Hydrogen-releasing nano vaccine for enhancing anti-tumor immunity as well as preparation method and application of hydrogen-releasing nano vaccine

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a hydrogen-releasing nano vaccine for enhancing anti-tumor immunity and a preparation method and application thereof.The hydrogen-releasing nano vaccine comprises an inner core and an outer core, the inner core is composed of a silicon dioxide nano frame loading an immunologic adjuvant and encapsulating a hydrogen prodrug, and the silicon dioxide nano frame is bridged through diselenide bonds; the shell is composed of a cancer cell membrane wrapping the surface of the inner core; the hydrogen prodrug comprises ammonia borane; the immunologic adjuvant comprises a CpG oligonucleotide. According to the invention, hydrogen therapy and the nano vaccine are creatively integrated; and hydrogen can be used as an immunologic adjuvant to be introduced into the nano vaccine, so that the mitochondrial function is improved, and the core problem of immune aging is directly solved. The nano vaccine shows excellent immune activation and tumor treatment effects in an old mouse model, and an effective scheme is provided for solving the clinical problem of low cancer immunotherapy response rate of old people.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a hydrogen-releasing nanovaccine for enhancing anti-tumor immunity, its preparation method, and its application. Background Technology

[0002] Cancer vaccines aim to activate the host's own immune system by delivering tumor antigens and immune adjuvants, thereby generating a specific anti-tumor immune response, and represent an important direction in the field of tumor immunotherapy. Nanoparticle vaccines, by co-delivering antigens and adjuvants to antigen-presenting cells, especially dendritic cells, can effectively enhance immunogenicity. However, the clinical efficacy of nanoparticle vaccines, especially in the elderly, is often unsatisfactory. This is mainly attributed to age-related immunosenescence, characterized by a decline in the function of cells such as dendritic cells, with oxidative stress and mitochondrial dysfunction being key factors leading to impaired immune cell function.

[0003] Molecular hydrogen possesses selective antioxidant, anti-inflammatory, and cytoprotective effects, specifically neutralizing highly toxic reactive oxygen species such as hydroxyl radicals without affecting reactive oxygen species with signal transduction functions. However, the application of hydrogen in vivo is limited by its low solubility and rapid diffusion, making it difficult to achieve and maintain effective concentrations at target sites.

[0004] How to solve the clinical challenge of low response rates to cancer immunotherapy in the elderly is a technical problem that needs to be addressed. Summary of the Invention

[0005] The purpose of this section is to outline some aspects of the embodiments of the present invention and to briefly describe some preferred embodiments.

[0006] As one aspect of the present invention, the present invention provides a hydrogen-releasing nanovaccine for enhancing anti-tumor immunity, comprising,

[0007] Core: Composed of a silica nanoframework loaded with an immune adjuvant and encapsulated with a hydrogen prodrug, wherein the silica nanoframework is bridged by diselenyl bonds;

[0008] The outer shell consists of a cancer cell membrane that surrounds the surface of the core.

[0009] The hydrogen prodrug includes ammoniaborane; the immune adjuvant includes CpG oligonucleotide.

[0010] As a preferred embodiment of the hydrogen-releasing nanovaccine for enhancing anti-tumor immunity described in this invention, the CpG oligonucleotide sequence is: 5'-TGACTGTGAACGTTCGAGATGA-3', 5'-TCCATGACGTTCCTGACGTT-3'.

[0011] As a preferred embodiment of the hydrogen-releasing nanovaccine for enhancing anti-tumor immunity described in this invention, the silica nanoframework is prepared by co-condensation of an inorganic silicon source and an organosilicon source bridged by diselenylene bonds via a sol-gel method.

[0012] As a preferred embodiment of the hydrogen-releasing nanovaccine for enhancing anti-tumor immunity described in this invention: the inorganic silicon source includes tetraethyl orthosilicate, and the organic silicon source includes bis-[3-(triethoxysilane)propyl]-diselenide.

[0013] As a preferred embodiment of the hydrogen-releasing nanovaccine for enhancing anti-tumor immunity described in this invention, the particle size of the hydrogen-releasing nanovaccine is 80-120 nm.

[0014] The present invention also provides a method for preparing the hydrogen-releasing nanovaccine for enhancing anti-tumor immunity, comprising the following steps:

[0015] (1) Preparation of diselenylene bond-bridged silica nanoframework: In the presence of an alkaline catalyst, an inorganic silicon source, a diselenylene bond-bridged organosilicon source and a hydrogen prodrug are mixed and reacted in an alcohol-water solution to obtain a silica nanoframework encapsulating a hydrogen prodrug.

[0016] (2) Loading immune adjuvant: The diselenylene bond-bridged silica nanoframework obtained in step (1) is mixed with the immune adjuvant solution to obtain nanoparticles loaded with immune adjuvant;

[0017] (3) Coating cancer cell membrane: The nanoparticles loaded with immune adjuvants obtained in step (2) are mixed with cell membrane vesicles isolated from cancer cells, and a biomimetic nanovaccine coated with cancer cell membrane is prepared by extrusion.

[0018] As a preferred embodiment of the method for preparing the hydrogen-releasing nanovaccine for enhancing anti-tumor immunity according to the present invention, the protein mass ratio of the nanoparticles loaded with immune adjuvant to cell membrane vesicles is 0.5-2:1.

[0019] As a preferred embodiment of the method for preparing the hydrogen-releasing nanovaccine for enhancing anti-tumor immunity according to the present invention, the mass ratio of the inorganic silicon source to the diselenylene bond-bridged organosilicon source is 1-5:1.

[0020] The present invention also provides the use of the hydrogen-releasing nanovaccine described above for enhancing anti-tumor immunity in the preparation of medicaments for the prevention and / or treatment of tumors, including melanoma.

[0021] The present invention also includes the use of the drug in combination with an immune checkpoint inhibitor; the immune checkpoint inhibitor includes an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0022] Beneficial effects of the invention: The invention is the first to integrate hydrogen therapy with nano-vaccines: The invention discovers that hydrogen can be used as an immune adjuvant to introduce nano-vaccines, improve mitochondrial function and directly solve the core problem of immune aging.

[0023] Intelligent response release: This invention utilizes the reactive oxygen species response characteristics of diselenylene bonds to achieve the synergistic and controllable release of hydrogen, antigens and adjuvants in target cells, thereby improving efficacy and reducing side effects.

[0024] Highly efficient targeting and activation: The cancer cell membrane encapsulation endows the nanovaccine with homologous targeting and good biocompatibility, enabling it to accumulate efficiently in lymphatic organs and be taken up by dendritic cells. The released hydrogen can effectively alleviate oxidative stress, promote mitochondrial biosynthesis and function, and strongly drive dendritic cell maturation and cytotoxic T cell activation.

[0025] Reversing immune aging: The nanovaccine of this invention has shown remarkable immune activation and tumor treatment effects in an aged mouse model, providing an effective solution to the clinical challenge of low response rates to cancer immunotherapy in the elderly population. Attached Figure Description

[0026] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below, wherein:

[0027] Figure 1 The hydrogen-releasing nanovaccine (MHF@CpG@CM) prepared in Example 1 of this invention and its physicochemical characterization.

[0028] in, Figure 1 The transmission electron microscope (TEM) image of A:MHF nanoparticles shows their uniform spherical morphology, with a scale bar of 100 nm.

[0029] Figure 1 The TEM image of the B:MHF@CpG@CM nanovaccine clearly shows its core-shell structure, with an outer cell membrane coating. The scale bar is 100 nm.

[0030] Figure 1 Comparison of Zeta potentials of C: MHF@CpG, cell membrane (CM) vesicles, and MHF@CpG@CM (n=3).

[0031] Figure 1 Hydrodynamic size distribution of D: MHF@CpG, cell membrane (CM) vesicles and MHF@CpG@CM (n=3).

[0032] Figure 1 Long-term stability of E:MHF@CpG and MHF@CpG@CM in cell culture medium.

[0033] Figure 1 TEM images of the degradation of F:MHF@CpG@CM after incubation for 2 days in a medium containing 0 or 100 µM H2O2, with a scale bar of 100 nm.

[0034] Figure 1 G in MHF@CpG@CM: Cumulative release curves of H2 and CpG in MHF@CpG@CM with or without 100 µM H2O2.

[0035] Figure 2 This image shows the detection of cellular uptake, hydrogen release, and lymph node targeting of the nanovaccine.

[0036] in, Figure 2 A: Confocal microscopy images of BMDCs after incubation with FITC-labeled MHF@CpG, MSF@CpG@CM and MHF@CpG@CM for 3 hours, showing cell uptake (blue: cell nuclei, green: nanoparticles), scale bar 10 µm.

[0037] Figure 2 The quantitative analysis of intracellular H2 release after treatment with B:BMDCs was performed to compare the sustained release capacity of free ammonia borane (AB) and MHF@CpG@CM (n=3).

[0038] Figure 2 C: Ex vivo fluorescence imaging of lymph nodes 24 hours after inoculation with Cy5.5-labeled MSF@CpG@CM and MHF@CpG@CM.

[0039] Figure 2 D in the figure: Quantitative analysis of fluorescence signals of Cy5.5 labeled MSF@CpG@CM and MHF@CpG@CM in lymph nodes at different time points (n=3).

[0040] Figure 2 E in the figure: Flow cytometry analysis of the internalization of Cy5.5-labeled nanovaccine by different immune cell subsets in lymph nodes 24 hours after vaccination (n=3).

[0041] Figure 3 This image shows the results of dendritic cell maturation, mitochondrial function, and oxidative stress detection.

[0042] in, Figure 3 A and B in the figure: Flow cytometry analysis of the expression levels of surface co-stimulatory molecules (CD80, CD86, CD40) in young (A) and old (B) BMDCs after treatment with different formulations (n=3).

[0043] Figure 3C: ELISA was used to detect the amount of IL-12 secreted in the supernatant of young and old BMDCs 24 hours after treatment with different preparations (n=4).

[0044] Figure 3 The expression levels of PGC1α, a key gene in mitochondrial biogenesis, were analyzed by D: Western Blot or real-time quantitative PCR after treatment of young and aged BMDCs with different formulations (n=4).

[0045] Figure 3 E: Flow cytometry was used to detect the mean fluorescence intensity (MFI) of MitoTracker DeepRed (MTDR) staining after treating young and aged BMDCs with different formulations, reflecting mitochondrial quality (n=4).

[0046] Figure 3 F: Flow cytometry was used to detect the mean fluorescence intensity (MFI) of MitoSOX staining after treatment of young and aged BMDCs with different formulations, reflecting mitochondrial ROS levels (n=4).

[0047] Figure 3 G: Extracellular flux analysis (mitochondrial stress test) shows the oxygen consumption rate (OCR) kinetic curves after treatment of aged BMDCs with different formulations, as well as the quantitative analysis of basal respiration, maximal respiration, reserve respiration capacity and ATP production (n=3).

[0048] Figure 3 H in the figure: High-resolution transmission electron microscopy observation of the morphology and structure of mitochondrial cristae after treatment with different formulations in aged BMDCs, scale bar is 500 nm.

[0049] Figure 3 I and J: JC-1 staining was used to assess changes in mitochondrial membrane potential after treatment of young (I) and old (J) BMDCs with different formulations (red: J-aggregates, representing high membrane potential; green: J-monomers, representing low membrane potential), with a scale bar of 10 µm.

[0050] Figure 3 Quantitative analysis of the ratio of fluorescence intensity of J-aggregates to J-monomers of JC-1 dye in young (K) and old (L) BMDCs (n=3).

[0051] Figure 4 This image shows the detection of dendritic cell recruitment and activation within lymph nodes.

[0052] in, Figure 4A: Immunofluorescence staining shows the aggregation of DCs in lymph nodes of young and old mice after inoculation with MSF@CpG@CM and MHF@CpG@CM (green: CD11c⁺ DCs), with a scale bar of 100 µm.

[0053] Figure 4 B: Flow cytometry analysis of the expression level of CCR7 on the surface of lymph node DCs in young and old mice after treatment with different preparations (n=4).

[0054] Figure 4 C and F: Flow cytometry quantitative analysis of the proportion of mature DCs (highly expressing CD80, CD86, CD40) in lymph nodes of young (C) and old (F) mice after treatment with different preparations (n=4).

[0055] Figure 4 The D, E and G, H: ELISA method was used to detect the cytokine levels of TNF-α and IL-12 in serum or lymph node homogenates of young (DE) and aged (GH) mice after treatment with different preparations (n=4).

[0056] Figure 5 This is a graph used for evaluating T-cell function and detecting preventive anti-tumor effects.

[0057] in, Figure 5 A: Flow cytometry analysis of CD8 levels in the spleen of aged mice after treatment with different formulations. + The proportion of T cells (n=4).

[0058] Figure 5 In B: Splenic T cells from aged mice treated with different formulations were co-cultured with B16OVA tumor cells for 24 hours, and the expression level of CD107a on the surface of T cells was detected by flow cytometry (n=4).

[0059] Figure 5 C in the above co-culture system: The killing effect of tumor cells was detected by the LDH release assay (n=4).

[0060] Figure 5 D in the diagram represents the timeline of preventative experimental design.

[0061] Figure 5 E and F in the figure: Tumor growth curves of young (E) and old (F) mice after prophylactic immunization with different formulations (n=5).

[0062] Figure 5 G and H in the figure: Survival curves of young (G) and old (H) mice after prophylactic immunization with different formulations (n=5).

[0063] Figure 6 Images showing the anti-tumor effects and tumor-infiltrating lymphocyte detection in different treatment groups.

[0064] in, Figure 6 A in the diagram represents a timeline of the therapeutic experimental design (time points for tumor inoculation, vaccination, and antibody administration).

[0065] Figure 6 B and C in the figure: Tumor growth curves of young (B) and old (C) mice in different treatment groups (n=5).

[0066] Figure 6 D and E in the figure: Survival curves of young (D) and old (E) mice in different treatment groups (n=5).

[0067] Figure 6 F in the image: Immunohistochemical or immunofluorescence staining shows CD8 in tumor tissues of young and aged mice in different treatment groups. + T cell infiltration, scale bar 100 µm. Detailed Implementation

[0068] To make the above-mentioned objectives, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to specific examples.

[0069] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0070] Example 1: Preparation and characterization of hydrogen-releasing nanovaccine (MHF@CpG@CM):

[0071] As one aspect of the technical solution of the present invention, a hydrogen-releasing nanovaccine is disclosed, which can be loaded with an immune adjuvant. Exemplarily, this embodiment uses diselenylene-bridged mesoporous silica nanoparticles loaded with the immune adjuvant CpG (5'-TCCATGACGTTCCTGACGTT-3') as the core and the tumor cell membrane as the outer shell.

[0072] Ammonia-borane hybridized diselenylene-bridged mesoporous silica was prepared using the sol-gel method, ultimately yielding ammonia-borane hybridized diselenylene-bridged mesoporous silica nanoparticles (MHF) with a mesoporous structure. The morphology, size, elemental composition, stability, surface potential, and mesoporous properties of the MHF were characterized using transmission electron microscopy (TEM), scanning electron microscopy (SEM), particle size potential analysis, and nitrogen adsorption-desorption apparatus. The TEM image is shown in Figure 1A. The prepared MHF exhibits a uniform morphology, good dispersibility, and a diameter of 60–100 nm. Specifically, 100 mg of ammonia-borane was dissolved in 52 mL of a mixed solution containing ethanol, deionized water, and 28% ammonia (volume ratio 20:5:1), and stirred vigorously for 15 minutes. Tetraethyl orthosilicate and bis-[3-(triethoxysilyl)propyl]-diselenide were prepared into a silicon precursor solution at a mass ratio of 3:1. The solution was added dropwise to the reaction mixture and stirred continuously at room temperature for 12 hours. The mixture was collected by high-speed centrifugation (10000 g, 5 min), washed three times with deionized water and ethanol, and then collected again to obtain diselenide-bridged mesoporous silica nanoparticles (MHF) with a mesoporous structure.

[0073] Preparation of mesoporous silica nanoparticles (MSF): 100 mg of ammonia borane was dissolved in 52 mL of a mixed solution containing ethanol, deionized water, and 28% ammonia (volume ratio 20:5:1), and stirred vigorously for 15 minutes. Tetraethyl orthosilicate was added dropwise to the reaction mixture, and the mixture was stirred continuously at room temperature for 12 hours. The mixture was collected by high-speed centrifugation (10000 g, 5 minutes), washed three times with deionized water and ethanol, and then collected again to obtain mesoporous silica nanoparticles (MSF) with a mesoporous structure.

[0074] To load CpG, 10 mg of the synthesized MHF nanoparticles were accurately weighed and dispersed in 10 mL of PBS (pH 7.4). 1 mg of CpG ODN1826 was added to the dispersion, and the mixture was incubated at 4°C with low-speed shaking (approximately 100 rpm) in the dark for 24 hours. After incubation, the mixture was centrifuged at 12,000 × g for 10 minutes at 4°C, and the precipitate (MHF@CpG) was collected and gently washed once with PBS. The CpG loading and encapsulation efficiency were calculated by measuring the UV absorbance of the supernatant at 260 nm. The results showed that the CpG loading of MHF@CpG was approximately 7.7 μg / mg.

[0075] To prepare the hydrogen-release vaccine, a large number of B16-F10 cells (mouse melanoma cells) were cultured to approximately 80% confluence. After washing the cells twice with pre-chilled PBS, the cells were collected using a cell scraper. The cells were resuspended in RIPA lysis buffer containing a protease inhibitor (phenylmethylsulfonyl fluoride, 1 mM) and incubated on ice for 30 minutes. Subsequently, the cells were first centrifuged at 4°C and 800 × g for 10 minutes to remove the nuclei and unlysaturated cells. The supernatant was collected and then ultracentrifuged at 4°C and 20,000 × g for 45 minutes; the precipitate was the cell membrane component. The membrane precipitate was resuspended in a small amount of pre-chilled deionized water and then squeezed through 400 nm and 200 nm polycarbonate membranes to form homogeneous cell membrane vesicles (CMs). The membrane protein concentration was determined using the BCA method, and the precipitate was aliquoted and stored at -80°C. The prepared MHF@CpG nanoparticles and B16-F10 cell membrane vesicles were mixed in PBS at a membrane protein to nanoparticle mass ratio of 1:1. The mixture was sonicated in an ice-water bath for 20 minutes to allow for initial fusion of the membrane and nanoparticles. Subsequently, a small liposome extruder (Avanti Research) was used. TM The nanoparticles were sequentially extruded 11 times each through polycarbonate membranes with pore sizes of 800 nm, 400 nm, and 200 nm at room temperature to form a core-shell structured hydrogen-releasing nanovaccine, MHF@CpG@CM. Uncoated membrane fragments or excessively large aggregates were removed by centrifugation (8,000 × g, 10 min), and the purified nanovaccine was collected, resuspended in PBS, and stored at 4°C for later use. Transmission electron microscopy was then performed. Figure 1 B shows that the hydrogen-releasing nanovaccine has a distinct core (dark) and outer shell (light) structure. Dynamic light scattering (DLS) Figure 1 C and Figure 1 The results showed that after coating the cell membrane, the hydrodynamic size of the hydrogen-releasing nanovaccine increased significantly, and the Zeta potential decreased due to the negative charge of the cell membrane, confirming successful coating. Figure 1 E showed that after incubation for 7 days in cell culture medium containing 10% fetal bovine serum, the particle size of the hydrogen-released nanovaccine did not change significantly, while the uncoated MHF@CpG showed obvious aggregation, indicating that the cell membrane coating significantly improved the colloidal stability of the nanoparticles. Figure 1 F showed that after incubation in PBS containing 100 μM H2O2 for 48 hours, TEM revealed significant degradation and fragmentation of the hydrogen-releasing nanovaccine structure. (Release study) Figure 1 G indicates that CpG accumulates and releases approximately 70% within 48 hours in the presence of H2O2, while release is minimal in PBS. Furthermore, MHF@CpG@CM continuously releases H2 for up to 48 hours under H2O2 stimulation, while the H2 release is less than 10% in the absence of H2O2, demonstrating its good stability and ROS-triggered release characteristics.

[0076] Example 2:

[0077] This embodiment verifies the in vitro biosafety, cellular uptake, and lymphatic distribution of the hydrogen-releasing nanovaccine, and explores the in vitro effects of the hydrogen-releasing nanovaccine on antibody-presenting cell activation and mitochondrial function.

[0078] RAW 264.7 macrophages and mouse bone marrow-derived dendritic cells (BMDCs) were cultured at 5 × 10⁶ cells per well. 3 Cells were seeded at a density of [number] cells per well in 96-well plates. After overnight culture, the medium was replaced with culture medium containing different concentrations (2.5, 5, 10, 20, 50, 100 μg / mL) of MHF@CpG@CM or a control formulation, and cultured for another 24 hours. Subsequently, 10 μL of CCK-8 solution was added to each well, and after incubation for 2 hours, the absorbance at 450 nm was measured using a microplate reader. Cell viability was calculated. The results showed that even at a concentration as high as 20 μg / mL, the cell viability remained above 85%, indicating that the nanovaccine has good biocompatibility. Subsequent in vitro experiments used 20 μg / mL as the working concentration.

[0079] BMDCs were inoculated into 24-well plates (1×10⁻⁶). 5 Cells / well were incubated with FITC-labeled hydrogen-releasing nanovaccine (20 μg / mL) for 3 hours. After washing with PBS, cell nuclei were fixed with 4% paraformaldehyde and stained with Hoechst 34580. Cells were then analyzed using a confocal microscope. Figure 2 A) Observation and flow cytometry revealed that the cellular uptake efficiency of the hydrogen-released nanovaccine was significantly higher than that of the uncoated MHF@CpG, demonstrating that the cell membrane coating can effectively promote the internalization of the nanovaccine by APCs.

[0080] C57BL / 6 mice were used as experimental subjects. Hydrogen-releasing nanovaccines were labeled with Cy5.5 and subcutaneously injected. At 0, 1, 4, 8, 12, 24, and 48 hours post-administration, the accumulation of the hydrogen-releasing nanovaccines in lymph nodes was observed and quantitatively analyzed using a small animal in vivo imaging system. The results, shown in Figures 2C and 2D, indicate that the hydrogen-releasing nanovaccines can effectively accumulate in lymph nodes.

[0081] BMDCs were extracted from the femur and tibia of 8-week-old (young) and 90-week-old (old) C57BL / 6 mice. They were co-incubated with 20 μg / mL of hydrogen-released nanovaccine for 3 hours, followed by the addition of CD11c-APC, CD80-FITC, CD86-PE, and CD40-PercP fluorescent antibodies, and incubated at 4°C in the dark for 30 minutes. After washing twice with PBS, the cells were resuspended in 300 μL of PBS and immediately analyzed by flow cytometry. CD11c-positive cell populations were delineated, and the percentage of CD80, CD86, and CD40 co-positive cells within these populations was statistically analyzed. Flow cytometry results. Figure 3 A and B show that hydrogen-released nanovaccines can induce BMDC maturation more efficiently than non-hydrogen-released nanovaccines.

[0082] The processed BMDCs were collected and incubated with 100 nM MitoTracker Deep Red FM working solution at 37°C in the dark for 30 minutes. After washing with PBS, their fluorescence intensity was detected by flow cytometry. Figure 3 E showed that the hydrogen-released nanovaccine improved the mitochondrial quality of BMDCs. Furthermore, the treated BMDCs were collected and incubated with 5 μM MitoSOX Red working solution at 37°C in the dark for 30 minutes. After washing with PBS, they were immediately analyzed by flow cytometry. Figure 3 F shows that hydrogen nanovaccines can reduce reactive oxygen species production in BMDCs mitochondria.

[0083] Example 3: This example explores the in vivo immune activation, anti-tumor effect evaluation, and safety of the nanovaccine.

[0084] Using young (8 weeks) and aged (90 weeks) C57BL / 6 mice as examples, PBS, MHF@CpG, MSF@CpG@CM, or MHF@CpG@CM (dose 10 mg / kg) were subcutaneously injected into the paw pads, and the mice were immunized three times on days -21, -14, and -7. On day 0, 2 × 10⁻⁶ PBS was subcutaneously injected into the right back of the mice. 4 One B16-F10 tumor cell. The tumor's major and minor axes were measured every 3 days using calipers, and the tumor volume was calculated (volume = length × width). 2 ×0.52). The survival status of the mice was also recorded. Figure 5 As shown in the EH results, the MHF@CpG@CM group exhibited the strongest tumor growth inhibition effect and the highest survival rate in both young and aged mice. Particularly in aged mice, the protective effect of MSF@CpG@CM was weak, while MHF@CpG@CM resulted in 80% of mice showing no tumor growth within 45 days, demonstrating a significant effect.

[0085] Taking young (8 weeks) and aged (90 weeks) C57BL / 6 mice as examples, 1×10⁻⁶ mice were subcutaneously injected into the right back of both young and aged mice on day 0. 5 B16-F10 cells were used. Mice were randomly assigned to receive: PBS, αPD-1 antibody (Bio X Cell), MSF@CpG@CM, MSF@CpG@CM + αPD-1, MHF@CpG@CM, and MHF@CpG@CM + αPD-1. The nanovaccine was administered subcutaneously via the footpad (10 mg / kg, days 7, 14, and 21), and the αPD-1 antibody was administered via tail vein injection (100 μg / dose, days 3 and 9). Tumor volume, survival rate, and tumor invasion CD8 were observed. + T cells, serum cytokines (such as IFN-γ, TNF-α). Figure 6 As shown in the BE study, MHF@CpG@CM monotherapy significantly inhibited tumor growth, while the combination of MHF@CpG@CM and αPD-1 showed the best efficacy, achieving 100% long-term survival in young mice and 80% long-term survival in older mice, significantly superior to other groups. The combination group also exhibited the highest levels of CD8⁺ T cell infiltration and pro-inflammatory cytokine levels within the tumor. After the therapeutic experiment, blood samples from mice in each group were collected for blood biochemical analysis (detecting ALT, AST, BUN, CR, TC, etc.), and major organs (heart, liver, spleen, lung, and kidney) were collected for paraffin embedding, sectioning, and H&E staining. Compared with the PBS control group, mice treated with MHF@CpG@CM monotherapy or in combination with αPD-1 showed serum biochemical indicators within the normal range, and no obvious pathological damage was observed in major organs. This indicates that the nanovaccine described in this invention has good in vivo safety.

[0086] In summary, this invention discloses a hydrogen-releasing biomimetic nanovaccine, its preparation method, and its application in the preparation of antitumor drugs. The nanovaccine comprises: a core, which is a silica nanoframework loaded with an immune adjuvant and encapsulating the hydrogen prodrug ammoniaborane, the silica being bridged by diselenyl bonds; and a shell, which is a cancer cell membrane encapsulating the surface of the core. This unique structure enables the nanovaccine to remain stable during in vivo circulation, and upon entering antigen-presenting cells with high levels of reactive oxygen species, the diselenyl bonds break, the framework degrades, thereby achieving the synergistic release of hydrogen, adjuvant, and antigen.

[0087] The nanovaccine of this invention can target lymphoid organs and degrade in response to their highly reactive oxygen species (ROS) environment, thereby synergistically releasing hydrogen, immune adjuvants, and tumor antigens. The released hydrogen can scavenge ROS within dendritic cells, improve mitochondrial function, reverse immunosenescence, and significantly promote dendritic cell maturation and CD8+. +T-cell activation. This nanovaccine demonstrated excellent efficacy in preventing and treating melanoma in both young and aged mouse models, and when combined with an anti-PD-1 antibody, it achieved complete regression of established tumors, providing a new strategy for enhancing cancer immunotherapy in elderly individuals.

[0088] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A hydrogen-releasing nanovaccine for enhancing anti-tumor immunity, characterized in that: include, Core: Composed of a silica nanoframework loaded with an immune adjuvant and encapsulated with a hydrogen prodrug, wherein the silica nanoframework is bridged by diselenyl bonds; The outer shell consists of a cancer cell membrane that surrounds the surface of the core. The hydrogen prodrug includes ammoniaborane; the immune adjuvant includes CpG oligonucleotide.

2. The hydrogen-releasing nanovaccine for enhancing anti-tumor immunity according to claim 1, characterized in that: The CpG oligonucleotide sequence is: 5'-TGACTGTGAACGTTCGAGATGA-3', 5'-TCCATGACGTTCCTGACGTT-3'.

3. The hydrogen-releasing nanovaccine for enhancing anti-tumor immunity according to claim 1 or 2, characterized in that: The silica nanoframework was prepared by co-condensation of an inorganic silicon source and an organosilicon source bridged by diselenylene bonds via a sol-gel method.

4. The hydrogen-releasing nanovaccine for enhancing anti-tumor immunity according to claim 3, characterized in that: The inorganic silicon source includes tetraethyl orthosilicate, and the organic silicon source includes bis-[3-(triethoxysilane)propyl]-diselenide.

5. The hydrogen-releasing nanovaccine for enhancing anti-tumor immunity according to claim 4, characterized in that: The hydrogen-releasing nanovaccine has a particle size of 80-120 nm.

6. The method for preparing the hydrogen-releasing nanovaccine for enhancing anti-tumor immunity according to claim 1, characterized in that: Includes the following steps, (1) Preparation of diselenylene bond-bridged silica nanoframework: In the presence of an alkaline catalyst, an inorganic silicon source, a diselenylene bond-bridged organosilicon source and a hydrogen prodrug are mixed and reacted in an alcohol-water solution to obtain a silica nanoframework encapsulating a hydrogen prodrug. (2) Loading immune adjuvant: The diselenylene bond-bridged silica nanoframework obtained in step (1) is mixed with the immune adjuvant solution to obtain nanoparticles loaded with immune adjuvant; (3) Coating cancer cell membrane: The nanoparticles loaded with immune adjuvants obtained in step (2) are mixed with cell membrane vesicles isolated from cancer cells, and a biomimetic nanovaccine coated with cancer cell membrane is prepared by extrusion.

7. The method for preparing the hydrogen-releasing nanovaccine for enhancing anti-tumor immunity according to claim 6, characterized in that: The ratio of the protein content of the nanoparticles loaded with the immune adjuvant to that of the cell membrane vesicles is 0.5-2:

1.

8. The method for preparing a hydrogen-releasing nanovaccine for enhancing anti-tumor immunity according to claim 6 or 7, characterized in that: The mass ratio of the inorganic silicon source to the diselenylene bond-bridged organic silicon source is 1-5:

1.

9. The application of the hydrogen-releasing nanovaccine for enhancing anti-tumor immunity according to claim 1 in the preparation of drugs for the prevention and / or treatment of tumors, characterized in that: The tumors include melanoma.

10. The application according to claim 9, characterized in that: The drug is used in combination with an immune checkpoint inhibitor; the immune checkpoint inhibitor includes an anti-PD-1 antibody or an anti-PD-L1 antibody.

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