratio of TM-NSs, Y27632, and OVA is 43.14:6.86:25). Centrifuge the solution at 10,000 rpm for 15 minutes to remove unencapsulated proteins and resuspend it in PBS for subsequent experiments. A simplified diagram of the preparation process is shown below. Figure 3 As shown.

[0066] (3) Synthesis of OVA@TM-NSs: Mix 50 μL of OVA solution (0.5 μg / μL, or 25 μg, in PBS) with 50 μL of TM-NSs solution (1 mg / mL, or 50 μg, in PBS) and incubate for 30 minutes to obtain OVA@TM-NSs. Centrifuge the solution at 10,000 rpm for 15 minutes to remove unencapsulated proteins and resuspend it in PBS for subsequent experiments.

[0067] The OVA-FITC used in subsequent experiments was commercially available fluorescein isothiocyanate-labeled ovalbumin. The preparation methods of OVA-FITC@TM-NSs and OVA-FITC-Y27632@TM-NSs were the same as above.

[0068] Example 2 Material Characterization and Effect Evaluation 1. Experimental Methods 1.1 Characterization of Nanosheets Scanning electron microscopy (SEM), SEM elemental distribution maps, and energy dispersive spectroscopy (EDS) (ZEISS Gemini 300, Carl Zeiss, Germany) were used to characterize the morphology and elemental distribution of the nanosheets; transmission electron microscopy (TEM) (Hillsboro, FEI, USA) was used to analyze the size and morphology of the prepared nanosheets; X-ray diffraction (XRD) (EMPYREAN, Panaco, Netherlands) was used to analyze the surface crystal phase of these nanosheets; and a Zeta potentiometer (nano-ZS ZEN 3600, Malvern Instruments, UK) was used to analyze the surface charge of the nanosheets.

[0069] The release profiles of OVA and Y27632 were determined as follows: OVA-Y27632@TM-NSs were dispersed in phosphate-buffered saline (PBS) at two different pH values ​​(7.4 and 5.5) and incubated with gentle shaking at 37°C. At set time intervals (0 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 16 h, 24 h, 48 h), the samples were centrifuged at 10,000 rpm for 15 min to obtain the supernatant. The mass of OVA in the supernatant was then determined using a BCA protein assay kit. The concentration of Y27632 was determined by high performance liquid chromatography (HPLC) (flow rate 1 mL / min; mobile phase: solvent A + solvent B, solvent A being an aqueous solution containing 0.1% formic acid, solvent B being an acetonitrile solution containing 0.1% formic acid; volume ratio of solvent A to solvent B 20:80).

[0070] 1.2 In vitro cytotoxicity detection and ROS generation evaluation of TM-NSs The cytotoxic effects of TM-NSs on antigen-presenting cells (APCs), including DC 2.4 cells, Raw 264.7 cells, and bone marrow-derived dendritic cells (BMDCs), were analyzed using the MTT assay. Briefly, DC 2.4 cells (1 × 10⁶ cells per well) were used to analyze the cytotoxic effects of TM-NSs on antigen-presenting cells (APCs), including DC 2.4 cells, Raw 264.7 cells, and bone marrow-derived dendritic cells (BMDCs). 4 Raw 264.7 cells (8 × 10⁶ cells per well) 3 (cells) or BMDCs (1×10 cells per well) 4Cells were seeded in 96-well plates and cultured for 24 hours in RPMI-1640 medium containing 10% FBS, DMEM high-glucose medium containing 10% FBS, and RPMI-1640 medium containing 10% FBS (10 ng / mL rmIL-4, 20 ng / mL rm-GM-CSF, and 50 μM β-mercaptoethanol). Then, the cells were treated with different concentrations of TM-NSs (0, 3.125, 6.25, 12.5, 25, 50, 100, and 200 μg / mL) and incubated for another 24 hours. The medium was then replaced with 100 μL containing MTT (0.5 mg / mL). Fresh culture medium ( / mL) was used to incubate the cells for 4 hours. The absorbance at 570 nm was measured using a microplate reader. Cell viability was calculated using the following formula: Cell viability (%) = (Absorbance at 570 nm of treatment group / Absorbance at 570 nm of control group) × 100%.

[0071] The ROS generated in vitro by TM-NSs was measured using a ROS detection kit. DC 2.4 cells (2 × 10⁴ cells per well) were used. 6 (100 μg / mL) cells were seeded in 6-well plates and incubated for 6 hours each with the following formulations: physiological saline, TM-NSs (100 μg / mL), and Mn. 2+ Cells were pretreated with NH4Cl (20 μg / mL) and then co-incubated with TM-NSs for 6 hours. Afterwards, the cells were washed twice with PBS buffer and incubated at 37°C with 2',7'-dichlorofluorescein diacetate (DCFH-DA) (20 μmol / L) for 15 minutes. Finally, the cells were washed and analyzed by flow cytometry (ACEA NovoCyte). TM-NSs-induced ROS production in Raw 264.7 cells was detected using the same method.

[0072] 1.3 Evaluation of cGAS-STING pathway activation in antigen-presenting cells (APCs) Western blot analysis was used to evaluate the effect of TM-NSs on STING pathway activation. Specifically, Raw264.7 cells (2 × 10⁶ cells) were used. 5 Cells (per mL) were seeded in 6-well plates and incubated at 37°C for 2 hours and 4 hours, respectively, using the following formulation. The formulation included: physiological saline, Mn... 2+Cells were then washed and lysed with TM-NSs (100 μg / mL) and 2'3'-cyclic guanylate-adenosine monophosphate synthase (cGAMP) (20 μg / mL). The cell lysates were loaded onto a 10% SDS-PAGE gel and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore). The blot was blocked with bovine serum albumin (BSA) solution and incubated overnight at 4°C with primary antibody (TBK-1, pTBK-1, NF-κB, pIRF-3, IRF-3, p-STING, or GAPDH), followed by binding to a horseradish peroxidase (HRP)-labeled secondary antibody. The WB band signal was visualized using Pierce ECL Western blot substrates.

[0073] 1.4 Evaluation of TM-NSs' promotion of APC cell maturation and activation Immature BMDCs (1×10 per well) 6 (1,000 cells) were seeded in a 24-well plate and treated with Mn. 2+ Cells were stimulated for 24 hours with TM-NSs (20 μg / mL), 2'3'-cyclic guanylate-adenosine monophosphate synthase (cGAMP, 20 μg / mL), and then collected. The expression of co-stimulatory molecules on BMDCs was analyzed by flow cytometry. Simultaneously, the supernatant of BMDCs was collected, and the levels of cytokines tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) were detected using a commercial enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's instructions. The effect of TM-NSs on BMDM maturation was assessed using the same method.

[0074] Evaluation of the ability of 1.5 nanometer vaccines to promote antigen uptake and cross-presentation in APC cells In vitro antigen uptake assay: DC 2.4 cells (1 × 10⁴ cells per well) were used. 6 Cells were seeded in 24-well plates and treated with ovalbumin-fluorescein isothiocyanate (OVA-FITC), OVA-FITC@TM-NSs, OVA-FITC-Y27632, OVA-FITC-Y27632@TM-NSs, and OVA-FITC-Mn. 2+ Cells were co-incubated at 37°C for 2 hours with OVA-FITC at a concentration of 50 μg / mL, TM-NSs at a concentration of 100 μg / mL, and Y27632 at a concentration of 30 μM. After incubation, cells were collected and antigen uptake was analyzed using flow cytometry (ACEANovoCyte) and multiphoton confocal microscopy (Leica, Germany).

[0075] APC cell in vitro antigen cross-presentation capacity assay: Immature bone marrow-derived dendritic cells (BMDCs, 1×10⁻⁶) were used. 6 Cells per mL were seeded in 24-well plates and seeded with various complexes (OVA-FITC, OVA@TM-NSs, OVA-Y27632, OVA-Y27632@TM-NSs, OVA-Mn). 2+ After stimulation with OVA-2'3'-cyclic guanylate adenosine monophosphate (cGAMP) for 24 hours, BMDCs were washed twice with phosphate-buffered saline (PBS) and stained at 4°C for 30 minutes with fluorescein isothiocyanate-labeled anti-mouse CD11c antibody and allophycocyanin-labeled anti-mouse SIINFEKL-H-2Kb antibody. Similarly, after washing BMDMs with PBS, they were stained at 4°C for 30 minutes with PE-labeled anti-mouse CD11b antibody, fluorescein isothiocyanate-labeled anti-mouse F4 / 80 antibody and allophycocyanin-labeled anti-mouse SIINFEKL-H-2Kb antibody. BMDCs and BMDMs were then washed twice with PBS and analyzed by flow cytometry (ACEA NovoCyte).

[0076] 1.6 Assessment of Antigen-Specific Humoral and Cellular Immune Responses To investigate the effects of TM nanosheet-based nanovaccines on antigen-specific humoral and cellular immunity, we administered two subcutaneous immunizations via the groin to 6-8 week old female C57BL / 6 mice. The inoculum included OVA (ovalbumin), OVA@TM-NSs (TM-NSs being the nanocarrier), OVA-Y27632 (Y27632 being a ROCK inhibitor), OVA-Y27632@TM-NSs, and OVA-Mn. 2+ (Manganese ions) and OVA-cGAMP. The dosages of OVA and TM-NSs were 20 μg and 200 μg per mouse, respectively; Y-27632, Mn 2+ The doses of cGAMP were 50 μg, 28 μg, and 20 μg per mouse, respectively. At specified time points (i.e., day 10 and day 21), we collected serum from these immunized mice and detected OVA-specific antibodies by enzyme-linked immunosorbent assay (ELISA), specifically including IgG and its subtypes (IgG1, IgG2b, and IgG2c).

[0077] To assess antigen-specific cellular immune responses, we isolated single-cell suspensions from mouse spleens 7 days after the last immunization and seeded them into 24-well plates (5 × 10⁶ cells per well). 6(100 μg per well) Then, these spleen cells were treated with OVA (100 μg per well) at 37°C for 72 hours. After treatment, the spleen cells were centrifuged and washed twice, and then stained on ice with APC-labeled anti-mouse CD4 antibody, FITC-labeled anti-mouse CD8 antibody, and PE-labeled anti-mouse IFN-γ antibody (or PE-labeled anti-mouse CD69 antibody) for 30 minutes. After that, the cells were analyzed by flow cytometry (ACEA NovoCyte). For PE-labeled anti-mouse IFN-γ staining, APC and FITC-labeled cells were first fixed with 4% paraformaldehyde at room temperature for 30 minutes, then the cells were perforated with 0.1% Triton X-100 (purchased from Sigma-Aldrich, Missouri, USA), and then stained with PE-labeled anti-mouse IFN-γ antibody.

[0078] In addition, we collected cell culture medium and analyzed the secretion of IL-5 and IFN-γ using an ELISA kit. To assess the proliferation capacity of spleen cells, we seeded spleen cells (5 × 10^5 cells per well) in 96-well plates and restimulated the cells for 48 hours with culture medium, OVA (5 μg per well), or bovine serum albumin (BSA, 5 μg per well). After stimulation, we added 10 μL of CCK-8 solution (purchased from Beyotime Biotech, Shanghai, China) to each well and measured the cell proliferation at 450 nm using a microplate reader.

[0079] Study on the anti-tumor effect of 1.7 nanometer vaccine To evaluate the antitumor efficacy of TM-NSs-based nanovaccines, we administered 1×10⁻⁶ nanoparticles on day 0. 6 One B16F10-OVA cell was subcutaneously injected into female C57BL / 6 mice. Five days later, the mice were randomly divided into 6 groups (OVA group, OVA@TM-NSs group, OVA-Y27632 group, OVA-Y27632@TM-NSs group, OVA-Mn group, etc.). 2+ Mice in each group (OVA-cGAMP group and OVA-cGAMP group) were subcutaneously immunized three times at 5-day intervals with different formulations (OVA dose: 20 μg per mouse; TM-NSs dose: 200 μg per mouse; Y-27632 dose: 50 μg per mouse; Mn...). 2+ The dosage was 28 μg per mouse; the cGAMP dosage was 20 μg per mouse. Tumor volume was measured every two days and calculated using the following formula: Tumor volume V (mm³) = 0.5 × length (mm) × width (mm) 2After 15 days of observation, the mice were euthanized for further analysis.

[0080] To assess the safety of the nanovaccine, we euthanized mice in the B16F10-OVA model to collect major organs (including heart, liver, spleen, lungs, and kidneys) and serum. The major organs were fixed in 4% paraformaldehyde and stained with hematoxylin and eosin. The serum samples were stored at -80°C for biochemical analysis.

[0081] 1.8 Tumor immune microenvironment (TIME) and TDLN cell analysis in mice after immunization To investigate the effects of nanovaccines on the recruitment, accumulation, and activation of antigen-presenting cells (APCs) in tumor draining lymph nodes (TDLNs), we obtained TDLNs from immunized mice in a melanoma treatment model and prepared them into single-cell suspensions. Subsequently, we stained the cells with CD11c, CD8a, and CD103 flow cytometry antibodies to analyze the proportion of cDC1 cells with cross-presentation function; and stained them with CD11c, CD80, and CD86 flow cytometry antibodies to analyze the activation of dendritic cells (DCs) in the TDLNs. CD80 and CD86 activation was measured using flow cytometry (ACEA NovoCyte). + CD103 + DCs (cDC1), CD80 + DCs and CD86 + The proportion of DCs.

[0082] To evaluate the effects of nanovaccines on the infiltration of various immune cells in the tumor immune microenvironment (TIME), we excised B16F10-OVA tumors three days after the third immunization of mice in a melanoma treatment model. First, the tumor tissue was minced, then digested with type I collagenase and DNase, followed by erythrocyte lysis buffer. Finally, the tissue was prepared into a single-cell suspension (at a concentration of 2 × 10⁻⁶ in PBS containing 2% fetal bovine serum (FBS)). 7100 μL of single-cell suspension was stained with the following conjugated flow cytometry antibodies provided by Biolegend: (1) CD4, CD8 and CD3; (2) CD3, CD4, CD25 and Foxp3; (3) CD11b, F4 / 80 and CD86; (4) CD11b, F4 / 80 and CD206; (5) CD45 and CD11c; (6) CD11b, Ly-6C and Ly-6G. Cells were stained at 4°C for 30 minutes to label surface markers. For intracellular staining (such as Foxp3), cells were first stained with surface markers, then fixed and perforated using a fixation / perforation kit (eBioscience), and finally stained with anti-mouse Foxp3 antibody. The labeled cells were then measured by flow cytometry (ACEA NovoCyte).

[0083] 2. Experimental Results 2.1 Characterization of TM-NSs materials First, metal-organic framework nanosheets TM-NSs were synthesized and characterized. Transmission electron microscopy (TEM) images showed that TM-NSs possessed a typical two-dimensional nanosheet morphology, with an average particle size between 200 and 300 nanometers. Figure 4 a) The particle size is suitable, making it easy for antigen-presenting cells (APCs) to take up, thus improving antigen capture efficiency and enhancing immune presentation. SEM analysis of elemental mapping of TM-NSs using energy-scattered X-ray spectroscopy (EDS) showed that C, O, N, and Mn elements were uniformly distributed ( Figure 5 a and Figure 4 (b) Simultaneously, the contents of C, O, Mn, and N were calculated to be 59.95%, 20.10%, 6.14%, and 12.22% (weight percentage), respectively (see Table 1), indicating that Mn successfully coordinated with TCPP(Mn), resulting in a uniform material composition, stable structure, and elemental proportions consistent with the target Mn-TCPP(Mn) MOF. The X-ray diffraction (XRD) spectrum of TM-NSs has two typical peaks, at 28.5998° and 47.2865° (…). Figure 4 d) indicates that TM-NSs has good crystallinity and a crystalline MOF structure, which is beneficial for loading antigens and Y27632. Zeta potential analysis shows that the charge of TM-NSs in water is -16.6 mV ( Figure 4 c) indicates that the material has good dispersibility in aqueous solution and can self-assemble with antigen and Y27632 through electrostatics, hydrogen bonding, π-π stacking.

[0084] Table 1. Proportions of C, O, Mn, N, P and Cl elements in TM-NSs

[0085] The characterization results above demonstrate that this invention successfully prepared two-dimensional Mn-TCPP (Mn) metal-organic framework nanosheets TM-NSs with uniform morphology, good crystallinity, uniform elemental distribution, and negatively charged surface. This two-dimensional nanostructure is advantageous for uptake by antigen-presenting cells, and its negatively charged surface can efficiently load tumor antigens and the ROCK inhibitor Y27632 through multiple interactions.

[0086] 2.2 Evaluation of ROS generation and in vitro cytotoxicity detection of TM-NSs To verify the ability of TM-NSs to generate reactive oxygen species (ROS) in antigen-presenting cells (APCs) endosomes / lysosomes. DC2.4 and Raw 264.7 cells were used to evaluate the degradation of TM-NSs in acidic endosomes / lysosomes, producing ROS and Mn. 2+ The ability to detect ROS was demonstrated by using 2′,7′-dichlorofluorescein diacetate (DCFH-DA) as a fluorescent probe. Figure 5 As shown in bc, DC 2.4 and Raw 264.7 cells co-incubated with TM-NSs showed better performance than Mn. 2+ The group showed higher intracellular ROS production; however, in the presence of the acidification inhibitor NH4Cl, intracellular ROS production was significantly reduced, indicating that TM-NSs do indeed produce ROS and Mn in an acidic environment via the Russell mechanism. 2+ ion.

[0087] This invention also evaluated the cytotoxicity of TM-NSs against DC cells and Raw 264.7. For example... Figure 5 As shown in de, even at a concentration of 200 μg / mL, the survival rate of DC2.4 cells and Raw 264.7 exceeded 85%, and similar results were observed in bone marrow-derived dendritic cells (BMDCs). Figure 6 ).

[0088] The above results demonstrate that the TM-NSs of the present invention can effectively generate ROS and release Mn in the acidic endosome / lysosome environment of antigen-presenting cells via the Russell mechanism. 2+ This provides a crucial foundation for activating the cGAS-STING pathway and promoting antigen cross-presentation. Simultaneously, TM-NSs exhibited no significant cytotoxicity to DC2.4, Raw 264.7, and BMDC within the effective concentration range, demonstrating high cell viability and excellent biosafety. This indicates that TM-NSs possess both excellent immune activation capabilities and good biocompatibility, enabling them to safely and efficiently initiate anti-tumor immune responses, making them highly suitable as carriers and adjuvants for tumor nanovaccines.

[0089] 2.3 Activation of the cGAS-STING pathway in APCs by TM-NSs Western blot (WB) was used to assess the activation of the cGAS-STING pathway. Raw264.7 cells were treated with different formulations (Mn... 2+ After co-incubation with ions, TM-NSs, and cGAMP for different times (2 hours and 4 hours), the TM-NSs group significantly promoted the expression of phosphorylated STING (p-STING) and greatly increased the expression of downstream proteins in the cGAS-STING pathway, including TBK1, p-TBK1, NF-κB, p-IRF-3, and IRF-3 (…). Figure 5 f and Figure 7 a). Compared to TM-NSs, Mn 2+ The expression of p-STING in the ionosphere was slightly increased, consistent with previous reports. Furthermore, this further demonstrates that intracellularly generated ROS can interact with Mn. 2+ It binds to and activates the STING pathway in antigen-presenting cells (APCs).

[0090] To further investigate the mechanism by which TM-NSs regulate cGAS-STING pathway activation, Raw 264.7 cells treated with saline (NS) or TM-NSs were sequenced for RNA. Compared with the NS group, the TM-NSs group showed upregulation of 464 genes and downregulation of 516 genes. Figure 5 g). Meanwhile, Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that TM-NSs significantly affected the tumor necrosis factor (TNF) signaling pathway, inflammation-related responses, and chemotaxis. Figure 5 h and Figure 7 b).

[0091] 2.4 TM-NSs promote the maturation and activation of dendritic cells (DCs) and macrophages. Precise regulation of antigen-presenting cells (APCs) maturation and activation is crucial and essential for maintaining a positive cycle of vaccine-mediated cancer immunity. APCs, particularly dendritic cells and macrophages, are two major professional antigen-presenting cells that must mature and secrete relevant cytokines before cross-presenting antigens to T cells. We further analyzed the adjuvant immunogenicity of TM-NSs on bone marrow-derived dendritic cells (BMDCs) and bone marrow-derived macrophages (BMDMs). First, BMDCs or BMDMs were co-incubated with different formulations for 24 hours and analyzed by flow cytometry. TM-NSs-treated BMDCs showed a significant increase in the expression of CD40, CD80, CD83, and CD86, while Mn... 2+ It also increased the expression of co-stimulatory molecules. As a classic STING agonist, cGAMP slightly increased CD40 expression, while having a negligible effect on CD83 expression. Figure 8 ad, Figure 9 We also examined the expression of major histocompatibility complex class I (MHC-I) and class II (MHC-II) on BMDCs. Compared with the control group, BMDCs treated with TM-NSs showed higher expression of MHC-I and MHC-II, while Mn... 2+ The treatment had little effect on MHC-I expression in the cells. Figure 8 (ef), indicating that TM-NSs promote the conversion of endogenous antigens to CD8. + T cell presentation is compared to Mn 2+ It has even greater advantages. Furthermore, activation of the cGAS-STING pathway in APCs can induce the secretion of pro-inflammatory cytokines. After treatment with TM-NSs, the production of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and IL-1β by BMDCs was significantly increased. Figure 8 gh, Figure 10 ).

[0092] Similar effects of TM-NSs were also observed in BMDMs, indicating that Mn 2+ The synergistic effect with ROS strongly induced the expression of co-stimulatory molecules. Figure 8 in, Figure 11 These results indicate that TM-NSs activation of the cGAS-STING pathway is beneficial for enhancing APC maturation. (See schematic diagram below.) Figure 8 o.

[0093] 2.5 Characterization of OVA-Y27632@TM-NSs nanovaccine materials Given the significant adjuvant effect of TM-NSs, we co-loaded the Rho-associated protein kinase (ROCK) inhibitor Y27632 and the model antigen OVA into TM-NSs, forming the nanovaccine OVA-Y27632@TM-NSs through self-assembly. Transmission electron microscopy (TEM) images showed that OVA-Y27632@TM-NSs exhibited sheet-like structures with dimensions ranging from 200 to 300 nanometers. Figure 12 a) The regular morphology and uniform size indicate that the carrier structure was not destroyed after loading the antigen and drug, and the target nanovaccine was successfully constructed. Furthermore, C, O, N, and Mn are uniformly distributed in the nanosheets ( Figure 14 a), accounting for 54.14%, 19.00%, 23.27% and 2.29% of the weight, respectively. Figure 12 (b and Table 2) Compared with TM-NSs, OVA-Y27632@TM-NSs has a higher N element content, which is due to the abundant OVA content, indicating that the material composition is uniform, the loading process is uniform and stable, and the OVA antigen has been successfully loaded into the nano-vaccine. The X-ray diffraction (XRD) pattern of OVA-Y27632@TM-NSs has four peaks at 28.4894, 29.9134, 33.0286, and 47.4169, indicating its crystallization characteristics ( Figure 12 c) indicates that after loading antigen and drug, the nanovaccine still maintains good crystallinity and the MOF backbone structure is intact. The Fourier transform infrared (FT-IR) spectra of TM-NSs and OVA-Y27632@TM-NSs were also characterized; the N-Mn stretching vibrations of both are located at 10¹⁰.57 cm⁻¹. Figure 12 d) indicates that the coordination structure between Mn and the ligand remains unchanged, and the loading of OVA and Y27632 did not disrupt the MOF core coordination structure, further proving the stability of the vector structure.

[0094] Table 2. Proportions of C, O, Mn, N, P and Cl elements in OVA-Y27632@TM-NSs

[0095] According to the Russell mechanism, nanovaccines degrade under acidic conditions, forming peroxides and releasing drugs and proteins. Based on this theory, we used the quinoline carboxylic acid (BCA) method and high-performance liquid chromatography (HPLC) to study the release of OVA and Y27632 under acidic conditions. Figure 13 As shown, under pH 5.5 conditions, nearly 90% of Y27632 and 80% of OVA were released from the metal-organic framework (MOF)-based nanovaccine within 16 hours. All data combined indicate that a MOF-based nanovaccine has been successfully prepared for further research.

[0096] Evaluation of the ability of 2.6-nanometer vaccines to promote antigen uptake and cross-presentation in APC cells Antigen uptake and cross-presentation by antigen-presenting cells (including dendritic cells and macrophages) are key steps in the effective maintenance of cancer immune circulation by vaccines. Therefore, we further evaluated the ability of the OVA-Y27632@TM-NSs nanovaccine to be uptaken by DC cells and macrophages for OVA antigens. Figure 14 b and Figure 15 The results showed that the OVA-Y27632@TM-NSs nanovaccine significantly enhanced the uptake of OVA-FITC in DC2.4s and Raw264.7 cells. Compared with free OVA-FITC, the OVA-Y27632@TM-NSs nanovaccine increased OVA uptake by 42.65 times in macrophages and by 4.7 times in DC2.4s cells, while TM-NSs only increased OVA uptake by 1.57 times. Figure 14 Further research indicates that ROCK blockade in BMDCs can increase antigen phagocytosis (cf). Figure 16 ).

[0097] Next, the cross-presentation efficiency of the MOF-based nanovaccine on BMDC and BMDM cells was evaluated using SIINFEKL-H-2Kb flow cytometry. After 24 hours of incubation, the OVA-Y27632@TM-NSs nanovaccine increased the expression of SIINFEKL antigen peptide on the surface of BMDC by approximately 2.4-fold and on the surface of BMDM by 4.0-fold, primarily attributed to the ability of TM-NSs to generate reactive oxygen species (ROS). Figure 14 Although manganese ions and cGAMP are comparable to MOF-based nanovaccines in promoting APC cell maturation and activation, their ability to phagocytose, process, and present antigens is significantly higher due to the advantages of enhanced ROS-mediated cross-presentation and increased Y27632-mediated uptake. Figure 17 This lays the foundation for effectively initiating an adaptive immune response.

[0098] 2.7 Evaluation of cellular immune response to OVA-Y27632@TM-NSs nanovaccine The efficacy of the metal-organic framework (MOF)-based OVA-Y27632@TM-NSs nanovaccine in inducing adaptive immune responses was investigated using C57BL / 6 mice. C57BL / 6 mice were administered different formulations (ovalbumin (OVA) as a negative control, OVA@TM, OVA-Y27632, OVA-Y72632@TM-NSs, OVA-Mn) on days 0 and 14. 2+Subcutaneous immunization (as a positive control, and OVA-cGAMP as another positive control) Figure 18 a). Mice serum was collected on days 10 and 14 and then assessed for primary and secondary antibody titers by enzyme-linked immunosorbent assay (ELISA). For primary antibody production, compared with soluble OVA, the MOF-based nanovaccine (OVA-Y72632@TM-NSs) increased anti-OVA immunoglobulin G (IgG) titers by approximately 1170-fold, anti-OVA IgG1 titers by 768-fold, anti-OVA IgG2b titers by 140-fold, and anti-OVA IgG2c titers by 160-fold, indicating that the MOF-based nanovaccine rapidly initiates effective antigen-specific humoral immunity after a single dose. Figure 18 be). With cGAMP adjuvant and soluble Mn 2+ In comparison, the OVA-Y72632@TM-NSs nanovaccine slightly increased the production of anti-OVA IgG, IgG2b, and IgG2c by 1 to 3 times, respectively, meaning its immunostimulatory effect was more significant than that of commercial STING agonists. Figure 18 be).

[0099] Regarding secondary antibody production, the MOF-based nanovaccine (OVA-Y72632@TM-NSs) increased IgG secretion by 1755-fold, while OVA@TM-NSs increased IgG titer by 1097-fold. The effect of OVA-Y27632 was relatively minor, indicating a synergistic effect between Mn-TCPP (Mn) nanosheets and the ROCK inhibitor Y27632 in generating humoral immunity. Figure 18 f). Regarding IgG subtypes, compared to the soluble OVA group, OVA-Y72632@TM-NSs increased the secretion of IgG1 and IgG2b by 1408-fold and 165-fold, respectively. Figure 18 Regarding IgG2c, compared with other administration methods, the serum of mice treated with OVA-Y72632@TM-NSs had the highest IgG2c content. Figure 18 i). It is worth noting that T helper cell type 1 (Th1) cytokines (such as IFN-γ) and T helper cell type 2 (Th2) cytokines (such as IL-5) can promote the production of antigen-specific IgG antibodies and subtypes. We further examined the effect of MOF-based nanovaccines on the production of cytokines secreted by splenocytes. Compared with the soluble OVA group, the MOF-based nanovaccines significantly promoted IL-5 secretion, increasing it by 32-fold, which is consistent with its effect on the IgG1 subtype. Figure 18Furthermore, compared to the OVA group, the MOF-based nanovaccine significantly increased IFN-γ production by 50-fold, which is beneficial for the differentiation of cytotoxic T lymphocytes (CTLs). Figure 18 k).

[0100] Given that the proliferation and activation of antigen-specific T cells are key factors in the adaptive immune response, we prepared single-cell suspensions of lymphocytes from immunized mice seven days after the second vaccination and restimulated these cells with ovalbumin (OVA). The proliferation of antigen-specific T cells was detected using CCK8 assays. Figure 18 As shown in Figure 1, the metal-organic framework (MOF)-based nanovaccine induced significant T lymphocyte proliferation upon restimulation with OVA, but this trend disappeared upon stimulation with bovine serum albumin (BSA), indicating that the nanovaccine induced an antigen-specific T cell response. Further flow cytometry was used to detect the typical cytotoxic effects of the MOF-based nanovaccine on CD8 cells. + T cells and cytotoxic CD4 + The effects of T cells, such as Figure 18 As shown in mn, compared with other treatment methods, OVA-Y27632@TM-NSs significantly improved IFN-γ + CD4 + The proportion of T cells (Th1 cytotoxic immune cells), and in addition, OVA-Y72632@TM-NSs significantly improved the immune response. Figure 18 (op). Additionally, the expression of CD69, another biomarker of T cell activation, was assessed, as well as CD4 in the OVA-Y72632@TM-NSs treatment group. + CD69 + (Activated CD4) + T cells and CD8 + CD69 + (Activated CD8) + The proportion of T cells was higher in all groups than in other groups. Figure 19 This suggests that activation of the cGAS-STING pathway and enhanced antigen processing capabilities contribute to triggering a robust T-cell-mediated adaptive immune response.

[0101] 2.8 Evaluation of the antitumor efficacy of OVA-Y27632@TM-NSs nanovaccine Encouraged by the excellent performance of MOF-based nanovaccines in maintaining antigen-antigen-presenting cell (APC) contact and inducing adaptive immune responses, we further evaluated the antitumor efficacy of the OVA-Y72632@TM-NSs nanovaccines in melanoma. A B16F10-OVA tumor-bearing model was established by subcutaneously inoculating 1×10^6 B16F10-OVA cells into the right flank of C57BL / 6 mice. Then, various formulations were administered on days 5, 10, and 15. Figure 20 a). For example Figure 20 As shown in bc, mice treated with the OVA-Y72632@TM-NSs nanovaccine had smaller tumor volumes compared to other treatments, a phenomenon that persisted throughout the observation period. This was primarily due to the cytotoxic CD8+. + T cells and cytotoxic CD4 + T cells are enhanced ( Figure 18 mp).

[0102] Given that biosafety is a well-known requirement for tumor vaccines, we also evaluated the effects of the nanovaccine on blood and major organs. Serum biochemical assays and hematoxylin and eosin (H&E) staining of major organs showed no significant differences among the six treatment groups, indicating that the OVA-Y72632@TM-NSs vaccine did not exhibit significant systemic toxicity. Figure 21-22 ).

[0103] 2.9 Tumor immune microenvironment (TIME) and TDLN cell analysis in mice after vaccination The accumulation and activation level of antigen-presenting cells (APCs) in lymph nodes are key factors affecting the sustained operation of vaccine-mediated cancer immune cycles. Activated APCs in lymph nodes can effectively initiate primary T cells and guide their migration to the tumor site, enhancing the infiltration of cytotoxic T lymphocytes (CTLs) into the tumor, ultimately mediating CTL recognition and killing of cancer cells. Therefore, we investigated the effects of MOF-based nanovaccines on APCs in lymph nodes. Compared with other treatments, the OVA-Y72632@TM-NSs nanovaccines significantly increased the number of dendritic cells (DCs, CD11c) in lymph nodes. + accumulation of () Figure 23 Regarding DC subtypes, CD8a was present in the OVA-Y72632@TM-NSs nanovaccine treatment group. + DCs and CD103 + The proportion of dendritic cells (DCs) was significantly higher in the OVA-Y72632@TM-NSs nanovaccine treatment group than in other treatment groups. This facilitates the effective identification of dead tumor cells and the efficient presentation of tumor-associated antigen information to T cells. Furthermore, compared to other groups, the OVA-Y72632@TM-NSs nanovaccine treatment group showed a higher proportion of CD8a.+ CD103 + The highest proportion of DCs confirms that CD8 generates potent cytotoxicity via the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-interferon gene stimulating factor (STING) signaling pathway. + T cells and cytotoxic CD4 + T cell-mediated immune response ( Figure 20 (de), in addition, CD80 in lymph nodes + DCs and CD86 + The number of DCs also increased, indicating that the OVA-Y72632@TM-NSs nanovaccine can induce strong DC maturation ( Figure 20 fg and Figure 24 ).

[0104] On the one hand, various immune cells in the tumor microenvironment affect the killing efficiency of the vaccine-induced cancer immune cycle. On the other hand, in the cancer immune cycle, the migration and infiltration of CTLs reduce some of the immunosuppressive factors caused by direct tumor cell killing, which is beneficial for reshaping the tumor immune microenvironment. Therefore, we next evaluated the changes of various immune cells in the tumor immune microenvironment after vaccination. Figure 20 As shown in the figure, compared with other groups, the OVA-Y72632@TM-NSs nanovaccine significantly promoted CD4. + T cells and CD8 + T cell infiltration indicates that MOF-based nanovaccines lead to higher CTL production and migration. Furthermore, as a prognostic indicator of immunotherapy, we further calculated CD8... + T cells and CD4 + The proportion of T cells was highest in the OVA-Y72632@TM-NSs nanovaccine treatment group. Figure 20 As the main immunosuppressive immune cells, we analyzed conventional T cells (Tregs, CD4+) + CD25 + Foxp3 + The proportion of T cells was found to be significantly lower in the OVA-Y72632@TM-NSs nanovaccine group. Figure 20 k, Figure 25 We also evaluated DCs (CD11c). + Regarding the infiltration of immune cells, compared with other treatments, the DC infiltration in the OVA-Y72632@TM-NSs group increased by approximately 0.5 to 3 times ( Figure 20 l, Figure 26 ).

[0105] Since the polarization of tumor-associated macrophages (TAMs) affects the efficacy of immunotherapy, we investigated the changes in M1 and M2 TAMs after various vaccinations. Compared with the OVA group, the OVA-Y72632@TM-NSs nanovaccine significantly increased M1 TAMs (CD11b). + F4 / 80 + CD86 + It reduced the infiltration of M2 type TAMs (CD11b cells) in the tumor microenvironment, while also decreasing the infiltration of these cells. + F4 / 80 + CD206 + The proportion of cells Figure 20 Furthermore, the OVA-Y72632@TM-NSs nanovaccine had minimal effect on myeloid-derived suppressor cells (MDSCs) and neutrophils, including monocyte MDSCs (mMDSCs, CD11b). + Ly6c + Ly6g − ), granulocyte MDSCs (gMDSCs, CD11b + Ly6c − Ly6g + SSC high ) and neutrophils (CD11b + Ly6c + Ly6g + SSC low () Figure 20 p, Figure 27 ).

[0106] These results demonstrate that MOF-based nanovaccines significantly enhance anti-tumor immune responses by precisely maintaining the cancer immune cycle.

[0107] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An anti-tumor nanovaccine, characterized in that, The metal-organic framework nanosheets include a tetra(4-carboxyphenyl)porphyrin ligand with a centrally chelated divalent manganese ion coordinated with manganese ions, and ROCK inhibitors and tumor antigens loaded on the nanosheets.

2. The anti-tumor nanovaccine according to claim 1, characterized in that, The ROCK inhibitor is Y27632 or a pharmaceutically acceptable salt thereof.

3. The anti-tumor nanovaccine according to claim 1 or 2, characterized in that, In the anti-tumor nanovaccine, the mass ratio of the metal-organic framework nanosheets, ROCK inhibitors, and tumor antigens is (43~44):(6~7):(24~26).

4. The anti-tumor nanovaccine according to claim 1 or 2, characterized in that, The metal-organic framework nanosheets are formed by coordination of manganese salt, tetrakis(4-carboxyphenyl)manganese porphyrin, and organic amine ligands under the regulation of surfactant.

5. A method for preparing an anti-tumor nanovaccine according to any one of claims 1 to 4, characterized in that, include: The metal-organic framework nanosheets were prepared, and then ROCK inhibitors and tumor antigens were loaded onto the nanosheets through self-assembly to obtain a nanovaccine.

6. The preparation method according to claim 5, characterized in that, The preparation method includes the following steps: (1) Preparation of metal-organic framework nanosheets: After dispersing manganese salt, organic amine ligand and surfactant in solvent, tetrakis(4-carboxyphenyl)manganese porphyrin solution is added to obtain the reaction system; (2) Loaded ROCK inhibitor: The solution containing ROCK inhibitor is added to the reaction system of step (1), dispersed, and then dried to remove impurities, thus obtaining metal-organic framework nanosheets loaded with ROCK inhibitor. (3) Loaded with tumor antigens: The solution containing tumor antigen is mixed with the solution containing metal-organic framework nanosheets loaded with ROCK inhibitor from step (2) and incubated. The resulting product is obtained after removing impurities.

7. The use of an antitumor nanovaccine according to any one of claims 1 to 4 or the preparation method according to claim 5 or 6 in the preparation of tumor therapeutic drugs.

8. The application according to claim 7, characterized in that, The tumor treatment drug is a drug that promotes the antigen uptake and cross-presentation capabilities of APC cells.

9. The application according to claim 7, characterized in that, The tumor treatment drug is one that activates the cGAS-STING pathway.

10. A tumor treatment drug, characterized in that, Including the anti-tumor nanovaccine as described in any one of claims 1 to 4.

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