Tumor vaccine containing heterogeneous fusion membrane as well as preparation method and application of tumor vaccine

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a tumor vaccine containing a heterogeneous fusion membrane as well as a preparation method and application of the tumor vaccine. The tumor specific antigen vaccine with self-adjuvant activity is designed by fusing extracellular vesicles (EV) from bacteria and cell membranes (CM) from tumor cells, shows a strong prevention effect in a tumor model, and can inhibit the occurrence of tumors. Furthermore, in order to enhance the limited tumor eradication capability of the heterogeneous fusion membrane, the tumor vaccine is also provided with an acoustic / chemical dynamic therapy (SDT / CDT) module, so that local tumor eradication and lasting anti-tumor immunity are integrated, solid tumors can be effectively damaged, and tumor metastasis can be inhibited.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a tumor vaccine comprising a heterogeneous fusion membrane, and a preparation method and application thereof. Background Art

[0002] Tumor immunotherapy is becoming an effective treatment after surgery, chemotherapy, and radiotherapy. Personalized tumor vaccines can not only prevent the occurrence of tumors, but also provide long-term protection as a particularly promising approach in combination with other treatments to eradicate established tumors by triggering immune responses mediated by tumor-specific immune cells. Tumor cell membrane (CM)-based biomimetic nanoplatforms break through traditional vaccine design protocols by providing targeted delivery, antigen presentation, and immune stimulation. Many studies have shown that CM express rich antigens, including personalized new antigens, which can be recognized and taken up by antigen-presenting cells (APCs) to further activate tumor-specific T cells in anti-tumor immune responses. However, the low immunogenicity of single CM hinders efficient immune activation of APCs and fails to induce effective anti-tumor immune responses. Therefore, many improved strategies based on CM are being widely studied.

[0003] In the past few years, hybrid biomimetic strategies have been described by fusing homogeneous cell or membrane components to achieve co-delivery of tumor antigens and immunostimulants, which is a new approach to construct nanovaccines for tumor immunotherapy. For example, some natural biomembranes have been used as components to fuse with CM to design vaccines, including platelets, erythrocytes, leukocytes, and dendritic cells. These hybrid membranes exhibit integrated functions and promote the delivery of tumor antigens to APCs. Given the complexity, high cost, and non-replicability of isolating and culturing immune or blood cells, simpler hybrid manufacturing methods have been explored to enhance immunological functions. Bacterial extracellular vesicles (EVs), membrane vesicles spontaneously formed by bacteria, are considered ideal immunostimulants because they inherit many natural adjuvant components from parent bacteria and exist in a non-replicating form. Compared with Food and Drug Administration (FDA)-approved adjuvants (e.g., aluminum salts, Tween 80, CpG 1018), EVs, as a natural adjuvant, can overcome immunosuppressive barriers and effectively stimulate innate and adaptive immunity. They can be produced through simple fermentation and purification procedures. Recently, a number of studies have investigated the immune function of fused membranes prepared from CM and EVs from Gram-negative bacteria (e.g., Escherichia coli, Salmonella) and obtained exciting results. Notably, recent studies have shown that Gram-positive bacteria can also secrete EVs. Unlike EVs of Gram-negative bacteria that are mainly derived from the outer membrane (so-called outer membrane vesicles, OMVs), EVs of Gram-positive bacteria originate from the cytoplasmic membrane. Staphylococcus aureus (S. aureus), as an important Gram-positive pathogen, has been reported to have great application prospects in disease treatment. In addition, EVs from S. aureus induce the production of interleukins (IL-1β, IL-6) and interferon-γ (IFN-γ), which are closely related to anti-tumor immunity. These intrinsic properties make EVs of S. aureus attractive candidates for vaccine preparation.

[0004] Although CM-based fusion membranes have great potential in initiating anti-tumor immunity, single immunotherapy remains difficult to eradicate established tumor models due to the diversity, complexity, and low immunogenicity of tumors. Recently, CM-based fusion membrane strategies that integrate immunotherapy with other treatment modalities have made it possible to achieve effective tumor ablation. For example, photothermal therapy, radiotherapy, or chemotherapy. However, these approaches still face problems such as weak light penetration, high cost, and side effects, which to some extent limit their scalability. Recent studies have shown that emerging reactive oxygen species (ROS)-based therapies can not only directly reduce tumor burden, but also cause immunogenic cell death (ICD) and increase the immunogenicity of established tumor tissues, thereby inducing secondary killing of tumor cells by immune cells, a mechanism that may synergize with fusion membrane vaccines to enhance anti-tumor immunity. Among ROS-based therapies, sonodynamic therapy (SDT) has been selected as a potential clinical approach for the treatment of a variety of tumors due to its unique deep tissue penetration, non-invasiveness, economy, and convenience. By temporally and spatially managing the irradiation site of ultrasound, ROS can be precisely generated in tumor tissues rather than normal tissues. In addition, chemodynamic therapy (CDT) is also a promising ROS-based treatment modality that fights tumors by catalyzing the production of ROS from overexpressed H2O2 in the tumor microenvironment without the need for external energy stimulation. Summary of the invention

[0005] The present invention designs a tumor-specific antigen vaccine with self-adjuvant activity by fusing extracellular vesicles (EV) from bacteria and cell membranes (CM) from tumor cells, which shows a strong preventive effect in tumor models and can inhibit tumorigenesis. Furthermore, in order to enhance the limited tumor eradication ability of heterogeneous fusion membranes, this type of tumor vaccine is also equipped with a sono / chemodynamic therapy (SDT / CDT) module, so that it integrates local tumor eradication with persistent anti-tumor immunity, which can effectively destroy solid tumors and inhibit tumor metastasis.

[0006] Specifically, in one aspect, the present invention provides a tumor vaccine comprising a fusion membrane formed by the fusion of a cell membrane derived from a tumor cell and an extracellular vesicle derived from a bacterium.

[0007] Furthermore, the cell membrane derived from tumor cells substantially retains cell membrane proteins on the surface of tumor cells, especially tumor-specific antigen proteins. The extracellular vesicles derived from bacteria substantially retain cell membrane proteins on the surface of bacteria, especially bacterial antigen proteins.

[0008] Furthermore, the bacteria is Staphylococcus aureus.

[0009] Furthermore, the tumor cells include breast cancer cells, colon cancer cells, liver cancer cells, gastric cancer cells, kidney cancer cells, pancreatic cancer cells, ovarian cancer cells, lymphoma cells, osteosarcoma cells, glioma cells, prostate cancer cells, melanoma cells or any combination thereof.

[0010] Furthermore, the protein mass ratio of the cell membrane derived from tumor cells to the extracellular vesicles derived from bacteria is 1:1 to 3:1.

[0011] The present invention has found that the tumor vaccine containing the fusion membrane shows a strong preventive effect on tumors and can inhibit tumor occurrence. However, its effect on the treatment and eradication of tumors is limited.

[0012] To this end, in another aspect, the present invention also provides a tumor vaccine, which, in addition to the fusion membrane formed by the fusion of the cell membrane derived from tumor cells and the extracellular vesicles derived from bacteria as described above, also includes a sono / chemodynamic therapeutic substance that forms a core-shell structure together with the fusion membrane, wherein the fusion membrane is the shell and the sono / chemodynamic therapeutic substance is the core.

[0013] Furthermore, the mass ratio of the fusion membrane to the sono / chemodynamic therapeutic substance is 1:1 to 1:5.

[0014] Furthermore, the sono / chemodynamic therapeutic substance comprises a metal-porphyrin coordination polymer.

[0015] Furthermore, the metal-porphyrin coordination polymer is a coordination polymer formed by metal, tri(pyridin-4-yl)amine and 5,10,15,20-tetra(4-pyridinyl)-21H,23H-porphyrin through metal-N coordination.

[0016] Furthermore, the metal is Pt.

[0017] Furthermore, the molar ratio of the metal to tri(pyridin-4-yl)amine and 5,10,15,20-tetra(4-pyridinyl)-21H,23H-porphyrin is 1:0.4-0.6:0.4-0.6.

[0018] In other aspects, the present invention also provides a method for preparing a tumor vaccine, which comprises the following steps:

[0019] (1) preparing cell membranes derived from tumor cells;

[0020] (2) preparing extracellular vesicles derived from bacteria;

[0021] (3) The cell membrane derived from tumor cells is fused with the extracellular vesicles derived from bacteria to obtain a fusion membrane.

[0022] Furthermore, step (1) includes: obtaining tumor cells, obtaining tumor cell precipitates by centrifugation, resuspending the tumor cell precipitates, disrupting the tumor cells, and separating the obtained cell membranes.

[0023] Furthermore, the resuspending is performed using a hypotonic solution.

[0024] Furthermore, the hypotonic solution comprises 5-10 mM MgCl2, 5-10 mM HCl and a protease inhibitor.

[0025] Furthermore, the crushing includes ultrasonic crushing.

[0026] Furthermore, the cell membrane obtained by separation includes collecting the supernatant by centrifuging the disrupted tumor cell solution, and collecting the precipitate after centrifuging the supernatant again, thereby obtaining the cell membrane.

[0027] Further, the first centrifugation includes centrifugation at 3000-6000 g for 5-15 min, and the second centrifugation includes centrifugation at 13000-18000 g for 20-50 min.

[0028] Furthermore, step (2) comprises culturing the bacteria, collecting the supernatant by centrifugation, and collecting the precipitate after centrifuging the supernatant again, thereby obtaining bacterial outer membrane vesicles.

[0029] Further, the first centrifugation includes centrifugation at 3000-6000 g for 10-30 min, and the second centrifugation includes centrifugation at 130000-180000 g for 1-3 h.

[0030] Furthermore, step (3) comprises mixing the cell membranes derived from tumor cells and the extracellular vesicles derived from bacteria in a buffer, and sonicating to achieve fusion.

[0031] Furthermore, the buffer is PBS buffer.

[0032] Furthermore, the bacteria is Staphylococcus aureus.

[0033] Furthermore, the tumor cells include breast cancer cells, colon cancer cells, liver cancer cells, gastric cancer cells, kidney cancer cells, pancreatic cancer cells, ovarian cancer cells, lymphoma cells, osteosarcoma cells, glioma cells, prostate cancer cells, melanoma cells or any combination thereof.

[0034] Furthermore, the protein mass ratio of the cell membrane derived from tumor cells to the extracellular vesicles derived from bacteria is 1:1 to 3:1.

[0035] Furthermore, the preparation method further comprises:

[0036] (4) The fusion membrane obtained in step (3) is coated on the surface of the sono / chemodynamic therapeutic substance to obtain a tumor vaccine with a core-shell structure.

[0037] Furthermore, step (4) comprises mixing the fusion membrane obtained in step (3) with the sono / chemodynamic therapeutic substance in a buffer solution, ultrasonically treating, collecting the precipitate by centrifugation, and washing to obtain the tumor vaccine having a core-shell structure.

[0038] Furthermore, the mass ratio of the fusion membrane to the sono / chemodynamic therapeutic substance is 1:1 to 1:5.

[0039] Furthermore, the buffer is PBS buffer.

[0040] Furthermore, the sono / chemodynamic therapeutic substance comprises a metal-porphyrin coordination polymer.

[0041] Furthermore, the metal-porphyrin coordination polymer is a coordination polymer formed by metal, tri(pyridin-4-yl)amine and 5,10,15,20-tetra(4-pyridinyl)-21H,23H-porphyrin through metal-N coordination.

[0042] Furthermore, the metal-porphyrin coordination polymer is prepared as follows: tri(pyridin-4-yl)amine, 5,10,15,20-tetra(4-pyridinyl)-21H,23H-porphyrin and a surfactant are uniformly mixed in solvent A to obtain solution A; a metal source is dissolved in solvent B to obtain solution B; solution A and solution B are mixed and reacted, and the precipitate is collected by centrifugation to obtain the metal-porphyrin coordination polymer.

[0043] Furthermore, the molar ratio of the metal to tri(pyridin-4-yl)amine and 5,10,15,20-tetra(4-pyridinyl)-21H,23H-porphyrin is 1:0.4-0.6:0.4-0.6.

[0044] Furthermore, the metal source is a metal salt or hydrate.

[0045] Furthermore, the metal is Pt.

[0046] Furthermore, the metal source is K2PtCl6.

[0047] Furthermore, the surfactant is polyvinyl pyrrolidone.

[0048] Furthermore, the solvent A is an acidic aqueous solution, preferably a hydrochloric acid aqueous solution.

[0049] Furthermore, the solvent B is water.

[0050] Furthermore, the reaction is carried out under stirring, the reaction temperature is 15-35° C., and the reaction time is 1-6 h.

[0051] In other aspects, the present invention also provides the use of the tumor vaccine as described herein or the tumor vaccine obtained by the preparation method described herein in the preparation of a medicament for preventing tumor occurrence, metastasis and / or recurrence.

[0052] In other aspects, the present invention also provides use of the tumor vaccine as described herein or the tumor vaccine obtained by the preparation method described herein in the preparation of a drug for treating tumors.

[0053] Furthermore, the treatment includes combined treatment with ultrasound.

[0054] Furthermore, the tumor includes breast cancer, colon cancer, liver cancer, gastric cancer, kidney cancer, pancreatic cancer, ovarian cancer, lymphoma, osteosarcoma, glioma, prostate cancer or melanoma.

[0055] Beneficial effects of the present invention

[0056] The present invention designs a tumor-specific antigen vaccine with self-adjuvant activity by fusing EV from Staphylococcus aureus and CM from tumor cells (taking melanoma cells as an example). The heterogeneous fusion membrane tumor vaccine EV-CM inherits and enhances the immunological functions of the two parental components, not only showing efficient innate immune activation marked by dendritic cell (DC) activation, but also showing powerful cytotoxic T lymphocyte (CTL)-derived tumor-specific immunity. Vaccination with this integrated heterogeneous fusion membrane can provide a powerful immune response to fight cancer cells. In addition, in order to enhance the limited tumor eradication ability of the heterogeneous fusion membrane, the EV-CM nanovaccine is equipped with an SDT / CDT module, and the platinum-porphyrin coordination polymer (CPIP) is implanted into the EV-CM nanoplatform (CPIP@EV-CM) as an immunopotentiator. Inspired by the excellent catalytic properties and stability of natural metal-porphyrin coordination polymers (CPs), and the fact that porphyrins are the most frequently explored biocompatible sonosensitizers, a novel platinum-porphyrin coordination polymer Pt-Pta / Por (CPIP) was designed to achieve the combination of SDT and CDT. Under ultrasound (US) irradiation, platinum-porphyrin CP can produce abundant ROS to directly kill tumor cells and induce ICD as an immunopotentiator (CPIP). This feedback strengthens the immunotherapeutic effect of EV-CM heterogeneous fusion membranes and achieves enhanced anticancer vaccination. Validated by a melanoma model, EV-CM heterogeneous nanovaccines show a strong preventive effect and can inhibit tumorigenesis. At the same time, combined with SDT / CDT, CPIP@EV-CM integrates local tumor eradication with persistent antitumor immunity, effectively destroys solid tumors and inhibits tumor metastasis. The present invention confirms the immunotherapeutic potential of the heterogeneous fusion membrane strategy and ultrasound-enhanced nanoplatform, and its flexibility for designing tumor immunogenicity, self-adjuvants, and scalable vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 :(A) Fluorescence co-localization images of EV-CM prepared from DiI-labeled EV and DiO-labeled CM.(B) SDS-PAGE protein band analysis of CM, EV and EV-CM.

[0058] Figure 2 :(A) Quantitative analysis of surface molecules CD86 and CD80 reflecting the maturity of BMDCs. (B) Secretion of IL-6 and IL-12 in the supernatant of BMDCs.

[0059] Figure 3 :(A) In vivo fluorescence images of CM, EV / CM and EV-CM accumulation in lymph nodes at different time points after footpad injection.(B) Quantitative analysis of the fluorescence intensity of DiR(CM) in inguinal lymph nodes.

[0060] Figure 4 :(A) Relative tumor volume of different nanovaccine immunization groups (n=3). (B) Percentage of tumor-free mice after tumor inoculation. (C) CD3 + CD8 + T cell percentage. (D)CD8 + CD69 + (E) Percentage of effector memory T cells.

[0061] Figure 5 :(A) SEM image of CPIP. (B) TEM image of CPIP. (C-D) TEM image of CPIP@EV-CM. (E) Energy dispersive X-ray spectroscopy (EDS) elemental mapping image of CPIP. (F) N1s XPS spectra of Por, Pta and Pt-Pta / Por(CPIP). (G) Pt 4f XPS spectrum of Pt-Pta / Por. (H) SDS-PAGE protein analysis of proteins retained on EV-CM and CPIP@EV-CM.

[0062] Figure 6 :(A) Schematic diagram of POD enzyme catalytic activity and UV absorption diagram of TMB oxidation assay. (B) Detection of ·OH generation using TA+H2O2 as substrate. (C) Detection of ·O2 generation using HE+H2O2 as substrate - (D) SOSG detection of the acoustic dynamic performance of CPIP. -2 ) 1 SOSG fluorescence intensity of O2. DPA degradation detection of CPIP@EV-CM sonodynamic performance, time-dependent (E) and power-dependent (F) sonodynamic effects. (G) Flow cytometry analysis of DCFH-DA stained B16F10 cells to detect intracellular ROS generation. (H) Quantitative analysis of Calcein-AM / PI live-dead staining. (I) Bio-TEM images of B16F10 cells treated with CPIP and CPIP@EV-CM for 6 hours.

[0063] Figure 7 :(A) Expression of CRT on the surface of tumor cells. (B) Contents of ATP and HMGB1 in the supernatant of tumor cell culture. (C) Expression of CD80 and CD86 on the surface of BMDCs by flow cytometry. (D) ELISA kit was used to measure the secretion of IL-6 and IL-12 in the supernatant of BMDCs.

[0064] Figure 8:Evaluation of the in vivo therapeutic effect of CPIP@EV-CM. (A) B16F10 tumor weight at the end of treatment. (B) HE staining of tumor sections. (C) Quantitative analysis of CRT immunofluorescence staining of tumor tissues after different treatments. (D) Quantitative analysis of HMGB1 staining after different treatments. (E) FCM evaluation of the maturity of tumor-infiltrating DCs. (F) FCM analysis of CD3 + CD8 + The percentage of T cells.

[0065] Fig. 9 :(A) Survival curves of mice in different treatment groups. (B) Number of lung metastatic nodules in different groups. (C) HE staining of lung sections in PBS and CPIP@EV-CM+US groups. (D) FCM evaluation of DC maturation in LNs. (E) FCM analysis of CD3 + CD8 + (F) FCM analysis of T cells in LNs EM Cells (CD3 + CD8 + CD44 + CD62 - ) ratio. DETAILED DESCRIPTION

[0066] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0067] The following examples prepare a tumor vaccine (hereinafter referred to as EV-CM) comprising a fusion membrane formed by the fusion of a cell membrane derived from a tumor cell (hereinafter referred to as CM) and an extracellular vesicle (hereinafter referred to as EV) derived from bacteria, and a tumor vaccine (hereinafter referred to as CPIP@EV-CM) comprising a sono / chemodynamic therapeutic substance (i.e., a platinum-porphyrin coordination polymer (hereinafter referred to as CPIP)) constituting a core-shell structure together with the fusion membrane, and characterize the structure and function thereof, wherein the characterization methods adopted are summarized as follows:

[0068] In vivo lymph node imaging. To evaluate the migration and accumulation of CM and EV-CM in lymph nodes, CM was previously labeled with DiR to prepare EV-CM. Labeled CM (250 μg per mouse) and EV-CM (250 μg per mouse) were subcutaneously injected into the right hind limb footpad. In vivo live imaging was performed at predetermined time intervals using an IVIS imaging system.

[0069] Preventive effect of EV-CM on B16F10 tumors. C57BL / 6 mice were randomly divided into groups and vaccinated three times by subcutaneous injection of 100 μL of different membrane preparations at intervals of 7 days. On the 7th day after the last vaccination, 2×10^5 B16F10 cells were injected subcutaneously into the right hind buttocks of mice. Tumor volume was measured every two days. Tumor volume was calculated according to the formula = 0.5×width^2×length. After treatment, T cell subsets in the spleen were detected using anti-CD3 antibodies, anti-CD4 antibodies, and anti-CD8a antibodies. CD8 was detected using anti-CD69 antibodies. + Anti-CD44 and anti-CD62L antibodies were used to detect the proportion of memory T cells.

[0070] Structural characterization of CPIP@EV-CM. XPS spectra were obtained using X-ray photoelectron spectroscopy (XPS, XSAM800, Kratos Analytical, UK) to detect the composition. Transmission electron microscopy (TEM) was performed on a calibrated probe FEITitan80-300S / TEM equipped with a Gatan EELS detector. Scanning electron microscopy (SEM) was performed using a ThermoFisherScientific ApreoSHiVoc. Membrane proteins were characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE): all samples were mixed with loading buffer and heated at 100°C for 10 min; after that, equal amounts of protein samples (30 μg per well) were loaded on SDS-PAGE and run at 120 V for 4 h. The gel was stained with Coomassie brilliant blue dye for 4 h and imaged using an exposure instrument ChemiDoc MP (Bio-Rad, USA) after washing with glacial acetic acid.

[0071] Catalytic performance of CPIP@EV-CM. The peroxidase (POD) activity was determined by colorimetry. 25 μL of catalyst (4 mg mL -1 ), 25 μL of TMB (10 mg mL -1 ) and 25 μL of H2O2 (0.1 M) were added to 2 mL of sodium acetate-acetic acid (NaOAc / HOAc) buffer [100 mM (pH 4.5)]. The catalytic oxidation of TMB (oxTMB) was studied by measuring the change in light absorption of the oxidized form of TMB at λmax=652 nm.

[0072] Detect ROS types. HE can be combined with O2 - The reaction generates fluorescent ethidium, which is excited at 470 nm and emits light at 610 nm. First, 1.5 mL of the catalyst (100 μg mL -1) solution, 1.5 mL of H2O2 (0.1 M) and 1.5 mL of HE-ethanol solution (1 mg mL -1 ) were mixed and vortexed, and then left to stand at 37°C for 40 min before fluorescence measurement (SynergyMx, Bio Tek, USA). TA probe can react with ·OH to form a fluorescent product (TAOH). The catalyst (100 μg mL -1 , 0.3 mL), TA solution (5 mM, 0.3 mL) and H2O2 (1 mM, 0.3 mL) were added to distilled water (1.8 mL), and then slowly stirred at 37 ° C in the dark for 12 h. Finally, the reaction solution was centrifuged at 10000 rpm for 2 min, and the supernatant was aspirated for fluorescence detection (Synergy Mx, Bio Tek, USA).

[0073] Acoustic dynamic testing. 1 For O2 detection, 5 μL of catalyst (2 mg mL -1 ) and 30 μL of DPA-DMSO (1 mg mL -1 ) solution was added to 165 μL of PBS and then analyzed using a UV-visible spectrophotometer. -2 ) time or different US irradiation powers (1 min) for the decomposition rate of DPA, and the relative absorbance change of DPA at 378 nm was used to quantify the decomposition rate.

[0074] In vitro targeting of B16F10 cells. B16F10 cells were seeded in a 24-well plate and cultured overnight. CPIP@EV-CM and CPIP were added and incubated for 6 hours. Then the cell pellets were collected and fixed with paraformaldehyde fixative for 24 hours. BioTEM was used to observe the uptake of nanoparticles by B16F10 cells.

[0075] Intracellular ROS generation. DCFH-DA reagent was used to detect the ROS level of B16F10 cells after treatment with CPIP@EV-CM nanoformulation. B16F10 cells were seeded in 24-well plates overnight, and then CPIP@EV-CM (100 μg mL -1 ) for 8 h, followed by US irradiation (1 MHz, 1 W cm -2 , 30% duty cycle) for 1 min. Subsequently, DCFH-DA was used as a ROS fluorescent probe for incubation for 30 min. After PBS washing, ROS was quantitatively analyzed by flow cytometry (Beckman Coulter, USA).

[0076] Live / dead cell fluorescence imaging. B16F10 cells in the logarithmic growth phase were incubated with CPIP@EV-CM (100 μg mL -1 ) for 24 h, followed by US irradiation (1 MHz, 1 W cm -2 , 30% duty cycle) for 1 min. The cells were then stained with Calcein-AM (AM, live cells, green) and propidium iodide (PI, dead cells, red), washed with PBS, and observed using an Olympus IX 83 microscope.

[0077] CRT immunofluorescence staining. To evaluate the expression of CRT, B16F10 cells were seeded in 24-well plates with 5 × 10^4 cells per well and cultured for 12 h. Then, CPIP@EV-CM (100 μg mL -1 ) and continued incubation for 24 h, followed by US irradiation (1 MHz, 1 W cm -2 , 30% duty cycle) for 1 min. After washing three times with PBS, the cells were fixed with 4% formaldehyde and blocked with immunostaining blocking buffer for 1 h. They were then incubated with rabbit monoclonal anti-CRT antibody at 4 °C overnight. The cells were then washed three times with TBST and subsequently incubated with FITC-labeled goat anti-rabbit IgG (H+L) antibody for 1 h. After staining with DAPI, the cells were observed using a microscope (IX 83, Olympus, Japan) and quantified using Image J software.

[0078] Detection of HMGB1 and ATP release. The HMGB1 concentration in the cell supernatant after different treatments was measured using an ELISA kit. The ATP concentration in the cell supernatant after different treatments was measured using an ATP assay kit, and the luminescence intensity was quantified using a fluorescent microplate reader (Synergy Mx, Bio Tek, USA).

[0079] Flow cytometric analysis of in vitro BMDC activation. BMDCs were isolated from the bone marrow of 6-week-old C57BL / 6 mice. To investigate the effects of different membrane preparations on BMDC maturation, CM (100 μg mL -1 )、EV(50μg mL -1 )、EV-CM(100μg mL -1 ) and EV / CM (100 μg mL -1 ) were incubated with BMDCs for 12 h. The supernatant was collected for further cytokine analysis. The cells were washed three times with PBS and then incubated with anti-CD11c, anti-CD86 and anti-CD80 antibodies for 15 min at 4°C. After washing three times with ice-cold PBS, they were analyzed by flow cytometry.

[0080] To investigate the effects of EV-CM in combination with SDT / CDT on BMDC maturation, B16F10 cells were first seeded on 24-well plates with 5×10^4 cells per well and cultured overnight. Then, the cells were incubated with CPIP@EV-CM (100 μg mL -1 ) for 24 h, followed by US irradiation (1 MHz, 1 W cm -2 , 30% duty cycle) for 1 min. Then the supernatants of tumor cells with different treatments were collected and added to BMDCs culture medium. BMDCs were cultured for another 12 h and then incubated with anti-mouse CD11c, anti-mouse CD80, and anti-mouse CD86 antibodies for 30 min. After washing three times, the cells were analyzed using a flow cytometer (Beckman CytoFLEX, USA).

[0081] Antitumor immune effect in B16F10 orthotopic tumor model. Six-week-old C57BL / 6 mice were purchased. Approximately 3×10^5 B16F10 cells were injected subcutaneously into the right hind hip of the mice to construct a mouse melanoma orthotopic tumor model. When the tumor volume reached approximately 100 mm 3 The mice were randomly divided into six groups (n=3 in each group) and treated with tumors every 3 days. Ultrasound irradiation conditions: intensity (2.5 W cm -2 ), frequency (1 MHz), duty cycle (30%), and time (3 min). Tumor volume (calculated as length × width^ 2 / 2). The relative tumor volume change curve was drawn. Tumor growth inhibition (TGI) was calculated by the formula: TGI = (tumor volume of the control group - tumor volume of the experimental group) / tumor volume of the control group × 100%; After the treatment, the mice were killed and the tumor tissues were stained with HE to analyze tissue damage. HMGB1 and CRT staining were performed to verify immunogenic cell death. In order to more comprehensively evaluate tumor-infiltrating immune cells, the tumor tissues of each group were digested into single-cell suspensions, and then stained for various immune indicators, and detected and analyzed using flow cytometry (BD Symphony A5, USA).

[0082] Tumor metastasis inhibition in B16F10 cell-bearing mice. To evaluate the function of CPIP@EV-CM in inhibiting tumor metastasis, a lung metastasis model was established by tail vein injection of B16F10-Luc cells (1×10^6 cells per mouse). Subsequent treatments were the same as the above procedures. At the end of treatment, the mice were killed and the lungs were photographed and recorded to assess the number of metastatic nodules on the lung surface (front and back). HE staining of lung tissue was performed to verify the extent of lung metastasis. To evaluate systemic anti-tumor immunity, lymph nodes were isolated and ground. Single cell suspensions were collected and then flow stained according to standard procedures.

[0083] Example 1: Preparation of heterogeneous fusion membrane tumor vaccine (hereinafter referred to as EV-CM) of cell membrane derived from tumor cells (hereinafter referred to as CM) and extracellular vesicles (hereinafter referred to as EV) derived from bacteria

[0084] In this example, an EV-CM heterogeneous fusion membrane tumor vaccine was prepared by fusing B16F10 melanoma cell membranes with EVs from the Gram-positive bacterium Staphylococcus aureus.

[0085] Preparation of B16F10 cell membranes: Preparation of B16F10 cell membranes (CM) and EVs was performed according to known methods. In brief, B16F10 cells in the logarithmic growth phase were collected and centrifuged (1500 rpm, 4°C, 5 min) to obtain cell pellets. The cells were then resuspended using reagents (10 mM MgCl2, 10 mM HCl, and 1× protease inhibitor cocktail). Afterwards, the cells were ultrasonically treated on ice for 30 min and then centrifuged at 4000 g for 10 min at 4°C. The supernatant was collected and centrifuged at 16000 g for 30 min at 4°C. Finally, the cell membrane pellet was stored at -80°C.

[0086] Preparation of bacterial EVs: A single colony of Staphylococcus aureus was inoculated in beef peptone medium (BPM) medium (200 mL) and cultured overnight at 37°C on a shaker. Then, the bacterial solution was added to fresh BPM medium at a ratio of 1:100 and cultured on a shaker for 15 h. The bacterial suspension was centrifuged at 5000 g for 20 min at 4°C, and the supernatant was collected and filtered with a 0.45 μm vacuum filter to remove bacteria. To obtain EVs, the filtered medium was centrifuged at 150,000 g for 2 h at 4°C. The obtained EVs were stored at -80°C.

[0087] Construction of EV-CM fusion membrane: The membrane protein mass was quantified using the BCA protein kit. Fusion was performed at a protein mass ratio of 2:1 (CM:EV). First, EVs resuspended in PBS and CM resuspended in PBS were mixed and sonicated on ice for 10 min. The mixture was then placed on a shaker at 37°C for 40 min to promote membrane fusion, resulting in EV-CM fusion membranes that were used directly for subsequent operations or frozen at -80°C for later use.

[0088] To demonstrate the successful fusion of EV and CM, EV and CM were stained with DiI (red signal) and DiO (green signal), respectively. The labeled EV, CM, and EV-CM fusion membranes were then observed by CLSM (N-SIM S, Nikon, Japan). Figure 1As shown in the figure, EVs isolated from the culture supernatant of Staphylococcus aureus showed spherical membrane vesicles and SDS-PAGE bands consistent with the literature. Immunoblotting analysis revealed the antigen gp100 specifically expressed on the B16F10 membrane, indicating that the malignant melanoma cell membrane was successfully extracted. Figure 1 As shown in A, EV and CM showed red and green fluorescence signals, respectively. The prepared EV-CM heterogeneous fusion membrane showed significant colocalization of fluorescence signals (yellow), and fluorescence colocalization analysis showed a high degree of fluorescence overlap (colocalization coefficient r = 0.91), indicating successful fusion. In addition, SDS-PAGE protein analysis showed that the EV-CM heterogeneous fusion membrane inherited most of the cell membrane proteins of EV and CM. Therefore, the functions of EV and CM can be retained in the heterogeneous fusion membrane ( Figure 1 B).

[0089] Example 2: Preventive effect of EV-CM on melanoma

[0090] To evaluate the ability of EV-derived fusogenic membranes to activate DCs in vitro, bone marrow dendritic cells (BMDCs) were incubated with different membrane preparations, and the expression of co-stimulatory molecules and secretion of cytokines on BMDCs, which are essential for DCs to initiate strong antigen-specific CTL responses, were subsequently examined. After incubation, the expression of CD80 or CD86 was only moderately upregulated in the CM-treated group; in contrast, EV-containing membrane preparations, including EV, CM / EV (physically mixed EV and CM), and EV-CM, significantly induced an upregulation of CD80 or CD86 expression ( Figure 2 A), indicating that EV components have potent adjuvant activity. EVs from S. aureus express pathogen-associated molecular patterns (PAMPs) on their surface, which can directly interact with pattern recognition receptors on BMDCs to stimulate DC maturation. Proinflammatory cytokines are the third signal required for DCs to elicit T cell antitumor responses via MHC-antigen complexes and co-stimulatory signals. The levels of proinflammatory cytokines IL-6 and IL-12 secreted by BMDCs further demonstrate the ability of EVs to promote DC maturation ( Figure 2 B). In addition, EV-CM is more effective than CM / EV in stimulating DC maturation. Previous studies have shown that delivering tumor autologous antigens and immune adjuvants together to the same dendritic cells (DC) can improve immune responses more effectively than a mixture of the individual components. Since the fusion structure of EV-CM will allow the spatial and temporal co-delivery of antigens and adjuvants to the same dendritic cells (DC), it will enhance the ability of CM to stimulate anti-tumor immune responses.

[0091] Migration of vaccine preparations to draining lymph nodes (LNs) and uptake by APCs is a prerequisite for immunization. To evaluate the migration ability of vaccines, different membrane preparations (CM pre-labeled with DIR) were injected subcutaneously in the right footpad of C57BL / 6 mice to analyze the accumulation of CM antigens in regional draining LNs. In vivo migration of membrane preparations was observed within 24 h after injection by small animal live fluorescence imaging, and the results showed that the fluorescence signals in regional draining LNs (popliteal and inguinal) of EV-CM or CM / EV groups were much higher than those of CM groups ( Figure 3 A), the presence of EVs can promote the delivery of CM antigens. Quantitative analysis of fluorescence signals in inguinal lymph nodes ( Figure 3 B) The results also showed that the fluorescence signal of the EV-CM group was the highest at any time point, and more significant contralateral drainage was observed in the EV-CM group than in the CM / EV group at 6 h after injection, which further demonstrated that the heterogeneous fusion membrane strategy has a higher antigen delivery efficiency than the physical mixture.

[0092] Next, we conducted a tumor prevention experiment using the B16F10 model to confirm the effectiveness of EV-CM in initiating anti-tumor immune responses and preventing tumorigenesis. Mice were subcutaneously immunized with different membrane preparations (CM, EV, CM / EV, and EV-CM) and then inoculated with B16F10 tumor cells. The CM or CM / EV preparation groups had only a slight tumor growth inhibitory effect. The EV group had little effect due to the lack of tumor antigens. In contrast, the EV-CM vaccine significantly inhibited tumorigenesis, with an inhibition rate of 78.69% ( Figure 4 A). Furthermore, EV-CM vaccination prolonged the tumor-free time (19 days) compared with the EV (7 days), CM (14 days), and CM / EV groups (15 days) ( Figure 4 B), indicating that EV-CM has superior tumor prevention ability.

[0093] To investigate the preventive mechanism, we applied flow cytometry to analyze T cell subsets. The results showed that vaccination with CM preparations only slightly increased CD8+CTLs, which was only 1.11 times higher than the control group (32.1%); enhanced CTLs levels were found in both CM / EV and EV-CM groups, with the percentages of CTLs reaching 43.4% and 49.2%, respectively ( Figure 4 C). In addition, CD69 is one of the markers of T lymphocyte activation, and CD8 + CD69 + The percentage of T cells increased significantly (29.4%) ( Figure 4 D). Given the key role of immune memory effect in tumor prevention, we also detected the effector memory T cells (T EM ) ratio. Compared with other groups, EV-CM vaccination induced TEM The significant increase in EV-CM-mediated tumor prevention revealed the mechanism behind the powerful Figure 4 E). These results collectively indicate that the EV-CM fusion nanovaccine effectively initiates tumor-specific immunity and achieves superior preventive efficacy against melanoma.

[0094] Example 3: Preparation of platinum-porphyrin coordination polymer (hereinafter referred to as CPIP) and preparation of EV-CM fusion membrane tumor vaccine implanted with CPIP (hereinafter referred to as CPIP@EV-CM)

[0095] In order to enhance the tumor eradication ability, the present invention also equips the EV-CM nanovaccine with an SDT / CDT module. The SDT / CDT core Pt-Pta / Por (CPIP) is synthesized by using potassium chloroplatinate as a donor of the Pt active site, tri(pyridin-4-yl)amine (Pta) and 5,10,15,20-tetra(4-pyridinyl)-21H,23H-porphyrin (Por) as molecular ligands, and polyvinylpyrrolidone (PVP) as a morphology regulator. The details are as follows.

[0096] Synthesis of platinum-porphyrin coordination polymer Pt-Pta / Por (CPIP): Pt-Pta / Por was synthesized using a simple one-pot method. Briefly, 5,10,15,20-tetra(4-pyridyl)-21H,23H-porphyrin (Por) (12.09 mg, 0.019 mM), tri(pyridin-4-yl)amine (4.85 mg, 0.0195 mM) and polyvinylpyrrolidone (PVP) (10 mg) were dissolved in hydrochloric acid (0.01 M, 10 mL) and sonicated for 30 min to form solution A. K2PtCl6 (19 mg, 0.039 mM) was dissolved in deionized water (10 mL) to form solution B. After that, solution A was quickly added to solution B and stirred vigorously for 4 h to form solution C. Then, the C solution was centrifuged at 11000 rpm for 10 min; the obtained nanoparticles were washed three times with deionized water and dried under vacuum at 60° C. overnight.

[0097] Typical SEM and TEM images of Pt-Pta / Por show that it has an almost monodisperse size distribution with an average diameter of about 200 nm ( Figure 5 AB). Energy dispersive X-ray spectroscopy (EDS) elemental mapping showed that Cl, C, N, O, and Pt elements were uniformly distributed on the Pt-Pta / Por nanoformulation ( Figure 5E). The chemical structure of Pt-Pta / Por was further analyzed by X-ray photoelectron spectroscopy (XPS). The N1s spectrum in Pt-Pta / Por shows that Pt-N has two structures in Pt-Pta / Por. The obvious N1s peak of Pt-Pta / Por at 399.4 eV is the Pt-pyridinic N structure, which is also the main active site structure. Due to the formation of the coordination bond of Pt-pyridinic N, it moves 0.7 eV to the high field compared with the initial pyridinic N (398.7 eV) of porphyrin and tri(pyridin-4-yl)amine. The secondary structure is Pt-pyrrolic N (401.2 eV), which moves 1.1 eV to the high field compared with the pyrrolic N in the pure ligand (Figure F). In addition, in the Pt 4f spectrum of Pt-Pta / Por, Pt-pyridinic N is at 72.3 and 75.5 eV, and Pt is a divalent cation. Similarly, the Pt-pyrrole N is at 74.6 and 77.8, with Pt as a tetravalent cation (Figure G).

[0098] Preparation of CPIP@EV-CM: CPIP (2.0 mL, 2 mg / mL) resuspended in PBS was mixed with fusion membranes (2.0 mL, 2 mg / mL) resuspended in PBS. The mixture was sonicated for 30 min, centrifuged at 10,000 rpm for 10 min, and then resuspended and washed twice with PBS.

[0099] Compared with CPIP, CPIP@EV-CM exhibited a spherical core-shell structure with an average particle size of approximately 252 nm and a coating film thickness of approximately 10 nm (Figures CD). In addition, SDS-PAGE also showed that the EV-CM membrane was successfully coated on CPIP (Figure H).

[0100] Example 4: Antitumor effects of CPIP@EV-CM in vitro and in vivo

[0101] First, the biocatalytic activity of CPIP@EV-CM related to ROS was explored. The peroxidase-like (POD) catalytic activity of CPIP@EV-CM in converting H2O2 into ROS was evaluated using the classic colorimetric probe 3,3',5,5'-tetramethylbenzidine (TMB). The ability of CPIP@EV-CM to catalyze the oxidation of TMB (oxTMB) was comparable to that of CPIP ( Figure 6 A), EV-CM coating does not affect the catalytic activity of the enzyme. Different ROS fluorescent probe detection also showed that ·OH( Figure 6 B) and O2 - ( Figure 6 The yield of C) was not affected by membrane coating. In addition, the sonodynamic performance of CPIP was verified by singlet oxygen fluorescence probe (SOSG). The results showed that after ultrasound irradiation, SOSG fluorescence increased sharply ( Figure 6D). And with the increase of US irradiation time or power, the characteristic absorption peak of DPA at 378nm gradually decreases ( Figure 6 EF), indicating that US irradiation can still significantly enhance the production of CPIP@EV-CM 1 These data demonstrate that the EV-CM coating has no significant effect on the chemodynamic and sonodynamic properties of CPIP.

[0102] Inspired by the excellent chemodynamic / sonic dynamic catalysis of ROS generation by CPIP@EV-CM, the generation of intracellular ROS and the tumor cell killing effect were subsequently explored. ROS were labeled with DCFH-DA green fluorescent probe and the generation of intracellular ROS was evaluated by flow cytometry. The results showed that after ultrasound irradiation, more ROS were generated in the cells of CPIP and CPIP@EV-CM groups. Figure 6 G). Next, we evaluated the direct toxicity of ROS to cancer cells. CPIP and CPIP@EV-CM induced more tumor cell death after US irradiation, indicating the synergistic effect of chemodynamic and sonodynamic therapy in antitumor treatment ( Figure 6 H). Notably, CPIP@EV-CM and CPIP@EV-CM+US groups induced more cell death compared to CPIP and CPIP+US, respectively. We attribute this to the homotypic targeting ability of CM disguised by EV-CM, which would increase the uptake of nanoparticles by tumor cells and subsequently exert better chemokinetic and sonodynamic effects. This view was supported by the bioTEM results of B16F10 cells, where more nanoparticles were observed in tumor cells in the CPIP@EV-CM group ( Figure 6 I).

[0103] Potent immune activation of CPIP@EV-CM in vitro

[0104] For anti-tumor immunotherapy, immunogenic cell death (ICD) induced by chemodynamic / sonodynamic therapy killing tumor cells will enhance the immunogenicity of tumors. To further understand the immune response driven by chemodynamic / sonodynamic therapy, we designed a two-step co-culture experiment using nanoparticles, B16F10 cells and BMDCs. First, the expression levels of ICD-related markers, including high-mobility group protein B1 (HMGB1), adenosine triphosphate (ATP), and calreticulin (CRT), were tested after co-incubation of nanoparticles with tumor cells. ATP is a chemokine that can recruit antigen-presenting cells such as dendritic cells; the exposure of CRT and HMGB1 promotes antigen-presenting cells to phagocytose dying tumor cells and subsequently present antigens to T cells. When US irradiation was introduced, the CRT green fluorescence expressed by tumor cells in the CPIP@EV-CM+US group was the brightest, indicating the abundant expression of CRT in B16F10 cells ( Figure 7A). Similarly, the secretion of HMGB1 and ATP by tumor cells in the CPIP@EV-CM+US group increased significantly ( Figure 7 B). The above results indicate that the synergistic effects of chemodynamic and sonodynamic therapy induce enhanced ICD, which lays the foundation for the subsequent activation of APCs and the enhancement of tumor-specific immunity.

[0105] Dendritic cells (DCs) play a key role in innate and adaptive immunity. Once exposed to antigens, immature DCs phagocytose antigens and process them into peptides. At the same time, immature DCs undergo maturation and present peptides to naive T cells. Therefore, we evaluated the synergistic effect of EV-CM shell and CPIP-derived chemodynamic / sonodynamic therapy on DCs activation by analyzing the expression of co-stimulatory molecules CD80 / CD86 by BMDC after co-incubation of tumor cell supernatants treated in the previous step. These molecules are considered to be representative markers of DCs maturity. Flow cytometry showed that the maturation of DCs in the treated groups was improved compared with the control group, and the highest maturation was induced by CPIP@EV-CM+US ( Figure 7 C). It is noteworthy that the DCs in CPIP@EV-CM were more mature than those in the CPIP+US group, which is in contrast to the results of the ICD experiment. This suggests that the effect of the EV-CM shell on BMDC activation is stronger than that of the ICD induced by chemodynamic / sonodynamic therapy. This further illustrates the necessity of introducing the EV-CM shell into the therapeutic system. In addition, as representative immunostimulatory cytokines produced by mature DCs, ELISA analysis of IL-6 and IL-12 showed similar results ( Figure 7 D). All the above results verified that CPIP@EV-CM can enhance immature DCs into a mature state, which can then induce downstream immune responses for immunotherapy.

[0106] Antitumor effect of CPIP@EV-CM on the established B16F10 mouse tumor model

[0107] Inspired by the in vitro tumor killing and immune activation effects, the in vivo antitumor effect of CPIP@EV-CM was then studied. Initially, a unilateral B16F10 tumor-bearing mouse model was established. The mice were treated with intratumoral injections every 3 days according to the grouping shown in the figure, for a total of 4 treatments, in which the nanoformulation was administered at 10 mg / kg body weight, and the US combination group was irradiated with ultrasound immediately after administration. Ultrasound conditions: intensity (2.5W cm -2 ), frequency (1MHz), duty cycle (30%), and time (3min). The CPIP@EV-CM+US treatment group showed the highest tumor growth inhibition (TGI) rate, which was about 74.4% ( Figure 8A). At the histological level, HE staining of tumor tissues showed that the tumor cells in the CPIP@EV-CM+US group were obviously separated, sparse, and fragmented ( Figure 8 B). Subsequently, the immune activation effect at the tumor site was evaluated. Immunofluorescence staining of ICD markers including CRT and HMGB1 was performed in tumor tissues. The CPIP@EV-CM+US group had the highest exposure of CRT on the tumor cell membrane ( Figure 8 C). Meanwhile, the expression of CRT in CPIP@EV-CM+US and CPIP+US was higher than that in CPIP@EV-CM and CPIP groups, indicating the synergistic effect of chemodynamic / sonodynamic therapy. In addition, the expression of nuclear HMGB1 was also consistent with this explanation ( Figure 8 D), HMGB1 will be released from the nucleus to the extracellular space during ICD, so nuclear expression will be reduced. Flow cytometry analysis of tumor-infiltrating DCs and T lymphocytes showed that the CPIP@EV-CM+US group induced the most mature DCs (42.8%) ( Figure 8 E), the proportion of CD3+CD8+T cells in tumor tissues increased significantly after CPIP@EV-CM+US treatment (46.7%) ( Figure 8 F). A large number of CD3+CD8+ T cells were detected in the CPIP (30.7%), CPIP+US (35.9%), and CPIP@EV-CM (38.7%) groups, indicating that SDT and CDT can trigger the infiltration of T cells and synergize with the EV-CM shell to further enhance the immune effect.

[0108] Anti-lung metastasis ability of CPIP@EV-CM in vivo

[0109] Afterwards, we investigated whether the CPIP@EV-CM formulation could inhibit tumor metastasis. A mouse model of lung metastasis was established by tail vein injection of B16F10-luc cells. The mice were treated with intratumoral injection every 3 days according to the groups shown in the figure, for a total of 4 times, in which the nanoformulation was administered at 10 mg / kg body weight, and the US combination group was irradiated with ultrasound immediately after administration. Ultrasound conditions: intensity (2.5 W cm -2 ), frequency (1MHz), duty cycle (30%), and time (3min). The survival rate of mice in the CPIP@EV-CM+US group increased to about 66% ( Fig. 9 A). After the experiment, the mouse lung tissue was removed and fixed with 4% paraformaldehyde to count the number of metastatic nodules. The results showed that the number of lung metastatic tumor nodules in the CPIP@EV-CM+US group was the least ( Fig. 9 B). HE staining of lung tissue also showed that mice treated with CPIP@EV-CM+US had less lung metastasis ( Fig. 9 C), can effectively inhibit tumor metastasis.

[0110] To further explore the therapeutic mechanism and synergistic effect of chemodynamic / sonodynamic therapy combined with EV-CM shell, we performed flow cytometry analysis on lymph node cells. Similar to the in vitro results, we further demonstrated that the integration of EV-CM shell endowed CPIP@EV-CM with a stronger ability to promote DC maturation, which makes it possible to combine immunotherapy with SDT / CDT against solid tumors. Specifically, CPIP@EV-CM+US induced a high level of DC maturation (21.1%), which was much higher than that of CPIP@EV-CM (15.6%) and CPIP+US (13.1%) alone, indicating the activation of innate immune response ( Fig. 9 D) T lymphocytes are the main effector cells of anti-tumor immunity. The CD3 + CD8 + The proportion has been greatly increased ( Fig. 9 E), indicating that T lymphocytes in the lymph nodes were significantly activated. In addition, to further evaluate the mechanism of the anti-metastatic effect of CPIP@EV-CM, we also detected effector memory T cells (T EM Compared with PBS (3.1%), CD44 + CD62L - T EM The number of cells increased significantly (11.1%) ( Fig. 9 F), indicating that CPIP@EV-CM+US can produce a strong immune memory effect and protect mice from tumor metastasis and recurrence.

[0111] In summary, the present invention prepared EV-derived fusion membranes of melanoma cell membranes and Staphylococcus aureus extracellular vesicles, and encapsulated SDT / CDT modules to design a biogenic nanovaccine (CPIP@EV-CM). The EV-CM fusion membrane integrates tumor-specific antigens and natural adjuvants, showing strong potential to activate the immune system and induce anti-tumor immune responses. In vivo vaccination showed that the fusion membrane protected mice from melanoma challenges, showing potential as a preventive cancer vaccine. After being equipped with the SDT / CDT module (CPIP), the chemodynamic / sonodynamic therapy synergistic immunotherapy of CPIP@EV-CM significantly enhanced the therapeutic effect on established tumors, which was attributed to the direct toxicity of ROS to tumor cells and enhanced immune stimulation. Chemodynamic / sonodynamic therapy can not only directly cause apoptosis and necrosis of cancer cells, but also release markers related to ICD, synergize the immunostimulatory effects of EV-CM, and induce strong systemic anti-tumor immune responses to primary and metastatic tumor foci to fight primary tumors and metastases. After intratumoral injection, CPIP@EV-CM activated the immune response by stimulating DC maturation and T cell activation. Here, the SDT / CDT module was used for therapeutic purposes. Obviously, other materials with different properties (e.g., size and morphology) and different functions (e.g., imaging, drug loading) can be freely selected according to complex requirements.

[0112] It should be noted that the preferred embodiments of the present invention are given in the specification and drawings of the present invention. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. These embodiments are not used as additional limitations on the content of the present invention. The purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. In addition, the above-mentioned technical features continue to be combined with each other to form various embodiments not listed above, which are all regarded as the scope of the present invention; further, for ordinary technicians in this field, they can be improved or transformed according to the above description, and all these improvements and transformations should belong to the protection scope of the claims attached to the present invention.

Claims

1. A tumor vaccine, characterized in that: The tumor vaccine comprises a fusion membrane formed by the fusion of a cell membrane derived from a tumor cell and an extracellular vesicle derived from bacteria.

2. The tumor vaccine according to claim 1, characterized in that The tumor vaccine further comprises a sono / chemodynamic therapeutic substance that forms a core-shell structure together with the fusion membrane, wherein the fusion membrane is the shell and the sono / chemodynamic therapeutic substance is the core; Furthermore, the mass ratio of the fusion membrane to the sono / chemodynamic therapeutic substance is 1:1 to 1:

5.

3. The tumor vaccine according to claim 1 or 2, characterized in that The bacteria is Staphylococcus aureus; Further, the tumor cells include breast cancer cells, colon cancer cells, liver cancer cells, gastric cancer cells, kidney cancer cells, pancreatic cancer cells, ovarian cancer cells, lymphoma cells, osteosarcoma cells, glioma cells, prostate cancer cells, melanoma cells or any combination thereof; Furthermore, the protein mass ratio of the cell membrane derived from tumor cells to the extracellular vesicles derived from bacteria is 1:1 to 3:

1.

4. The tumor vaccine according to claim 2, characterized in that The sono / chemodynamic therapeutic substance includes a metal-porphyrin coordination polymer; Further, the metal-porphyrin coordination polymer is a coordination polymer formed by metal and tri(pyridin-4-yl)amine and 5,10,15,20-tetra(4-pyridinyl)-21H,23H-porphyrin through metal-N coordination; Further, the metal is Pt; Furthermore, the molar ratio of the metal to tri(pyridin-4-yl)amine and 5,10,15,20-tetra(4-pyridinyl)-21H,23H-porphyrin is 1:0.4-0.6:0.4-0.

6.

5. A method for preparing a tumor vaccine, characterized in that: The following steps are involved: (1) preparing cell membranes derived from tumor cells; (2) preparing extracellular vesicles derived from bacteria; (3) The cell membrane derived from tumor cells is fused with the extracellular vesicles derived from bacteria to obtain a fusion membrane.

6. The method according to claim 5, characterized in that Step (1) comprises: obtaining tumor cells, obtaining tumor cell precipitates by centrifugation, resuspending the tumor cell precipitates, disrupting the tumor cells, and separating the obtained cell membranes; Further, the resuspension is performed using a hypotonic solution; Further, the hypotonic solution comprises 5-10 mM MgCl2, 5-10 mM HCl and a protease inhibitor; Further, the crushing includes ultrasonic crushing; Furthermore, the cell membrane obtained by separation includes centrifuging the broken tumor cell solution to collect the supernatant, centrifuging the supernatant again, and collecting the precipitate to obtain the cell membrane; Further, the first centrifugation includes centrifugation at 3000-6000 g for 5-15 min, and the second centrifugation includes centrifugation at 13000-18000 g for 20-50 min; Furthermore, step (2) comprises culturing the bacteria, collecting the supernatant by centrifugation, and collecting the precipitate after centrifuging the supernatant again, thereby obtaining bacterial outer membrane vesicles; Further, the first centrifugation includes centrifugation at 3000-6000 g for 10-30 min, and the second centrifugation includes centrifugation at 130000-180000 g for 1-3 h; Further, step (3) comprises mixing the cell membrane derived from the tumor cell and the extracellular vesicle derived from the bacteria in a buffer, and sonicating to achieve fusion; Further, the bacteria is Staphylococcus aureus; Further, the tumor cells include breast cancer cells, colon cancer cells, liver cancer cells, gastric cancer cells, kidney cancer cells, pancreatic cancer cells, ovarian cancer cells, lymphoma cells, osteosarcoma cells, glioma cells, prostate cancer cells, melanoma cells or any combination thereof; Furthermore, the protein mass ratio of the cell membrane derived from tumor cells to the extracellular vesicles derived from bacteria is 1:1 to 3:

1.

7. The method according to claim 5, characterized in that The preparation method further comprises: (4) coating the fusion membrane obtained in step (3) on the surface of the sono / chemodynamic therapeutic substance to obtain a tumor vaccine with a core-shell structure; Furthermore, step (4) comprises mixing the fusion membrane obtained in step (3) with the sono / chemodynamic therapeutic substance in a buffer, ultrasonically treating, collecting the precipitate by centrifugation, and washing to obtain the tumor vaccine having a core-shell structure; Furthermore, the mass ratio of the fusion membrane to the sono / chemodynamic therapeutic substance is 1:1 to 1:

5.

8. The method according to claim 7, characterized in that The sono / chemodynamic therapeutic substance includes a metal-porphyrin coordination polymer; Further, the metal-porphyrin coordination polymer is a coordination polymer formed by metal and tri(pyridin-4-yl)amine and 5,10,15,20-tetra(4-pyridinyl)-21H,23H-porphyrin through metal-N coordination; Further, the metal-porphyrin coordination polymer is prepared as follows: tri(pyridin-4-yl)amine, 5,10,15,20-tetra(4-pyridinyl)-21H,23H-porphyrin and a surfactant are uniformly mixed in a solvent A to obtain a solution A; a metal source is dissolved in a solvent B to obtain a solution B; solution A and solution B are mixed for reaction, and a precipitate is collected by centrifugation to obtain the metal-porphyrin coordination polymer; Further, the molar ratio of the metal to tris(pyridin-4-yl)amine and 5,10,15,20-tetra(4-pyridinyl)-21H,23H-porphyrin is 1:0.4-0.6:0.4-0.6; Further, the metal source is a metal salt or hydrate; Further, the metal is Pt; Further, the metal source is K2PtCl6; Further, the surfactant is polyvinyl pyrrolidone; Further, the solvent A is an acidic aqueous solution, preferably a hydrochloric acid aqueous solution; Further, the solvent B is water; Furthermore, the reaction is carried out under stirring, the reaction temperature is 15-35° C., and the reaction time is 1-6 h.

9. Use of the tumor vaccine according to any one of claims 1 to 4 or the tumor vaccine obtained by the preparation method according to any one of claims 5 to 8 in the preparation of a medicament for preventing tumor occurrence, metastasis and / or recurrence.

10. Use of the tumor vaccine according to any one of claims 1 to 4 or the tumor vaccine obtained by the preparation method according to any one of claims 5 to 8 in the preparation of a drug for treating tumors; Furthermore, the treatment includes combined treatment with ultrasound.

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