CN115607660A

CN115607660A - Preparation method and application of vaccine loaded with cancer cell whole cell component and mixed membrane component

Info

Publication number: CN115607660A
Application number: CN202210758781.5A
Authority: CN
Inventors: 刘密
Original assignee: Suzhou Ersheng Biopharmaceutical Co Ltd
Current assignee: Suzhou Ersheng Biopharmaceutical Co Ltd
Priority date: 2022-06-30
Filing date: 2022-06-30
Publication date: 2023-01-17
Also published as: WO2024000724A1

Abstract

The invention relates to a preparation method and application of a vaccine loaded with cancer cell whole cell components and mixed membrane components, wherein the preparation method comprises the following steps: s1, obtaining a membrane component of a cancer cell; s2, activating antigen presenting cells to obtain membrane components; s3, obtaining a membrane component of bacteria; and S4, enabling products of the S1 and the S2 and/or products of the S1 and the S3 to act with the second particles, and enabling the membrane components to be loaded on the second particles to obtain the vaccine. The invention realizes that the vaccine derived from tumor tissues or cancer cells loads broad-spectrum and diverse cancer cell antigens and loads mixed membrane components on the surface, thereby endowing the vaccine with proper bionic membrane characteristics. The method can prepare the cancer vaccine loaded with broad-spectrum cancer cell epitope, and can be used for preventing and treating various cancers.

Description

Preparation method and application of vaccine loaded with cancer cell whole cell component and mixed membrane component

Technical Field

The invention relates to the field of immunotherapy, in particular to a preparation method and application of a vaccine loaded with cancer cell whole cell components and mixed membrane components.

Background

Cancer immunotherapy is one of the most important treatments for cancer, and among them, cancer vaccine is one of the important treatments for cancer immunotherapy. At present, nanoparticles coated with cancer cell membranes as cancer vaccines for preventing and treating tumors are very extensive, for example, zhu J Y et al (preferred cancer cell self-recognition and tumor self-targeting by coating nanoparticles with a host cell membrane) provide nanoparticles coating cancer cell membranes specifically derived from homologous tumors, specifically, a magnetic iron oxide nanoparticle (Fe iron oxide nanoparticle) is prepared ₃ O ₄ MNPs) platform, CMBMNPs (cell membrane bionic modified nanoparticles) from various cancer cell lines are used for researching the homologous targeting capability of the CMBMNPs, and experimental results show that the cell membrane bionic modified Fe ₃ O ₄ MNPs can achieve highly specific self-recognition of a source cancer cell line in vitro and have excellent targeting ability to a homologous tumor. Even when heterotypic tumor competition exists, the NPs still selectively target homologous tumors. In addition, researches disclose cancer vaccines of various cancer cell membrane coated nanoparticles, wherein the cancer cell membrane coated gelatin nanoparticles (PDTC @ GNPs) are used for treating tumors, and the MIA-PaCa-2 pancreatic cancer cell membrane coated gold nanoparticles are used for treating pancreatic cancer. However, because the types of antigens loaded by the nano-antibacterial material are limited, the types of membranes loaded by the nano-antibacterial material are limited, and the loading capacity of the traditional naked magnetic nano-particles for bactericidal drugs is limited, or in some cases, the nano-antibacterial material can also generate active oxygen substances such as superoxide anion free radicals, hydroxyl free radicals, hydrogen peroxide and singlet molecular oxygen in bacterial cells, and the excessive accumulation of the substances can cause the apoptosis of the bacterial cells, at present, a plurality of cells are not loaded simultaneouslyA membrane component, a cancer cell whole cell antigen and a cancer vaccine which is loaded with anti-tumor bacteria.

Disclosure of Invention

In order to solve the technical problems, the invention provides a nano-vaccine or a micro-vaccine which is internally loaded with tumor tissues and/or cancer cell whole-cell components, is loaded with cancer cell membrane and/or extracellular vesicle membrane on the surface, and is activated with antigen presenting cell membrane components and/or membrane components derived from bacteria, and after the nano-vaccine or the micro-vaccine is injected into a human body, the nano-vaccine has better stability and easier activation of cancer cell specific immune response due to biomimetic simulation of a membrane structure, so that the nano-vaccine or the micro-vaccine can better play a role in preventing or treating cancer.

The invention provides a preparation method of a vaccine loaded with cancer cell whole cell components and mixed membrane components, which comprises the following steps:

s1, obtaining a cell membrane component of a cancer cell and/or a cancer cell extracellular vesicle membrane component from the cancer cell;

s2, co-incubating the antigen presenting cells and the first particles to activate the antigen presenting cells, and obtaining cell membrane components of the activated antigen presenting cells; wherein the first particles are loaded with a cancer-associated antigen;

s3, obtaining a cell membrane component and/or a bacterial extracellular vesicle membrane component of the bacteria from the bacteria;

s4, the first mixed membrane component and/or the second mixed membrane component and the second particles are/is subjected to combined action, so that the mixed membrane component is loaded on the second particles, and the vaccine loaded with the cancer cell whole cell component and the mixed membrane component is obtained; wherein the second particles are loaded with a cancer cell whole cell component, the first mixed membrane component is a mixture of S1 and S2 products, and the second mixed membrane component is a mixture of S1 and S3 products;

wherein the cancer cell whole-cell component comprises a water-soluble component and a water-insoluble component which are obtained by water splitting of cancer cells and/or tumor tissues, and the water-insoluble component is dissolved by a dissolving agent and then loaded on the second particles; or the cancer cell whole cell component comprises a soluble component obtained by dissolving cancer cells and/or tumor tissues after lysis by a dissolving solution containing a dissolving agent.

Further, in step S1, the method further comprises a step of pretreating the cancer cells before obtaining the membrane fraction, wherein the pretreatment is to culture the cancer cells in a culture medium containing adriamycin, a tinib drug, chloroquine or azacytidine.

Further, the tinib-based drugs include gefitinib, imatinib mesylate, nilotinib, sunitinib, lapatinib, and the like.

Further, in step S2, the cancer-associated antigen is a polypeptide antigen or a cancer cell whole cell fraction.

Further, in step S3, before obtaining the membrane fraction, the method further comprises a step of pretreating the bacteria by culturing the bacteria in a culture medium containing doxorubicin, a tinib drug, chloroquine, or azacytidine.

Further, the second particles are also loaded with a bacterial component obtained by lysing the bacteria or bacterial outer vesicles with a lysis solution containing a lytic agent, and then lysing the lysate with the lysis solution.

Further, the means for obtaining membrane fractions from cancer cells, antigen presenting cells, bacteria include sonication, homogenization, high speed stirring, high pressure disruption, high shear disruption, swelling, chemicals, shrinking, and the like.

Further, the means by which the second particles interact with the cell membrane fraction include co-incubation, sonication, stirring, homogenization, co-extrusion, ultrafiltration, dialysis, homogenization, and the like.

Further, the bacteria include bcg, probiotics, oncolytic bacteria, and the like. Including but not limited to BCG vaccine, escherichia coli, bifidobacterium longum, bifidobacterium breve, bifidobacterium lactis, lactobacillus rhamnosus, lactobacillus acidophilus, lactobacillus gasseri, lactobacillus reuteri, etc.

Further, the second particle loaded with the cancer cell whole cell component and the bacterial component is prepared by the following steps:

(1) Tumor tissues and/or cancer cells are cracked by a dissolving solution containing a dissolving agent, and then dissolved by the dissolving solution; or swelling, repeatedly freezing and thawing and/or ultrasonically lysing tumor tissue and/or cancer cells, collecting water-soluble components in the lysate, and dissolving water-insoluble components in the lysate with a dissolving solution containing a dissolving agent;

(2) Cracking bacteria by using a dissolving solution containing a dissolving agent to obtain a bacteria lysate, and dissolving the bacteria lysate by using the dissolving solution; or swelling, repeatedly freezing and melting and/or ultrasonically lysing the bacteria, collecting water-soluble components of the bacteria, and dissolving the water-insoluble components in the bacteria with a dissolving solution containing a dissolving agent;

(3) Loading (1) and/or (2) into the interior and/or the surface of the nano-particle or the micro-particle.

Further, the solubilizing agent may be urea, guanidine hydrochloride, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, albumin, lecithin, inorganic salt, triton, tween, amino acid, glycoside, choline, etc.

Furthermore, the antigen presenting cells are dendritic cells or mixed antigen presenting cells containing Dendritic Cells (DC), namely the mixed antigen presenting cells can also contain one or two of B cells and macrophages.

Further, the material for preparing the first particles or the second particles may be any material capable of being vegetated with nanoparticles or microparticles, such as natural polymer materials, organic synthetic polymer materials, inorganic materials, and the like.

The organic synthetic high molecular material is selected from at least one of polylactic acid-glycolic acid copolymer, polylactic acid, polyglycolic acid, polyethylene glycol, polycaprolactone, poloxamer, polyvinyl alcohol, polyvinylpyrrolidone, polyethyleneimine, polytrimethylene carbonate, polyanhydride, poly-p-dioxanone, polymethyl methacrylate, PLGA-PEG, PLA-PEG, PGA-PEG, polyamino acid, synthetic polypeptide and synthetic lipid; the natural polymer material is selected from at least one of lecithin, cholesterol, alginate, albumin, collagen, gelatin, cell membrane, starch, saccharide and polypeptide; the inorganic material is at least one of ferric oxide, ferroferric oxide, calcium carbonate and calcium phosphate.

Further, the first particle and/or the second particle are also loaded with an immunopotentiating adjuvant. Immune enhancing adjuvants include, but are not limited to, at least one of immunopotentiators of microbial origin, products of the human or animal immune system, innate immune agonists, adaptive immune agonists, chemically synthesized drugs, fungal polysaccharides, traditional Chinese medicines, and others; immune enhancing adjuvants include, but are not limited to, at least one of pattern recognition receptor agonists, BCG (BCG), manganese related adjuvants, BCG cell wall backbone, BCG methanol extraction residues, BCG muramyl dipeptide, mycobacterium phlei, polyoxin, mineral oil, virus-like particles, immune enhanced reconstituted influenza virosomes, cholera enterotoxin, saponin and its derivatives, resiquimod, thymosin, neonatal bovine liver active peptide, mi Kuimo tex, polysaccharides, curcumin, immune adjuvant CpG, immune adjuvant poly (I: C), immune adjuvant poly ICLC, corynebacterium parvum, hemolytic streptococcal preparations, coenzyme Q10, levamisole, polycytidylic acid, manganese adjuvants, aluminum adjuvants, calcium adjuvants, various cytokines, interleukins, interferons, polyinosinic acid, polyanosinic acid, alum, aluminum phosphate, lanolin, squalene, cytokines, vegetable oils, endotoxins, adjuvants, liposomes, MF59, double stranded ginseng RNA, double stranded DNA, aluminum related adjuvants, CAF01, astragalus effective ingredients.

Preferably, the immune enhancing adjuvant is a Toll-like receptor agonist; more preferably, two or more Toll-like receptor agonists are combined to ensure that the nanoparticles or microparticles can better activate cancer cell-specific T cells after being phagocytosed by antigen presenting cells.

Further, two or more Toll-like receptor agonists are used in combination with Poly (I: C)/Poly (ICLC) and CpG-ODN (CpG oligodeoxynucleotide). Preferably, the CpG-ODN is two or more CpG-ODN.

Further, the adjuvant may be supported inside and/or on the surface of the first particle or the second particle.

Further, the first particle or the second particle is also loaded with a substance that enhances lysosomal escape, such as amino acids, polyamino acids (e.g., arginine, polyarginine, lysine, polylysine, histidine, polyhistidine), nucleic acids, positively charged polypeptides (e.g.KALA polypeptide, RALA polypeptide, melittin, etc.), lipids, saccharides, and inorganic substances (such as NH) with proton sponge effect ₄ HCO ₃ ) Protamine, histone, and the like.

Furthermore, the nanoparticles or microparticles can be prepared by the existing preparation methods, including but not limited to common solvent evaporation method, dialysis method, microfluidic method, extrusion method, and hot melt method.

Furthermore, the nanoparticles or microparticles may not be modified during the preparation process, and suitable modification techniques may also be employed to increase the antigen loading of the nanoparticles or microparticles. Modification techniques include, but are not limited to, biomineralization (e.g., silicidation, calcification, magnesiation), gelation, crosslinking, chemical modification, addition of charged species, and the like.

Further, the antigen can be loaded on the surface of the nanoparticle or microparticle by means including, but not limited to, adsorption, covalent attachment, charge interaction (e.g., addition of positively charged species, addition of negatively charged species), hydrophobic interaction, one or more steps of immobilization, mineralization, encapsulation, and the like.

Furthermore, one or more layers of water-soluble antigens and/or water-insoluble antigens loaded on the surfaces of the nano particles or the micro particles are loaded, and when a plurality of layers of water-soluble antigens and/or water-insoluble antigens are loaded on the surfaces, modifiers are arranged between the layers.

Further, the first and second particles are each independently selected from nanoparticles or microparticles, which ensure that the particles are phagocytosed by antigen presenting cells, and the particle size is in a suitable range for improved phagocytosis efficiency. The particle size of the nanoparticles is 1nm-1000nm, more preferably, the particle size is 30nm-1000nm, and most preferably, the particle size is 100nm-600nm; the microparticles have a particle size of 1 μm to 1000 μm, more preferably a particle size of 1 μm to 100 μm, more preferably a particle size of 1 μm to 10 μm, most preferably a particle size of 1 μm to 5 μm.

Further, in the process of activating the antigen presenting cells, the incubation system contains cytokines and/or antibodies; the cell factor is at least one of interleukin, tumor necrosis factor, interferon and colony stimulating factor; antibodies include, but are not limited to, α CD-3 antibodies, α CD-4 antibodies, α CD-8 antibodies, α CD-28 antibodies, α CD-40 antibodies, α OX-40L antibodies.

Further, cytokines include, but are not limited to, interleukin 2 (IL-2), interleukin 7 (IL-7), interleukin 14 (IL-14), interleukin 4 (IL-4), interleukin 15 (IL-15), interleukin 21 (IL-21), interleukin 17 (IL-17), interleukin 12 (IL-12), interleukin 6 (IL-6), interleukin 33 (IL-33), gamma interferon (IFN- γ), TNF- α, granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF).

Further, the activated antigen-presenting cells, cancer cells or bacteria may be washed appropriately before being prepared into nano-or micro-vaccines, and the washing solution used in the washing may contain protease inhibitors and/or phosphatase inhibitors.

Furthermore, the first particle or the second particle is modified with a target with active targeting function, and the target can be mannose, mannan, a CD19 antibody, a CD20 antibody, a BCMA antibody, a CD32 antibody, a CD11c antibody, a CD103 antibody, a CD44 antibody and the like.

The nanometer vaccine or the micrometer vaccine is applied to preparing the medicine for treating or preventing cancer.

Further, the antigen presenting cells are derived from one or more of autologous, allogeneic, cell-line, or stem cell differentiation.

By the scheme, the invention at least has the following advantages:

the surface of the nano-scale vaccine or the micron-scale vaccine not only loads the membrane component of the cancer cell, but also loads the membrane component of other cells, such as antigen presenting cell membrane or extracellular vesicle membrane component, or bacterial cell membrane or extracellular vesicle membrane component, the load of the mixed membrane component, and the load of the internal whole cell component and/or bacteria, so that the vaccine of the invention has more diversified and broader-spectrum antigen epitopes, has the bionic membrane characteristic, enhances the stability, and simultaneously overcomes the problem that the live cell vaccine is difficult to store and maintain the activity.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following description is made with reference to the preferred embodiments of the present invention and the accompanying detailed drawings.

Drawings

In order that the present disclosure may be more readily and clearly understood, reference will now be made in detail to the present disclosure, examples of which are illustrated in the accompanying drawings.

FIG. 1 is a schematic diagram of the preparation process and application of the nano-or micro-vaccine of the present invention; wherein a is a schematic diagram of collecting and preparing nano particles or micro particles of water-soluble antigens and water-insoluble antigens respectively; b is a schematic diagram of dissolving tumor tissues and/or cancer cells or bacteria whole cell components and preparing nano particles or micro particles by using a dissolving solution containing a dissolving agent; c is a schematic diagram for preparing the nano vaccine or the micro vaccine.

FIGS. 2 to 11 are experimental results of the growth rate and survival time of mouse tumor in the case of cancer prevention or treatment using nano-or micro-vaccine in examples 1 to 10, respectively; in FIGS. 2-4 and 6-11, a represents the experimental results of tumor growth rate (n.gtoreq.8) in the prevention or treatment of cancer; b represents the survival experimental results of mice in the prevention or treatment of cancer (n.gtoreq.8), and each data point is the mean. + -. Standard error (mean. + -. SEM); the significant difference of the tumor growth inhibition experiment in the step a is analyzed by an ANOVA method, and the significant difference in the step b is analyzed by Kaplan-Meier and log-rank test; * Indicates a significant difference with p < 0.005 compared to PBS blank control;&&&shows that compared with a nano vaccine/micro vaccine control group prepared after blank nano particles/micro particles are loaded with cancer cell membrane components on the surface, the p is less than 0.005, and the difference is significant; delta represents that the p is less than 0.05 and has significant difference compared with the nano vaccine/micron vaccine prepared by loading cancer cell membrane components on the surfaces of the nanoparticles/micron particles loaded with cancer cell whole cell components inside; delta represents that the p is less than 0.01 and has significant difference compared with the nano vaccine/micron vaccine prepared by the nano particle/micron particle surface loaded with cancer cell membrane components; mu represents and internally loads cancer cell whole cellsThe components are loaded on the surface of the nano vaccine/micro vaccine group with the cancer cell membrane component and the activated antigen presenting cell membrane component, and compared with the nano vaccine/micro vaccine group, the p is less than 0.05, and the significant difference exists; # shows that there is a significant difference compared to the nano/micro vaccine group with cancer cell membrane components, unactivated antigen presenting cell membrane components and bacterial extracellular vesicle components loaded on the surface, with p < 0.05; the # indicates that compared with the nano/micro vaccine group which is internally loaded with cancer cell whole cell components and is loaded with cancer cell membrane components, unactivated antigen presenting cell membrane components and bacterial extracellular vesicle components on the surface, the p is less than 0.01, and the significant difference exists; eta represents that the p is less than 0.05 and has significant difference compared with a nano vaccine/micron vaccine group which is internally loaded with cancer cell whole cell components, simultaneously loaded with cancer cell membrane components on the surface, activated by nano particles loaded with 4 antigen polypeptides, and bacterial extracellular vesicle components; the lambda represents a significant difference from the nano vaccine which only loads the outer vesicle component of bacteria and the immunologic adjuvant in the interior and loads the mixed membrane component on the surface at the same time, wherein the ratio p of the nano vaccine is less than 0.005; the lambda is obviously different from the nano vaccine which only loads the outer vesicle component of bacteria and the immunologic adjuvant inside and loads the mixed membrane component on the surface at the same time, wherein the ratio p is less than 0.01; gamma represents that the nano vaccine loaded with cancer cell whole cell components and immune adjuvants inside and mixed membrane components on the surface has significant difference with p less than 0.05; omega represents that the ratio p of the nano vaccine loaded with the cancer cell component and the bacterial component inside and the cancer cell membrane component on the surface is less than 0.05, and has significant difference; rho represents the significant difference with the nano vaccine/micron vaccine ratio of the cancer cell component and the bacterial component loaded inside and the cancer cell membrane component and the lactobacillus acidophilus loaded on the surface, and p is less than 0.05; phi represents the ratio of nano vaccine/micron vaccine of the internal loaded cancer cell component and the bacterial component cracked and dissolved by 8M urea and the surface loaded bacterial outer vesicle component, and p is less than 0.05, so that the difference is significant; beta represents the ratio of nano vaccine/micron vaccine of the bacterial component cracked and dissolved by the cancer cell component and Tween 80 loaded inside and the bacterial component loaded on the surface and the cancer cell outer vesicle component, and p is less than 005, significant difference; theta represents the ratio of the nano vaccine/micron vaccine which is internally loaded with cancer cell whole cell components and is only loaded with antigen presenting cell membrane components on the surface, and the p is less than 0.05, so that the significant difference exists; omega represents that the p is less than 0.05 and has significant difference compared with the nano vaccine/micron vaccine group which internally loads cancer cell whole cell components and externally loads cancer cells and DC cell membrane components; pi represents that the p is less than 0.005 and has significant difference compared with the nano vaccine/micron which only loads adjuvant inside and loads activated antigen presenting cell membrane component, treated cancer cell membrane component and treated bacterial membrane component on the surface; tau represents that compared with the nano vaccine/micron of which the internal is loaded with cancer cell whole cell components and the surface is loaded with activated antigen presenting cell membrane components, untreated cancer cell membrane components and untreated bacterial membrane components, p is less than 0.05, and has significant difference;

the vaccine represents that the p is less than 0.05 per micron compared with the nano vaccine/micron of which the cancer cell membrane component and the antigen presenting cell membrane component are loaded on the surface, the cancer cell whole cell component, 1 CpG + Poly (I: C) mixed adjuvant and the lysosome escaping substance are loaded inside, and the significant difference exists; xi represents that the p of the nano vaccine/micron vaccine loaded with the cancer cell membrane component and the antigen presenting cell membrane component on the surface is less than 0.05 and has significant difference compared with the nano vaccine/micron vaccine loaded with the cancer cell whole cell component and 2 CpG + Poly (I: C) mixed adjuvants; delta represents a significant difference with p < 0.05 per micrometer of nano-vaccine loaded with Triton lysed and lysed bacterial fraction and cancer cell whole cell fraction, while surface loaded with activated antigen presenting cell membrane fraction, treated cancer cell membrane fraction and treated bacterial membrane fraction.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

The vaccine for preventing or treating cancer of the present invention is loaded with cancer cells and/or tumor tissue whole cell components and/or bacterial components inside and mixed membrane components on the surface. The surface-loaded mixed membrane components comprise membrane components of cell membranes or extracellular vesicles of cancer cells, antigen-presenting cells and/or bacteria, and the preparation process and application fields thereof are shown in fig. 1.

In preparing nanoparticles or microparticles for internally loading cancer cells and/or tumor tissue whole cell components, it is necessary to prepare the whole cell components first. When preparing the whole cell component, cells or tissues can be cracked, then water-soluble antigens and water-insoluble antigens are respectively collected, and nano-particle systems or micro-particle systems are respectively prepared; or directly using a dissolving solution containing a dissolving agent to directly crack cells or tissues and dissolve cell whole cell components and prepare nano or micro particles. The cell whole cell component can be subjected to inactivation or (and) denaturation, solidification, biomineralization, ionization, chemical modification, nuclease treatment and other treatments before or (and) after lysis to prepare nano particles or micro particles; the nano-particles or the micro-particles can also be directly prepared before cell lysis or (and) after cell lysis without any inactivation or (and) denaturation, solidification, biomineralization, ionization, chemical modification and nuclease treatment. In some embodiments of the present invention, the cells are inactivated or (and) denatured before being lysed, or the cells may be inactivated or (and) denatured after being lysed during the actual use, or both before and after being lysed; in some embodiments of the present invention, the inactivation or (and) denaturation treatment before or (and) after cell lysis is uv irradiation and high temperature heating, and during actual use, treatment methods including but not limited to radiation irradiation, high pressure, solidification, biomineralization, ionization, chemical modification, nuclease treatment, collagenase treatment, freeze drying, etc. may also be used. Those skilled in the art can understand that in the practical application process, the skilled person can make appropriate adjustments according to specific situations.

When the mixed membrane component is loaded on the surface, the mixed cells of the cancer cells, the antigen presenting cells and/or the bacteria are firstly mechanically destroyed by using the methods of ultrasound, stirring, high pressure, high shearing force, homogenization and the like, then the membrane component is collected by centrifugation and/or filtration and/or coextrusion and/or ultrafiltration and/or dialysis through filter membranes with different pore diameters in sequence, and then the membrane component is interacted with the nano particles or the micro particles which are loaded with the tumor tissues and/or the cancer cell whole cell components and/or the bacteria components, so that the membrane component is loaded on the surface of the nano particles or the micro particles.

If the antigen-presenting cells used are activated antigen-presenting cells, cytokines and/or antibodies may be used to help increase the efficiency of activation when the nanoparticle or microparticle is used to activate the antigen-presenting cells in vitro, and the antigen-presenting cells may be derived from autologous or allogeneic sources, or from cell lines or stem cells. The antigen presenting cells can be DC cells, B cells, macrophages or any mixture of the three, and can also be other cells with antigen presenting functions.

If the cancer cells are treated appropriately to increase antigen content, the treatment includes but is not limited to incubation with chemicals such as doxorubicin for a period of time, an environment of high calcium ions to increase cell stress, and the like.

In some embodiments, a solvent evaporation process is used to prepare the first and second particles.

Taking the preparation of the first and second particles by the multiple emulsion method as an example, the specific preparation method of the nano-or micro-vaccine by using the nano-or micro-particle activated antigen presenting cells loaded with the cancer cell whole cell component and/or the bacterial component is as follows:

step 1, adding a first predetermined volume of an aqueous phase solution containing a first predetermined concentration to a second predetermined volume of an organic phase containing a prepared particle raw material of a second predetermined concentration.

In some embodiments, the aqueous phase solution may contain components of cancer cells and/or bacterial lysates, as well as an immune enhancing adjuvant; the components of cancer cell and/or bacterial lysate are water-soluble antigens or non-water-soluble antigens dissolved in a lytic agent such as urea or guanidine hydrochloride. The aqueous solution contains a concentration of water-soluble antigen or a concentration of antigen that is not water-soluble, i.e., a first predetermined concentration that requires a protein polypeptide concentration of greater than 1ng/mL, sufficient to carry the whole cell fraction of the cancer cell to activate the relevant cell. The concentration of the immunopotentiating adjuvant in the initial aqueous phase is greater than 0.01ng/mL.

In some embodiments, the aqueous phase solution contains components of tumor tissue and/or cancer cell lysate and an immune enhancing adjuvant; the components of the tumor tissue and/or cancer cell lysate are water-soluble antigens or original water-insoluble antigens dissolved in a lytic agent such as urea or guanidine hydrochloride. The aqueous solution contains a concentration of water-soluble antigen or a concentration of antigen that is not water-soluble, i.e., a first predetermined concentration that requires a protein polypeptide concentration of greater than 0.01ng/mL, sufficient to carry the whole cell fraction of the cancer cell to activate the relevant cell. The concentration of the immunopotentiating adjuvant in the initial aqueous phase is greater than 0.01ng/mL.

In some embodiments, the aqueous phase solution contains the components of the bacterial lysate and an immunopotentiating adjuvant; the components of the bacterial lysate are water-soluble antigens or original water-insoluble antigens dissolved in a dissolving agent such as urea or guanidine hydrochloride. The aqueous phase contains a concentration of water-soluble antigen or a concentration of original water-insoluble antigen, i.e., a first predetermined concentration, that requires a protein polypeptide concentration level of greater than 0.01ng/mL, sufficient bacterial composition to be loaded to activate the relevant cells. The concentration of the immunopotentiating adjuvant in the initial aqueous phase is greater than 0.01ng/mL.

In some embodiments, the particle is prepared from PLGA or PLA, and the organic solvent is dichloromethane. Additionally, in some embodiments, the second predetermined concentration of the starting material for preparing the particles ranges from 0.5mg/mL to 5000mg/mL, preferably 100mg/mL.

In the present invention, PLGA or modified PLGA is selected because the material is biodegradable and has been approved by the FDA for use as a drug dressing. Research shows that PLGA has certain immunoregulation function, so that it is suitable for use as supplementary material for preparing nanometer particle and micron particle. In practical application, suitable materials can be selected according to practical situations.

In practice, the second predetermined volume of the organic phase is set according to its ratio to the first predetermined volume of the aqueous phase, and in the present invention the ratio between the first predetermined volume of the aqueous phase and the second predetermined volume of the organic phase ranges from 1.1 to 5000, preferably from 1. The first predetermined volume, the second predetermined volume, and the ratio of the first predetermined volume to the second predetermined volume can be adjusted as needed to adjust the size of the nanoparticles or microparticles produced during the implementation.

Preferably, when the aqueous phase solution is a lysate component solution, the concentration of the protein and the polypeptide is more than 1ng/mL, preferably 1 mg/mL-100 mg/mL; when the aqueous phase solution is lysate component/immunoadjuvant solution, the concentration of protein and polypeptide is more than 1ng/mL, preferably 1 mg/mL-100 mg/mL, and the concentration of immunoadjuvant is more than 0.01ng/mL, preferably 0.01 mg/mL-20 mg/mL. In the organic phase solution, the solvent is DMSO, acetonitrile, ethanol, chloroform, methanol, DMF, isopropanol, dichloromethane, propanol, ethyl acetate, etc., preferably dichloromethane; the concentration of the organic phase is 0.5mg/mL to 5000mg/mL, preferably 100mg/mL.

And 2, carrying out ultrasonic treatment for more than 2 seconds or stirring for more than 1 minute or homogenizing treatment or microfluidic treatment on the mixed solution obtained in the step 1. Preferably, when the stirring is mechanical stirring or magnetic stirring, the stirring speed is greater than 50rpm, and the stirring time is greater than 1 minute, for example, the stirring speed is 50rpm to 1500rpm, and the stirring time is 0.1 hour to 24 hours; during ultrasonic treatment, the ultrasonic power is more than 5W, and the time is more than 0.1 second, such as 2-200 seconds; the homogenizing treatment is carried out by using a high pressure/ultrahigh pressure homogenizer or a high shear homogenizer, wherein the pressure is more than 5psi, such as 20 psi-100 psi, when the high pressure/ultrahigh pressure homogenizer is used, and the rotating speed is more than 100rpm, such as 1000 rpm-5000 rpm, when the high shear homogenizer is used; microfluidic processing flow rates of greater than 0.01mL/min, such as 0.1mL/min to 100mL/min, are used. The nano-grade and/or micron-grade particles are subjected to ultrasonic treatment, stirring treatment, homogenizing treatment or microfluidic treatment, the size of the prepared micro-nano particles can be controlled by the ultrasonic time or the stirring speed or the homogenizing treatment pressure and time, and the particle size can be changed when the particles are too large or too small.

And 3, adding the mixture obtained after the treatment in the step 2 into a third preset volume of aqueous solution containing a third emulsifier with a preset concentration, and performing ultrasonic treatment for more than 2 seconds or stirring for more than 1 minute or performing homogenization treatment or microfluidic treatment. Adding the mixture obtained in the step 2 into an emulsifier aqueous solution, and continuing to carry out ultrasonic treatment or stirring for nano-crystallization or micro-crystallization. In the present invention, the sonication time is greater than 0.1 seconds, such as from 2 to 200 seconds, the agitation speed is greater than 50rpm, such as from 50rpm to 500rpm, and the agitation time is greater than 1 minute, such as from 60 to 6000 seconds. Preferably, when the stirring is mechanical stirring or magnetic stirring, the stirring speed is greater than 50rpm, and the stirring time is greater than 1 minute, for example, the stirring speed is 50rpm to 1500rpm, and the stirring time is 0.5 hour to 5 hours; during ultrasonic treatment, the ultrasonic power is 50W-500W, and the time is more than 0.1 second, such as 2-200 seconds; the homogenizing treatment is carried out by using a high pressure/ultrahigh pressure homogenizer or a high shear homogenizer, wherein the pressure is more than 20psi, such as 20 psi-100 psi, when the high pressure/ultrahigh pressure homogenizer is used, and the rotating speed is more than 1000rpm, such as 1000 rpm-5000 rpm, when the high shear homogenizer is used; microfluidic processing flow rates of greater than 0.01mL/min, such as 0.1mL/min to 100mL/min, are used. The nano-or micro-scale treatment is carried out by ultrasonic treatment, stirring, homogenizing treatment or micro-fluidic treatment, the size of the prepared nano-or micro-particles can be controlled by the ultrasonic time or the stirring speed or the homogenizing treatment pressure and time, and the change of the particle size can be brought by the over-large or over-small of the ultrasonic time or the stirring speed or the homogenizing treatment pressure and time.

In some embodiments, the aqueous emulsifier solution is an aqueous polyvinyl alcohol (PVA) solution, the third predetermined volume is 5mL, and the third predetermined concentration is 20mg/mL. The third predetermined volume is adjusted according to its ratio to the second predetermined volume. In the present invention, the range between the second predetermined volume and the third predetermined volume is 1.1-1, and preferably may be 2:5. The ratio of the second predetermined volume to the third predetermined volume may be adjusted during implementation to control the size of the nanoparticles or microparticles. Similarly, the ultrasonic time or stirring time, the volume of the emulsifier aqueous solution and the concentration in the step are all taken according to the obtained nano particles or micro particles with proper size.

And 4, adding the liquid obtained after the treatment in the step 3 into a fourth preset volume of emulsifier aqueous solution with a fourth preset concentration, and stirring until preset stirring conditions are met.

In this step, the aqueous emulsifier solution is a PVA solution or other solution.

The fourth predetermined concentration is 5mg/mL, and the fourth predetermined concentration is selected based on obtaining nanoparticles or microparticles of a suitable size. The fourth predetermined volume is selected based on a ratio of the third predetermined volume to the fourth predetermined volume. In the present invention, the ratio of the third predetermined volume to the third predetermined volume is in the range 1.5-1, preferably 1. The ratio of the third predetermined volume to the fourth predetermined volume may be adjusted during the implementation to control the size of the nanoparticles or microparticles.

In the present invention, the predetermined stirring condition in this step is until the volatilization of the organic solvent is completed, that is, the volatilization of dichloromethane in step 1 is completed.

And 5, centrifuging the mixed solution which is processed in the step 4 and meets the preset stirring condition at the rotating speed of more than 100RPM for more than 1 minute, removing the supernatant, and resuspending the remaining precipitate in a fifth preset volume of aqueous solution containing the lyoprotectant with a fifth preset concentration or a sixth preset volume of PBS (or physiological saline).

In some embodiments of the present invention, the pellet obtained in step 5 is resuspended in the sixth predetermined volume of PBS (or physiological saline) without lyophilization, and subsequent experiments relating to the interaction of nanoparticles or microparticles with membrane components can be directly performed.

In some embodiments of the present invention, the pellet obtained in step 5 is re-suspended in the aqueous solution containing the lyoprotectant by freeze-drying, and then subjected to subsequent experiments after freeze-drying.

In the invention, the freeze-drying protective agent is Trehalose (Trehalose).

In the present invention, the fifth predetermined concentration of the lyoprotectant in this step is 4% by mass, and is set so as not to affect the lyophilization effect in the subsequent lyophilization.

And 6, freeze-drying the suspension containing the freeze-drying protective agent obtained in the step 5, and then keeping the freeze-dried substance for later use.

And 7, mechanically destroying the cancer cells, the antigen presenting cells or the bacteria or the extracellular vesicles by adopting the methods of low-power ultrasound, mechanical stirring, homogenization, high shear force, high pressure, swelling and the like, and collecting membrane components of the destroyed cells or vesicles.

And 8, co-acting the membrane component prepared in the step 7 with the prepared nano particles and/or micro particles for a certain time. The tumor tissue and/or cancer cells and cancer cells, antigen presenting cells from which the nanoparticles and/or microparticles are prepared may be autologous or allogeneic.

And 9, collecting the nanoparticles or the microparticles after the combined action, purifying the nanoparticles or the microparticles by adopting a centrifugal method, an ultrafiltration method or a dialysis method and the like to obtain the nano vaccine or the micron vaccine, and directly using, freezing for later use or preparing freeze-dried powder for later use after freeze drying.

In other embodiments, the specific method for preparing the nano-or micro-vaccine after preparing the antigen-loaded nanoparticles or microparticles using the multiple emulsion method is as follows:

the steps 1-4 are the same as above.

And 5, centrifuging the mixed solution which is processed in the step 4 and meets the preset stirring condition for more than 1 minute at the rotating speed of more than 100RPM, removing the supernatant, and re-suspending the remaining precipitate in a fifth preset volume of solution containing the water-soluble and/or non-water-soluble antigen in the cancer cell whole cell component at a fifth preset concentration, or re-suspending the remaining precipitate in a fifth preset volume of solution containing the water-soluble and/or non-water-soluble antigen in the cancer cell whole cell component at a fifth preset concentration and the adjuvant.

And 6, centrifuging the mixed solution which is processed in the step 5 and meets the preset stirring condition for more than 1 minute at the rotating speed of more than 100RPM, removing the supernatant, resuspending the remaining precipitate in a sixth preset volume of solidification treatment reagent or mineralization treatment reagent, performing centrifugal washing after acting for a certain time, and adding a seventh preset substance containing positively charged or negatively charged substances and performing the action for a certain time.

In some embodiments of the present invention, the pellet obtained in step 6 may be resuspended in the seventh predetermined volume of the charged substance without lyophilization, and then the subsequent experiments related to the interaction of the nanoparticles or microparticles with the membrane components may be directly performed.

In some embodiments of the present invention, the precipitate obtained in step 6 is re-suspended in an aqueous solution containing a lyoprotectant and then dried under vacuum or freeze-dried at room temperature, and then dried before further experiments.

In the invention, the freeze-drying protective agent is Trehalose (Trehalose) or a mixed solution of mannitol and sucrose. In the present invention, the concentration of the drying protective agent in this step is set so as not to affect the drying effect in the subsequent drying, because the concentration is 4% by mass.

And 7, drying the suspension containing the drying protective agent obtained in the step 6, and then keeping the dried substance for later use.

In the present invention, the modification and antigen loading steps of steps 5-7 can be repeated multiple times to increase the antigen loading. When a substance having a positive or negative charge is added, a substance having the same charge may be added a plurality of times or a substance having different charges may be added alternately.

And 8, mechanically destroying the cancer cells, the antigen presenting cells or the bacteria or the extracellular vesicles by adopting the methods of low-power ultrasound, mechanical stirring, homogenization, high shear force, high pressure, swelling and the like, and collecting membrane components of the destroyed cells or vesicles.

And 9, co-acting the membrane component prepared in the step 8 with the prepared nano particles and/or micro particles for a certain time. The tumor tissue and/or cancer cells and cancer cells, antigen presenting cells from which the nanoparticles and/or microparticles are prepared may be autologous or allogeneic.

And step 10, collecting the nanoparticles or microparticles after the combined action, purifying the nanoparticles or microparticles by methods such as centrifugation, ultrafiltration or dialysis and the like to obtain the nano vaccine or the micron vaccine, which can be directly used, frozen for later use or prepared into freeze-dried powder for later use after freeze drying.

Example 1 Nanoprotein for prevention of melanoma

This example uses mouse melanoma as a cancer model to illustrate how to prevent melanoma using nano-vaccines. In this embodiment, a B16F10 melanoma tumor tissue is lysed to prepare a water-soluble antigen and a water-insoluble antigen of the tumor tissue, then, a nanoparticle loaded with the water-soluble antigen and the water-insoluble antigen of the tumor tissue is prepared by a solvent evaporation method using organic polymer material PLGA as a nanoparticle framework material and Polyinosinic-polycystic acid (poly (I: C)) as an immunoadjuvant, and then, a cancer cell membrane component and/or an activated antigen presenting cell membrane component and a bacterial extracellular vesicle component are loaded on the surface of the nanoparticle for preventing cancer.

(1) Lysis of tumor tissue and Collection of fractions

Subcutaneous inoculation of 1.5X 10 in the back of each C57BL/6 mouse ⁵ B16F10 cells, which grow to a volume of about 1000mm in the tumor ³ Mice were sacrificed and tumor tissue was harvested. Tumor tissues were cut into pieces and ground, and a suitable amount of ultrapure water was added through a cell filtration screen and freeze-thawed repeatedly for 5 times with ultrasound to destroy lysed cells. After the cells are cracked, the lysate is centrifuged for 5 minutes at the rotating speed of 5000g, and the supernatant is taken as the water-soluble antigen which can be dissolved in pure water; adding 8M urea aqueous solution into the obtained precipitation part to dissolve the precipitation part so as to convert the water-insoluble antigen insoluble in pure water into the one soluble in 8M urea aqueous solution. Mixing water-soluble antigen and water-insoluble antigen according to the mass ratio of 1:1 to obtain the antigen raw material source for preparing the nano particles.

(2) Preparation of nanoparticles with whole-cell components loaded inside

In this embodiment, the nanoparticles and the blank nanoparticles used as a control are prepared by a multiple emulsion method in a solvent evaporation method. The molecular weight of PLGA used as a material for preparing the nano particles is 7-17 KDa, and the adopted immunologic adjuvant is poly (I: C) and the poly (I: C) is encapsulated in the nano particles. The preparation method is as described above, during the preparation process, the whole cell lysate component and the adjuvant are loaded inside the nanoparticles by the double emulsion method, then 100mg of the nanoparticles are centrifuged at 10000g for 20 minutes, and after being resuspended by 10mL of ultrapure water containing 4% trehalose, the nanoparticles are freeze-dried for 48 hours. The average particle diameter of the nano particles 1 is about 260nm, and the surface potential is about minus 9 mV; each 1mg of PLGA nanoparticle 1 was loaded with about 100. Mu.g of protein or polypeptide component, and each 1mg of PLGA nanoparticle 1 was loaded with 0.02mg of poly (I: C) immunoadjuvant. The blank nanoparticle preparation material and the preparation method are the same, the particle size is about 260nm, and the same amount of adjuvant is loaded but no lysate component is loaded.

(3) Preparation of cancer cell membrane fraction

The B16F10 cancer cells were harvested, then washed twice with saline, resuspended in saline and sonicated at 7.5W for 20 minutes. The sample was then centrifuged at 2000g for 20 minutes and the supernatant collected, the supernatant collected after centrifugation at 7000g for 20 minutes, then centrifuged at 15000g for 120 minutes and the supernatant collected and the pellet discarded, and the pellet resuspended in PBS for use.

(4) Preparation and activation of bone marrow-derived dendritic cells (BMDCs)

This example illustrates how to prepare BMDCs by taking dendritic cells prepared from mouse bone marrow cells as an example. Firstly, 1C 57 mouse of 6-8 weeks old is taken out and killed by dislocation of cervical vertebrae, tibia and femur of hind leg are taken out by operation and put into PBS, and muscle tissue around the bone is removed by scissors and tweezers. The two ends of the bone are cut off by scissors, the PBS solution is extracted by a syringe, the needles are respectively inserted into the marrow cavity from the two ends of the bone, and the marrow is repeatedly washed into a culture dish. Bone marrow solution was collected, centrifuged at 400g for 3min, and then 1mL of erythrocyte lysate was added to lyse red blood. Add 3mL of RPMI1640 (10% FBS) medium to terminate lysis, centrifuge at 400g for 3min and discard the supernatant. Culturing the cells in 10mm culture dish, using RPMI1640 (10% FBS) medium, adding recombinant mouse GM-CSF (20 ng/mL), 37 degree, 5% CO ₂ The culture was carried out for 7 days. The flasks were gently shaken on day 3 and supplemented with the same volume of medium containing GM-CSF (20 ng/mL) RPMI1640 (10% FBS). On day 6, medium was subjected to half-volume change. On day 7, a small number of suspended and semi-adherent cells were collected and assayed by flow,when CD86 ⁺ CD80 ⁺ Cells in CD11c ⁺ The proportion of cells is between 15 and 20 percent, and the BMDCs cultured by induction can be used for the next experiment.

Co-incubation of nanoparticles (800. Mu.g) loaded with cancer cell whole cell fractions derived from tumor tissue with BMDCs (1000 ten thousand) in 15mL RPMI1640 complete medium for 48 hours (37 ℃,5% ₂ ) The incubation system contained granulocyte-macrophage colony stimulating factor (GM-CSF, 1000U/mL), IL-2 (500U/mL), IL-7 (1000U/mL) and IL-15 (500U/mL). The activated DCs were then harvested and centrifuged at 400g for 5 minutes, then the cells were washed twice with 4 ℃ Phosphate Buffered Saline (PBS) containing protease inhibitors, resuspended in PBS water and sonicated at 4 ℃ for 1 minute with low power (22.5W). Centrifuging the sample at 3000g for 15 minutes and collecting supernatant, centrifuging the supernatant at 8000g for 15 minutes, collecting supernatant, centrifuging at 16000g for 90 minutes, collecting the supernatant, discarding the supernatant, collecting precipitate, resuspending the precipitate in PBS, and filtering the sample with a membrane filter to obtain the nanoparticle based on the antigen presenting cell membrane component, wherein the particle size is 130 nm.

(5) Preparation of bacterial extracellular vesicles (OMVs)

The cultured E.coli (BL 21) was collected, centrifuged at 3000g for 10 minutes, the supernatant was removed and centrifuged at 13000g for 90 minutes, and the pellet was resuspended in PBS to obtain the collected bacterial extracellular vesicles (3 mg/mL).

(6) Preparation of nano-vaccine

And (3) resuspending 50mg of blank nanoparticles prepared in the step (2) in 9mL of PBS, mixing the blank nanoparticles with 1mL of 3mg of cancer cell membrane components prepared in the step (3), incubating at room temperature for 15 minutes, repeatedly co-extruding through an extrusion membrane of 0.45 mu m, centrifuging at 13000g for 25 minutes, discarding supernatant, and then resuspending precipitate by using PBS to obtain the nano vaccine 1 with the particle size of 270nm.

Or 50mg of the nanoparticles loaded with the whole cell components prepared in the step (2) are resuspended in 9mL of LPBS, then mixed with 1mL of 3mg of the cancer cell membrane components prepared in the step (3), incubated at room temperature for 15 minutes, then repeatedly co-extruded through an extrusion film of 0.45 mu m, then centrifuged at 13000g for 25 minutes, and after supernatant liquid is discarded, the nanoparticles are resuspended and precipitated by PBS, namely the nano vaccine 2, and the particle size is 270nm.

Or mixing 1mL of 1.5mg of the cancer cell membrane fraction prepared in step (3) with 1.5mg of the antigen-presenting cell membrane fraction-based nanoparticles prepared in step (4), incubating for 10 minutes, and repeatedly co-extruding through a 0.22 μm extrusion membrane. Then, 100mg of the whole cell component-loaded nanoparticles prepared in the step (2) are resuspended in 9ml PBS, then mixed with the mixed membrane component and incubated for 15 minutes at room temperature, then extruded through an extrusion membrane of 0.45 μm repeatedly, then centrifuged at 13000g for 25 minutes, and after supernatant is discarded, the nanoparticles are resuspended and precipitated by PBS to obtain the nano vaccine 3 with the particle size of 270nm.

Or mixing 1mL of 1mg of the cancer cell membrane fraction prepared in step (3) with 1mg of the antigen-presenting cell membrane fraction-based nanoparticles prepared in step (4) and 1mg of the bacterial extracellular vesicles prepared in step (5), incubating for 10 minutes, and repeatedly co-extruding through a 0.22 μm extrusion membrane. Then, 100mg of the whole-cell component-loaded nanoparticles prepared in the step (2) are resuspended in 9ml PBS, then mixed with the mixed membrane component and incubated for 15 minutes at room temperature, then extruded through an extrusion membrane of 0.45 μm repeatedly, then centrifuged at 13000g for 25 minutes, and after supernatant is discarded, the nanoparticles are resuspended and precipitated by PBS to obtain the nano vaccine 4 with the particle size of 270nm.

(7) Nano-vaccine for cancer prevention

Female C57BL/6 of 6-8 weeks is selected as a model mouse to prepare a melanoma-bearing mouse. Mice were subcutaneously injected with 1mg of nano-vaccine 1, or with 1mg of nano-vaccine 2, or with 1mg of nano-vaccine 3, or with 1mg of nano-vaccine 4, or with 100 μ LPBS on days-42, 35, 28, 21, 14, and 7, respectively, prior to inoculation of the mice with cancer cells. On day 0, each mouse was subcutaneously inoculated at 1.5X 10 in the lower right back ⁵ And B16F10 cells. The tumor growth rate and survival time of the mice were monitored. In the experiment, the size of the tumor volume of the mice was recorded every 3 days from day 3. Tumor volume was calculated using the formula v =0.52 × a × b ² (iii) calculating, wherein v is the tumor volume,a is the tumor length and b is the tumor width. For ethics of animal experiments, when the tumor volume of the mice exceeds 2000mm in the survival period test of the mice ³ I.e., mice were considered dead and were euthanized.

(8) Results of the experiment

As shown in fig. 2, the mice in the PBS control group showed a fast tumor growth rate and a short survival time; the tumor growth rate of the mice inoculated with the nano vaccine group is obviously reduced. Moreover, the effect of the nano vaccine 2, the nano vaccine 3 and the nano vaccine 4 is better than that of the nano vaccine 1; the effect of the nano vaccine 3 and the nano vaccine 4 is better than that of the nano vaccine 2; the effect of the nano vaccine 4 is better than that of the nano vaccine 3. This demonstrates that the addition of cancer cell membrane components, membrane components of antigen-presenting cells activated by nanoparticles loaded with cancer cell whole cell components, and membrane components of bacterial extracellular vesicles can improve the efficacy of the nano-vaccine. In conclusion, the nano vaccine provided by the invention has a good prevention effect on melanoma.

In this embodiment, the membrane components of the cancer cell membrane and the antigen-presenting cell membrane are prepared by using an ultrasonic method, and in practical application, the cells may be mechanically destroyed by using methods such as stirring, high pressure, high shear force, homogenization and the like, and then the membrane components may be collected by centrifugation and/or filtration through filter membranes with different pore sizes and/or ultrafiltration and/or dialysis in sequence. In this embodiment, a mixed cell membrane of cancer cell membrane components, mixed cell membranes of cancer cells and antigen-presenting cells, or mixed cell membranes of cancer cells, antigen-presenting cells, and extracellular vesicles of bacteria is used, and in practical applications, a mixed membrane component of extracellular vesicles of cancer cells, mixed membrane components of extracellular vesicles of antigen-presenting cells, and bacteria, or mixed membrane components of extracellular vesicles of any of these types of cells may also be used. This example employs co-incubation and co-extrusion to co-act the nanoparticles with the membrane fraction, and in practice one or more of stirring, homogenization, sonication, ultrafiltration, dialysis and homogenization may be used. In the embodiment, the nanoparticles loaded with the whole cell component and the membrane component are used for preparing the nano vaccine in a coaction mode, and the microparticles loaded with the whole cell component and the membrane component can be used for preparing the micro vaccine in a coaction mode in practical application.

Example 2 Nanoprotein for treatment of melanoma

This example illustrates how a nano-vaccine can be used to treat melanoma in mice as a cancer model. In this embodiment, a B16F10 melanoma tumor tissue is lysed to prepare a water-soluble antigen and a water-insoluble antigen of the tumor tissue, then PLGA is used as a nanoparticle framework material, poly (I: C), cpG BW006, and CpG2395, which are Toll-like receptor agonists, are used as a mixed immunoadjuvant, and the water-soluble antigen and the water-insoluble antigen of the tumor tissue are loaded in the nanoparticle by a solvent evaporation method, and then cell membrane components of cancer cells and antigen presenting cells are loaded on the surface of the nanoparticle, thereby obtaining the melanoma vaccine.

(1) Lysis of tumor tissue and Collection of fractions

Subcutaneous inoculation of 1.5X 10 in the back of each C57BL/6 mouse ⁵ B16F10 cells, which grow to a volume of about 1000mm in the tumor ³ Mice were sacrificed and tumor tissue was harvested. Tumor tissue is cut into pieces and ground, and a proper amount of pure water is added through a cell filter screen and freeze thawing is repeated for 5 times, and ultrasonic waves are accompanied to destroy the lysed cells. After the cells are cracked, the lysate is centrifuged for 5 minutes at the rotating speed of 5000g, and the supernatant is taken as the water-soluble antigen which can be dissolved in pure water; the addition of 8M urea to the resulting precipitate converts the water-insoluble antigen insoluble in pure water to soluble in an 8M aqueous urea solution by dissolving the precipitate. The above is the source of the antigen raw material for preparing the nanoparticle system.

(2) Preparation of nanoparticle systems

In this embodiment, the nanoparticles are prepared by a solvent evaporation method. The nanoparticles carrying the water-soluble antigen in the cancer cell whole cell fraction and the nanoparticles carrying the water-insoluble antigen in the cancer cell whole cell fraction are prepared separately and then used together at the time of use. Nanoparticle 1 preparation material PLGA molecular weight is 24Da-38KDa, the adopted immune adjuvants are poly (I: C), cpG BW006 and CpG2395, and the adjuvants are loaded in the nanoparticle. As mentioned above, in the preparation process, the antigen and adjuvant are loaded inside the nanoparticles by using a multiple emulsion method, after the antigen (lysis component) is loaded inside, 100mg of the nanoparticles are centrifuged at 10000g for 20 minutes, and are resuspended by using 10mL of ultrapure water containing 4% trehalose, and then are freeze-dried for 48 hours. The average particle diameter of the nano particles is about 320 nm; each 1mg PLGA nanoparticle 1 was loaded with about 100. Mu.g of protein and polypeptide components, and 0.025mg each of poly (I: C), cpG BW006 and CpG2395 immunoadjuvants.

Control polypeptide nanoparticle preparation materials and methods were as above, and the four polypeptide neoantigens loaded were B16-M20 (Tubb 3, FRRKAFLHWYTGEAMDEMEFTEAESNM), B16-M24 (Dag, TAVITPPTTTTKKARVSTPKPATPSTD), B16-M46 (Actn 4, NHSGLVTFQAFIDVMSRETTDTDTADQ) and TRP2:180-188 (SVYDFFVWL). Each 1mgPLGA nanoparticle loads 100 ug of polypeptide component, and each of poly (I: C), cpG BW006 and CpG2395 is 0.025mg, and the average particle size is about 320 nm.

(3) Preparation of antigen-presenting cells and membrane fractions thereof

This example uses mixed antigen presenting cells of BMDCs and B cells. BMDC was prepared as in example 1. The method for isolating B cells was as follows: after C57BL/6 mice were sacrificed, spleens of the mice were harvested to prepare single cell suspensions of splenocytes of the mice, and CD19 among live cells in the splenocytes (dead cells were labeled with a live-dead cell dye to remove dead cells) was separated by magnetic bead sorting ⁺ B cells. Then, BMDC and B cells were mixed at a ratio of 1:1 and used as mixed antigen-presenting cells.

1mg of cancer cell whole cell component-loaded nanoparticle 1 (in which water-soluble component-loaded nanoparticle 500. Mu.g + water-insoluble component-loaded nanoparticle 500. Mu.g) or 1mg of polypeptide nanoparticle and 2000 ten thousand of mixed antigen-presenting cells (in which DC1000 ten thousand + B cells 1000 ten thousand) were mixed in 20mL of RPMI1640 complete medium and then incubated for 48 hours (37 ℃,5% CO) ₂ ) The incubation system contained IL-2 (1000U/mL), IL-7 (1000U/mL), IL-12 (200U/mL), GM-CSF (500U/mL), and albumin (50 ng/mL). After incubation, the incubated cells were centrifuged at 400g for 5 minutes, the supernatant was discarded, and the cells were washed twice with PBS to obtain activated antigen-presenting cells.

In this example, antigen-presenting cells that are not activated by any nanoparticles or antigen-presenting cells that are activated by nanoparticles are used to prepare membrane fractions, respectively. In preparing the membrane fraction, the mixed antigen-presenting cells that were not activated (2000 ten thousand) or the mixed antigen-presenting cells that had been activated by the nanoparticles (2000 ten thousand) were collected and centrifuged at 400g for 5 minutes, and then the corresponding mixed antigen-presenting cells were washed twice with 4 ℃ Phosphate Buffered Saline (PBS) containing a protease inhibitor, and the cells were resuspended in PBS water and then sonicated at 4 ℃ for 3 minutes with low power (12W). Centrifuging the sample at 3000g for 15 min, collecting supernatant, centrifuging the supernatant at 8000g for 15 min, collecting supernatant, repeatedly filtering the supernatant with a 0.22 μm membrane filter, centrifuging at 16000g for 90 min, collecting the supernatant, discarding the supernatant, collecting precipitate, and resuspending the precipitate in PBS to obtain the cell membrane component of the antigen presenting cell.

(4) Preparation of bacterial extracellular vesicles (OMVs)

The cultured Bifidobacterium longum (BL 21) was collected, centrifuged at 3000g for 10 minutes, the supernatant was centrifuged at 15000g for 90 minutes after removing the pellet, and the pellet was resuspended in PBS to obtain the collected bacterial extracellular vesicles (3 mg/mL).

(5) Preparation of cancer cell membrane fraction

(6) Preparation of Mixed Membrane Components

Mixing the unactivated mixed antigen presenting cell membrane component collected in the step (3) or the mixed antigen presenting cell membrane component activated by the nano particles, the bacterial extracellular vesicle component collected in the step (4) and the cancer cell membrane component collected in the step (5) according to a mass ratio of 2.

(7) Preparation of nano-vaccine

And (3) mixing 100mg of the nanoparticles 1 (50 mg of nanoparticles loaded with water-soluble components and 50mg of nanoparticles loaded with water-insoluble components) prepared in the step (2) and 10mg of the mixed membrane component prepared in the step (6), incubating for 20 minutes, repeatedly filtering with a 0.45-micrometer filter membrane, centrifuging the filtrate at 13000g for 20 minutes, discarding the supernatant, then re-suspending and precipitating with 4% trehalose aqueous solution, and freeze-drying for 48 hours to obtain freeze-dried powder, namely the nano vaccine. Wherein, the particle diameter of the nano vaccine 1 prepared by mixing the mixed antigen presenting cell membrane component activated by the nano particle 1 internally loaded with the whole cell component, the cancer cells and the bacterial extracellular vesicles is 340nm; the particle size of the nano vaccine 2 prepared by mixing the nanoparticle activated mixed antigen presenting cell membrane component internally loaded with four polypeptide antigens with cancer cells and bacterial extracellular vesicles is 340nm; the nano-vaccine 3 prepared by mixing the inactivated mixed antigen-presenting cell membrane fraction with cancer cells and bacterial extracellular vesicles has a particle size of 340nm.

(8) Nano-vaccine for cancer treatment

Selecting female C57BL/6 of 6-8 weeks as model mouse, preparing melanoma tumor-bearing mouse, and inoculating 1.5 × 10 subcutaneous inoculation to the lower right back of each recipient mouse on day 0 ⁵ And B16F10 cells. Each mouse was inoculated with 1mg of nano-vaccine 1 or nano-vaccine 2 or nano-

vaccine

3 or 100 μ L of PBS 5 days, 8 days, 11 days, 15 days and 20 days before and after inoculation of the mice with cancer cells, respectively. The mouse tumor growth rate and the mouse survival time are monitored as above.

(9) Results of the experiment

As shown in FIG. 3, the growth rate of the tumors was very fast in the PBS control group mice, and the survival time of the mice was very short. The growth speed of the tumor of the mice with the nano vaccine group is obviously slowed down, and the life cycle of the mice is obviously prolonged. Wherein the effect of the nano vaccine 1 and the nano vaccine 2 is better than that of the nano vaccine 3; the effect of the nano vaccine 1 is better than that of the nano vaccine 2. This demonstrates that the antigen loaded nanoparticle activated mixed antigen presenting cell membrane fraction performed better when added to the mixed membrane fraction than when not activated by any nanoparticles; and the effect of the nano-particles internally loaded with cancer cell whole-cell components after activating the mixed antigen presenting cell membrane components and adding the mixed antigen presenting cell membrane components into the mixed membrane components is better than that of the nano-particles internally loaded with polypeptide antigens.

Example 3 Nanoprotein for treatment of melanoma

This example illustrates how to treat cancer using nano-vaccines, using mouse melanoma as a cancer model. In this example, B16F10 melanoma tumor tissue and cancer cells were first lysed to prepare a water-soluble antigen mixture (mass ratio 1:1) and a water-insoluble antigen mixture (mass ratio 1:1) of tumor tissue and cancer cells, and the water-soluble antigen mixture and the water-insoluble antigen mixture were mixed at a mass ratio of 1:1. Then, PLGA is used as a nanoparticle framework material, poly (I: C), cpG2006 and CpG2395 are used as adjuvants to prepare nanoparticles loaded with lysate components inside, and then the nanoparticles and mixed cell membranes of cancer cells and activated antigen presenting cells are subjected to coaction for a certain time to prepare the nano vaccine for treating cancer.

(1) Lysis of tumor tissue and cancer cells and collection of fractions

Tumor tissue was collected by first subcutaneously inoculating 1.5X 10 dorsal cells of each C57BL/6 mouse ⁵ B16F10 cells, which grow to a volume of about 1000mm in the tumor ³ Killing mice and picking tumor tissues, cutting the tumor tissues into blocks, grinding, adding a proper amount of pure water through a cell filter screen, repeatedly freezing and thawing for 5 times, and destroying samples obtained by lysis with ultrasound; when the cultured B16F10 cancer cell line was collected, the culture medium was removed by centrifugation, then washed twice with PBS and centrifuged to collect cancer cells, the cancer cells were resuspended in ultrapure water, freeze-thawed repeatedly 3 times, and lysed with ultrasonic disruption. After the tumor tissue or the cancer cells are cracked, centrifuging the lysate for 5 minutes at the rotating speed of 5000g and taking supernatant fluid as water-soluble antigen which can be dissolved in pure water; the addition of 8M urea to the resulting precipitate converts the water-insoluble antigen insoluble in pure water to soluble in an 8M aqueous urea solution by dissolving the precipitate. Mixing water-soluble antigens of tumor tissues and water-soluble antigens of cancer cells according to a mass ratio of 1:1; the water-insoluble antigen of tumor tissue and the water-insoluble antigen of cancer cells are mixed in a mass ratio of 1:1. Mixing water soluble antigensThe material and the water-insoluble antigen mixture are mixed according to the mass ratio of 1:1, namely the antigen raw material source for preparing the nano particles.

(2) Preparation of bacterial extracellular vesicles (OMVs)

The cultured E.coli (BL 21) was collected, centrifuged at 5000 Xg for 10min at 4 ℃ and the resulting supernatant (200 mL) was filtered through a 0.45 μm EPS filter (Millipore) and concentrated to 50mL using a 50K ultrafiltration tube. The concentrate was further filtered through a 0.22 μm EPS membrane (micropore), and then OMV was collected from the filtrate by ultracentrifugation using 150,000 Xg at 4 ℃ for 1 hour, and after discarding the supernatant, the resulting precipitate was dissolved using a 10% sodium deoxycholate aqueous solution to be completely dissolved in the aqueous solution so as to be supported inside the nanoparticles.

(3) Preparation of nanoparticles

In this embodiment, the nanoparticles are prepared by a solvent evaporation method. The molecular weight of PLGA material prepared from the nanoparticle 1 is 24Da-38KDa, the adopted immunologic adjuvants are poly (I: C), cpG2006 and CpG2395, and the adjuvants are loaded in the nanoparticle; when preparing the nanoparticles, the mass ratio of the whole cell lysate component and the bacterial extracellular vesicle component contained in the first internal aqueous phase is 3:1. As mentioned above, during the preparation process, the lysate component containing the whole cell antigen prepared in step (1), the bacterial extracellular vesicle component prepared in step (2) and the adjuvant are loaded inside the nanoparticles by a multiple emulsion method, then 100mgPLGA nanoparticles are centrifuged at 10000g for 20 minutes, and 10mL of ultrapure water containing 4% trehalose is used for resuspension, and then the resuspension is frozen and dried for 48 hours. The average particle diameter of the nano particles 1 is about 350 nm; each 1mg PLGA nanoparticle 1 was loaded with about 100. Mu.g of protein and polypeptide components, and 0.025mg each of poly (I: C), cpG2006 and CpG2395 immunoadjuvants.

The preparation material and the preparation method of the nano particle 2 are the same, only the adjuvant and the cancer cell whole cell lysate component are loaded inside the nano particle 2, but no bacterial extracellular vesicle component is loaded, the average particle size of the nano particle 2 is about 350nm, about 100 mug of protein and polypeptide component are loaded in each 1mg of PLGA nano particle 2, and 0.025mg of each immune adjuvant, namely poly (I: C), cpG2006 and CpG2395, is loaded.

The preparation material and the preparation method of the nano particle 3 are the same, only the adjuvant and the bacterial extracellular vesicle component are loaded inside the nano particle 3, but no cancer cell lysate component is loaded, the average particle size of the nano particle 3 is about 350nm, each 1mg of PLGA nano particle 1 is loaded with about 100 mug of protein and polypeptide components, and each 1mg of PLGA nano particle is loaded with 0.025mg of poly (I: C), cpG2006 and CpG2395 immunologic adjuvants.

(4) Preparation of antigen-presenting cells and membrane fractions thereof

This example uses mixed antigen presenting cells of BMDCs and B cells. BMDC was prepared as in example 1. The method for isolating B cells was as follows: after C57BL/6 mice were sacrificed, spleens of the mice were harvested to prepare single cell suspensions of splenocytes of the mice, and CD19 among live cells in the splenocytes (dead cells were labeled with a live-dead cell dye to remove dead cells) was separated by magnetic bead sorting ⁺ B cells. Then, BMDCs and B cells were mixed at a quantitative ratio of 1:1 and used as mixed antigen-presenting cells.

1mg of cancer cell whole cell component-loaded nanoparticle 1 was mixed with 2000 ten thousand of mixed antigen-presenting cells (1000 ten thousand of DC + 1000 ten thousand of B cells) in 20mL of RPMI1640 complete medium and incubated for 48 hours (37 ℃,5% CO) ₂ ) The incubation system contained IL-2 (1000U/mL), IL-7 (1000U/mL), IL-12 (200U/mL), GM-CSF (500U/mL), and albumin (50 ng/mL). After incubation, the incubated cells were centrifuged at 400g for 5 minutes, the supernatant was discarded, and the cells were washed twice with PBS to obtain activated antigen-presenting cells.

In preparing the membrane fraction, the mixed antigen-presenting cells (2000 ten thousand) that had been activated by the nanoparticles were collected and centrifuged at 400g for 5 minutes, and then the corresponding mixed antigen-presenting cells were washed twice with Phosphate Buffered Saline (PBS) containing a protease inhibitor at 4 ℃ and then resuspended in PBS water and then sonicated at low power (12W) for 3 minutes at 4 ℃. Then centrifuging the sample for 15 minutes at 3000g and collecting the supernatant, centrifuging the supernatant for 15 minutes at 8000g and collecting the supernatant, repeatedly filtering the supernatant by using a 0.22 mu m membrane filter, centrifuging the filtered supernatant for 90 minutes at 16000g, collecting the supernatant, discarding the supernatant, collecting the precipitate, and re-suspending the precipitate in PBS to obtain the cell membrane component of the antigen presenting cell.

(5) Preparation of cancer cell membrane fraction

The B16F10 cancer cells were collected, centrifuged at 400g for 5 minutes, the supernatant was discarded, and the precipitated cells were resuspended using PBS, then washed three times with 30mM pH 7.0Tris-HCl buffer containing 0.0759M sucrose and 0.225M mannitol for 5 minutes by centrifugation at 400g, then washed twice with PBS containing phosphatase inhibitor and protease inhibitor, and then mechanically disrupted by high pressure homogenization (5 mPa) for 1 minute. Then, after the sample was filtered through membranes having a pore size of 30 μm, 10 μm, 5 μm, 2 μm, and 0.45 μm in this order, the filtrate was centrifuged at 18000g for 30 minutes, the supernatant was discarded, and the precipitate was collected and resuspended in PBS for use.

(6) Preparation of Mixed Membrane Components

And (3) mixing the mixed antigen presenting cell membrane component activated by the nanoparticles collected in the step (4) and the cancer cell membrane component collected in the step (5) according to the mass ratio of 1:1, stirring at room temperature at 1500RPM for 3 minutes, and repeatedly co-extruding by using a membrane extruder to obtain the mixed membrane component of the two.

(7) Preparation of nano-vaccine

And (3) resuspending 100mg of the

nanoparticle

1 or 2 or 3 prepared in the step (3) in 9mL of LPBS, mixing with 1mL of the mixed membrane component prepared in the step (6) of 5mg, mechanically stirring at 1500rpm for 10 minutes, repeatedly co-extruding through a 0.45-micrometer membrane, collecting an extruded liquid, centrifuging at 13000g for 25 minutes, removing a supernatant, and then resuspending and precipitating with PBS to obtain the nano vaccine. Wherein the nano-particle 1 is used for preparing the nano-vaccine 1, and the particle size is 370nm; the nano-particle 2 is used for preparing the nano-vaccine 2, and the particle size is 370nm; the nano-particle 3 is used for preparing the nano-vaccine 3, and the particle size is 370nm.

(8) Nano-vaccine for cancer treatment

Female C57BL/6 of 6-8 weeks is selected as a model mouse to prepare a melanoma tumor-bearing mouse. Each mouse was inoculated subcutaneously on day 0, at the lower right back, at 1.5X 10 ⁵ And B16F10 cells. Respectively injecting 500 mu g of nano vaccine 1 or nano vaccine 2 or nano vaccine 3 subcutaneously per mouse on 4 th, 7 th, 10 th, 15 th, 20 th and 25 th days after melanoma inoculation100 μ L PBS. In the experiment, the tumor volume was recorded every 3 days from day 3, and the tumor volume and survival time were monitored as described above.

(9) Results of the experiment

As shown in a and b of FIG. 4, the growth rate of the tumor was high in the mice of the PBS control group, and the survival time of the mice was short. The nano vaccine 1, the nano vaccine 2 and the nano vaccine 3 can obviously inhibit the growth of tumors and prolong the life cycle of mice. Wherein, the effect of the nano vaccine 1 is better than that of the nano vaccine 2 and the nano vaccine 3. The results show that the internal loading of the bacterial extracellular vesicle component and the internal loading of the cancer cell whole-cell component are beneficial to improving the effect of the cancer nano-vaccine with the mixed membrane component loaded on the surface.

Example 4 Nanoprotein for prevention of melanoma Lung metastasis

This example illustrates the use of a nano-vaccine to prevent cancer metastasis in a mouse melanoma lung model. In this example, the B16F10 melanoma tumor tissue was first lysed to prepare water-soluble and water-insoluble antigens of the tumor tissue; then, a nanoparticle system loaded with water-soluble and water-insoluble antigens of tumor tissue is prepared. In this example, the method of silicification and addition of charged substances was used to increase the antigen loading, and only one cycle of mineralization treatment was performed. In this example, the nanoparticles are used to activate antigen-presenting cells, and then the antigen-presenting cells are used to prepare a nano-vaccine for preventing cancer metastasis.

(1) Lysis of tumor tissue and Collection of fractions

Subcutaneous inoculation of 1.5X 10 in the back of each C57BL/6 mouse ⁵ B16F10 cells, which grow to a volume of about 1000mm in the tumor ³ Mice were sacrificed and tumor tissue was harvested. Cutting tumor tissue into pieces, grinding, adding collagenase, incubating in RPMI1640 culture medium for 30min, adding appropriate amount of pure water through cell filter screen, freeze thawing for 5 times, and optionally ultrasonic treating to destroy lysed cells. After the cells are cracked, the lysate is centrifuged for 5 minutes at the rotating speed of 5000g, and the supernatant is taken as the water-soluble antigen which can be dissolved in pure water; adding 10% sodium deoxycholate to the obtained precipitate to dissolve the precipitateThe water-insoluble antigen which is not dissolved in pure water can be converted into the antigen which is soluble in 10 percent sodium deoxycholate water solution, and the water-soluble antigen and the water-insoluble antigen are mixed according to the mass ratio of 2:1, thus obtaining the antigen raw material source for preparing the particles.

(2) Preparation of bacterial Membrane fraction

Collecting cultured Bifidobacterium longum, centrifuging at 5000g for 30 minutes, centrifuging and washing twice by PBS, then suspending in PBS, then using 20W ultrasonic treatment at 4 ℃ for 5 minutes, then filtering and extruding by 20 mu m, 10 mu m, 5 mu m, 1 mu m and 0.45 mu m filter membranes in sequence, removing precipitates, centrifuging the supernatant at 13000g for 90 minutes, and suspending the precipitates in PBS to obtain the collected Bifidobacterium longum bacterial membrane component.

Collecting cultured lactobacillus acidophilus, centrifuging for 30 minutes at 5000g, centrifuging and washing twice by PBS, then suspending in PBS, then using 20W ultrasonic wave to process for 5 minutes at 4 ℃, then filtering and extruding through 20 mu m, 10 mu m, 5 mu m, 1 mu m and 0.45 mu m filter membranes in sequence, removing precipitates, centrifuging the supernatant for 90 minutes at 13000g, and suspending the precipitates in PBS to obtain the collected lactobacillus acidophilus bacterial membrane components.

(3) Preparation of nanoparticles

In the embodiment, the nanoparticles and the blank nanoparticles serving as a reference are prepared by a solvent volatilization method, appropriate modification and improvement are performed, and two modification methods of low-temperature silicification and charged substance addition are adopted in the preparation process of the nanoparticles to improve the antigen loading capacity. In order to load the bacterial cell membrane fraction to the nanoparticles, the bacterial membrane fraction was first solubilized using 8M urea, and then the solubilized bacterial cell membrane fraction was mixed with the tumor tissue whole cell fraction. The mass ratio of the tumor tissue whole cell component and the bacterial membrane component (bifidobacterium longum or lactobacillus acidophilus) used for preparing the nano particles is 1:1. The molecular weight of PLGA material for preparing the nano particles is 24-38 KDa, and the adopted immunologic adjuvant is poly (I: C), cpG1018 and CpG2395. Preparation method As described previously, in the preparation process, the tumor tissue lysate component, the bacterial membrane (Bifidobacterium longum or Lactobacillus acidophilus) component and the adjuvant are loaded inside the nanoparticles by the double emulsion method, then 100mg of the nanoparticles are centrifuged at 10000g for 20 minutes, then 7mL of PBS is used to resuspend the nanoparticles and mixed with 3mL of PBS solution containing cell lysate (60 mg/mL), then 10000g is centrifuged for 20 minutes, then 10mL of silicate solution (containing 150mM NaCl, 80mM tetramethyl orthosilicate and 1.0mM HCl, pH 3.0) is used to resuspend the nanoparticles and fixed at room temperature for 10min, then-80 ℃ is fixed for 24h, after centrifugation washing with ultrapure water, 3mL of PBS containing protamine (5 mg/mL) and polylysine (10 mg/mL) is used to resuspend and act for 10min, then 10000g is centrifuged for 20min for washing, 10mL of PBS solution containing cell lysate (50 mg/mL) is used to act for 10min, then 10000g is centrifuged for 20min and re-suspended with 4% PBS containing cell lysate (50 mg/mL) and then dried after centrifugation for 48h; before using, the particles are resuspended by 7mL PBS and then added with 3mL cancer tissue lysate component (protein concentration is 50 mg/mL) containing adjuvant and acted for 10min at room temperature, so as to obtain the modified nano particles which are loaded with lysate inside and outside and are subjected to frozen silicification and cationic substance addition. The nano particle loaded with the tumor tissue lysate component and the bifidobacterium longum is a nano particle 1, the average particle size is about 350nm, about 300 mu g of protein or polypeptide component is loaded on each 1mg of PLGA nano particle, and each 1mg of PLGA nano particle 1 is loaded with 0.02mg of poly (I: C), cpG1018 and CpG2395. The nano particle loaded with the tumor tissue lysate component and the lactobacillus acidophilus is a nano particle 2, the average particle diameter is about 350nm, about 300 mu g of protein or polypeptide component is loaded on each 1mg of PLGA nano particle, and each 1mg of PLGA nano particle 2 is loaded with 0.02mg of poly (I: C), cpG1018 and CpG2395.

(4) Preparation of cancer cell and bacterial cell membrane fractions

The cell-cultured B16F10 melanoma cells were collected, centrifuged at 400g for 5 minutes, the supernatant was discarded, and then washed and mixed twice with Phosphate Buffered Saline (PBS) at 4 ℃ containing a protease inhibitor, and the cells were resuspended in PBS. Performing ultrasonic treatment on 1000 ten thousand B16F10 cells at 4 ℃ for 1 minute by low power (15W), centrifuging a sample at 3000g for 15 minutes, collecting supernatant, centrifuging the supernatant at 8000g for 15 minutes, collecting supernatant, mixing the obtained supernatant with 3mg of bacterial (bifidobacterium longum or lactobacillus acidophilus) membrane components prepared in the step (2), co-incubating at room temperature for 10 minutes, repeatedly co-extruding by using a filter membrane with the diameter of 0.22 mu m, collecting extruded liquid, centrifuging the extruded liquid at 16000g for 90 minutes, collecting supernatant, discarding precipitate, and re-suspending the precipitate in PBS to obtain the mixed cell membrane components.

As a control membrane fraction, only cancer cell membrane fraction was used. Cell-cultured B16F10 melanoma cells were harvested, centrifuged at 400g for 5 minutes and the supernatant discarded, followed by washing and mixing twice with 4 ℃ Phosphate Buffered Saline (PBS) containing protease inhibitors and resuspending the cells in PBS. Then, cancer cells are subjected to low-power (15W) ultrasonic treatment at 4 ℃ for 1 minute, then a sample is centrifuged at 3000g for 15 minutes, supernatant is collected, the supernatant is centrifuged at 8000g for 15 minutes, then the supernatant is collected, a filter membrane with the diameter of 0.22 mu m is used for repeated extrusion, the extruded liquid is collected, centrifuged at 16000g for 90 minutes, supernatant is collected, precipitate is discarded, and the precipitate is resuspended in PBS to obtain a cancer cell membrane component.

(5) Preparation of nano-vaccine

Suspending 100mg of the nanoparticle 1 prepared in step (3) in 9mL of pbs, and then mixing with 10mL of 20mg of the cancer cells prepared in step (4) and the bifidobacterium longum bacterial membrane component; resuspending 100mg of nanoparticles 2 prepared in step (3) in 9mL of pbs and then mixing with 10mL of 20mg of cancer cells prepared in step (4) and the lactobacillus acidophilus bacterial membrane fraction; or 100mg of the nanoparticle 1 prepared in step (3) was resuspended in 9mL of PBS and then mixed with 10mL of 20mg of the membrane fraction of the cancer cell membranes prepared in step (4). And (3) treating the mixture of the nanoparticles and the membrane components by using a homogenizer at 1500rpm for 5 minutes, then repeatedly co-extruding by using a filter membrane with the diameter of 0.45 mu m, collecting the extruded liquid, centrifuging the extruded liquid at 13000g for 20 minutes, removing the supernatant, and then re-suspending and precipitating the precipitate by using PBS to obtain the nano vaccine. Wherein the nano vaccine prepared by using the mixed membrane component of the cancer cells and the bifidobacterium longum is nano vaccine 1, and the particle size is 370nm; the nano vaccine prepared by using the mixed membrane component of the cancer cells and the lactobacillus acidophilus is nano vaccine 2, and the particle size is 370nm; the nano vaccine prepared by using the cancer cell membrane component is nano vaccine 3, and the particle size is 370nm.

(6) Nano-vaccine for preventing cancer metastasis

Selecting female C57BL/6 of 6-8 weeks as model mouse preparationMelanoma tumor-bearing mice. 200 μ g of Nanoprotein 1 or Nanoprotein 2 or

Nanoprotein

3 or 100 μ L of PBS was injected subcutaneously per mouse at-35, -28, -21, -14 and-7 days before mouse cancer molding. Each mouse was inoculated intravenously at day 0 with 1X 10 ⁵ Individual B16F10 cells, mice were sacrificed on day 16 and the number of melanoma foci in the lungs of the mice was visually recorded.

(7) Results of the experiment

As shown in fig. 5, PBS control mice had more and larger foci, while nano-vaccine treated mice had significantly reduced numbers of cancers. Moreover, the prevention effect of the nano vaccine 1 and the nano vaccine 2 on the cancer lung metastasis is obviously better than that of the nano vaccine 3; and the prevention effect of the nano vaccine 1 on the cancer lung metastasis is obviously better than that of the nano vaccine 2.

EXAMPLE 5 Nanoprotein for treatment of Colon cancer

This example begins by lysing colon cancer tumor tissue to prepare a mixture of water soluble antigen (1:1 mass ratio) and water insoluble antigen (1:1 mass ratio) and mixing the mixture of water soluble antigen and water insoluble antigen in a mass ratio of 1:1. Then, PLA is used as a nanoparticle framework material, cpGSL03 and Poly ICLC are used as immune adjuvants to prepare nanoparticles, and the surface of the nanoparticles is loaded with cancer cell extracellular vesicles and bacterial extracellular vesicles to prepare the nano vaccine for treating colon cancer.

(1) Lysis of tumor tissue and cancer cells and collection of fractions

Each C57BL/6 mouse was inoculated subcutaneously into the back of 2X 10 mice ⁶ The MC38 cells in the tumor grow to a volume of about 1000mm ³ Mice were sacrificed and tumor tissue was harvested. Tumor tissue is cut into pieces and ground, and a proper amount of pure water is added through a cell filter screen and is repeatedly frozen and thawed for 5 times, and ultrasonic waves are carried out to destroy the cracked cells. After the cells are cracked, centrifuging the lysate for 5 minutes at the rotating speed of more than 5000g and taking supernatant fluid as water-soluble antigen which can be dissolved in pure water; the precipitation fraction is dissolved by adding 8M urea aqueous solution to the precipitation fraction, thereby converting the water-insoluble antigen insoluble in pure water into soluble antigen in aqueous solution. Will be derived from colon cancer tumor tissue and lungMixing water-soluble antigens of cancer cells according to a mass ratio of 1:1; the mixture is a raw material source for preparing the nano particles.

(2) Preparation of bacterial Membrane fraction

Selecting live BCG vaccine, centrifuging for 30 minutes at 5000g, then centrifuging and washing twice by PBS, then suspending in PBS, carrying out ultrasonic treatment for 5 minutes at 4 ℃ by 20W, then filtering and extruding by 20 mu M, 10 mu M, 5 mu M, 1 mu M and 0.45 mu M filter membranes in sequence, centrifuging for 90 minutes at 13000g after removing precipitate, obtaining the collected bacterial membrane component after suspending the precipitate in PBS, and then cracking and dissolving the bacterial membrane component by 8M urea aqueous solution.

Or selecting live BCG vaccine, centrifuging for 30 minutes at 5000g, then centrifuging and washing twice by using PBS, then suspending in the PBS, carrying out ultrasonic treatment for 5 minutes at 4 ℃ by using 20W, then filtering and extruding by sequentially passing through 20 mu m, 10 mu m, 5 mu m, 1 mu m and 0.45 mu m filter membranes, centrifuging for 90 minutes at 13000g after removing precipitate, obtaining the collected bacterial membrane component after suspending the precipitate in the PBS, and then cracking and dissolving the bacterial membrane component by using Tween 80 aqueous solution.

(3) Preparation of nanoparticles

In this embodiment, the nanoparticles are prepared by a solvent evaporation method, and the tumor tissue lysate component, the bacterial membrane component lysed and dissolved in step (2), and the immunoadjuvant are loaded in the nanoparticles. The molecular weight of PLA which is a material for preparing the nano particles is 20KDa, the adopted immune adjuvants are CpG SL03 and Poly ICLC, and the adjuvants are distributed in the nano particles. Preparation method As mentioned above, in the preparation process, firstly, the lysate mixture and the adjuvant are loaded inside the nanoparticles by using the multiple emulsion method, after the lysate and the adjuvant are loaded inside, 100mg of the nanoparticles are centrifuged at 10000g for 20 minutes, and are resuspended by using 10mL of ultrapure water containing 4% trehalose, and then are freeze-dried for 48 hours. The nano particle prepared by using 8M urea-dissolved bacterial membrane component is nano particle 1, the average particle size of the nano particle is about 280nm, about 90 mug of protein or polypeptide component is loaded on each 1mg of PLGA nano particle, and each 1mg of PLGA nano particle contains 0.03mg of CpGSL03 and Poly ICLC immunologic adjuvant respectively; the nano particle prepared by using the Tween 80 dissolved bacterial membrane component is nano particle 2, the average particle diameter of the nano particle is about 280nm, about 90 mu g of protein or polypeptide component is loaded on each 1mg of PLGA nano particle, and each 1mg of PLGA nano particle contains 0.03mg of CpGSL03 and Poly ICLC immunologic adjuvant respectively.

(4) Preparation of bacterial outer vesicles (OMVs) and cancer extracellular vesicles

Culturing live BCG vaccine in LB culture medium, collecting cultured sample, centrifuging for 30min at 5000g, discarding precipitate, collecting supernatant, performing ultrasonic treatment at 15W for 10min, centrifuging the supernatant at 16000g for 90 min, and resuspending the precipitate in PBS to obtain the collected bacterial outer vesicle component.

Culturing MC38 cells in DMEM high-sugar medium containing 50ng/mL adriamycin for 6 days, changing the culture solution every 2 days, collecting the cultured MC38 cells, washing twice with 4 ℃ Phosphate Buffer Solution (PBS) containing protease inhibitor, centrifuging for 5 minutes at 400g, removing the precipitate, ultrasonically treating the supernatant at 15W for 10 minutes, centrifuging for 90 minutes at 15000g, and re-suspending the precipitate in PBS to obtain the collected cancer cell outer vesicle membrane component.

(5) Preparation of nano-vaccine

And (3) suspending 100mg of the nanoparticle 1 prepared in the step (3) in 9mL of PBS, mixing with 1mL of the cancer cell extracellular vesicle (5 mg) prepared in the step (4) and the bacterial extracellular vesicle (5 mg), performing ultrasonic action at 4 ℃ for 2 minutes, repeatedly co-extruding through a 0.45-micrometer filter membrane, centrifuging the extruded liquid at 13000g for 30 minutes, discarding the supernatant, and then re-suspending and precipitating the precipitate by using PBS to obtain the nano vaccine 1 with the particle size of 300nm.

And (3) suspending 100mg of the nanoparticles 2 prepared in the step (3) in 9mL of PBS, mixing with 1mL of the cancer cell extracellular vesicles (5 mg) prepared in the step (4) and the bacterial extracellular vesicles (5 mg), performing ultrasonic action at 4 ℃ for 2 minutes, repeatedly co-extruding through a 0.45-micrometer filter membrane, centrifuging the extruded liquid at 13000g for 30 minutes, discarding the supernatant, and then re-suspending and precipitating the precipitate by using PBS to obtain the nano vaccine 2 with the particle size of 300nm.

And (3) suspending 100mg of the nanoparticles 1 prepared in the step (3) in 9mL of LPBS, mixing with 1mL of the bacterial outer vesicles (10 mg) prepared in the step (4), performing ultrasonic action at 4 ℃ for 10 minutes, centrifuging at 13000g for 20 minutes, discarding supernatant, and then suspending the precipitate with PBS to obtain the nano vaccine 3 with the particle size of 300nm.

(6) Nano-vaccine for cancer treatment

Selecting female C57BL/6 of 6-8 weeks as a model mouse to prepare a colon cancer tumor-bearing mouse. Each mouse was inoculated subcutaneously 2X 10 on day 0 ⁶ Each mouse was injected subcutaneously with 800 μ g nano-

vaccine

1 or 800 μ g nano-

vaccine

2 or 800 μ g nano-

vaccine

3 or 100 μ LPBS on

days

4, 7, 10, 15 and 20, respectively. Tumor growth and mouse survival monitoring were as above.

(7) Results of the experiment

As shown in FIG. 6, the tumors of the control mice grew rapidly, while the tumors of the mice treated with the three nano-vaccines all grew significantly slower or disappeared. Moreover, nano-vaccine 1 is better than nano-vaccine 2 and nano-vaccine 3. Therefore, the addition of the cancer cell extracellular vesicle membrane component is beneficial to the efficacy of the vaccine, and the bacterial membrane components obtained by cracking different lysates are loaded in the nano vaccine prepared in the nano vaccine, so that the effect is different.

EXAMPLE 6 Nanoprotein for treatment of melanoma

This example uses melanoma as a cancer model to illustrate how to treat melanoma using a nano-vaccine loaded with the whole cell fraction of melanoma tumor tissue internally, and lung cancer cells and antigen presenting cell membrane fraction on the surface. In this example, the water soluble and water insoluble components of B16F10 melanoma tumor tissue were first lysed. Then PLGA is used as a nanoparticle framework material, manganese particles and CpG2395 are used as immune adjuvants to prepare nanoparticles, and then membrane components are loaded on the surfaces of the nanoparticles for treating cancers.

(1) Lysis of tumor tissue and Collection of fractions

C57BL/6 mice were each inoculated subcutaneously on the back with 1.5X 10 ⁵ B16F10 cells grown to a volume of about 1000mm in tumors ³ Mice were sacrificed and tumor tissue was harvested. Cutting tumor tissue into small pieces, grinding, and filtering with cell screenAdding ultrapure water into the tumor tissue single cell suspension, repeatedly freezing and thawing the tumor tissue single cell suspension for 5 times, and performing ultrasonic lysis. Nuclease was added to the lysate and allowed to act at 37 ℃ for 10 minutes, and then the lysate containing the nuclease was heated at 95 ℃ for 5 minutes. Then centrifuging at 5000g for 5min to obtain supernatant as water-soluble component, and dissolving the precipitate with 10% octyl glucoside to obtain dissolved original water-insoluble component. Mixing the water-soluble components from melanoma tumor tissues with the original water-insoluble components dissolved in the octyl glucoside aqueous solution according to the mass ratio of 3:1 to obtain the antigen source for preparing the nano particles.

(2) Preparation of nanoparticles

In this example, the nanoparticles were prepared by a multiple emulsion method. The molecular weight of the adopted nano particle preparation material PLGA is 24-38 KDa, and the adopted immunologic adjuvant is a mixed adjuvant of a STING stimulant and a Toll-like receptor stimulant: manganese colloidal particles (STING agonists) and CpG2395 (Toll-like receptor agonists). Firstly, preparing a manganese adjuvant, then mixing the manganese adjuvant with a water-soluble component or a water-insoluble component in a cancer cell whole-cell component, and taking the mixture as a first water phase to prepare the nanoparticle internally loaded with the lysate component and the adjuvant by adopting a multiple emulsion method. In the preparation of the manganese adjuvant, firstly, 1mL of 0.3M Na ₃ PO ₄ The solution was added to 9mL of physiological saline, followed by 2mL of 0.3M MnCl ₂ Standing the solution overnight to obtain Mn ₂ OHPO ₄ The particle size of the colloidal manganese adjuvant is about 13nm. Then mixing the manganese adjuvant with a water-soluble component (60 mg/mL) or a non-water-soluble component (60 mg/mL) of the cancer cell whole-cell component according to a volume ratio of 1:3, and loading the antigen and the manganese adjuvant into the nanoparticle by adopting a multiple emulsion method. After loading the lysis component and adjuvant inside, 100mg of nanoparticles were centrifuged at 10000g for 20 minutes, resuspended in 10mL of ultrapure water containing 4% trehalose, and lyophilized for 48h for use. The average particle size of the nanoparticles is about 370nm, and the surface potential of the nanoparticles is about-5 mV; each 1mg PLGA nano particle is loaded with about 120 mug protein and polypeptide components, and each 1mg PLGA nano particle uses 0.04mg CpG2395 adjuvant.

(3) Preparation and activation of antigen presenting cells

The antigen presenting cells are mixed antigen presenting cells derived from peripheral blood B cells and BMDCs. BMDC was prepared as above. Peripheral blood was collected from mice after C57BL/6 sacrifice, peripheral Blood Mononuclear Cells (PBMC) were isolated from peripheral blood, and CD19 was then sorted from PBMC using flow cytometry ⁺ B cells. Mixing BMDC and B cells according to the quantity ratio of 1:1 to obtain the mixed antigen presenting cells.

1mg of the cancer cell whole cell fraction-loaded nanoparticles prepared in step (2) were mixed with 2000 ten thousand of mixed antigen-presenting cells (1000 ten thousand of BMDC + 1000 ten thousand of B cells) in 20mL of RPMI1640 complete medium and incubated for 48 hours (37 ℃,5% CO) ₂ ) The incubation system contained IL-21 (500U/mL), IL-2 (500U/mL), IL-7 (500U/mL) and CD80 antibody (10 ng/mL). After incubation, the incubated cells were centrifuged at 400g for 5 minutes, and the supernatant was discarded, followed by washing twice with PBS to obtain activated antigen-presenting cells.

(4) Preparation of cancer cell and antigen presenting cell membrane fraction

Collecting B16F10 melanoma cells cultured by cells, centrifuging for 5 minutes at 400g, discarding supernatant, resuspending precipitated cells by PBS, mixing the melanoma cells with activated mixed antigen presenting cells according to the quantity ratio of 2:1, washing twice by PBS containing phosphatase inhibitor and protease inhibitor, mechanically destroying the cells by ultrasonic treatment for 2 minutes at 15W, centrifuging the sample for 5 minutes at 3000g, discarding the precipitate, centrifuging the supernatant for five minutes at 8000g, discarding the precipitate, centrifuging the supernatant for 60 minutes at 15000g, discarding the supernatant, collecting the precipitate, and resuspending the precipitate in PBS to obtain mixed cell membrane components.

As a control membrane fraction, only activated mixed antigen-presenting cells were used for preparation. The mixed antigen-presenting cells were washed twice with PBS containing phosphatase and protease inhibitors, and then mechanically disrupted by sonication at 15W for 2 minutes. Centrifuging the sample at 3000g for 5 minutes, discarding the precipitate, centrifuging the supernatant at 8000g for five minutes, discarding the precipitate, centrifuging the supernatant at 15000g for 60 minutes, discarding the supernatant, collecting the precipitate, and resuspending the precipitate in PBS to obtain the mixed antigen presenting cell membrane component.

(5) Preparation of nano vaccine

And (3) resuspending the nanoparticles (100 mg) prepared in the step (2) in 9mL of PBS, mixing with 1mL of mixed cell membrane component (2 mg) of the cancer cells and the antigen presenting cells prepared in the step (4), adding DSPE-PEG-CD32 monoclonal antibody (0.1 mg) into the mixture, carrying out ultrasonic treatment at 50W for 2 minutes, repeatedly co-extruding by using a filter membrane with the diameter of 0.45 mu m, centrifuging the extruded liquid at 12000g for 20 minutes, removing supernatant, collecting precipitate, and resuspending the precipitate in PBS to obtain the nano vaccine 1, wherein the particle size of the nano vaccine is 390 nanometers, and the surface potential is-4 mV.

And (3) resuspending the nanoparticles (100 mg) prepared in the step (2) in 9mL of PBS, mixing with 1mL of the mixed antigen presenting cell membrane component (2 mg) prepared in the step (4), adding DSPE-PEG-CD32 monoclonal antibody (0.1 mg) into the mixture, carrying out ultrasonic treatment at 50W for 2 minutes, repeatedly co-extruding by using a filter membrane with the diameter of 0.45 mu m, centrifuging the extruded liquid at 12000g for 20 minutes, removing supernatant, collecting precipitate, and carrying out resuspension on the precipitate in PBS to obtain the nano vaccine 2, wherein the particle size of the nano vaccine is 390 nanometers, and the surface potential is-4 mV.

(6) Nano-vaccine for cancer treatment

Female C57BL/6 of 6-8 weeks is selected as a model mouse to prepare a melanoma-bearing mouse. Each mouse was inoculated subcutaneously 1.5X 10 on day 0 ⁵ B16F10 cells were injected subcutaneously with 500 μ g nano-

vaccine

1 or 500 μ g nano-

vaccine

2 or 100 μ LPBS on

days

5, 8, 11, 16 and 21, respectively. Mouse tumor volume and survival were monitored as above.

(7) Results of the experiment

As shown in FIG. 7, the tumors of the PBS control mice grow up rapidly, and the tumor velocity of the mice treated with the nano-vaccine is significantly slower than that of the control mice, and the survival time is prolonged. In conclusion, the nano vaccine provided by the invention has a treatment effect on cancer. Moreover, the effect of the nano vaccine 1 loaded with cancer cells and mixed antigen presenting cell membrane components on the surface is better than that of the nano vaccine 2 loaded with only mixed antigen presenting cell membrane components on the surface. In the nano vaccine of the present embodiment, the CD32 monoclonal antibody is used as the target head for active targeting, and in practical application, any target head having the ability to target cells, such as mannose, mannan, CD205 monoclonal antibody, CD19 monoclonal antibody, etc., may be used.

Example 7 micron vaccine for prevention of breast cancer

(1) Lysis of cancer cells

The cultured 4T1 cells were centrifuged at 400g for 5 minutes, then washed twice with PBS and resuspended in ultrapure water. The obtained cancer cells are respectively inactivated and denatured by ultraviolet rays and high-temperature heating, and then a proper amount of 6M guanidine hydrochloride aqueous solution is adopted to crack the breast cancer cells and dissolve a lysate, namely the antigen raw material source.

(2) Preparation of microparticle systems

In the embodiment, the preparation of the microparticles adopts a multiple emulsion method, the molecular weight of PLGA serving as a microparticle framework material is 38KDa-54KDa, and the adopted immune adjuvants are CpG2395, cpG1018 and Poly (I: C). The preparation method comprises the steps of firstly preparing the micron particles internally loaded with the lysate component and the adjuvant by a multiple emulsion method, then centrifuging 100mg of the micron particles for 20 minutes at 9000g, using 10mL of ultrapure water containing 4% trehalose for resuspension, and drying for 48 hours for later use. The average particle diameter of the micron particle system is about 2.10 mu m, and the surface potential is about-6 mV; each 1mg PLGA microparticle was loaded with about 110. Mu.g of protein or polypeptide fraction, containing 0.03mg each of CpG2395, cpG1018 and Poly (I: C).

(3) Preparation and activation of antigen presenting cells

The antigen presenting cells are mixed antigen presenting cells derived from peripheral blood B cells and BMDCs. BMDC was prepared as described above. Peripheral blood was collected from mice after C57BL/6 sacrifice, peripheral Blood Mononuclear Cells (PBMC) were isolated from peripheral blood, and CD19 was then sorted from PBMC using flow cytometry ⁺ B cells. Mixing BMDC and B cells according to the quantity ratio of 1:1 to obtain the mixed antigen presenting cells.

1mg of the cancer cell whole cell fraction-loaded microparticles prepared in step (2) were mixed with 2000 ten thousand of mixed antigen-presenting cells (of which 1000 ten thousand of BMDC + 1000 ten thousand of B cells) in 20mL of RPMI1640 complete medium, and incubated for 48 hours (37 ℃,5% CO) ₂ ) The incubation system contained IL-2 (500U/mL) and IL-7 (5)00U/mL), IL-15 (500U/mL), GM-CSF (500U/mL). After incubation, the incubated cells were centrifuged at 400g for 5 minutes, and the supernatant was discarded, followed by washing twice with PBS to obtain activated antigen-presenting cells.

Or mixing 1mg of the cancer cell whole cell fraction-loaded microparticles prepared in step (2) with 2000 ten thousand BMDCs in 20mL RPMI1640 complete medium and incubating for 48 hours (37 ℃,5% CO) ₂ ) The incubation system contained IL-2 (500U/mL), IL-7 (500U/mL), IL-15 (500U/mL), and GM-CSF (500U/mL). After incubation, the incubated cells were centrifuged at 400g for 5 minutes, and the supernatant was discarded, followed by washing twice with PBS to obtain activated antigen-presenting cells.

(4) Preparation of cancer cell and antigen presenting cell membrane fraction

Collecting cell culture 4T1 cells, centrifuging for 5 minutes at 400g, discarding supernatant, resuspending precipitated cells by using PBS, mixing breast cancer cells and the activated mixed antigen presenting cells in the step (3) according to the quantity ratio of 1:1, washing twice by using PBS containing protease inhibitors, mechanically destroying cells by ultrasonic treatment at 10W for 5 minutes, centrifuging the sample for 5 minutes at 2000g, centrifuging for 10 minutes at 6000g after discarding precipitates, centrifuging the supernatant for 10 minutes at 15000g after discarding precipitates, discarding supernatant, collecting precipitates, and resuspending the precipitates in PBS to obtain the mixed cell membrane component.

As a control membrane fraction, cancer cells and BMDC cells activated in step (3) were used for preparation. 4T1 cells and BMDCs were mixed in a quantitative ratio of 1:1, and then the mixed cells were washed twice with PBS containing a phosphatase inhibitor and a protease inhibitor, and then mechanically disrupted by 10W sonication for 5 minutes. Centrifuging the sample at 2000g for 5 minutes, discarding the precipitate, centrifuging the supernatant at 6000g for 10 minutes, then discarding the precipitate, centrifuging the supernatant at 15000g for 60 minutes, discarding the supernatant, collecting the precipitate, and resuspending the precipitate in PBS to obtain the mixed cell membrane component of the cancer cells and the DCs.

(5) Preparation of a micro-vaccine

And (3) resuspending the microparticles (100 mg) prepared in the step (2) in 9mL of PBS, mixing with 1mL of the cancer cells prepared in the step (4) and the mixed antigen presenting cell membrane component (2 mg), carrying out ultrasonic treatment at 20W for 5 minutes, centrifuging at 10000g for 15 minutes, removing supernatant, collecting precipitate, and resuspending the precipitate in PBS to obtain the micrometer vaccine 1, wherein the particle size of the micrometer vaccine 1 is 2.12 mu m, and the surface potential is-4 mV.

And (3) resuspending the microparticles (100 mg) prepared in the step (2) in 9mL of PBS, mixing with 1mL of mixed cell membrane components (2 mg) of the cancer cells and the DCs, carrying out ultrasonic treatment for 5 minutes at 20W, centrifuging for 15 minutes at 10000g, removing supernate, collecting precipitates, and resuspending the precipitates in the PBS to obtain the micrometer vaccine 2, wherein the particle size of the micrometer vaccine 2 is 2.12 micrometers, and the surface potential is-4 mV.

(6) Micron vaccine for preventing cancer

Female BALB/c of 6-8 weeks is selected as a model mouse to prepare a breast cancer tumor-bearing mouse. The mice were vaccinated with either 800 μ g of the

micro vaccine

1 or 800 μ g of the

micro vaccine

2 or 100 μ LPBS per mouse on days-35, 28, 21, 14 and 7, respectively, before they were vaccinated with cancer cells. Each mouse was inoculated subcutaneously with 1X 10 injection on day 0 ⁶ 4T1 cells, mouse tumors and survival monitoring were as described above.

(7) Results of the experiment

As shown in fig. 8, the tumor growth rate and survival time of the mice treated with the micron vaccine were significantly slower than those of the PBS control group. Therefore, the micro vaccine provided by the invention has a prevention effect on breast cancer. Moreover, the effect of the micron vaccine 1 is better than that of the micron vaccine 2, which shows that the effect of the cell membrane component of the cancer cell and the mixed antigen presenting cell loaded on the surface of the micron vaccine is better than that of the cell membrane component of the cancer cell and the single antigen presenting cell loaded on the surface of the micron vaccine. Theoretically DC, the process of T cell activation after uptake of a vaccine by antigen presenting cells, is the most important antigen presenting cell: (

The primary activation of T cells), the DC cell membrane can better guide the phagocytosis of the micro vaccine by the DC, and the experiment proves that the cell membrane component of the B cells can improve the guide of the phagocytosis of the micro vaccine by the DC and the subsequent primary activation of the T cells.

Example 8 Nanoprovacines of calcified nanoparticles surface-Supported Membrane fraction for prevention of cancer

This example illustrates the use of calcified nanoparticle surface loaded membrane components to prepare a nano vaccine for cancer prevention, and other biomineralization techniques, cross-linking, gelation, etc. can be used to modify the particles for practical use. In this example, a mouse melanoma tumor tissue was lysed with a 10% sodium deoxycholate aqueous solution (containing 8M arginine) and then dissolved, the tumor tissue lysate was loaded onto nanoparticles, and a membrane fraction was loaded onto the surface of the nanoparticles to prepare a nano vaccine for cancer prevention.

(1) Lysis of tumor tissue and cancer cells

Mouse B16F10 melanoma tumor tissue was collected, cut into small pieces, and lysed with 10% sodium deoxycholate aqueous solution (containing 8M arginine) to dissolve the tumor tissue whole cell fraction.

(2) Preparation of nanoparticles

This example of a biocalcification nanoparticle after loading the cancer cell whole cell component inside and on the surface of the nanoparticle. In the embodiment, the nano particle 1 is prepared by a solvent volatilization method, the molecular weight of PLGA (polylactic-co-glycolic acid) serving as a nano particle preparation material is 7-17 KDa, and immune adjuvants CpG2006 and Poly (I: C) are loaded in the nano particle. The preparation method is as follows, firstly loading lysate components in the nanoparticles by a multiple emulsion method, then suspending 100mg PLGA nanoparticles by 18mL PBS after 13000g is centrifuged for 20min, then adding 2mL tumor tissue lysate (60 mg/mL) dissolved in 8M urea, acting for 10min at room temperature, centrifuging 12000g for 20min, and collecting the precipitate. The 100mg PLGA nanoparticles were then resuspended in 20mL DMEM medium, followed by 200. Mu.L of CaCl ₂ (1 mM) and reacted at 37 ℃ for two hours. The precipitate was then collected after centrifugation at 10000g for 20 minutes and resuspended with ultrapure water and washed twice by centrifugation. Nanoparticle 1, with an average particle size of about 240nm, loaded with about 140 μ g of protein or polypeptide component per 1mg of PLGA nanoparticle, cpG2006 and Poly (I: C) each 0.03mg.

The blank nano particle 2 is prepared from the same materials and by the same method as the nano particle 1, but only carries the immunologic adjuvant but not the cancer cell lysate component, the average particle size is about 240nm, about 140 mu g of protein or polypeptide component is carried by each 1mg of PLGA nano particle, and each 0.03mg of CpG2006 and Poly (I: C).

(3) Preparation of antigen-presenting cell membrane fraction

This example uses the DC2.4 cell line and BMDM as antigen presenting cells. The BMDM was prepared as follows: anaesthetizing C57 mice, dislocating, killing, sterilizing the mice with 75% ethanol, cutting a small opening on the back of the mice with scissors, directly tearing the skin to the position of the lower leg joint of the mice with hands, and removing the foot joints and the skin of the mice. The hind limb was removed with scissors along the greater trochanter at the base of the mouse thigh, the muscle tissue was removed, the rat was placed in a 75% ethanol-containing petri dish and soaked for 5min, and the new 75% ethanol-containing petri dish was transferred to a clean bench. The leg bone soaked in ethanol is transplanted into cold PBS for soaking, and the ethanol on the surfaces of the tibia and the femur is washed off, and the process can be repeated for 3 times. The cleaned femur and tibia are separated, the two ends of the femur and tibia are cut off by scissors, the cold induction medium is sucked by a 1mL syringe to blow out bone marrow from the femur and tibia, and purging is carried out for 3 times until the leg bone is not obviously red. The culture medium containing bone marrow cells was repeatedly blown up with a 5mL pipette to disperse the cell clumps, then the cells were sieved using a 70 μm cell filter, transferred into a 15mL centrifuge tube, centrifuged at 1500rpm/min for 5min, the supernatant was discarded, the erythrocyte lysate was added, resuspended and left to stand for 5min at 1500rpm/min, centrifuged for 5min after which the supernatant was discarded, resuspended with a cold prepared bone marrow macrophage induction medium (DMEM high-glucose medium containing 15% L929 medium), and plated. Cells are cultured overnight to remove other contaminating cells that adhere faster such as fibroblasts and the like. Collecting non-adherent cells, and arranging the cells into a dish or a cell culture plate according to the experimental design. Macrophage colony-stimulating factor (M-CSF) was used at a concentration of 40ng/mL to stimulate differentiation of bone marrow cells to mononuclear macrophages. Culturing for 8 days, and observing the macrophage morphological change under a light microscope. After 8 days the cells were harvested by digestion, incubated with anti-mouse F4/80 antibody and anti-mouse CD11b antibody at 4 ℃ for 30min in the dark, and the proportion of successfully induced macrophages was identified using flow cytometry.

Co-incubation of nanoparticle 1 (1000. Mu.g) prepared in step (2) with DC2.4 (500 million) and BMDM cells (500 million) in 15mL high-glucose DMEM complete medium for 48 hours (37 ℃,5% ₂ ) (ii) a The incubation system contained GM-CSF (2000U/mL), IL-2 (500U/mL), IL-7 (200U/mL), IL-12 (200U/mL), and CD40 antibody (20 ng/mL). The incubated cells were collected, centrifuged at 400g for 5 minutes, resuspended using PBS and washed twice. Thus obtaining the mixed antigen presenting cells.

Mechanically destroying cells of the mixed antigen presenting cells by ultrasonic treatment at 20W for 2 minutes, centrifuging a sample at 2000g for 5 minutes, centrifuging a supernatant at 6000g for 10 minutes after removing precipitates, then centrifuging the supernatant at 15000g for 60 minutes after removing the precipitates, removing the supernatant, collecting the precipitates, and re-suspending the precipitates in PBS to obtain the cell membrane component of the mixed antigen presenting cells.

Culturing lactobacillus rhamnosus in an LB culture medium containing 30 mu M adriamycin, collecting a cultured sample, centrifuging for 30 minutes at 5000g, discarding the precipitate, collecting a supernatant, carrying out ultrasonic treatment for 10 minutes at 15W, centrifuging for 90 minutes at 16000g, and carrying out heavy suspension on the precipitate in PBS to obtain the collected bacterial outer vesicle component 1.

The B16F10 cells were cultured in high-glucose DMEM complete medium containing 30. Mu.M of doxorubicin for 6 days, with the medium being changed every 2 days. Collecting cell culture B16F10 cells, centrifuging at 400g for 5 minutes, then removing supernatant, resuspending precipitated cells by using PBS, mechanically destroying cells by carrying out ultrasonic treatment on cancer cells at 20W for 2 minutes, centrifuging a sample at 2000g for 5 minutes, removing precipitate, then centrifuging at 6000g for 10 minutes, then removing precipitate, centrifuging at 15000g for 60 minutes, then removing supernatant, collecting precipitate, and resuspending the precipitate in PBS to obtain the cancer cell membrane component 1.

Or culturing lactobacillus rhamnosus in an LB culture medium, collecting a cultured sample, centrifuging for 30 minutes at 5000g, discarding the precipitate, collecting a supernatant, performing ultrasonic treatment for 10 minutes at 15W, centrifuging for 90 minutes at 16000g, and resuspending the precipitate in PBS to obtain the collected bacterial outer vesicle component 2.

Alternatively, B16F10 cells were cultured in high-glucose DMEM complete medium for 6 days with a change every 2 days. Collecting cell culture B16F10 cells, centrifuging for 5 minutes at 400g, then discarding supernatant, resuspending precipitated cells by using PBS, mechanically destroying cells by carrying out ultrasonic treatment on cancer cells for 2 minutes at 20W, centrifuging for 5 minutes at 2000g, discarding precipitates, centrifuging for 10 minutes at 6000g, then discarding precipitates, centrifuging for 60 minutes at 15000g, then discarding supernatant, collecting precipitates, and resuspending the precipitates in PBS to obtain cancer cell membrane component 2.

(5) Preparation of nano-vaccine

And (3) resuspending the nanoparticles 1 (100 mg) prepared in the step (2) in 9mL of PBS, mixing with 1mL of the mixed antigen presenting cell membrane component (5 mg) prepared in the step (3), the cancer cell membrane component 1 (5 mg) prepared in the step (4) and the bacterial outer vesicle component 1 (5 mg), carrying out ultrasonic treatment at 20W for 5 minutes, repeatedly co-extruding by using a filter membrane with the diameter of 0.45 mu m, centrifuging at 13000g for 30 minutes, removing supernatant, collecting precipitate, and resuspending the precipitate in PBS to obtain the nano vaccine 1, wherein the particle size of the nano vaccine is 260nm.

Resuspending the nanoparticles 2 (100 mg) prepared in the step (2) in 9mL of PBS, mixing with 1mL of the mixed antigen presenting cell membrane component (5 mg) prepared in the step (3), the cancer cell membrane component 1 (5 mg) prepared in the step (4) and the bacterial outer vesicle component 1 (5 mg), carrying out ultrasonic treatment at 20W for 5 minutes, repeatedly co-extruding through a 0.45-micrometer filter membrane, centrifuging at 13000g for 30 minutes, removing a supernatant, collecting a precipitate, and resuspending the precipitate in PBS to obtain the nano vaccine 2, wherein the particle size of the nano vaccine is 260nm.

And (3) resuspending the nanoparticles 1 (100 mg) prepared in the step (2) in 9mL of PBS, mixing with 1mL of the mixed antigen presenting cell membrane component (5 mg) prepared in the step (3), the cancer cell membrane component 2 (5 mg) prepared in the step (4) and the bacteria outer vesicle component 2 (5 mg), carrying out ultrasonic treatment at 20W for 5 minutes, repeatedly co-extruding by using a filter membrane with the diameter of 0.45 mu m, centrifuging at 13000g for 30 minutes, removing supernatant, collecting precipitate, and resuspending the precipitate in PBS to obtain the nano vaccine 3, wherein the particle size of the nano vaccine is 260nm.

(6) Nano-vaccine cancer prevention

Selecting female C57BL/6 of 6-8 weeks as a model mouse to prepare a melanoma-bearing mouse, and respectively inoculating 800 mu g of

nano vaccine

1, or 800 mu g of

nano vaccine

2, or 800 mu g of

nano vaccine

3, or 100 mu L of PBS to each mouse at the-35 th day, the-28 th day, the-21 th day, the-14 th day and the-7 th day before the mice are inoculated with cancer cells. On day 0, each recipient mouse was subcutaneously inoculated at 1.5X 10 in the lower right back ⁵ And B16F10 cells. Mouse tumor volume and survival monitoring methods are as above.

(7) Results of the experiment

As shown in fig. 9, the tumors grew quickly and the mice died quickly in the PBS group of mice. The 3 kinds of nano vaccines can obviously slow down the growth speed of the tumor of the mouse and prolong the life cycle of the mouse. The effect of the nano vaccine 1 is better than that of the nano vaccine 2 and the nano vaccine 3, which shows that the cancer cell whole cell component is loaded in the nano vaccine to be beneficial to improving the vaccine effect, and the cancer cell pretreated by the adriamycin and the cell membrane component prepared by the bacteria can also improve the curative effect of the nano vaccine.

Example 9 Nanoprotein for treatment of melanoma

(1) Lysis of tumor tissue and cancer cells and collection of fractions

Tumor tissue was collected by first subcutaneously inoculating 1.5X 10 dorsal cells of each C57BL/6 mouse ⁵ B16F10 cells, which grow to a volume of about 1000mm in the tumor ³ Killing mice and picking tumor tissues, cutting the tumor tissues into blocks, grinding, passing through a cell filter screen to prepare single cell suspension, adding ultrapure water, repeatedly freezing and thawing and carrying out ultrasonic lysis on the cells, adding nuclease for 5 minutes, and then inactivating the nuclease at 95 ℃ for 10 minutes. Then centrifuging at 8000g for 3min to obtain supernatant as water-soluble antigen; the precipitated fraction was dissolved with a 10% aqueous solution of sodium deoxycholate to dissolve the water-insoluble antigen. The water-soluble antigen and the water-insoluble antigen dissolved by the sodium deoxycholate are mixed and dissolved according to the mass ratio of 1:1 to obtain the antigen raw material source for preparing the nanoparticle system.

(2) Preparation of nanoparticles

In this example, the nanoparticles were prepared by a multiple emulsion method. The nano-particle 1 is prepared from PLGA with the molecular weight of 24KDa-38KDa, the adopted immunologic adjuvant is poly (I: C), cpG1018 and CpG2216, the substance for increasing the immune escape of lysosome is KALA polypeptide (WEAKLAKALAKALAKHLAKALAKALKACEA), and the adjuvant and the KALA polypeptide are loaded in the nano-particle. The preparation method is as described above, in the preparation process, firstly, the lysate component, the adjuvant and the KALA polypeptide are loaded inside the nanoparticles by a multiple emulsion method, then 100mg of the nanoparticles are centrifuged at 12000g for 25 minutes, and after being resuspended by 10mL of ultrapure water containing 4% trehalose, the nanoparticles are freeze-dried for 48 hours. The average particle diameter of the nano particles is about 250nm, and the surface potential is about-5 mV; each 1mg PLGA nano particle is loaded with about 100 mug protein or polypeptide component, each 1mg PLGA nano particle is loaded with 0.02mg of poly (I: C), cpG1018 and CpG2216 immunologic adjuvant, and each 1mg PLGA nano particle is loaded with 0.05mg of KALA polypeptide.

The preparation material and the preparation method of the nano particle 2 are the same as those of the nano particle, the particle size is about 250nm, the surface potential is about minus 5mV, KALA polypeptide is not loaded, and the same amount of adjuvant and cell lysis component are loaded.

The preparation material and the preparation method of the nano-particles 3 are the same, the particle size is about 250nm, and the surface potential is about-5 mV; each 1mg PLGA nanoparticle is loaded with about 100 mug protein and polypeptide components, each 1mg PLGA nanoparticle is loaded with 0.02mg poly (I: C), 0.04mg CpG1018 and 0.05mg KALA polypeptide.

(3) Preparation of cancer cell membrane fraction and antigen-presenting cell membrane fraction

Collecting cell culture B16F10 cells, centrifuging for 5 minutes at 400g, discarding supernatant, resuspending the precipitated cells by PBS, mechanically destroying the cells by ultrasonic treatment at 20W for 2 minutes, centrifuging the sample for 5 minutes at 2000g, discarding the precipitate, centrifuging the supernatant for 10 minutes at 6000g, discarding the precipitate, centrifuging the supernatant for 60 minutes at 15000g, discarding the supernatant, collecting the precipitate, and resuspending the precipitate in PBS to obtain the cancer cell membrane fraction.

The antigen presenting cells are BMDCs and the preparation method is the same as above. 2mg of nanoparticles 1 prepared in step (2) were mixed with 3000 ten thousand BMDCs in 20mL RPMI1640 complete mediumPost-incubation for 48 hours (37 ℃,5% CO) ₂ ) The incubation system contained IL-2 (500U/mL), IL-7 (500U/mL), IL-15 (500U/mL), and GM-CSF (500U/mL). After finishing incubation, centrifuging 400g of the incubated cells for 5 minutes, discarding supernatant, then washing twice by PBS containing phosphatase inhibitor and protease inhibitor, then mechanically destroying cells by ultrasonic treatment at 15W for 2 minutes, centrifuging the sample at 3000g for 5 minutes, discarding the precipitate, centrifuging the supernatant at 8000g for five minutes, then discarding the precipitate, repeatedly co-extruding the supernatant by using a 0.22 mu m filter membrane, centrifuging the extruded liquid at 15000g for 60 minutes, discarding supernatant, collecting the precipitate, and re-suspending the precipitate in PBS to obtain the antigen presenting cell membrane component.

Mixing the cancer cell membrane component and the antigen presenting cell membrane component according to the mass ratio of 3:1, then repeatedly co-extruding by using a filter membrane with the thickness of 0.22 mu m, centrifuging the extruded liquid at 15000g for 60 minutes, removing supernatant, collecting precipitate, and re-suspending the precipitate in PBS to obtain the mixed cell membrane component of the cancer cell and the antigen presenting cell.

(4) Preparation of nano-vaccine

And (3) resuspending the nanoparticles (100 mg) prepared in the step (2) in 9mL PBS, mixing with 1mL of the cancer cell membrane component (2 mg) prepared in the step (3), performing low-power (20W) ultrasonic treatment at 4 ℃ for 3 minutes, centrifuging at 12000g for 60 minutes, collecting supernatant, discarding the precipitate, and resuspending the precipitate in PBS to obtain the nano vaccine. The vaccine prepared from the nano particles 1 is nano vaccine 1, the particle size is 260 nanometers, and the surface charge is-5 mV; the vaccine prepared from the nano-particles 2 is nano-vaccine 2, the particle size is 260 nanometers, and the surface charge is-5 mV; the vaccine prepared from the nano-particles 3 is the nano-vaccine 3, the particle size is 260 nanometers, and the surface charge is-5 mV.

And (3) carrying out low-power (20W) ultrasonic treatment on the cancer cell membrane component (2 mg) prepared in the step (3) at 4 ℃ for 3 minutes, then centrifuging at 12000g for 60 minutes, collecting and removing supernatant, collecting and precipitating, and carrying out heavy suspension on the precipitate in PBS to obtain the nano vaccine 4, wherein the particle size is 160 nanometers, and the surface charge is-5 mV.

(5) Nano-vaccine for treating cancer

Selecting female C57BL/6 of 6-8 weeks as model mouse to prepare blackA melanoma-bearing mouse. Each mouse was inoculated subcutaneously on day 0, at the lower right back, at 1.5X 10 ⁵ And B16F10 cells. 60 μ g of one of the nano-vaccines 1-4 or 100 μ LPBS subcutaneously on

days

6, 10, 15 and 20 after melanoma vaccination, respectively. In the experiment, the mouse tumor volume and survival monitoring method is the same as above.

(6) Results of the experiment

As shown in fig. 10, tumors grew rapidly in the PBS control group. Compared with a control group, the growth speed of the tumor of the mouse treated by the nano vaccine is obviously slowed down, and the survival period is obviously prolonged. Moreover, the nano-vaccine 1, the nano-vaccine 2 and the nano-vaccine 3 which load cancer cell membrane components and antigen presenting cell membrane components on the surfaces of the nano-particles which load cancer cell whole cell components inside are obviously better than the nano-vaccine 4 which is prepared by cancer cell membrane and antigen presenting cell membrane components. Moreover, the nano vaccine 1 prepared by adding the nano particles added with the lysosome escape substance is better than the nano vaccine 2 prepared by adding the nano particles without lysosome escape. Moreover, the nano-vaccine 1 prepared by using the nanoparticles with two kinds of CpG and Poly (I: C) as mixed adjuvants has better treatment effect than the nano-vaccine 3 prepared by using only one kind of nanoparticles with CpG and Poly (I: C) mixed adjuvants. This demonstrates that internal loading of cancer cell whole cell components is critical for the preparation of cancer cell membrane components into nano-vaccines. In conclusion, the nano vaccine provided by the invention has a good treatment effect on cancer. In the embodiment, KALA polypeptide is used as lysosome escape substance loaded in the nano-particle or micro-particle, and any lysosome escape increasing substance, such as polypeptide, amino acid, organic high molecular substance, inorganic substance with proton sponge effect, etc., can be used in practical use.

Example 10 Nanoprovacines of nanoparticles surface-Supported Membrane fraction for prevention of cancer

(1) Lysis of cancer cells

Mouse Pan02 pancreatic cancer cells were collected, lysed with 10% aqueous sodium dodecyl sulfate and the whole cell fraction of the cancer cells was lysed.

(2) Preparation of bacterial Membrane fraction

Centrifuging the cultured bifidobacterium breve for 30 minutes at 5000g, then centrifuging and washing twice by using PBS, then suspending in the PBS, carrying out ultrasonic treatment for 5 minutes at 4 ℃ by using 20W, then filtering and extruding by sequentially passing through 20 mu M, 10 mu M, 5 mu M, 1 mu M and 0.45 mu M filter membranes, centrifuging the supernatant for 90 minutes at 13000g after removing precipitates, obtaining the collected bacterial membrane components after suspending the precipitates in the PBS, and then cracking and dissolving the bacterial membrane components by using 8M urea aqueous solution.

Centrifuging the cultured bifidobacterium breve for 30 minutes at 5000g, then centrifuging and washing twice by using PBS, then suspending in the PBS, carrying out ultrasonic treatment for 5 minutes at 4 ℃ by using 20W, then filtering and extruding by sequentially passing through 20 mu m, 10 mu m, 5 mu m, 1 mu m and 0.45 mu m filter membranes, centrifuging the supernatant for 90 minutes at 13000g after removing precipitates, obtaining the collected bacterial membrane components after suspending the precipitates in the PBS, and then using 1% Triton110 aqueous solution to crack and dissolve the bacterial membrane components.

(3) Preparation of nanoparticles

In the embodiment, the nano particles are prepared by a solvent volatilization method, the molecular weight of PLGA serving as a nano particle preparation material is 7KDa-17KDa, and immune adjuvants CpG2395 and Poly (I: C) are adopted. The immune adjuvant is loaded inside the nanoparticle together with a cancer cell lysate fraction, 8M urea or Triton solubilized bacterial membrane fraction. The mass ratio of the cancer cell lysate fraction to the 8M urea or Triton solubilized bacterial membrane fraction is 1:1. The preparation method is as follows, firstly loading lysate components in the nanoparticles by a double emulsion method in the preparation process, then after centrifuging 13000g for 20min, 100mg PLGA nanoparticles are resuspended by 10mL of aqueous solution containing 2% sucrose and 2% mannitol, and then freeze-drying for 48 hours. The nanoparticle loaded with the immune adjuvant, the cancer cell lysate component and the 8M urea-dissolved bacterial membrane component is nanoparticle 1, the average particle size is about 240nm, each 1mg of PLGA nanoparticle is loaded with about 140 mug of protein or polypeptide component, and each of CpG2006 and Poly (I: C) is 0.03mg. The nanoparticle loaded with the immune adjuvant, the cancer cell lysate component and the Triton-dissolved bacterial membrane component is nanoparticle 2, the average particle size is about 240nm, each 1mg of PLGA nanoparticle is loaded with about 140 mug of protein or polypeptide component, and each of CpG2006 and Poly (I: C) is 0.03mg.

(4) Preparation of antigen-presenting cell membrane fraction

This example uses the DC2.4 cell line and BMDM as antigen presenting cells. BMDM was prepared as in example 8.

Co-incubation of nanoparticle 1 (1000. Mu.g) prepared in step (3) with DC2.4 (500 ten thousand) and BMDM cells (500 ten thousand) in 15mL high-glucose DMEM complete medium for 48 hours (37 ℃,5% ₂ ) (ii) a The incubation system contained GM-CSF (2000U/mL), IL-2 (500U/mL), IL-7 (200U/mL), IL-12 (200U/mL), and CD40 antibody (20 ng/mL). The incubated cells were collected, centrifuged at 400g for 5 minutes, resuspended using PBS and washed twice. Thus obtaining the mixed antigen presenting cells.

(5) Preparation of bacterial outer vesicles (OMVs) and cancer extracellular vesicles

Culturing the bifidobacterium breve in an LB culture medium containing 10nM sunitinib, collecting a cultured sample, centrifuging for 30 minutes at 5000g, discarding the precipitate, collecting the supernatant, carrying out ultrasonic treatment for 10 minutes at 15W, centrifuging for 90 minutes at 16000g, and carrying out heavy suspension on the precipitate in PBS to obtain the collected bacterial outer vesicle component 1.

B16F10 cells were cultured in high-glucose DMEM complete medium containing 10nM sunitinib for 6 days, with changes every 2 days. Collecting cell culture B16F10 cells, centrifuging for 5 minutes at 400g, then discarding supernatant, resuspending precipitated cells by using PBS, mechanically destroying cells by carrying out ultrasonic treatment on cancer cells for 2 minutes at 20W, centrifuging for 5 minutes at 2000g, discarding precipitates, centrifuging for 10 minutes at 6000g, then discarding precipitates, centrifuging for 60 minutes at 15000g, then discarding supernatant, collecting precipitates, and resuspending the precipitates in PBS to obtain the cancer cell membrane component 1.

Or culturing the bifidobacterium breve in an LB culture medium, collecting a cultured sample, centrifuging for 30 minutes at 5000g, discarding the precipitate, collecting a supernatant, carrying out ultrasonic treatment for 10 minutes at 15W, centrifuging for 90 minutes at 16000g, and carrying out heavy suspension on the precipitate in PBS to obtain the collected bacterial outer vesicle component 2.

(6) Preparation of nano-vaccine

And (3) resuspending the nanoparticle 1 (100 mg) prepared in the step (3) in 9mL of PBS, mixing with 1mL of the mixed antigen presenting cell membrane component (5 mg) prepared in the step (4), the cancer cell membrane component 1 (5 mg) prepared in the step (5) and the bacterial outer vesicle component 1 (5 mg), carrying out ultrasonic treatment for 5 minutes at 20W, repeatedly co-extruding by using a filter membrane with the diameter of 0.45 mu m, centrifuging for 30 minutes at 13000g, removing supernatant, collecting precipitate, and resuspending the precipitate in PBS to obtain the nano vaccine 1, wherein the particle size of the nano vaccine is 260nm.

And (3) resuspending the nanoparticles 2 (100 mg) prepared in the step (3) in 9mL of PBS, mixing with 1mL of the mixed antigen presenting cell membrane component (5 mg) prepared in the step (4), the cancer cell membrane component 1 (5 mg) prepared in the step (5) and the bacterial outer vesicle component 1 (5 mg), carrying out ultrasonic treatment for 5 minutes at 20W, repeatedly co-extruding by using a filter membrane with the diameter of 0.45 mu m, centrifuging for 30 minutes at 13000g, removing supernatant, collecting precipitate, and resuspending the precipitate in PBS to obtain the nano vaccine 2, wherein the particle size of the nano vaccine is 260nm.

And (3) resuspending the nanoparticles 1 (100 mg) prepared in the step (3) in 9mL of PBS, mixing with 1mL of the mixed antigen presenting cell membrane component (5 mg) prepared in the step (4), the cancer cell membrane component 2 (5 mg) prepared in the step (5) and the bacteria outer vesicle component 2 (5 mg), carrying out ultrasonic treatment at 20W for 5 minutes, repeatedly co-extruding by using a filter membrane with the diameter of 0.45 mu m, centrifuging at 13000g for 30 minutes, removing supernatant, collecting precipitate, and resuspending the precipitate in PBS to obtain the nano vaccine 3, wherein the particle size of the nano vaccine is 260nm.

(7) Nano-vaccine cancer prevention

Selecting female C57BL/6 of 6-8 weeks as a model mouse to prepare a Pan02 pancreatic cancer tumor-bearing mouse, and respectively inoculating 800 mu g of

nano vaccine

1, or 800 mu g of

nano vaccine

2, or 800 mu g of

nano vaccine

3, or 100 mu L of PBS to each mouse at-35 days, 28 days, 21 days, 14 days and 7 days before the mice are inoculated with cancer cells. On day 0, each recipient mouse was inoculated subcutaneously at 2X 10 in the lower right back ⁶ And Pan02 cells. Mouse tumor volume and survival monitoring methods are as above.

(8) Results of the experiment

As shown in fig. 11, the tumors grew quickly and the mice died quickly in the PBS group of mice. The 3 kinds of nano vaccines can obviously slow down the growth speed of the tumor of the mouse and prolong the life cycle of the mouse. The effect of the nano vaccine 1 is better than that of the nano vaccine 2 and the nano vaccine 3, which shows that the bacterial membrane component cracked by the specific lysate is loaded in the nano vaccine to be beneficial to improving the vaccine effect, and the curative effect of the nano vaccine can be improved by using the cancer cells pretreated by the sunitinib and the bacterial membrane component prepared by the bacteria.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.

Claims

1. A method for preparing a vaccine loaded with a cancer cell whole cell fraction and a mixed membrane fraction, comprising the steps of:

s4, enabling the first mixed membrane component and/or the second mixed membrane component to act together with the second particles, and enabling the mixed membrane component to be loaded on the second particles to obtain the vaccine loaded with the cancer cell whole cell component and the mixed membrane component; wherein the second particles are loaded with a cancer cell whole cell component, the first mixed membrane component is a mixture of S1 and S2 products, and the second mixed membrane component is a mixture of S1 and S3 products;

the cancer cell whole-cell component comprises a water-soluble component and a water-insoluble component which are obtained by water splitting of cancer cells and/or tumor tissues, and the water-insoluble component is dissolved by a dissolving agent and then loaded on the second particles; or the cancer cell whole cell component comprises a soluble component obtained by cracking and dissolving cancer cells and/or tumor tissues by a dissolving solution containing a dissolving agent.

2. The method of claim 1, wherein: in step S1, the method further comprises a step of pretreating the cancer cells by culturing the cancer cells in a culture medium containing doxorubicin, a tinib drug, chloroquine, or azacytidine before obtaining the membrane fraction.

3. The method of claim 1, wherein: in step S2, the cancer-associated antigen is a polypeptide antigen or a cancer cell whole cell fraction.

4. The method of claim 1, wherein: in step S3, before obtaining the membrane fraction, a step of pretreating the bacteria by culturing the bacteria in a culture medium containing doxorubicin, a tinib drug, chloroquine, or azacytidine is further included.

5. The method of claim 1, wherein: the second particles are also loaded with a bacterial component obtained by lysing bacteria or bacterial outer vesicles with a lysis solution containing a lytic agent, and then lysing the lysate with the lysis solution.

6. The method of claim 1, wherein: the bacteria are selected from one or more of bacillus calmette-guerin, probiotics and oncolytic bacteria.

7. The method of claim 1, wherein: the dissolving agent is selected from one or more of aqueous solutions of urea, guanidine hydrochloride, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, albumin, lecithin, inorganic salt, triton, tween, amino acid, glucoside and choline.

8. The method of claim 1, wherein: the first or second particles are loaded with an immunological adjuvant and/or a lysosomal escape-increasing substance.

9. A vaccine prepared by the method of any one of claims 1 to 8.

10. Use of the vaccine of claim 9 in the manufacture of a medicament for the treatment or prevention of cancer.