Bionic nano-vaccine with artificial mitochondria and preparation method and application thereof

Abstract

The application discloses a kind of artificial mitochondrion's biomimetic nano vaccine and its preparation method and application, belong to biological medicine technical field.The biomimetic nano vaccine is core-shell structure, and the core is active oxygen response mesoporous silica nanoparticles, and the shell is hybrid membrane wrapped outside the core.The biomimetic nano vaccine of the application simulates the core control function of natural mitochondrion, synergistically promotes mitochondrial autophagy and mitochondrial biosynthesis, thereby fundamentally restores the mitochondrial homeostasis and immune function of old DCs, and finally stimulates strong anti-tumor immune response.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a biomimetic nanovaccine integrating artificial mitochondria, its preparation method, and its application. Background Technology

[0002] Cancer immunotherapy, particularly cancer vaccines, aims to combat tumors by activating the body's specific immune response. Dendritic cells (DCs), as the most potent antigen-presenting cells, play a central role in initiating and regulating adaptive immune responses. However, in older individuals, DC function declines significantly, manifested as reduced antigen phagocytosis, cross-presentation, migration, and cytokine secretion. This directly leads to insufficient immunogenicity and limited efficacy of vaccines in older populations. This age-related immunosenescence is one of the major challenges currently facing cancer vaccine development.

[0003] Recent studies have shown that mitochondrial dysfunction is a key intrinsic mechanism leading to the functional decline of dendritic cells (DCs) in older cells. Mitochondria are not only the cell's energy factories but also play a wide role in cell signal transduction, metabolic regulation, and cell fate determination. Mitochondrial homeostasis imbalance is prevalent in older DCs, characterized by a reduction in the mass of functional mitochondria and the accumulation of dysfunctional mitochondria. This leads to increased levels of reactive oxygen species (ROS), decreased membrane potential, and insufficient energy metabolism, ultimately weakening the immune function of DCs.

[0004] Restoring mitochondrial homeostasis is considered a promising strategy for reversing immunosenescence. Current strategies mainly involve two aspects: first, promoting mitochondrial biosynthesis to increase the number of functional mitochondria, for example, using drugs such as metformin (Met); and second, clearing dysfunctional mitochondria, primarily through inducing mitophagy. However, existing technologies lack an integrated platform capable of efficiently and synergistically targeting and delivering these two functions to aging dendritic cells (DCs). For example, simple drug delivery may lack targeting specificity, and how to safely and effectively induce mitophagy in vivo remains a challenge.

[0005] Therefore, developing a novel vaccine platform that can mimic the natural regulatory functions of mitochondria, simultaneously "clearing" bad mitochondria and "generating" good mitochondria, and can target and deliver tumor antigens, adjuvants, and these mitochondrial regulators to elderly DCs is of great clinical need and scientific significance for improving the immunotherapy effect in elderly cancer patients. Summary of the Invention

[0006] The purpose of this invention is to provide a novel biomimetic nanovaccine NA / AM (Nanovaccine: NA; Artificial mitochondria: AM; NA / AM) integrating artificial mitochondria. The "artificial mitochondria" function is simulated by the outer shell MM (responsible for inducing mitophagy) and the core Met (responsible for promoting mitochondrial biosynthesis). This vaccine, by mimicking the core regulatory functions of natural mitochondria, synergistically promotes mitophagy and mitochondrial biosynthesis, thereby fundamentally restoring mitochondrial homeostasis and immune function in aged dendritic cells (DCs), ultimately stimulating a powerful anti-tumor immune response.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a biomimetic nanovaccine integrating artificial mitochondria, comprising: a core of mesoporous silica nanoparticles, and a hybridization membrane wrapped around the core; The mesoporous silica nanoparticles are loaded with immune adjuvants and mitochondrial biosynthesis promoters. The hybrid membrane is formed by the fusion of a cancer cell membrane (CM) and a mitochondrial membrane (MM).

[0008] Furthermore, the mesoporous silica nanoparticles are reactive oxygen species (ROS) responsive mesoporous silica nanoparticles.

[0009] Furthermore, the reactive oxygen species (ROS) responsive mesoporous silica nanoparticles were prepared using the following method: The template agent and catalyst were dissolved in deionized water. Under heating and stirring conditions, a mixed solution of silicon source and diselenylene bond-bridged organosilicon precursor was slowly added. After reacting for several hours, the product was collected by centrifugation, and the template agent was removed by reflux with ethanol / ammonium nitrate solution to obtain mesoporous silica nanoparticles with ROS responsiveness (due to diselenylene bonds) and good drug loading capacity.

[0010] Furthermore, the immune adjuvant is a Toll-like receptor 9 (TLR9) agonist CpG oligonucleotide (CpG ODN), and the mitochondrial biosynthesis promoter is metformin (Met).

[0011] Furthermore, the cancer cell membrane (CM) is derived from the target tumor cells.

[0012] Secondly, the present invention provides a method for preparing the above-mentioned biomimetic nanovaccine, comprising the following steps: Step 1, Synthesis of ROS-responsive mesoporous silica nanoparticles (MSF): A sol-gel method was employed. A template agent (e.g., CTAB) and a catalyst (e.g., triethanolamine) were dissolved in deionized water. A mixed solution of a silicon source (e.g., tetraethyl orthosilicate, TEOS) and a diselenylene-bridged organosilicon precursor (e.g., BTESePD) was slowly added under heating (e.g., 80°C) and stirring, and the reaction was allowed to proceed for several hours. The product was collected by centrifugation, and the template agent was removed by reflux using an ethanol / ammonium nitrate solution, yielding MSF nanoparticles with ROS responsiveness (due to diselenylene bonds) and good drug loading capacity.

[0013] Step 2, co-loading of immune adjuvant (CpG) and mitochondrial biosynthesis promoter (Met): The MSF nanoparticles obtained in Step 1 were co-incubated with CpG ODN and Met in a buffer solution (such as PBS or deionized water) (e.g., stirred at room temperature for 24 hours). Unloaded molecules were removed by centrifugation to obtain co-loaded nanoparticles MSF@CpG / Met.

[0014] Step 3, Extraction of cancer cell membrane (CM) and mitochondrial membrane (MM) and preparation of hybridization membranes: Cancer cell membrane (CM) extraction: Cell membranes are extracted from target tumor cells (e.g., B16-OVA). Differential centrifugation is used: after cell lysis, the cells are first centrifuged at low speed (e.g., 600×g, 10 min) to remove the nucleus and debris, then ultracentrifuged (e.g., 20,000×g, 45 min) to collect the membrane components. The membrane precipitate is resuspended and extruded through a polycarbonate membrane (e.g., 400 nm) to form uniform CM vesicles.

[0015] Mitochondrial membrane (MM) extraction: Mitochondria were extracted from dendritic cells (DCs) of young (e.g., 8-week-old) mice. Using a mitochondrial extraction kit, mitochondria were obtained by cell homogenization and differential centrifugation (first at low speed to remove debris, then at high speed, such as 11,000×g, for 10 min, to precipitate intact mitochondria). The mitochondria were then lysed and ultracentrifuged to obtain MMs. The MMs were then lyophilized and stored.

[0016] Hybrid membrane (CM / MM) preparation: The extracted CM vesicles and MM are mixed at a certain protein mass ratio (preferably 1:1), and the two are fused to form hybrid membrane vesicles (CM / MM) by ultrasonic treatment (ice bath) and / or repeated extrusion through a polycarbonate membrane (e.g., 200 nm).

[0017] Step 4, Final Construction of the Biomimetic Nanovaccine: The MSF@CpG / Met obtained in Step 2 is mixed with the hybridization membrane (CM / MM) vesicles prepared in Step 3 at a certain mass ratio (e.g., MSF:membrane protein = 2:1). The mixture is then extruded by sonication in an ice bath and sequentially through polycarbonate membranes with decreasing pore sizes (e.g., 800 nm, 400 nm, 200 nm) to coat the MSF@CpG / Met core, forming the final core-shell structured nanovaccine NA / AM. The mixture is purified by centrifugation and stored in PBS.

[0018] Thirdly, this invention provides the application of the above-mentioned biomimetic nanovaccine in the preparation of a drug, wherein the drug is used for: Prevention and / or treatment of cancer, especially melanoma.

[0019] Enhance the anti-tumor immune response in individuals, especially older individuals.

[0020] It can be used in combination with immune checkpoint inhibitors (such as anti-PD-1 antibodies and anti-PD-L1 antibodies) to synergistically enhance anti-tumor efficacy.

[0021] Restoring mitochondrial homeostasis in aging dendritic cells (DCs) includes simultaneously promoting mitophagy and mitochondrial biosynthesis.

[0022] Improve the function of aging dendritic cells (DCs), including enhancing their ability to phagocytose antigens, cross-present, migrate, mature, and secrete cytokines.

[0023] Activate antigen-specific T cell responses, including the activation, proliferation, and cytotoxicity of CD8⁺ T cells and CD4⁺ T cells.

[0024] The beneficial effects of this invention are: 1. Synergistic regulation of mitochondrial homeostasis: For the first time, mitochondrial membranes (MM, which induces mitophagy) from young DCs and metformin (Met, which promotes mitochondrial biosynthesis) are creatively integrated into the same nanoplatform, achieving synergistic regulation of mitochondria in aged DCs through "cleansing and replenishing" and fundamentally reversing mitochondrial dysfunction.

[0025] 2. Bionic Design and Highly Efficient Targeting: Utilizing MM derived from young DCs as the vaccine shell component not only provides a strong mitochondrial autophagy signal but also significantly enhances the efficiency of the nanovaccine being recognized and internalized by older DCs, overcoming the bottleneck of decreased uptake capacity in older APCs. The cancer cell membrane (CM) shell provides homologous tumor antigens and the inherent ability to target homologous tumors.

[0026] 3. Intelligent Release and Multiple Functions: The ROS-responsive MSF core ensures the specific release of CpG and Met within the high ROS environment of APCs, improving efficacy and reducing off-target effects. This platform simultaneously achieves three functions: antigen presentation, immune adjuvant activation, and mitochondrial functional remodeling.

[0027] 4. Highly effective reversal of immune aging: In an aged mouse model, the nanovaccine of this invention can significantly restore the mitochondrial mass, membrane potential and metabolic function of aged DCs, and effectively promote their maturation and migration. Ultimately, it successfully stimulates a strong antigen-specific CD8⁺ T cell response and compensatory cytotoxicity of CD4⁺ T cells, breaking through the limitations of the aged immune system.

[0028] 5. Superior anti-tumor efficacy: In both prophylactic and therapeutic melanoma models in young and aged mice, the nanovaccine of this invention demonstrated superior anti-tumor efficacy compared to traditional nanovaccines. Especially when used in combination with anti-PD-1 antibodies, it achieved complete tumor regression and long-term survival in aged mice.

[0029] 6. High safety: In vitro and in vivo safety evaluations of the system show that the nanovaccine has good biocompatibility and has not caused significant systemic toxicity or organ damage. Attached Figure Description

[0030] Figure 1 This study compares the mitochondrial quality and function of young and aged dendritic cells (DCs). A shows the mean fluorescence intensity (MFI) of young and aged DCs after MitoTracker Deep Red (MTDR) staining, analyzed by flow cytometry, reflecting mitochondrial quality (n=6); B shows transmission electron microscopy (TEM) images of young and aged DCs, showing the overall mitochondrial morphology, scale bar = 5 μm; C shows the MFI of young and aged DCs after MitoSOX Red staining, analyzed by flow cytometry, reflecting mitochondrial ROS levels (n=5); D shows confocal microscopy images of young and aged DCs after staining with the JC-1 probe (red: JC-1 aggregates, representing high membrane potential; green: JC-1 monomers, representing low membrane potential), scale bar = 10. μm; E represents the expression levels of genes related to mitochondrial biosynthesis and autophagy in young and aged DCs analyzed by qPCR (n=3); F represents the oxygen consumption rate (OCR) curves of young and aged DCs, assessed by mitochondrial stress test (n=3); G represents the expression levels of MHC-II and CD80 molecules on the surface of young and aged DCs analyzed by flow cytometry (n=3).

[0031] Figure 2This study aimed to restore mitochondrial homeostasis and function in aged dendritic cells (DCs) using artificial mitochondria (MM / Met). A shows a TEM image (scale bar = 500 nm) of mitochondria and autophagosomes co-localized after 24 hours of incubation of aged DCs with MM vesicles. B shows a confocal microscopy image (scale bar = 10 μm) of mitochondria and LC3B+ autophagosomes co-localized after incubation of aged DCs with MM vesicles. C and E show the expression of mitochondrial autophagy-related genes (C) and mitochondrial biosynthesis-related genes (E) in aged DCs after different treatments, analyzed by qPCR (n=3). D shows the mitosclerotic fibrillation (MFI) of aged DCs after different treatments stained with MitoSOX Red by flow cytometry, reflecting mitochondrial ROS levels (n=5). F shows the mitochondrial mean diastolic response (MTDR) of aged and young DCs after different treatments by flow cytometry. The relative changes in MFI (n=6); G represents the OCR curves of mitochondrial stress in aged DCs after different treatments (n=3); H represents the uptake capacity of aged DCs after different treatments for labeled B16 cell membrane (WGA) and cytoplasmic dye (CFSE) by flow cytometry (n=3); I represents the expression level of CCR7 on the surface of aged DCs after different treatments by flow cytometry (n=3); J represents the trajectory analysis of aged DCs after different treatments towards CCL21 in vitro.

[0032] Figure 3 Characterization of the integrated artificial mitochondria nanovaccine (NA / AM). A shows a TEM image of the NA / AM nanovaccine, displaying its core-shell structure (scale bar = 100 nm); B and C compare the hydrodynamic dimensions (B) and zeta potential (C) of MSF@CpG / Met, CM / MM hybrid membrane vesicles, and NA / AM, respectively (n=3); D shows the TEM mapping analysis of NA / AM; E shows a confocal microscopy image demonstrating the successful fusion of DiO (green) labeled MM and DiD (red) labeled CM to form hybrid vesicles (scale bar = 10 μm); F shows the cumulative release curves of Met and CpG in NA / AM with and without 100 µM H2O2 (n=3); G shows the long-term stability of MSF@CpG / Met and NA / AM after 7 days of storage in cell culture medium (n=3).

[0033] Figure 4This study investigates the cellular uptake of nanovaccines and their in vitro activation of aged DCs. In this table, A and B are confocal microscopy images of FITC-labeled NA and NA / AM after incubation with young (A) and aged (B) DCs for 2 hours, respectively, with a scale bar of 10 μm; C shows the expression of co-stimulatory molecules (CD40, CD80, CD86) on the surface of aged DCs after different treatments, analyzed by flow cytometry (n=3); D and E show the secretion levels of TNF-α (D) and IL-12 (E) in the culture supernatant of aged DCs after different treatments, respectively, detected by ELISA (n=5); F shows the relative changes in MTDR and MFI of aged DCs after different treatments, analyzed by flow cytometry (n=5); G shows confocal microscopy images of aged DCs after different treatments stained with JC-1, showing changes in mitochondrial membrane potential, with a scale bar of 10 μm; H shows the expression of the PGC1α gene in aged DCs after different treatments, analyzed by qPCR (n=5); and I shows the statistical count of the number of LC3B+ autophagosomes in aged DCs after different treatments (n=10).

[0034] Figure 5 This study evaluated the in vivo lymph node distribution and antigen-specific T cell response of the nanovaccine. A shows in vitro fluorescence imaging of lymph nodes in young and aged mice 24 hours after inoculation with Cy5.5-labeled NA and NA / AM; B and C show quantitative analysis of NA and NA / AM fluorescence signals in lymph nodes of young (B) and aged (C) mice at different time points (n=3); D shows immunofluorescence staining analysis of lymph nodes in aged mice 24 hours after inoculation with NA and NA / AM (scale bar = 100 μm), showing the uptake of the nanovaccine by DCs (CD11c⁺); E and F show the quantitative analysis of NA and NA / AM uptake rates by DCs in lymph nodes of young (E) and aged (F) mice by flow cytometry (n=4); G and H show the analysis of mature DCs (highly expressing CD40, CD80, ...) in lymph nodes of young (G) and aged (H) mice by flow cytometry. The proportion of CD86 (n=4); I represents the serum levels of IL-12 and TNF-α in mice 24 hours after vaccination by ELISA (n=4); J represents the expression of CD107a on the surface of T cells detected by flow cytometry after co-culturing CD8⁺ T cells and CD4⁺ T cells derived from the spleen of young and old mice with B16OVA cells for 6 hours (n=5).

[0035] Figure 6This study illustrates the preventive and therapeutic effects of nanovaccines in a mouse model of melanoma. A shows the timeline of the prophylactic experiment; B and C represent the tumor growth curves (B) and survival rate curves (C) of young mice after prophylactic immunization with different formulations (n=5); D and E represent the tumor growth curves (D) and survival rate curves (E) of aged mice after prophylactic immunization with different formulations (n=5); F shows the proportion of effector memory T cells in the spleen of mice analyzed by flow cytometry on day 60 (n=5); G shows the timeline of the therapeutic experiment (combined with anti-PD-1 antibody); H and I represent the tumor growth curves (H) and survival rate curves (I) of young mice in different treatment groups (n=5); J and K represent the tumor growth curves (J) and survival rate curves (K) of aged mice in different treatment groups (n=5); L and M represent the infiltration of CD8⁺ T cells in tumor tissues of mice in different treatment groups analyzed by flow cytometry (L) and immunofluorescence staining (M) (n=5, scale bar = 100 μm). Detailed Implementation

[0036] In a first aspect, the present invention provides a biomimetic nanovaccine integrating artificial mitochondria, the structure of which is a core-shell structure, comprising: (1) Core: Composed of reactive oxygen species (ROS) responsive mesoporous silica nanoparticles (MSF), the core is internally loaded with: Immunoadjuvant: preferably a Toll-like receptor 9 (TLR9) agonist CpG oligonucleotide (CpG ODN).

[0037] Mitochondrial biosynthesis promoter: preferably metformin (Met).

[0038] (2) Outer shell: a hybrid membrane that surrounds the core, the hybrid membrane being formed by the fusion of cancer cell membrane (CM) and mitochondrial membrane (MM).

[0039] Cancer cell membrane (CM): Derived from target tumor cells (e.g., B16-OVA melanoma cells), providing a complete set of homologous tumor-associated antigens (TAAs).

[0040] Mitochondrial membrane (MM): Derived from mitochondria of young, healthy dendritic cells (DCs). The MM membrane is rich in "eat-me" signals and membrane proteins that can initiate mitophagy, and can serve as a potent inducer of mitophagy.

[0041] The nanovaccine can be represented as NA / AM (Nanovaccine: NA; Artificial mitochondria: AM; NA / AM). The "artificial mitochondria" function is simulated by the outer shell MM (responsible for inducing mitophagy) and the core Met (responsible for promoting mitochondrial biosynthesis).

[0042] Secondly, the present invention provides a method for preparing the above-mentioned biomimetic nanovaccine, comprising the following steps: Step 1, Synthesis of ROS-responsive mesoporous silica nanoparticles (MSF): A sol-gel method was employed. A template agent (e.g., CTAB) and a catalyst (e.g., triethanolamine) were dissolved in deionized water. A mixed solution of a silicon source (e.g., tetraethyl orthosilicate, TEOS) and a diselenylene-bridged organosilicon precursor (e.g., BTESePD) was slowly added under heating (e.g., 80°C) and stirring, and the reaction was allowed to proceed for several hours. The product was collected by centrifugation, and the template agent was removed by reflux using an ethanol / ammonium nitrate solution, yielding MSF nanoparticles with ROS responsiveness (due to diselenylene bonds) and good drug loading capacity.

[0043] Step 2, co-loading of adjuvant (CpG) and mitochondrial biosynthesis promoter (Met): The MSF nanoparticles obtained in Step 1 were co-incubated with CpG ODN and Met in a buffer solution (such as PBS or deionized water) (e.g., stirred at room temperature for 24 hours). Unloaded molecules were removed by centrifugation to obtain co-loaded nanoparticles MSF@CpG / Met.

[0044] Step 3: Extraction of cancer cell membrane (CM) and mitochondrial membrane (MM) and preparation of hybridization membranes Cancer cell membrane (CM) extraction: Cell membranes are extracted from target tumor cells (e.g., B16-OVA). Differential centrifugation is used: after cell lysis, the cells are first centrifuged at low speed (e.g., 600×g, 10 min) to remove the nucleus and debris, then ultracentrifuged (e.g., 20,000×g, 45 min) to collect the membrane components. The membrane precipitate is resuspended and extruded through a polycarbonate membrane (e.g., 400 nm) to form uniform CM vesicles.

[0045] Mitochondrial membrane (MM) extraction: Mitochondria were extracted from dendritic cells (DCs) of young (e.g., 8-week-old) mice. Using a mitochondrial extraction kit, mitochondria were obtained by cell homogenization and differential centrifugation (first at low speed to remove debris, then at high speed, such as 11,000×g, for 10 min, to precipitate intact mitochondria). The mitochondria were then lysed and ultracentrifuged to obtain MMs. The MMs were then lyophilized and stored.

[0046] Hybrid membrane (CM / MM) preparation: The extracted CM vesicles and MM are mixed at a certain protein mass ratio (preferably 1:1), and the two are fused to form hybrid membrane vesicles by ultrasonic treatment (ice bath) and / or repeated extrusion through a polycarbonate membrane (e.g., 200 nm).

[0047] Step 4, Final Construction of the Biomimetic Nanovaccine: The MSF@CpG / Met obtained in Step 2 is mixed with the hybridization membrane (CM / MM) vesicles prepared in Step 3 at a certain mass ratio (e.g., MSF:membrane protein = 2:1). The mixture is then extruded by sonication in an ice bath and sequentially through polycarbonate membranes with decreasing pore sizes (e.g., 800 nm, 400 nm, 200 nm) to coat the MSF@CpG / Met core, forming the final core-shell structured nanovaccine NA / AM. The mixture is purified by centrifugation and stored in PBS.

[0048] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0049] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0050] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Example 1

[0051] Mitochondrial Function Characterization in Young and Old Dendritic Cells (DCs) Dendritic cells (DCs) were collected from young (8-week-old) and old (90-week-old) female C57BL / 6J mice. Young DCs were obtained from the bone marrow and lymph nodes of 8-week-old C57BL / 6J mice, while old DCs were obtained from the same tissues of 90-week-old C57BL / 6J mice. The procedure was as follows: First, mouse lymph nodes were mechanically separated, and a single-cell suspension of the lymph nodes was prepared using a fine metal mesh. The cells were washed and centrifuged using a gradient centrifugation method. The low-density layer rich in DCs at the interface was carefully collected, washed, and resuspended in DC growth medium containing 20 ng / mL GM-CSF. For bone marrow-derived DCs, the bone marrow was washed with warm DMEM (containing 10% FBS and penicillin-streptomycin), centrifuged for 5 minutes, and the cells were resuspended in DC growth medium containing 20 ng / mL GM-CSF. Cells were resuspended in preheated complete culture medium, and 100 nM MitoTracker Deep Red (MTDR, ThermoFisher) was added. The cells were incubated at 37°C in a 5% CO2 incubator for 30 minutes in the dark. Cells were washed twice with pre-chilled PBS, resuspended, and immediately analyzed by flow cytometry (e.g., BD FACS Celesta) to determine the mean fluorescence intensity (MFI) of the red fluorescence channel. Results are as follows: Figure 1 As shown in Figure A, the MTDR MFI of older DCs is significantly lower than that of younger DCs.

[0052] Young and aged DCs were collected, resuspended in HBSS buffer, and 2 μM MitoSOX Red (ThermoFisher) was added. The cells were incubated at 37°C in the dark for 20 minutes. Cells were washed twice with HBSS, resuspended, and the MFI (molecular fluorescence index) of the red fluorescence channel was detected by flow cytometry. Results are as follows: Figure 1 As shown in Figure C, the MitoSOX MFI of older DCs was significantly higher than that of younger DCs. The JC-1 mitochondrial membrane potential assay kit was used.

[0053] Young and aged dendritic cells (DCs) were collected, resuspended in JC-1 working solution, and incubated in a 37°C, 5% CO2 incubator in the dark for 20 minutes. Cells were washed twice with JC-1 staining buffer. One portion of the cells was observed using a confocal microscope (red: J-aggregates, ~590 nm emission; green: J-monomers, ~529 nm emission), while the other portion was analyzed by flow cytometry to detect red and green fluorescence intensity, and the J-aggregate / J-monomer ratio was calculated. Results are as follows: Figure 1 As shown in Figure D, the JC-1 red-green ratio of older DCs is significantly lower than that of younger DCs.

[0054] The Seahorse XF Cell Energy Metabolism Analyzer was used. Young and aged DCs were seeded at appropriate densities into Seahorse XF96 cell culture plates. The cells were hydrated in XF assay medium free of phenol red and containing 10 mM glucose, 1 mM sodium pyruvate, and 2 mM L-glutamine. Oligomycin (1.5 μM), FCCP (1 μM), and rotenone / antimycin A (0.5 μM each) were injected sequentially. Oxygen consumption rate (OCR) was monitored in real time, and basal respiration, maximum respiration, reserve respiration capacity, and ATP production were calculated. Results are as follows: Figure 1 As shown in Figure F, all respiratory parameters of older DCs were significantly lower than those of younger DCs.

[0055] B16-OVA cells were labeled with the cell membrane dye WGA-555 and the cytoplasmic dye CFSE. The labeled tumor cells were co-cultured with young or aged dendritic cells (DCs) for 12 hours, and the fluorescence intensity of the DCs, reflecting their phagocytic capacity, was detected by flow cytometry. The results showed that aged DCs had a significantly lower phagocytic capacity for tumor cells than young DCs. After co-culturing young and aged DCs with B16-OVA cells, their expression levels were analyzed by flow cytometry after staining with antibodies (such as FITC-anti-MHC-II and APC-anti-CD80). Figure 1 The results showed that after co-culturing with tumor cells, the maturation and activation of aged DCs were significantly lower than those of young DCs.

[0056] Therefore, the mitochondrial membrane of young DCs was subsequently selected as a component of the vaccine shell to provide a strong mitophagy signal and enhance the efficiency of the nanovaccine being recognized and internalized by older DCs. Example 2

[0057] Extraction of mitochondrial membrane (MM) and functional verification of artificial mitochondria (MM / Met) Young dendritic cells (DCs) were washed with PBS solution, and mitochondria were extracted from them using a cell mitochondrial isolation kit (C3602S, Beyotime) according to the manufacturer's instructions. The extracted mitochondria were lysed in hypotonic buffer, and the mitochondrial membranes (MMs) were collected by gradient centrifugation and stored at -80°C for later use.

[0058] Specifically, the mixture was incubated in an ice bath for 30 minutes, followed by homogenization to disrupt the cells. Next, the cell homogenate was centrifuged at 4°C and 600×g for 10 minutes. The homogenate was then subjected to differential centrifugation: first, low-speed centrifugation (600×g, 10 minutes) to remove cell debris, followed by high-speed centrifugation (11000×g, 10 minutes) to precipitate intact mitochondria. Finally, the resulting mitochondrial fractions were lysed and ultracentrifuged. The final mitochondrial membranes (MMs) were freeze-dried and stored at -80°C for later use.

[0059] MMs extracted from young DCs were resuspended in deionized water or PBS and passed sequentially through 400 nm and 200 nm polycarbonate membranes 11 times each using a microextruder to form uniform MM vesicles.

[0060] Aged dendritic cells (DCs) were seeded in 96-well plates (5000 cells per well) and treated with different concentrations (0, 10, 25, 50, 100, 200 μM) of metformin (Met) for 24 hours. Cytotoxicity was assessed using the CCK8 assay, and changes in mitochondrial mass were detected by MTDR staining and flow cytometry. The results showed that 50 μM Met maximally increased mitochondrial mass in aged DCs without significant cytotoxicity; therefore, 50 μM was selected as the working concentration for subsequent in vitro experiments.

[0061] Aged dendritic cells (DCs) were seeded in well plates (5000 cells per well) and divided into the following groups: PBS control group, MM vesicle group, Met (50 μM) group, and MM vesicle and Met combined group (MM / Met). After 24 hours of treatment, the expression of mitophagy-related genes (such as PINK1, Parkin, LC3B) and mitochondrial biosynthesis-related genes (such as PGC1α, NRF1, TFAM) was detected. Figure 2 C and E showed that the combined use of MM and Met significantly enhanced the expression of mitochondrial biosynthesis genes and mitophagy-related genes in aged DCs. Flow cytometry analysis was performed. Figure 2 The studies in D and F revealed that aged DCs showed a significant increase in mitochondrial mass and a significant decrease in mitochondrial ROS production levels after co-incubation with MM / Met. Figure 2 Detection of mitochondrial respiratory function in G. Figure 2 The antigen phagocytic capacity of DCs was assessed by the H study. The results showed that the combined treatment of MM / Met synergistically upregulated mitophagy and biosynthesis, and most effectively restored mitochondrial respiration, mitochondrial homeostasis and immune function in aged DCs. Example 3

[0062] Preparation and characterization of nanovaccines integrating artificial mitochondria (NA / AM) 0.8 g CTAB and 0.2 g triethanolamine were dissolved in 50 mL deionized water and stirred vigorously in an 80°C water bath for 20 minutes. 5.0 g TEOS was mixed with an appropriate amount of diselenylene-bridged organosilicon precursor (BTESePD, with a mass ratio to TEOS optimized to 1:4) and slowly added dropwise to the above reaction solution. The reaction was carried out with gentle stirring at 80°C for 6 hours. After the reaction was complete, the white precipitate was collected by centrifugation (6000 rpm, 10 min) and washed three times with deionized water and anhydrous ethanol. The product was dispersed in anhydrous ethanol containing 1% (w / v) NH4NO3 and refluxed at 80°C for 24 hours to remove the template agent CTAB. The final product was collected by centrifugation, washed with ethanol, and vacuum dried to obtain ROS-responsive mesoporous silica nanoparticles (MSF) powder. The morphology, pore structure, and elemental composition of MSF were characterized by TEM, BET / BJH, EDS, and TEM mapping, demonstrating that MSF has a uniform morphology, high specific surface area and pore volume, and contains silicon, oxygen, and selenium.

[0063] 5 mg of MSF nanoparticles were accurately weighed and dispersed in 10 mL of deionized water. 100 μg of CpG ODN1826 and 1.5 mg of metformin (Met) were added to the dispersion. The mixture was incubated on a shaker in the dark for 24 hours at room temperature. The precipitate (MSF@CpG / Met) was collected by centrifugation (12,000 × g, 10 min) and gently washed once with PBS. The drug loading was calculated by measuring the absorbance (CpG) of the supernatant at 260 nm and analyzing it using HPLC (Met). The final CpG loading was approximately 9.3 μg / mg MSF, and the Met loading was approximately 136.1 μg / mg MSF.

[0064] Cell membranes were extracted from B16-OVA cells and extruded through a 400 nm membrane to form CM vesicles. Mitochondrial membranes were extracted from young DCs as described in Example 2 and extruded through a 200 nm membrane to form vesicles. CM and MM vesicles were mixed at a 1:1 membrane protein ratio and sonicated in an ice-water bath (100 W, 2 seconds on, 3 seconds off) for 20 minutes. Subsequently, using a mini extruder, the membranes were extruded sequentially through 800 nm, 400 nm, and 200 nm polycarbonate membranes 11 times each to promote membrane fusion. Figure 3 The fusion of DiO (green) labeled MM and DiD (red) labeled CM was observed by confocal microscopy, proving the successful preparation of the hybridization membrane.

[0065] The prepared MSF@CpG / Met nanoparticles and CM / MM hybridization membrane vesicles were mixed in PBS at an MSF mass to membrane protein mass ratio of 2:1. The mixture was sonicated in an ice-water bath for 20 minutes. Subsequently, using a mini extruder, the mixture was extruded 11 times each through polycarbonate membranes with pore sizes of 800 nm, 400 nm, and 200 nm, respectively, to coat the nanoparticle cores with the hybridization membrane. Uncoated membrane material was removed by centrifugation (8,000 × g, 10 min), and the purified nanovaccine NA / AM was collected, resuspended in PBS, and stored at 4°C for later use.

[0066] TEM Figure 3 Observations in the middle anatomical region (MA) showed that the NA / AM has a distinct core (MSF) and shell (hybrid membrane) structure. DLS revealed that the hydrodynamic dimensions of the NA / AM increased after coating, and the zeta potential became more negative due to the presence of the membrane. Figure 3 (BC). In PBS containing 100 μM H2O2, NA / AM can be degraded, and the cumulative release of CpG and Met reaches 54.9% and 69.4%, respectively, within 48 hours, while the release in PBS is slow ( Figure 3 (F). After incubation in serum-containing medium for 7 days, NA / AM maintained good dispersibility, while uncoated MSF@CpG / Met aggregated (F). Figure 3 (G). Example 4

[0067] In vitro activity evaluation of nano-vaccines DCs and RAW 264.7 cells were seeded in 96-well plates and incubated with different concentrations (0-100 μg / mL) of NA or NA / AM for 24 hours. Cell viability was assessed using the MTT assay. The results showed that cell viability was above 80% at concentrations ≤50 μg / mL, indicating that the nanovaccine has good biocompatibility.

[0068] Young and aged DCs were incubated with FITC-labeled NA or NA / AM (20 μg / mL) for 2 hours. The samples were analyzed using a confocal microscope. Figure 4 Cellular uptake was observed and quantified using AB and flow cytometry. The results showed that NA / AM had significantly higher uptake efficiency than NA in both young and old DCs, especially in old DCs, where NA / AM effectively overcame the deficiency of decreased uptake capacity.

[0069] Young or aged DCs were incubated with 20 μg / mL NA / AM for 12 hours. The expression levels of CD40, CD80, and CD86 in the CD11c⁺ DC population were detected by flow cytometry. Figure 4(C). The concentrations of TNF-α and IL-12 in the culture supernatant were detected by ELISA. Figure 4 (Delta-methyl-D-methyl). Results confirmed that NA / AM most effectively promoted the maturation of young DCs and significantly reversed maturation barriers in older DCs. After incubating older DCs with 20 μg / mL NA / AM for 12 hours, MTDR staining and flow cytometry were used to detect mitochondrial quality (…). Figure 4 JC-1 staining and confocal observation (F). Figure 4 Mitochondrial membrane potential was detected by qPCR using G and flow cytometry. PGC1α (a biosynthetic protein) in aged DCs was detected by qPCR. Figure 4 (H) and PINK1 (mitochondrial autophagy, Figure 4 The expression results of related genes (I) demonstrate that NA / AM can simultaneously enhance mitochondrial biosynthesis and mitophagy in aged DCs, thereby synergistically improving mitochondrial function and providing an energy and metabolic basis for its full activation. Example 5

[0070] In vivo immune evaluation and anti-tumor effects of nano-vaccines 1. Distribution in vivo and DC uptake Cy5.5-labeled NA or NA / AM was subcutaneously injected into young and aged mice via the footpads. Popliteal lymph nodes were harvested at different time points (1, 4, 8, 12, 24, 48 h) for in vitro fluorescence imaging. Figure 5 (A) and fluorescence signal quantification ( Figure 5 (Middle BC). The results showed that NA / AM had a stronger accumulation capacity in lymph nodes, especially in the lymph nodes of aged mice.

[0071] Twenty-four hours after injection, lymph nodes were collected and prepared into a single-cell suspension. The suspension was stained with anti-CD11c antibody, and the uptake rate of the nanovaccine by DCs was analyzed by flow cytometry. Figure 5 (D-DF). The results confirmed that NA / AM can be taken up more efficiently by DCs in the lymph nodes of aged mice.

[0072] Mouse paw pads were subcutaneously inoculated with a nano-vaccine. Twenty-four hours later, draining lymph nodes were harvested, and the expression of CD40, CD80, and CD86 in CD11c⁺ cells was analyzed by flow cytometry. Figure 5 (G and H). Serum was collected, and IL-12 and TNF-α levels were detected by ELISA. Figure 5 One week after three immunizations (7 days apart), CD8⁺ and CD4⁺ T cells from the spleen were isolated and co-cultured with B16OVA cells for 6 hours. The expression of CD107a was detected by flow cytometry. Figure 5(J). The results showed that NA / AM can effectively promote the maturation of DCs in vivo and stimulate a strong antigen-specific T cell response, which is particularly significant in aged mice.

[0073] Young and aged mice were subcutaneously injected with Saline, NA, NA@MM (a nanovaccine with MM coating (without Met)), NA@Met (a nanovaccine with Met (without MM)), or NA / AM (equivalent CpG dose) in the paw pads, and immunized three times on days -21, -14, and -7. On day 0, 2 × 10⁻⁶ cells were subcutaneously injected into the right back of the mice. 4 Mice were euthanized with B16-F10 tumor cells. Tumor volume was measured every 2-3 days, and survival was recorded. On day 60, mice were sacrificed, and spleens were harvested for flow cytometry analysis of effector memory T cells. Figure 6 (Middle F). Figure 6 As shown in the median BE, the NA / AM group exhibited the strongest tumor growth inhibition and the highest survival rate in both young and aged mice. NA / AM successfully induced strong immune memory in aged mice.

[0074] 1×10⁻⁶ mice were subcutaneously injected into the right back of both young and aged mice. 5 B16-F10 cells were used. Treatment began when the tumor reached approximately 80 mm³ (around day 2). Groups were assigned: Saline, αPD-1, NA / AM, NA + αPD-1, and NA / AM + αPD-1. The nanovaccine was administered subcutaneously to the footpad (days 2, 4, and 7), and the αPD-1 antibody was administered via tail vein injection (100 μg / dose, days 3 and 9). Tumor volume, survival, and tumor-infiltrating lymphocytes were monitored. Results are as follows. Figure 6 As shown in the results from the Chinese chemoradiochemical chromatography (CIC) study, NA / AM monotherapy significantly inhibited tumor growth, while the combination of NA / AM and αPD-1 showed the best efficacy, achieving 100% long-term survival in young mice and approximately 67% (4 / 6) long-term survival in older mice, significantly superior to other groups. The results of CD8+ T cell infiltration in tumor tissues of mice in different treatment groups showed that the combination of NA / AM and αPD-1 significantly increased the proportion of CD8+ T cells in tumor tissues (flow cytometry). Figure 6 (Middle L), and promoted the deep infiltration of these effector T cells into the tumor parenchyma (immunofluorescence, Figure 6 (M), which enhances the anti-tumor immune response. Example 6

[0075] Systemic security evaluation After the therapeutic experiment in Example 5 (day 90), blood samples were collected from mice in each group for complete blood count and blood biochemical analysis (e.g., ALT, AST, BUN, CRE, TC, etc.). Major organs (heart, liver, spleen, lung, and kidney) were also collected for paraffin embedding, sectioning, and H&E staining. The results showed that, compared with the PBS control group, mice treated with NA / AM alone or in combination with αPD-1 had serum biochemical indicators and complete blood count parameters within the normal range, and no obvious pathological damage was observed in the major organs. This indicates that the nanovaccine described in this invention has good in vivo safety.

[0076] This invention successfully developed a biomimetic nanovaccine, NA / AM, integrating artificial mitochondria. This vaccine innovatively integrates mitochondrial membranes (MM, which induces mitophagy) derived from young dendritic cells (DCs) with metformin (Met, which promotes mitochondrial biosynthesis) on the same platform. It co-delivers CpG adjuvant and Met via a ROS-responsive MSF core and utilizes a cancer cell membrane / mitochondrial membrane hybrid shell to provide antigens and targeting / autophagy signals. This design can synergistically restore mitochondrial homeostasis in aged DCs, fundamentally reversing their functional aging. In both young and aged mouse models, NA / AM demonstrated excellent effects in promoting DC activation, stimulating T-cell immunity, and anti-tumor activity. Especially when used in combination with immune checkpoint inhibitors, it effectively overcomes age-related immune barriers and achieves regression of established tumors. This vaccine platform exhibits good safety and provides a novel strategy and approach for developing highly effective cancer immunotherapies for the elderly.

Claims

1. A biomimetic nanovaccine integrating artificial mitochondria, characterized in that, It has a core-shell structure, including: The core is composed of mesoporous silica nanoparticles, and the core is loaded with immune adjuvants and mitochondrial biosynthesis promoters. The outer shell is a hybrid membrane that surrounds the core, and the hybrid membrane is formed by the fusion of cancer cell membrane and mitochondrial membrane.

2. The biomimetic nanovaccine according to claim 1, characterized in that, The mesoporous silica nanoparticles are mesoporous silica frameworks with reactive oxygen species responsiveness; preferably, the mesoporous silica frameworks contain diselenide bond bridging structures.

3. The biomimetic nanovaccine according to claim 1, characterized in that, The immune adjuvant is CpG oligonucleotide, and the mitochondrial biosynthesis promoter is metformin.

4. The biomimetic nanovaccine according to claim 1, characterized in that, The cancer cell membrane is derived from melanoma cells, preferably B16-F10 or B16-OVA cells.

5. The biomimetic nanovaccine according to claim 1, characterized in that, The mitochondrial membrane originates from the mitochondria of young, healthy dendritic cells.

6. The method for preparing the biomimetic nanovaccine according to claim 1, characterized in that, Includes the following steps: Step 1: Prepare mesoporous silica nanoparticles; Step 2, co-loading of immune adjuvant and mitochondrial biosynthesis promoter: The mesoporous silica nanoparticles obtained in Step 1 are co-incubated with immune adjuvant and mitochondrial biosynthesis promoter in solution, and the co-loaded nanoparticles are obtained by centrifugation. Step 3, preparation of hybridization membrane: cancer cell membrane vesicles and mitochondrial membrane vesicles are mixed, and fused by ultrasonic treatment and membrane extrusion to form hybridization membrane vesicles; Step 4, constructing the biomimetic nanovaccine: The co-loaded nanoparticles obtained in step 2 are mixed with the hybridization membrane vesicles obtained in step 3, and then subjected to ultrasonic treatment and membrane extrusion to coat the surface of the co-loaded nanoparticles with the hybridization membrane, thus obtaining the biomimetic nanovaccine.

7. The preparation method according to claim 6, characterized in that, In step 3, the cancer cell membrane and mitochondrial membrane are mixed at a protein mass ratio of 1:1; in step 4, the mass ratio of the co-loaded nanoparticles to the hybrid membrane vesicles is 2:

1.

8. A pharmaceutical composition, characterized in that, It includes the biomimetic nanovaccine as described in any one of claims 1-5 and a pharmaceutically acceptable carrier.

9. The use of the biomimetic nanovaccine according to any one of claims 1-5 or the pharmaceutical composition according to claim 8 in the preparation of a medicament for the prevention and / or treatment of tumors.

10. The application according to claim 9, characterized in that, The drug is used to enhance the antitumor immune response in elderly individuals; and / or, the drug is used in combination with an immune checkpoint inhibitor; and / or, the mechanism of action of the drug includes restoring mitochondrial homeostasis in dendritic cells, which includes simultaneously promoting mitophagy and mitochondrial biosynthesis.

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