WO2024000724A1 - 负载癌细胞全细胞组分和混合膜组分的疫苗的制备方法及其应用 - Google Patents

负载癌细胞全细胞组分和混合膜组分的疫苗的制备方法及其应用 Download PDF

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WO2024000724A1
WO2024000724A1 PCT/CN2022/108965 CN2022108965W WO2024000724A1 WO 2024000724 A1 WO2024000724 A1 WO 2024000724A1 CN 2022108965 W CN2022108965 W CN 2022108965W WO 2024000724 A1 WO2024000724 A1 WO 2024000724A1
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components
cells
cancer
antigen
loaded
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French (fr)
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刘密
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苏州尔生生物医药有限公司
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/0005Vertebrate antigens
    • A61K39/0011Cancer antigens
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/13Tumour cells, irrespective of tissue of origin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66Microorganisms or materials therefrom
    • A61K35/74Bacteria
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/39Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/48Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005Wall or coating material
    • A61K9/5063Compounds of unknown constitution, e.g. material from plants or animals
    • A61K9/5068Cell membranes or bacterial membranes enclosing drugs
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/48Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51Nanocapsules; Nanoparticles
    • A61K9/5107Excipients; Inactive ingredients
    • A61K9/513Organic macromolecular compounds; Dendrimers
    • A61K9/5146Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A61K9/5153Polyesters, e.g. poly(lactide-co-glycolide)
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/48Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51Nanocapsules; Nanoparticles
    • A61K9/5107Excipients; Inactive ingredients
    • A61K9/5176Compounds of unknown constitution, e.g. material from plants or animals
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • A61P35/04Antineoplastic agents specific for metastasis
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/51Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515Animal cells
    • A61K2039/5154Antigen presenting cells [APCs], e.g. dendritic cells or macrophages
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/555Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511Organic adjuvants
    • A61K2039/55561CpG containing adjuvants; Oligonucleotide containing adjuvants

Definitions

  • the invention relates to the field of immunotherapy, and in particular to a preparation method and application of a vaccine loaded with cancer cell whole cell components and mixed membrane components.
  • Cancer immunotherapy is one of the most important treatments for cancer, and cancer vaccines are one of the important methods for cancer immunotherapy.
  • nanoparticles coated with cancer cell membranes are widely used as cancer vaccines for tumor prevention and treatment, as provided by Zhu J Y et al. (Preferential cancer cell self-recognition and tumor self-targeting by coating nanoparticles with homotypic cancer cell membranes)
  • a kind of nanoparticles coated with cancer cell membranes specifically derived from homologous tumors was prepared.
  • CMBMNPs CMBMNPs
  • Cell membrane biomimetic modified nanoparticles were used to study their homologous targeting ability.
  • Fe 3 O 4 MNPs biomimetically modified cancer cell membranes can achieve highly specific self-recognition of source cancer cell lines in vitro, and have the ability to target homologous tumors. Excellent targeting capabilities. The NPs still selectively target homologous tumors even when competition from heterotypic tumors exists.
  • various cancer cell membrane coatings such as cancer cell membrane-coated gelatin nanoparticles (PDTC@GNPs) for tumor treatment and MIA-PaCa-2 pancreatic cancer cell membrane-coated gold nanoparticles for pancreatic cancer treatment. Nanoparticle cancer vaccines.
  • the present invention provides a membrane component that carries tumor tissue and/or cancer cell whole cell components internally, a membrane component that carries cancer cell membranes and/or extracellular vesicle membranes on the surface, and an activated antigen-promoting method.
  • Nano-vaccines or micro-vaccines that are composed of cell membrane components and/or membrane components derived from bacteria are more stable and more likely to activate cancer cell-specific immune responses due to the bionic simulation of the membrane structure after injection into the body. Can better prevent or treat cancer.
  • the invention provides a method for preparing a vaccine loaded with cancer cell whole cell components and mixed membrane components, which includes the following steps:
  • the first mixed membrane component and/or the second mixed membrane component interact with the second particles to load the mixed membrane components on the second particles to obtain the cancer cell-loaded whole cell component and the mixed membrane component.
  • a vaccine wherein the second particle is loaded with cancer cell whole cell components, the first mixed membrane component is a mixture of S1 products and S2 products, and the second mixed membrane component is a mixture of S1 products and S3 products;
  • the whole cell components of cancer cells include water-soluble components and non-water-soluble components obtained by water lysis of cancer cells and/or tumor tissues, and the non-water-soluble components are loaded on the second particles after being dissolved by a dissolving agent; or
  • the whole cell components of cancer cells include soluble components obtained by lysing cancer cells and/or tumor tissues with a dissolving solution containing a dissolving agent.
  • step S1 before obtaining the membrane component, the step of pretreating the cancer cells is also included.
  • the pretreatment is to place the cancer cells in a culture medium containing doxorubicin, tinib drugs, chloroquine or azacitidine. culture in.
  • tinib drugs include gefitinib, imatinib, imatinib mesylate, nilotinib, sunitinib, lapatinib, etc.
  • the cancer-related antigen is a polypeptide antigen or a whole cell component of cancer cells.
  • step S3 before obtaining the membrane component, a step of pretreating the bacteria is also included.
  • the pretreatment is to culture the bacteria in a culture medium containing doxorubicin, tinib, chloroquine or azacitidine. .
  • the second particles are also loaded with bacterial components.
  • the bacterial components are obtained by lysing bacteria or bacterial outer vesicles with a dissolving solution containing a dissolving agent, and then dissolving the lysed product with the dissolving solution.
  • methods to obtain membrane components from cancer cells, antigen-presenting cells, and bacteria include ultrasound, homogenization, homogenization, high-speed stirring, high-pressure destruction, high-shear force destruction, swelling, chemical substances, shrinkage, etc. .
  • the ways in which the second particles interact with the cell membrane components include co-incubation, ultrasound, stirring, homogenization, co-extrusion, ultrafiltration, dialysis, homogenization, etc.
  • bacteria include BCG, probiotics, oncolytic bacteria, etc. Including but not limited to Bacillus Calmette-Guérin, Escherichia coli, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium lactis, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus gasseri, Lactobacillus reuteri, etc.
  • the second particles loaded with cancer cell whole cell components and bacterial components are prepared by the following steps:
  • the dissolving agent may be urea, guanidine hydrochloride, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, albumin, lecithin, inorganic salts, Triton, Tween, amino acids, glycosides, and choline. wait.
  • the antigen-presenting cells are dendritic cells or mixed antigen-presenting cells containing dendritic cells (DC), that is, the mixed antigen-presenting cells may also contain one or both of B cells and macrophages. kind.
  • the material for preparing the first particle or the second particle can be any material that can be planted with nanoparticles or microparticles, such as natural polymer materials, organic synthetic polymer materials, inorganic materials, etc.
  • the organic synthetic polymer material is selected from polylactic acid-glycolic acid copolymer, polylactic acid, polyglycolic acid, polyethylene glycol, polycaprolactone, poloxamer, polyvinyl alcohol, polyvinylpyrrolidone, polyethyleneimine, Polytrimethylene carbonate, polyanhydride, polydioxanone, polydioxanone, polymethyl methacrylate, PLGA-PEG, PLA-PEG, PGA-PEG, polyamino acids, synthetic peptides and at least one of synthetic lipids;
  • the natural polymer material is selected from at least one of lecithin, cholesterol, alginate, albumin, collagen, gelatin, cell membrane, starch, sugar and polypeptide;
  • the inorganic material is selected from From at least one of ferric oxide, ferric tetroxide, calcium carbonate and calcium phosphate.
  • first particle and/or the second particle are also loaded with an immune-enhancing adjuvant.
  • Immune-enhancing adjuvants include, but are not limited to, immune enhancers derived from microorganisms, products of the human or animal immune system, innate immune agonists, adaptive immune agonists, chemically synthesized drugs, fungal polysaccharides, traditional Chinese medicine and at least one of other categories.
  • Immune-enhancing adjuvants include but are not limited to pattern recognition receptor agonists, Bacillus Calmette-Guérin (BCG), manganese-related adjuvants, BCG cell wall skeleton, BCG methanol extraction residue, BCG muramyl dipeptide, Mycobacterium phlei, Polyclonal A, Mineral Oil, Virus-Like Particles, Immunoenhancing Reconstructed Influenza Virosomes, Cholera Enterotoxin, Saponins and Derivatives, Resiquimod, Thymosin, Neonatal Bovine Liver Peptide, Miquimod, Polysaccharide, Turmeric Factor, immune adjuvant CpG, immune adjuvant poly(I:C), immune adjuvant poly ICLC, Corynebacterium parvum vaccine, hemolytic streptococcus preparation, coenzyme Q10, levamisole, polycytidylic acid, manganese adjuvant, aluminum Adjuvants, calcium adjuvants, calcium
  • the immune-enhancing adjuvant is a Toll-like receptor agonist; more preferably, a combination of two or more Toll-like receptor agonists ensures that nanoparticles or microparticles can better activate cancer after being engulfed by antigen-presenting cells.
  • Cell-specific T cells are preferred.
  • the combination of two or more Toll-like receptor agonists is a combination of poly(I:C)/Poly(ICLC) and CpG-ODN (CpG oligodeoxynucleotide).
  • the CpG-ODN is two or more CpG-ODNs.
  • the adjuvant can be loaded on the interior and/or surface of the first particle or the second particle.
  • first particle or the second particle is also loaded with substances that enhance lysosomal escape, such as amino acids, polyamino acids (such as arginine, polyarginine, lysine, polylysine, histidine, Polyhistidine), nucleic acids, positively charged peptides (such as KALA peptides, RALA peptides, melittin, etc.), lipids, sugars, inorganic substances with proton sponge effect (such as NH 4 HCO 3 ), fish essence Proteins, histones, etc.
  • substances that enhance lysosomal escape such as amino acids, polyamino acids (such as arginine, polyarginine, lysine, polylysine, histidine, Polyhistidine), nucleic acids, positively charged peptides (such as KALA peptides, RALA peptides, melittin, etc.), lipids, sugars, inorganic substances with proton sponge effect (such as NH 4 HCO 3 ),
  • nanoparticles or microparticles can be prepared using existing preparation methods, including but not limited to common solvent evaporation methods, dialysis methods, microfluidic methods, extrusion methods, and hot melt methods.
  • nanoparticles or microparticles may not be modified during the preparation process, or appropriate modification technology may be used to increase the antigen loading capacity of the nanoparticles or microparticles.
  • Modification technologies include but are not limited to biomineralization (such as silicification, calcification, magnesization), gelation, cross-linking, chemical modification, addition of charged substances, etc.
  • the methods by which antigens are loaded on the surface of nanoparticles or microparticles include, but are not limited to, adsorption, covalent connection, charge interaction (such as adding positively charged substances, adding negatively charged substances), hydrophobic interactions, one-step or Multi-step curing, mineralization, wrapping, etc.
  • the water-soluble antigen and/or water-insoluble antigen loaded on the surface of the nanoparticles or microparticles is loaded into one or more layers.
  • the layers are Between them are modifiers.
  • the first particle and the second particle are independently selected from nanoparticles or microparticles, which can ensure that the particles are phagocytized by the antigen-presenting cells, and in order to improve the phagocytosis efficiency, the particle size must be within an appropriate range.
  • the particle size of nanoparticles is 1nm-1000nm, more preferably, the particle size is 30nm-1000nm, most preferably, the particle size is 100nm-600nm; the particle size of microparticles is 1 ⁇ m-1000 ⁇ m, more preferably, The particle size is 1 ⁇ m-100 ⁇ m, more preferably, the particle size is 1 ⁇ m-10 ⁇ m, and most preferably, the particle size is 1 ⁇ m-5 ⁇ m.
  • the incubation system contains cytokines and/or antibodies; the cytokines are selected from at least one of interleukin, tumor necrosis factor, interferon, and colony-stimulating factor; the antibodies include but are not Limited to ⁇ CD-3 antibody, ⁇ CD-4 antibody, ⁇ CD-8 antibody, ⁇ CD-28 antibody, ⁇ CD-40 antibody, ⁇ OX-40 antibody, and ⁇ OX-40L antibody.
  • 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- ⁇ ), TNF- ⁇ , granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF).
  • IL-2 interleukin 2
  • IL-7 interleukin 7
  • IL-14 interleukin 14
  • IL-4 interleukin 4
  • IL-15 interleukin 15
  • IL-21 interleukin 21
  • IL-17 interleukin 17
  • IL-12 interleukin 12
  • IL-6 interleukin 6
  • the activated antigen-presenting cells, cancer cells or bacteria can be appropriately washed before being prepared into nano-vaccines or micro-vaccines, and the washing liquid used in the washing process can contain protease inhibitors and/or phosphatase inhibitors.
  • first particle or the second particle is also modified with a target with active targeting function.
  • the target may be mannose, mannan, CD19 antibody, CD20 antibody, BCMA antibody, CD32 antibody, CD11c antibody, CD103 antibody. , CD44 antibodies, etc.
  • nano-vaccine or micro-vaccine of the present invention in the preparation of drugs for the treatment or prevention of cancer.
  • antigen-presenting cells are derived from one or more of autologous, allogeneic, cell line or stem cell differentiation.
  • the present invention at least has the following advantages:
  • the surface of the nano-vaccine or micro-vaccine of the present invention also loads membrane components of other cells, such as antigen-presenting cell membranes or extracellular vesicle membrane components, or bacterial cell membranes or extracellular membrane components.
  • membrane components of other cells such as antigen-presenting cell membranes or extracellular vesicle membrane components, or bacterial cell membranes or extracellular membrane components.
  • the load of vesicle membrane components, mixed membrane components, and the load of internal whole cell components and/or bacteria make the vaccine of the present invention have more diverse and broader antigenic epitopes, and have bionic membrane characteristics. The stability is enhanced and the problems of difficulty in preserving and maintaining activity of live cell vaccines are overcome.
  • Figure 1 is a schematic diagram of the preparation process and application of nano-vaccines or micro-vaccines of the present invention
  • a is a schematic diagram of collecting and preparing nanoparticles or micro-particles for water-soluble antigens and water-insoluble antigens respectively
  • b is a dissolving solution containing a dissolving agent
  • c is a schematic diagram of preparing nanovaccines or micron vaccines according to the present invention.
  • Figures 2-11 are respectively the experimental results of mouse tumor growth rate and survival time when nano vaccines or micro vaccines are used to prevent or treat cancer in Examples 1-10; in Figures 2-4 and 6-11, a represents the prevention or treatment of cancer.
  • the significant difference in the tumor growth inhibition experiment in medium was analyzed by ANOVA method, and the significant difference in b was analyzed by Kaplan-Meier and log-rank test; *** means p ⁇ 0.005 compared with the PBS blank control group, there is a significant difference; &&& means p ⁇ 0.005, there is a significant difference compared with the control group of nanovaccine/microvaccine prepared by loading cancer cell membrane components on the surface of blank nanoparticles/microparticles; ⁇ represents the difference between nanoparticles and nanoparticles loaded with cancer cell whole cell components
  • p ⁇ 0.05 there is a significant difference between the nanovaccine/micron vaccine prepared with cancer cell membrane components loaded on the surface of micron particles; ⁇ represents the difference between the nanoparticles/micron particles loaded with cancer cell membrane components on the surface There is a significant difference at p ⁇ 0.01 between the prepared nano-vaccine/micro-vaccine; ⁇ means that the nanoparticles are loaded with whole cell components of cancer cells internally and at the same time loaded with cancer cell membrane components and activated antigen-presenting cell membrane components on the surface. Compared with the vaccine/micron vaccine group, p ⁇ 0.05, there is a significant difference; # indicates that the whole cell components of cancer cells are loaded internally, and the cancer cell membrane components, unactivated antigen-presenting cell membrane components and bacterial cells are loaded on the surface.
  • nanovaccine/microvaccine group with outer vesicle components means that the whole cell components of cancer cells are loaded inside, and the cancer cell membrane components and unactivated antigen present are loaded on the surface at the same time.
  • p ⁇ 0.01 there is a significant difference between the nanovaccine/microvaccine group that contains cell membrane components and bacterial extracellular vesicle components; eta means that the whole cell components of cancer cells are loaded internally and the cancer cell membrane components are loaded on the surface.
  • the antigen-presenting cell membrane component and the bacterial extracellular vesicle component have a significant difference at p ⁇ 0.05; ⁇ indicates that it is different from the internal only
  • the ratio of nanovaccines loaded with bacterial outer vesicle components and immune adjuvants, and mixed membrane components loaded on the surface at the same time is p ⁇ 0.005, with a significant difference; ⁇ means that compared with those loaded with only bacterial outer vesicle components and immune adjuvants inside,
  • the ratio of the nanovaccine with mixed membrane components loaded on the surface at the same time is p ⁇ 0.01, which is a significant difference; ⁇ represents the ratio p of the nanovaccine with the whole cell components of cancer cells and immune adjuvants internally loaded with the mixed membrane components on the surface at the same time.
  • ⁇ 0.05 there is a significant difference; ⁇ means that the ratio p ⁇ 0.05, there is a significant difference compared with the nanovaccine loaded with cancer cell components and bacterial components internally, and cancer cell membrane components loaded on the surface; ⁇ means there is a significant difference with the nanovaccine loaded with cancer cells internally Components and bacterial components, the nanovaccine/microvaccine ratio of surface-loaded cancer cell membrane components and Lactobacillus acidophilus, p ⁇ 0.05, has a significant difference; ⁇ represents the lysis and dissolution of internally loaded cancer cell membrane components and 8M urea Bacterial components, the nanovaccine/microvaccine ratio of surface-loaded bacterial outer vesicle components, p ⁇ 0.05, has a significant difference; ⁇ represents the bacterial components lysed and dissolved with internally loaded cancer cell components and Tween 80 , the ratio of nano-vaccine/micro vaccine loaded with bacterial extravesicle components and cancer cell extracellular vesicle components on the surface, p ⁇ 0.05, has a significant
  • represents the whole cell components of cancer cells loaded internally, and the activated antigen-presenting cell membrane components, unprocessed cancer cell membrane components and surface-loaded components at the same time.
  • the comparison of nanovaccine/micron of untreated bacterial membrane components is p ⁇ 0.05, there is a significant difference; Represents a nano-vaccine/micron phase loaded with cancer cell whole cell components internally and a CpG+Poly(I:C) mixed adjuvant + lysosomal escape substance, and with cancer cell membrane components and antigen-presenting cell membrane components loaded on the surface.
  • Ratio p ⁇ 0.05 there is a significant difference; ⁇ represents the difference with the internal loaded cancer cell whole cell components and two kinds of CpG+Poly (I:C) mixed adjuvants, the surface loaded cancer cell membrane components and antigen-presenting cell membrane components Compared with nano vaccine/micro vaccine, there is a significant difference at p ⁇ 0.05; ⁇ represents the bacterial components and whole cell components of cancer cells that are lysed and dissolved by Triton loaded internally, and the activated antigen-presenting cell membrane components are loaded on the surface at the same time. There is a significant difference at p ⁇ 0.05 between the nanovaccine/micron of treated cancer cell membrane components and treated bacterial membrane components.
  • the vaccine for preventing or treating cancer according to the present invention is loaded with cancer cells and/or tumor tissue whole cell components and/or bacterial components internally, and at the same time, mixed membrane components are loaded on the surface.
  • the surface-loaded mixed membrane components include cell membranes of cancer cells, antigen-presenting cells and/or bacteria or membrane components of extracellular vesicles.
  • the preparation process and application fields are shown in Figure 1.
  • the whole cell component When preparing nanoparticles or microparticles for internally loading cancer cells and/or whole cell components of tumor tissue, the whole cell component needs to be prepared first.
  • cells or tissues When preparing whole cell components, cells or tissues can be lysed and water-soluble antigens and water-insoluble antigens can be collected separately and nano or micron particle systems can be prepared respectively; or the cells or tissues can be directly lysed using a lysing solution containing a dissolving agent and the cells lysed Whole cell fractions and preparation of nano or micro particles.
  • the whole cell components of the cells of the present invention can be processed before or (and) after lysis, including but not limited to inactivation or (and) denaturation, solidification, biomineralization, ionization, chemical modification, nuclease treatment, etc.
  • Nanoparticles or microparticles can then be prepared; nanoparticles can also be directly prepared before or (and) after cell lysis without any inactivation or (and) denaturation, solidification, biomineralization, ionization, chemical modification, or nuclease treatment. or micron particles.
  • cells are inactivated or/and denatured before lysis.
  • inactivation or/and denaturation can also be performed after cell lysis, or cells can be lysed before and/or denatured.
  • inactivation or (and) denaturation treatment is performed; in some embodiments of the present invention, the inactivation or (and) denaturation treatment method before or after cell lysis is ultraviolet irradiation and high-temperature heating.
  • treatment methods including but not limited to radiation irradiation, high pressure, curing, biomineralization, ionization, chemical modification, nuclease treatment, collagenase treatment, freeze-drying and other treatment methods can also be used.
  • radiation irradiation high pressure, curing, biomineralization, ionization, chemical modification, nuclease treatment, collagenase treatment, freeze-drying and other treatment methods
  • nuclease treatment collagenase treatment
  • freeze-drying and other treatment methods can also be used.
  • Nanoparticles or microparticles of cellular components and/or bacterial components work together to load the membrane components on the surface of the nanoparticles or microparticles.
  • antigen-presenting cells used are activated antigen-presenting cells, when using nanoparticles or microparticles to activate the antigen-presenting cells in vitro, cytokines and/or antibodies can be used to assist in improving the activation efficiency.
  • the antigen-presenting cells can be derived from Autologous or allogeneic, or derived from cell lines or stem cells.
  • Antigen-presenting cells can be DC cells, B cells, macrophages, or any mixture of the above three, or other cells with antigen-presenting functions.
  • the treatment methods include but are not limited to incubation with chemical drugs such as doxorubicin for a period of time, high calcium ion environment to increase cell pressure, etc.
  • a solvent evaporation method is used to prepare the first and second particles.
  • the specific steps for preparing nano-vaccines or micro-vaccines are using nanoparticles or micron particle-activated antigen-presenting cells loaded with cancer cell whole cell components and/or bacterial components.
  • the preparation method is as follows:
  • Step 1 Add 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 second predetermined concentration of the raw material for preparing particles.
  • the aqueous solution may contain each component of the cancer cell and/or bacterial lysate and an immune-enhancing adjuvant; each component of the cancer cell and/or bacterial lysate is water-soluble during preparation.
  • the antigen may be the original non-water-soluble antigen dissolved in a dissolving agent such as urea or guanidine hydrochloride.
  • concentration of the water-soluble antigen or the original non-water-soluble antigen contained in the aqueous phase solution that is, the first predetermined concentration requires the concentration of protein polypeptide to be greater than 1ng/mL, which can load enough whole cell components of cancer cells to activate related cells.
  • the concentration of the immune-enhancing adjuvant in the initial aqueous phase is greater than 0.01ng/mL.
  • the aqueous solution contains each component of the tumor tissue and/or cancer cell lysate and an immune-enhancing adjuvant; each component of the tumor tissue and/or cancer cell lysate is water-soluble during preparation.
  • the sexual antigen may be the original non-water-soluble antigen dissolved in a dissolving agent such as urea or guanidine hydrochloride.
  • the concentration of the water-soluble antigen or the original non-water-soluble antigen contained in the aqueous solution, that is, the first predetermined concentration requires the protein polypeptide concentration to be greater than 0.01ng/mL, which can load enough whole cell components of cancer cells to activate related cells. .
  • the concentration of the immune-enhancing adjuvant in the initial aqueous phase is greater than 0.01ng/mL.
  • the aqueous solution contains each component of the bacterial lysate and an immune-enhancing adjuvant; each component of the bacterial lysate is a water-soluble antigen or is dissolved in urea or guanidine hydrochloride during preparation.
  • the concentration of the water-soluble antigen or the original concentration of the non-water-soluble antigen contained in the aqueous phase solution, that is, the first predetermined concentration requires the protein polypeptide concentration to be greater than 0.01ng/mL, and can load enough bacterial components to activate relevant cells.
  • the concentration of the immune-enhancing adjuvant in the initial aqueous phase is greater than 0.01ng/mL.
  • the raw material for preparing particles is PLGA or PLA, and methylene chloride is used as the organic solvent.
  • the second predetermined concentration of raw materials for preparing particles ranges from 0.5 mg/mL to 5000 mg/mL, preferably 100 mg/mL.
  • PLGA or modified PLGA is selected because this material is a biodegradable material and has been approved by the FDA for use as a pharmaceutical dressing. Studies have shown that PLGA has certain immunomodulatory functions and is therefore suitable as an excipient in the preparation of nanoparticles or microparticles. In practical applications, appropriate materials can be selected according to actual conditions.
  • the second predetermined volume of the organic phase is set according to its ratio to the first predetermined volume of the aqueous phase.
  • the range of the ratio of the first predetermined volume of the aqueous phase to the second predetermined volume of the organic phase is It is 1:1.1-1:5000, preferably 1:10.
  • 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 prepared nanoparticles or microparticles.
  • the concentration of protein and polypeptide is greater than 1 ng/mL, preferably 1 mg/mL ⁇ 100 mg/mL; when the aqueous phase solution is a lysate component/immune adjuvant solution, wherein The concentration of protein and polypeptide is greater than 1ng/mL, preferably 1mg/mL ⁇ 100mg/mL, and the concentration of immune adjuvant is greater than 0.01ng/mL, preferably 0.01mg/mL ⁇ 20mg/mL.
  • the solvent is DMSO, acetonitrile, ethanol, chloroform, methanol, DMF, isopropyl alcohol, dichloromethane, propanol, ethyl acetate, etc., preferably dichloromethane;
  • the concentration of the organic phase is 0.5 mg/mL ⁇ 5000mg/mL, preferably 100mg/mL.
  • Step 2 subject the mixed solution obtained in Step 1 to ultrasonic treatment for more than 2 seconds or stirring or homogenization treatment or microfluidic treatment for more than 1 minute.
  • the stirring is mechanical stirring or magnetic stirring
  • the stirring speed is greater than 50 rpm
  • the stirring time is greater than 1 minute.
  • the stirring speed is 50 rpm ⁇ 1500 rpm
  • the stirring time is 0.1 hour ⁇ 24 hours
  • the ultrasonic power is greater than 5W
  • the time Greater than 0.1 seconds such as 2 to 200 seconds
  • the pressure is greater than 5 psi, such as 20 psi to 100 psi.
  • the rotation speed of the shear homogenizer is greater than 100rpm, such as 1000rpm to 5000rpm; the flow rate of microfluidic processing is greater than 0.01mL/min, such as 0.1mL/min-100mL/min.
  • Ultrasonic or stirring or homogenization treatment or microfluidic treatment can be used for nanonization and/or micronization.
  • the length of ultrasonic time or stirring speed or homogenization pressure and time can control the size of the prepared micro-nano particles. Too large or too small will cause to changes in particle size.
  • Step 3 Add the mixture obtained after step 2 to a third predetermined volume of aqueous solution containing a third predetermined concentration of emulsifier and perform ultrasonic treatment for more than 2 seconds or stirring for more than 1 minute or perform homogenization or microfluidic treatment. deal with.
  • the mixture obtained in step 2 is added to the aqueous emulsifier solution and continued to be ultrasonically or stirred to form nanometers or micrometers.
  • the ultrasonic time is greater than 0.1 seconds, such as 2 to 200 seconds
  • the stirring speed is greater than 50 rpm, such as 50 rpm to 500 rpm
  • the stirring time is greater than 1 minute, such as 60 to 6000 seconds.
  • the stirring speed is greater than 50rpm, and the stirring time is greater than 1 minute.
  • the stirring speed is 50rpm to 1500rpm, and the stirring time is 0.5 to 5 hours; during ultrasonic treatment, the ultrasonic power is 50W to 500W.
  • the time is greater than 0.1 seconds, such as 2 to 200 seconds; when homogenizing, use a high-pressure/ultra-high-pressure homogenizer or high-shear homogenizer.
  • the pressure is greater than 20 psi, such as 20 psi to 100 psi.
  • the rotation speed is greater than 1000rpm, such as 1000rpm to 5000rpm; when using microfluidic processing, the flow rate is greater than 0.01mL/min, such as 0.1mL/min-100mL/min.
  • Ultrasonic or stirring or homogenization treatment or microfluidic treatment can be used to nanonize or micronize the particles.
  • the length of ultrasonic time or stirring speed or homogenization process pressure and time can control the size of the prepared nano or micron particles. Too large or too small will cause Changes in particle size.
  • the emulsifier aqueous solution is a polyvinyl alcohol (PVA) aqueous solution
  • the third predetermined volume is 5 mL
  • the third predetermined concentration is 20 mg/mL.
  • the third predetermined volume is adjusted according to its ratio to the second predetermined volume.
  • the range between the second predetermined volume and the third predetermined volume is set to 1:1.1-1:1000, preferably 2:5.
  • the ratio of the second predetermined volume and the third predetermined volume can be adjusted.
  • the ultrasonic time or stirring time, the volume and concentration of the emulsifier aqueous solution in this step are all based on obtaining nanoparticles or microparticles of suitable size.
  • Step 4 Add the liquid obtained after the treatment in Step 3 to a fourth predetermined volume of the emulsifier aqueous solution with a fourth predetermined concentration, and stir until the predetermined stirring conditions are met.
  • the emulsifier aqueous solution is PVA solution or other solutions.
  • the fourth predetermined concentration is 5 mg/mL, and the selection of the fourth predetermined concentration is based on obtaining nanoparticles or microparticles of suitable size.
  • the selection of the fourth predetermined volume is determined based on the ratio of the third predetermined volume to the fourth predetermined volume.
  • the ratio of the third predetermined volume to the third predetermined volume is in the range of 1:1.5-1:2000, preferably 1:10.
  • the ratio of the third predetermined volume and the fourth predetermined volume can be adjusted in order to control the size of the nanoparticles or microparticles.
  • the predetermined stirring condition of this step is until the volatilization of the organic solvent is completed, that is, the volatilization of methylene chloride in step 1 is completed.
  • Step 5 After centrifuging the mixed liquid that meets the predetermined stirring conditions in Step 4 at a rotation speed of greater than 100 RPM for more than 1 minute, remove the supernatant, and resuspend the remaining sediment in a fifth predetermined volume of Five predetermined concentrations of an aqueous solution containing a lyoprotectant or a sixth predetermined volume of PBS (or physiological saline).
  • step 5 when the precipitate obtained in step 5 is resuspended in the sixth predetermined volume of PBS (or physiological saline), there is no need to freeze-dry, and the subsequent co-action of nanoparticles or microparticles with membrane components can be directly performed.
  • PBS physiological saline
  • the precipitate obtained in step 5 needs to be freeze-dried when resuspended in an aqueous solution containing a lyoprotectant, and then freeze-dried before conducting subsequent experiments.
  • Trehalose is selected as the freeze-drying protective agent.
  • the fifth predetermined concentration of the freeze-drying protective agent in this step is 4% by mass. The reason why this is set is to not affect the freeze-drying effect during subsequent freeze-drying.
  • Step 6 After freeze-drying the suspension containing the lyoprotectant obtained in Step 5, the freeze-dried material is used for later use.
  • Step 7 Mechanically destroy cancer cells, antigen-presenting cells, bacteria, or extracellular vesicles using methods such as low-power ultrasound, mechanical stirring, homogenization, high shear force, high pressure, swelling, etc., and collect the destroyed cells. or membrane components of vesicles.
  • Step 8 The membrane component prepared in step 7 is reacted with the nanoparticles and/or microparticles prepared above for a certain period of time.
  • Tumor tissue and/or cancer cells, cancer cells, and antigen-presenting cells for preparing nanoparticles and/or microparticles can be from autologous or allogeneic sources.
  • Step 9 Collect the co-acted nanoparticles or microparticles, and use centrifugation, ultrafiltration or dialysis to purify the nanoparticles or microparticles, which is the nanovaccine or micron vaccine. It can be used directly, frozen for later use, or prepared after freeze-drying. Freeze-dried powder is available for later use.
  • the specific preparation method for preparing nano-vaccines or micro-vaccines after using the double emulsion method to prepare antigen-loaded nanoparticles or microparticles is as follows:
  • Steps 1 to 4 are the same as above.
  • Step 5 After centrifuging the mixed liquid that meets the predetermined stirring conditions in Step 4 at a rotation speed of greater than 100 RPM for more than 1 minute, remove the supernatant, and resuspend the remaining sediment in a fifth predetermined volume of five predetermined concentrations of a solution containing water-soluble and/or non-water-soluble antigens in the whole cell fraction of cancer cells, or the remaining pellet is resuspended in a fifth predetermined volume of a fifth predetermined concentration containing all cancer cell components.
  • a solution in which water-soluble and/or non-water-soluble antigens in the cell component are mixed with adjuvants.
  • Step 6 After centrifuging the mixed solution that meets the predetermined stirring conditions in Step 5 at a rotation speed of greater than 100 RPM for greater than 1 minute, remove the supernatant, and resuspend the remaining sediment in a sixth predetermined volume of solidified liquid.
  • the treatment reagent or mineralization treatment reagent is centrifuged and washed after acting for a certain period of time, and then the seventh predetermined substance containing positively or negatively charged substances is added and acted for a certain period of time.
  • the precipitate obtained in step 6 does not need to be freeze-dried after being resuspended in the seventh predetermined volume of charged substance, and subsequent experiments related to the co-action of nanoparticles or microparticles with membrane components can be directly performed.
  • the precipitate obtained in step 6 is resuspended in an aqueous solution containing a drying protective agent and then subjected to room temperature vacuum drying or freeze vacuum drying, and subsequent experiments are performed after drying.
  • the freeze-drying protective agent is trehalose or a mixed solution of mannitol and sucrose.
  • concentration of the drying protective agent in this step is 4% by mass, which is set so as not to affect the drying effect during subsequent drying.
  • Step 7 After drying the suspension containing the drying protective agent obtained in Step 6, the dried material is used for later use.
  • the modification and antigen loading steps of steps 5 to 7 can be repeated multiple times to increase the loading capacity of the antigen.
  • substances with the same charge can be added multiple times or substances with different charges can be added alternately.
  • Step 8 Mechanically destroy cancer cells, antigen-presenting cells, bacteria, or extracellular vesicles using methods such as low-power ultrasound, mechanical stirring, homogenization, high shear force, high pressure, swelling, etc., and collect the destroyed cells. or membrane components of vesicles.
  • Step 9 The membrane component prepared in Step 8 is reacted with the nanoparticles and/or microparticles prepared above for a certain period of time.
  • Tumor tissue and/or cancer cells, cancer cells, and antigen-presenting cells for preparing nanoparticles and/or microparticles can be from autologous or allogeneic sources.
  • Step 10 Collect the co-acted nanoparticles or microparticles, and use centrifugation, ultrafiltration or dialysis to purify the nanoparticles or microparticles, which is the nanovaccine or micron vaccine. It can be used directly, frozen for later use, or prepared after freeze-drying. Freeze-dried powder is available for later use.
  • This example uses mouse melanoma as a cancer model to illustrate how to use nanovaccines to prevent melanoma.
  • B16F10 melanoma tumor tissue was lysed to prepare water-soluble antigen and water-insoluble antigen of the tumor tissue.
  • the organic polymer material PLGA was used as the nanoparticle skeleton material
  • Polyinosinic-polycytidylic acid (poly(I:C )) is an immune adjuvant that uses a solvent evaporation method to prepare nanoparticles loaded with water-soluble antigens and non-water-soluble antigens of tumor tissue, and then loads cancer cell membrane components and/or activated antigen-presenting cell membranes on the surface of the nanoparticles. Components and components of bacterial extracellular vesicles are subsequently used to prevent cancer.
  • the nanoparticles and the blank nanoparticles used as a control were prepared by the double emulsion method in the solvent evaporation method.
  • the molecular weight of PLGA, the material used to prepare the nanoparticles, is 7KDa-17KDa.
  • the immune adjuvant used is poly(I:C) and the poly(I:C) is contained in the nanoparticles.
  • the preparation method is as mentioned above. During the preparation process, the whole cell lysate components and adjuvants are first loaded inside the nanoparticles using the double emulsion method, and then 100 mg of the nanoparticles are centrifuged at 10,000 g for 20 minutes, and 10 mL of trehalose containing 4% is used.
  • the average particle size of the nanoparticles 1 is about 260nm, and the surface potential is about -9mV; each 1 mg PLGA nanoparticle 1 is loaded with approximately 100 ⁇ g of protein or peptide components, and the poly(I:C) immune adjuvant used per 1 mg PLGA nanoparticle 1 is 0.02mg.
  • the preparation materials and preparation methods of blank nanoparticles are the same, the particle size is about 260nm, and the same amount of adjuvant is loaded but no lysate component is loaded.
  • B16F10 cancer cells were collected, washed twice with physiological saline, resuspended in physiological saline and sonicated at 7.5W for 20 minutes. Then centrifuge the sample at 2000g for 20 minutes and collect the supernatant. Centrifuge the supernatant at 7000g for 20 minutes and collect the supernatant. Then collect the supernatant after centrifugation at 15000g for 120 minutes. Discard the supernatant and collect the pellet. Place the pellet in PBS. Resuspend and set aside.
  • BMDC bone marrow-derived dendritic cells
  • This example uses the preparation of dendritic cells from mouse bone marrow cells as an example to illustrate how to prepare BMDC.
  • a 6-8 week old C57 mouse was sacrificed by cervical dislocation.
  • the tibia and femur of the hind legs were surgically removed and placed in PBS.
  • the muscle tissue around the bones was removed with scissors and tweezers.
  • the needles are inserted into the bone marrow cavity from both ends of the bone, and the bone marrow is repeatedly flushed into the culture dish.
  • RPMI 1640 (10% FBS) medium to stop lysis, centrifuge at 400 g for 3 min, and discard the supernatant.
  • the cells were placed in a 10 mm culture dish and cultured in RPMI 1640 (10% FBS) medium with recombinant mouse GM-CSF (20 ng/mL) added at 37 degrees Celsius and 5% CO 2 for 7 days. On the third day, shake the culture bottle gently and supplement the same volume of RPMI 1640 (10% FBS) medium containing GM-CSF (20ng/mL).
  • Nanoparticles (800 ⁇ g) loaded with whole cell components of cancer cells derived from tumor tissue were incubated with BMDC (10 million) in 15 mL RPMI1640 complete medium for 48 hours (37°C, 5% CO 2 ), and 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). Then the activated DCs were collected and centrifuged at 400g for 5 minutes, and then washed twice with 4°C phosphate buffer solution (PBS) containing protease inhibitors.
  • PBS 4°C phosphate buffer solution
  • the cells were resuspended in PBS water and incubated at 4°C at low power (22.5 W) Ultrasound for 1 minute. Then centrifuge the sample at 3000g for 15 minutes and collect the supernatant. Centrifuge the supernatant at 8000g for 15 minutes and collect the supernatant. Then collect the supernatant after centrifugation at 16000g for 90 minutes. Discard the supernatant and collect the pellet. Place the pellet in PBS. After resuspension, use a membrane filter to filter the sample to obtain nanoparticles based on the cell membrane component of the antigen-presenting cells, with a particle size of 130 nanometers.
  • step (2) Resuspend 50 mg of the blank nanoparticles prepared in step (2) in 9 mL PBS, then mix with 1 mL of 3 mg cancer cell membrane components prepared in step (3) and incubate at room temperature for 15 minutes, and then repeatedly pass through 0.45 ⁇ m extrusion The membrane was co-extruded, and then centrifuged at 13,000g for 25 minutes. The supernatant was discarded and the pellet was resuspended in PBS to become nanovaccine 1, with a particle size of 270 nm.
  • 1.5 mg of the cancer cell membrane component prepared in step (3) and 1.5 mg of the nanoparticles based on the antigen-presenting cell membrane component prepared in step (4) are mixed and incubated for 10 minutes, and then repeatedly squeezed through 0.22 ⁇ m. Film co-extrusion.
  • 100 mg of the nanoparticles loaded with whole cell components prepared in step (2) were resuspended in 9 mL PBS, mixed with the aforementioned mixed membrane components and incubated at room temperature for 15 minutes, and then repeatedly co-extruded through a 0.45 ⁇ m extrusion membrane. Then centrifuge at 13000g for 25 minutes, discard the supernatant and resuspend the pellet in PBS, which is Nano Vaccine 3, with a particle size of 270 nm.
  • Nano Vaccine 2 shows that the tumor growth rate of mice in the PBS control group was very fast and their survival period was very short; the tumor growth rate of mice in the nanovaccine group was significantly slower.
  • Nano Vaccine 2 Nano Vaccine 3 and Nano Vaccine 4 are more effective than Nano Vaccine 1; Nano Vaccine 3 and Nano Vaccine 4 are more effective than Nano Vaccine 2; Nano Vaccine 4 is more effective than Nano Vaccine 3.
  • the nanovaccine of the present invention has a good preventive effect on melanoma.
  • the ultrasonic method is used to prepare cancer cell membranes and antigen-presenting cell membrane components.
  • stirring, high pressure, high shear force, homogenization and other methods can also be used to mechanically destroy the cells, and then use
  • the membrane components are collected by centrifugation and/or sequential filtration through membranes with different pore sizes and/or ultrafiltration and/or dialysis.
  • This embodiment uses 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 bacterial extracellular vesicles. In practical applications, extracellular vesicles of cancer cells can also be used.
  • the vesicle component is a mixed membrane component of antigen-presenting cells and bacteria, or a mixed membrane component of extracellular vesicles from any of the above types of cells.
  • This embodiment uses co-incubation and co-extrusion methods to co-act the nanoparticles with the membrane components. In practical applications, one of stirring, homogenization, ultrasound, ultrafiltration, dialysis and homogenization can also be used. Various. In this embodiment, nanoparticles loaded with whole cell components and membrane components are used to prepare nano vaccines. In practical applications, micron vaccines can also be prepared using microparticles loaded with whole cell components and membrane components.
  • This example uses mouse melanoma as a cancer model to illustrate how to use nanovaccines to treat melanoma.
  • B16F10 melanoma tumor tissue was lysed to prepare water-soluble antigen and water-insoluble antigen of the tumor tissue.
  • PLGA was used as the nanoparticle skeleton material, and poly(I: which is also a Toll-like receptor agonist) was used.
  • C), CpG BW006 and CpG2395 are mixed immune adjuvants that use a solvent evaporation method to load water-soluble antigens and non-water-soluble antigens of tumor tissues into nanoparticles, and then load the cell membrane components of cancer cells and antigen-presenting cells into nanoparticles.
  • the surface of the particles is the melanoma vaccine.
  • B16F10 cells 1.5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated on the back of each C57BL/6 mouse. When the tumor grew to a volume of approximately 1000 mm 3 , the mice were sacrificed and the tumor tissue was removed. Cut the tumor tissue into pieces and then grind it. Add an appropriate amount of pure water through a cell filter and freeze and thaw repeatedly 5 times. Ultrasound can be used to destroy the lysed cells. After the cells are lysed, centrifuge the lysate at 5000g for 5 minutes and take the supernatant to obtain the water-soluble antigen that is soluble in pure water; add 8M urea to the resulting precipitate to dissolve the precipitate and remove the insoluble antigen from pure water. The non-water-soluble antigen is converted into soluble in 8M urea aqueous solution.
  • the above are the sources of antigen raw materials for preparing nanoparticle systems.
  • the nanoparticles were prepared by solvent evaporation method.
  • nanoparticles loaded with water-soluble antigens in the whole cell component of cancer cells and nanoparticles loaded with non-water-soluble antigens in the whole cell component of cancer cells are prepared separately, and then used together.
  • the molecular weight of PLGA, the preparation material for nanoparticle 1, is 24Da-38KDa.
  • the immune adjuvants used are poly(I:C), CpG BW006 and CpG2395, and the adjuvants are contained inside the nanoparticles.
  • the preparation method is as described above. During the preparation process, the double emulsion method is first used to load the antigen and adjuvant inside the nanoparticles.
  • the nanoparticles After loading the antigen (cleavage component) inside, 100 mg of the nanoparticles are centrifuged at 10,000g for 20 minutes, and 10 mL containing Resuspend in 4% trehalose ultrapure water and freeze-dry for 48 h.
  • the average particle size of the nanoparticles is about 320nm; each 1 mg of PLGA nanoparticles is loaded with approximately 100 ⁇ g of protein and peptide components, and 0.025 mg each of poly(I:C), CpG BW006 and CpG2395 immune adjuvants.
  • the preparation materials and preparation methods of the control peptide nanoparticles are the same as above.
  • the four loaded peptide neoantigens are B16-M20 (Tubb3, FRRKAFLHWYTGEAMDEMEFTEAESNM), B16-M24 (Dag1, TAVITPPTTTTKKARVSTPKPATPSTD), B16-M46 (Actn4, NHSGLVTFQAFIDVMSRETTDTDTADQ) and TRP2:180 -188(SVYDFFVWL).
  • Each 1 mg of PLGA nanoparticles is loaded with 100 ⁇ g of polypeptide components, 0.025 mg each of poly(I:C), CpG BW006, and CpG2395, and the average particle size is about 320 nm.
  • This example uses a mixed antigen-presenting cell of BMDC and B cells.
  • the preparation method of BMDC is the same as in Example 1.
  • the isolation method of B cells is as follows: kill the C57BL/6 mice, remove the mouse spleen, prepare a single cell suspension of mouse splenocytes, and use magnetic bead sorting to separate the living cells in the splenocytes (use live and dead cell dye labeling dead cells to remove dead cells) CD19 + B cells. Then, BMDC and B cells are mixed at a quantity ratio of 1:1 and used as mixed antigen-presenting cells.
  • 1 mg of nanoparticles loaded with whole cell components of cancer cells 1 including 500 ⁇ g of nanoparticles loaded with water-soluble components + 500 ⁇ g of nanoparticles loaded with non-water-soluble components
  • 1 mg of polypeptide nanoparticles and 20 million mixed antigen-presenting cells (10 million DCs + 10 million B cells) were mixed in 20mL RPMI1640 complete medium and incubated for a total of 48 hours (37°C, 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 (50ng/mL). After the incubation is completed, centrifuge the incubated cells at 400g for 5 minutes, discard the supernatant, and wash twice with PBS to obtain activated antigen-presenting cells.
  • antigen-presenting cells that have not been activated by any nanoparticles or antigen-presenting cells that have been activated by nanoparticles are used to prepare membrane components.
  • PBS phosphate buffer solution
  • the supernatant was centrifuged at 8000g for 15 minutes and the supernatant was collected.
  • the supernatant was repeatedly filtered using a 0.22 ⁇ m membrane filter and the sample was centrifuged at 16000g for 90 minutes. Collect and discard the supernatant, collect the precipitate, and resuspend the precipitate in PBS to obtain the cell membrane fraction of the antigen-presenting cells.
  • B16F10 cancer cells were collected, washed twice with physiological saline, resuspended in physiological saline and sonicated at 7.5W for 20 minutes. Then centrifuge the sample at 2000g for 20 minutes and collect the supernatant. Centrifuge the supernatant at 7000g for 20 minutes and collect the supernatant. Then collect the supernatant after centrifugation at 15000g for 120 minutes. Discard the supernatant and collect the pellet. Place the pellet in PBS. Resuspend and set aside.
  • the collected cancer cell membrane components are mixed at a mass ratio of 2:1:1, then stirred at 1500RPM for 2 minutes at room temperature, and then repeatedly co-extruded using a membrane extruder to obtain the mixed membrane components of the three.
  • step (2) Mix 100 mg of nanoparticles 1 prepared in step (2) (50 mg of nanoparticles loaded with water-soluble components + 50 mg of nanoparticles loaded with non-water-soluble components) and 10 mg of the mixed membrane component prepared in step (6) and incubate them for 20 minutes, then filter repeatedly using a 0.45 ⁇ m filter membrane, centrifuge the filtrate at 13000g for 20 minutes, discard the supernatant, resuspend the pellet in 4% trehalose aqueous solution, and freeze-dry for 48 hours to obtain a lyophilized powder that is the nano vaccine.
  • the particle size of the nanovaccine 1 prepared by mixing activated mixed antigen-presenting cell membrane components with nanoparticles 1 loaded with whole cell components and extracellular vesicles of cancer cells and bacteria is 340nm; using nanoparticles 1 loaded internally with four polypeptide antigens
  • the nanoparticle-activated mixed antigen-presenting cell membrane component is mixed with the extracellular vesicles of cancer cells and bacteria.
  • the particle size of the nanovaccine 2 is 340nm; the unactivated mixed antigen-presenting cell membrane component is mixed with the extracellular vesicles of cancer cells and bacteria.
  • the particle size of the nanovaccine 3 prepared by mixing the vesicles is 340nm.
  • mice Female C57BL/6 mice aged 6-8 weeks were selected as model mice to prepare melanoma tumor-bearing mice.
  • 1.5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated into the lower right corner of the back of each recipient mouse.
  • the 8th day, the 11th day, the 15th day and the 20th day before and after the mouse was inoculated with cancer cells each mouse was respectively inoculated with 1 mg of Nano vaccine 1 or Nano vaccine 2 or Nano vaccine 3 or 100 ⁇ L of PBS.
  • the methods for monitoring mouse tumor growth rate and mouse survival are the same as above.
  • nano vaccine 1 and nano vaccine 2 are more effective than nano vaccine 3; nano vaccine 1 is more effective than nano vaccine 2.
  • This example uses mouse melanoma as a cancer model to illustrate how to use nanovaccines to treat cancer.
  • 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 then The water-soluble antigen mixture and the water-insoluble antigen mixture are mixed at a mass ratio of 1:1.
  • PLGA is used as the nanoparticle skeleton material
  • Poly(I:C), CpG2006 and CpG2395 are used as adjuvants to prepare nanoparticles internally loaded with lysate components, and then the nanoparticles are combined with cancer cells and activated antigen-presenting cells.
  • the mixed cell membranes work together for a certain period of time to prepare a ready-to-use nano-vaccine for the treatment of cancer.
  • B16F10 cells When collecting tumor tissue, 1.5 ⁇ 10 5 B16F10 cells were first subcutaneously inoculated on the back of each C57BL/6 mouse. When the tumor grew to a volume of approximately 1000 mm 3 , the mice were sacrificed and the tumor tissue was removed. The tumor tissue was cut into sections. Grind, add an appropriate amount of pure water through a cell strainer and freeze and thaw repeatedly 5 times, and can be accompanied by ultrasound to destroy the lysed sample; when collecting the cultured B16F10 cancer cell line, first centrifuge to remove the medium, then wash twice with PBS and centrifuge Cancer cells were collected, resuspended in ultrapure water, frozen and thawed three times, and destroyed and lysed by ultrasound.
  • the nanoparticles were prepared by solvent evaporation method.
  • the molecular weight of PLGA, the preparation material for nanoparticle 1, is 24Da-38KDa.
  • the immune adjuvants used are poly(I:C), CpG2006 and CpG2395, and the adjuvants are contained inside the nanoparticles; when preparing the nanoparticles, the first internal water phase
  • the mass ratio of whole cell lysate components and bacterial extracellular vesicle components contained in it is 3:1.
  • the preparation method is as described above.
  • the double emulsion method is first used to load the whole cell antigen-containing lysate component prepared in step (1) and the bacterial extracellular vesicle component prepared in step (2) inside the nanoparticles. and adjuvant, then 100 mg PLGA nanoparticles were centrifuged at 10,000 g for 20 min, resuspended in 10 mL of ultrapure water containing 4% trehalose and then freeze-dried for 48 h.
  • the average particle size of the nanoparticles 1 is about 350nm; each 1 mg of PLGA nanoparticles 1 is loaded with approximately 100 ⁇ g of protein and peptide components, and 0.025 mg each of poly(I:C), CpG2006 and CpG2395 immune adjuvants.
  • Nanoparticle 2 The preparation materials and preparation methods of Nanoparticle 2 are the same, except that only adjuvants and cancer cell whole cell lysate components are loaded internally, but no bacterial extracellular vesicle components are loaded.
  • the average particle size of Nanoparticle 2 is about 350nm, per 1mg PLGA nanoparticle 2 is loaded with approximately 100 ⁇ g of protein and peptide components, and 0.025 mg each of poly(I:C), CpG2006 and CpG2395 immune adjuvants.
  • Nanoparticle 3 The preparation materials and preparation methods of Nanoparticle 3 are the same, except that they only load adjuvants and bacterial extracellular vesicle components inside, but do not load any cancer cell lysate components.
  • the average particle size of Nanoparticle 3 is about 350nm, and 1 mg of PLGA nanometers Particle 1 is loaded with approximately 100 ⁇ g of protein and peptide components, and 0.025 mg each of poly(I:C), CpG2006 and CpG2395 immune adjuvants.
  • This example uses a mixed antigen-presenting cell of BMDC and B cells.
  • the preparation method of BMDC is the same as in Example 1.
  • the isolation method of B cells is as follows: kill the C57BL/6 mice, remove the mouse spleen, prepare a single cell suspension of mouse splenocytes, and use magnetic bead sorting to separate the living cells in the splenocytes (use live and dead cell dye labeling dead cells to remove dead cells) CD19 + B cells. Then, BMDC and B cells are mixed at a quantity ratio of 1:1 and used as mixed antigen-presenting cells.
  • 1 mg of nanoparticles 1 loaded with whole cell components of cancer cells were mixed with 20 million mixed antigen-presenting cells (including 10 million DCs + 10 million B cells) in 20 mL RPMI1640 complete medium and incubated for 48 hours (37°C , 5% CO 2 ), the incubation system contains IL-2 (1000U/mL), IL-7 (1000U/mL), IL-12 (200U/mL), GM-CSF (500U/mL) and albumin ( 50ng/mL). After the incubation is completed, centrifuge the incubated cells at 400g for 5 minutes, discard the supernatant, and wash twice with PBS to obtain activated antigen-presenting cells.
  • mixed antigen-presenting cells including 10 million DCs + 10 million B cells
  • the membrane fraction When preparing the membrane fraction, first collect the mixed antigen-presenting cells (20 million cells) that have been activated by nanoparticles, centrifuge them at 400g for 5 minutes, and then wash them with 4°C phosphate buffer solution (PBS) containing protease inhibitors. Mix the antigen-presenting cells accordingly twice, resuspend the cells in PBS water and sonicate at 4°C for 3 minutes at low power (12W). The sample was then centrifuged at 3000g for 15 minutes and the supernatant was collected. The supernatant was centrifuged at 8000g for 15 minutes and the supernatant was collected. The supernatant was repeatedly filtered using a 0.22 ⁇ m membrane filter and the sample was centrifuged at 16000g for 90 minutes. Collect and discard the supernatant, collect the precipitate, and resuspend the precipitate in PBS to obtain the cell membrane fraction of the antigen-presenting cells.
  • PBS phosphate buffer solution
  • Collect B16F10 cancer cells discard the supernatant after centrifugation at 400g for 5 minutes, and resuspend the precipitated cells in PBS, then centrifuge at 400g for 5 minutes in 30mM pH 7.0 Tris-HCl buffer containing 0.0759M sucrose and 0.225M mannitol. Wash three times and then twice with PBS containing phosphatase inhibitors and protease inhibitors, followed by high-pressure homogenization (5 mPa) for 1 min to mechanically disrupt the cells. The sample was then filtered through membranes with pore diameters of 30 ⁇ m, 10 ⁇ m, 5 ⁇ m, 2 ⁇ m, and 0.45 ⁇ m. The filtrate was centrifuged at 18,000g for 30 minutes and the supernatant was discarded to collect the precipitate. The precipitate was resuspended in PBS for later use.
  • Nanoparticle 1 or Nanoparticle 2 or Nanoparticle 3 prepared in step (3) Resuspend 100 mg of Nanoparticle 1 or Nanoparticle 2 or Nanoparticle 3 prepared in step (3) in 9 mL of PBS, then mix with 1 mL of 5 mg of the mixed membrane component prepared in step (6), and stir mechanically at 1500 rpm for 10 minutes. Then use repeated co-extrusion through a 0.45 ⁇ m membrane. Collect the extruded liquid, centrifuge it at 13,000g for 25 minutes, discard the supernatant, and resuspend the pellet in PBS to become the nanovaccine.
  • the one prepared using nanoparticles 1 is nanovaccine 1, with a particle size of 370nm;
  • the one prepared using nanoparticles 2 is nanovaccine 2, with a particle size of 370nm;
  • the one prepared using nanoparticles 3 is nanovaccine 3, with a particle size of 370nm.
  • Melanoma tumor-bearing mice were prepared by selecting 6-8 week old female C57BL/6 as model mice. On day 0, 1.5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated into the lower right side of the back of each mouse. On the 4th day, 7th day, 10th day, 15th day, 20th day and 25th day after melanoma inoculation, each mouse was subcutaneously injected with 500 ⁇ g Nano vaccine 1 or Nano vaccine 2 or Nano vaccine 3 or 100 ⁇ L PBS. . In the experiment, the mouse tumor volume was recorded every 3 days starting from the 3rd day. The method for monitoring the mouse tumor volume and survival period was the same as above.
  • Nanovaccine 1, nanovaccine 2 and nanovaccine 3 can significantly inhibit tumor growth and prolong the survival of mice.
  • Nano Vaccine 1 is more effective than Nano Vaccine 2 and Nano Vaccine 3. This shows that the internal loading of bacterial extracellular vesicle components and the internal loading of cancer cell whole cell components are beneficial to improving the effect of cancer nanovaccines loaded with mixed membrane components on the surface.
  • This example uses a mouse melanoma lung model to illustrate the use of nanovaccines to prevent cancer metastasis.
  • B16F10 melanoma tumor tissue is first lysed to prepare water-soluble antigens and water-insoluble antigens of the tumor tissue; then, a nanoparticle system loaded with water-soluble antigens and water-insoluble antigens of the tumor tissue is prepared.
  • siliconization and adding charged substances were used to increase the loading capacity of the antigen, and only one round of mineralization was performed.
  • nanoparticles are first used to activate antigen-presenting cells, and then the antigen-presenting cells are used to prepare nanovaccines and used to prevent cancer metastasis.
  • B16F10 cells 1.5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated on the back of each C57BL/6 mouse.
  • the mice were sacrificed and the tumor tissue was removed. Cut the tumor tissue into pieces and then grind it. Add collagenase and incubate in RPMI 1640 medium for 30 minutes. Then add an appropriate amount of pure water through a cell filter and freeze and thaw repeatedly 5 times. Ultrasound can be used to destroy the lysed cells. After the cells are lysed, centrifuge the lysate at 5000g for 5 minutes and take the supernatant, which is the water-soluble antigen soluble in pure water; add 10% sodium deoxycholate to the resulting precipitate to dissolve the precipitate.
  • the nanoparticles and the blank nanoparticles used as a control were prepared by the solvent evaporation method, and appropriate modifications and improvements were made.
  • two modification methods low-temperature siliconization technology and addition of charged substances, were used to increase the loading capacity of the antigen. .
  • the bacterial membrane components were first dissolved in 8M urea, and then the dissolved bacterial cell membrane components were mixed with the tumor tissue whole cell components.
  • the mass ratio of tumor tissue whole cell components and bacterial membrane components (Bifidobacterium longum or Lactobacillus acidophilus) used in preparing nanoparticles is 1:1.
  • the molecular weight of PLGA, the material used to prepare the nanoparticles, is 24KDa-38KDa, and the immune adjuvants used are poly(I:C), CpG1018 and CpG2395.
  • the preparation method is as mentioned above. During the preparation process, the double emulsion method is first used to load tumor tissue lysate components, bacterial membrane (Bifidobacterium longum or Lactobacillus acidophilus) components and adjuvants inside the nanoparticles, and then 100 mg nanoparticles are loaded into the nanoparticles.
  • the particles were centrifuged at 10,000 g for 20 min, and then the nanoparticles were resuspended in 7 mL of PBS and mixed with 3 mL of PBS solution containing cell lysate (60 mg/mL), followed by centrifugation at 10,000 g for 20 min, and then 10 mL of silicate solution (containing 150 mM NaCl , 80mM tetramethyl orthosilicate and 1.0mM HCl, pH 3.0), resuspended and fixed at room temperature for 10min, then fixed at -80°C for 24h, centrifuged and washed with ultrapure water, and then used 3mL containing protamine (5mg/mL ) and polylysine (10 mg/mL) in PBS and incubated for 10 min, then centrifuged at 10,000 g for 20 min for washing, resuspended in 10 mL of PBS solution containing cell lysate (50 mg/mL) and incubated for 10
  • the nanoparticles loaded with tumor tissue lysate components and Bifidobacterium longum are nanoparticles 1, with an average particle size of about 350nm.
  • Each 1 mg PLGA nanoparticle is loaded with approximately 300 ⁇ g of protein or peptide components.
  • Each 1 mg PLGA nanoparticle 1 is loaded with 0.02 mg each of poly(I:C), CpG1018 and CpG2395.
  • the nanoparticles loaded with tumor tissue lysate components and Lactobacillus acidophilus are nanoparticles 2, with an average particle size of about 350nm.
  • Each 1 mg PLGA nanoparticle is loaded with approximately 300 ⁇ g of protein or peptide components, and each 1 mg PLGA nanoparticle 2 is loaded with poly. (I:C), CpG1018 and CpG2395 0.02mg each.
  • B16F10 melanoma cells Collect the cultured B16F10 melanoma cells, discard the supernatant after centrifugation at 400 g for 5 minutes, wash and mix twice with 4°C phosphate buffer solution (PBS) containing protease inhibitors, and resuspend the cells in PBS. 10 million B16F10 cells were sonicated at 4°C for 1 minute at low power (15W), then the sample was centrifuged at 3000g for 15 minutes and the supernatant was collected. The supernatant was centrifuged at 8000g for 15 minutes and the supernatant was collected.
  • PBS phosphate buffer solution
  • the supernatant was mixed with the 3 mg bacterial (Bifidobacterium longum or Lactobacillus acidophilus) membrane component prepared in step (2) and incubated at room temperature for 10 minutes, and then repeatedly co-extruded using a 0.22 ⁇ m filter to collect the extruded liquid. Then, centrifuge at 16000 g for 90 minutes, collect and discard the supernatant, collect the precipitate, and resuspend the precipitate in PBS to obtain the mixed cell membrane components.
  • 3 mg bacterial (Bifidobacterium longum or Lactobacillus acidophilus) membrane component prepared in step (2) was incubated at room temperature for 10 minutes, and then repeatedly co-extruded using a 0.22 ⁇ m filter to collect the extruded liquid. Then, centrifuge at 16000 g for 90 minutes, collect and discard the supernatant, collect the precipitate, and resuspend the precipitate in PBS to obtain the mixed cell membrane components.
  • the cancer cells were sonicated at 4°C for 1 minute at low power (15W), then the sample was centrifuged at 3000g for 15 minutes and the supernatant was collected, the supernatant was collected after centrifugation at 8000g for 15 minutes, and then 0.22 ⁇ m
  • the filter membrane is extruded repeatedly, and the extruded liquid is collected and centrifuged at 16,000g for 90 minutes. The supernatant is discarded to collect the precipitate.
  • the precipitate is resuspended in PBS to obtain the cancer cell membrane components.
  • Nanoparticle 1 prepared in step (3) Resuspend 100 mg of Nanoparticle 1 prepared in step (3) in 9 mL PBS, and then mix with 10 mL of 20 mg of the cancer cell and Bifidobacterium longum bacterial membrane components prepared in step (4); Particle 2 is resuspended in 9 mL PBS, and then mixed with 10 mL of 20 mg of the cancer cell and Lactobacillus acidophilus bacterial membrane components prepared in step (4); or 100 mg of nanoparticle 1 prepared in step (3) is resuspended in 9 mL PBS, Then mix it with 10 mL of 20 mg of the cancer cell membrane component prepared in step (4).
  • the mixture of the above nanoparticles and membrane components was treated with a homogenizer at 1500 rpm for 5 minutes, and then repeatedly co-extruded using a 0.45 ⁇ m filter. The extruded liquid was collected and centrifuged at 13000g for 20 minutes, and the supernatant was discarded. Resuspend the pellet in PBS and it will be the nano vaccine.
  • the nanovaccine prepared using the mixed membrane components of cancer cells and Bifidobacterium longum is Nanovaccine 1, with a particle size of 370 nm; the nanovaccine prepared using the mixed membrane components of cancer cells and Lactobacillus acidophilus is Nanovaccine 2, with particles The particle size is 370nm; the nanovaccine prepared using cancer cell membrane components is Nanovaccine 3, and the particle size is 370nm.
  • mice were prepared by selecting 6-8 week old female C57BL/6 as model mice. On day -35, day -28, day -21, day -14 and day -7 before mouse cancer modeling, each mouse was subcutaneously injected with 200 ⁇ g of nano vaccine 1 or nano vaccine 2 or nano vaccine 3 or 100 ⁇ L PBS. On day 0, each mouse was intravenously inoculated with 1 ⁇ 10 5 B16F10 cells. On day 16, the mice were sacrificed, and the number of melanoma cancer foci in the lungs of the mice was observed and recorded.
  • mice in the PBS control group had more and larger cancer foci, while the number of cancers in mice treated with nanovaccine was significantly reduced.
  • the preventive effect of Nano Vaccine 1 and Nano Vaccine 2 on cancer lung metastasis is significantly better than that of Nano Vaccine 3; and the preventive effect of Nano Vaccine 1 on cancer lung metastasis is significantly better than that of Nano Vaccine 2.
  • colon cancer tumor tissue is first lysed to prepare a water-soluble antigen mixture (mass ratio 1:1) and a water-insoluble antigen mixture (mass ratio 1:1), and the water-soluble antigen mixture and the water-insoluble antigen mixture are divided according to mass Mix ratio 1:1.
  • PLA as the nanoparticle skeleton material
  • CpGSL03 and Poly ICLC as immune adjuvants to prepare nanoparticles
  • Pure water converts water-insoluble antigens into soluble ones in aqueous solution.
  • Water-soluble antigens from colon cancer tumor tissue and lung cancer cancer cells are mixed at a mass ratio of 1:1; this mixture is the source of raw materials for preparing nanoparticles.
  • the nanoparticles are prepared using a solvent evaporation method, and the tumor tissue lysate components, the bacterial membrane components lysed and dissolved in step (2), and the immune adjuvant are loaded into the nanoparticles.
  • the molecular weight of PLA, the material used to prepare the nanoparticles, is 20KDa.
  • the immune adjuvants used are CpG SL03 and Poly ICLC, and the adjuvants are distributed inside the nanoparticles.
  • the preparation method is as mentioned above. During the preparation process, the double emulsion method is first used to load the lysate mixture and adjuvant inside the nanoparticles.
  • nanoparticles After loading the lysate and adjuvant inside, 100 mg nanoparticles are centrifuged at 10000g for 20 minutes, and 10 mL containing Resuspend in 4% trehalose ultrapure water and freeze-dry for 48 h.
  • the nanoparticles prepared by using bacterial membrane components dissolved in 8M urea are nanoparticles 1.
  • the average particle size of the nanoparticles is about 280nm.
  • Each 1 mg PLGA nanoparticle is loaded with approximately 90 ⁇ g of protein or peptide components.
  • Each 1 mg PLGA nanoparticle contains CpGSL03 and Poly. 0.03mg each of ICLC immune adjuvants; the nanoparticles prepared using bacterial membrane components dissolved in Tween 80 are nanoparticles 2.
  • the average particle size of the nanoparticles is about 280nm, and each 1 mg of PLGA nanoparticles is loaded with approximately 90 ⁇ g of protein or peptide components. , each 1mg of PLGA nanoparticles contains 0.03mg each of CpGSL03 and Poly ICLC immune adjuvant.
  • MC38 cells were cultured in DMEM high-glucose medium containing 50 ng/mL doxorubicin for 6 days, during which the medium was changed every 2 days.
  • the cultured MC38 cells were collected and used in 4°C phosphate buffer solution (PBS) containing protease inhibitors.
  • PBS 4°C phosphate buffer solution
  • Nanoparticle 1 prepared in step (3) in 9 mL PBS, mix with 1 mL of cancer cell extracellular vesicles (5 mg) and bacterial extracellular vesicles (5 mg) prepared in step (4), and then incubate at 4°C. 10W ultrasonic for 2 minutes, then use 0.45 ⁇ m filter membrane to repeatedly co-extrude. Centrifuge the extrudate at 13000g for 30 minutes. Discard the supernatant and resuspend the pellet in PBS to obtain Nano Vaccine 1. The particle size is 300nm.
  • Nano Vaccine 3 Resuspend 100 mg of nanoparticle 1 prepared in step (3) in 9 mL PBS, then mix with 1 mL of bacterial outer vesicles (10 mg) prepared in step (4), and then ultrasonicate at 4°C for 10 minutes at 10 W, and then incubate at 13000 g Centrifuge for 20 minutes, discard the supernatant, and resuspend the pellet in PBS to obtain Nano Vaccine 3, with a particle size of 300 nm.
  • mice Female C57BL/6 mice aged 6-8 weeks were selected as model mice to prepare colon cancer tumor-bearing mice. On day 0, each mouse was subcutaneously inoculated with 2 ⁇ 10 6 MC38 cells, and on days 4, 7, 10, 15, and 20, each mouse was subcutaneously injected with 800 ⁇ g nanovaccine 1 Or 800 ⁇ g Nanovaccine 2 or 800 ⁇ g Nanovaccine 3 or 100 ⁇ g PBS. Tumor growth and mouse survival monitoring methods were the same as above.
  • Nanovaccine 1 is better than Nanovaccine 2 and Nanovaccine 3. It can be seen that the addition of extracellular vesicle membrane components of cancer cells is beneficial to the efficacy of the vaccine. Moreover, the effects of nanovaccines prepared internally by loading bacterial membrane components obtained by lysis with different lysis solutions into nanovaccines are different.
  • This example uses melanoma as a cancer model to illustrate how to treat melanoma using a nanovaccine loaded internally with whole-cell components of melanoma tumor tissue and with surface-loaded lung cancer cells and antigen-presenting cell membrane components.
  • the water-soluble components and non-water-soluble components in the B16F10 melanoma tumor tissue are first cleaved. Then use PLGA as the nanoparticle skeleton material, manganese particles and CpG2395 as immune adjuvants to prepare nanoparticles, and then load membrane components on the surface of the nanoparticles for cancer treatment.
  • B16F10 cells were subcutaneously inoculated on the back of each C57BL/6 mouse.
  • the mice were sacrificed and the tumor tissue was removed.
  • the tumor tissue was cut into small pieces and then ground and filtered through a cell mesh to obtain a tumor tissue single cell suspension.
  • the tumor tissue single cell suspension was repeatedly frozen and thawed 5 times, supplemented by ultrasonic lysis. Add nuclease to the lysis solution and incubate at 37°C for 10 minutes, and then heat the lysis solution containing nuclease at 95°C for 5 minutes.
  • the antigen source for preparing nanoparticles is obtained by mixing the water-soluble component from melanoma tumor tissue and the original non-water-soluble component dissolved in the octylglucoside aqueous solution at a mass ratio of 3:1.
  • the nanoparticles were prepared using the double emulsion method.
  • the molecular weight of the nanoparticle preparation material PLGA used is 24KDa-38KDa
  • the immune adjuvant used is a mixed adjuvant of STING agonist and Toll-like receptor agonist: manganese colloidal particles (STING agonist) and CpG2395 (Toll-like receptor agonist). body agonist).
  • the manganese adjuvant is first prepared, and then the manganese adjuvant is mixed with the water-soluble component or the non-water-soluble component in the whole cell component of the cancer cells and is used as the first aqueous phase to prepare the internal load lysate component and adjuvant using the double emulsion method. of nanoparticles.
  • manganese adjuvant When preparing manganese adjuvant, first add 1mL of 0.3M Na 3 PO 4 solution to 9mL of physiological saline, then add 2mL of 0.3M MnCl 2 solution, and leave it overnight to obtain Mn 2 OHPO 4 colloidal manganese adjuvant.
  • the manganese adjuvant particle size is approximately 13 nm. Then, mix the manganese adjuvant with the water-soluble component (60 mg/mL) or the non-water-soluble component (60 mg/mL) of the whole cell component of cancer cells at a volume ratio of 1:3, and then use the double emulsion method to combine the antigen and manganese adjuvant.
  • the agent is loaded into the nanoparticles.
  • the nanoparticles were centrifuged at 10,000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours before use.
  • the average particle size of the nanoparticles is about 370nm, and the surface potential of the nanoparticles is about -5mV; each 1 mg of PLGA nanoparticles is loaded with approximately 120 ⁇ g of protein and peptide components, and the CpG2395 adjuvant used per 1 mg of PLGA nanoparticles is 0.04 mg.
  • Antigen-presenting cells are mixed antigen-presenting cells derived from B cells and BMDCs from peripheral blood.
  • the preparation method of BMDC is the same as above.
  • Peripheral blood mononuclear cells (PBMC) were isolated from the peripheral blood of mice after killing C57BL/6, and then CD19 + B cells were sorted from PBMC using flow cytometry. Mix BMDC and B cells at a ratio of 1:1 to form mixed antigen-presenting cells.
  • step (2) Mix 1 mg of nanoparticles loaded with whole cell components of cancer cells prepared in step (2) with 20 million mixed antigen-presenting cells (including 10 million BMDCs + 10 million B cells) in 20 mL of RPMI1640 complete medium and incubate them together. 48 hours (37°C, 5% CO 2 ), the incubation system contains IL-21 (500U/mL), IL-2 (500U/mL), IL-7 (500U/mL) and CD80 antibody (10ng/mL) . After the incubation is completed, centrifuge the incubated cells at 400g for 5 minutes, discard the supernatant, and wash twice with PBS to obtain activated antigen-presenting cells.
  • mixed antigen-presenting cells including 10 million BMDCs + 10 million B cells
  • a control membrane fraction only activated mixed antigen-presenting cells were used.
  • the mixed antigen-presenting cells were washed twice with PBS containing phosphatase inhibitors and protease inhibitors, and then mechanically disrupted by sonication at 15W for 2 minutes. Centrifuge the sample at 3000g for 5 minutes, discard the precipitate, and centrifuge the supernatant at 8000g for five minutes, then discard the precipitate. Centrifuge the supernatant at 15000g for 60 minutes, discard the supernatant, and collect the precipitate. Resuspend the precipitate in PBS. After suspension, the cell membrane components of the mixed antigen-presenting cells are obtained.
  • nanovaccine 1 Resuspend the nanoparticles (100 mg) prepared in step (2) in 9 mL PBS, then mix with 1 mL of the mixed cell membrane fraction (2 mg) of cancer cells and antigen-presenting cells prepared in step (4), and add DSPE to the above mixture.
  • -PEG-CD32 monoclonal antibody 0.1mg
  • sonicated at 50W for 2 minutes and then repeatedly co-extruded using a 0.45 ⁇ m filter membrane. Centrifuge the extruded liquid at 12000g for 20 minutes and then discard the supernatant to collect the precipitate.
  • nanovaccine 1 After being resuspended in PBS, nanovaccine 1 is obtained.
  • the nanovaccine particle size is 390 nanometers and the surface potential is -4mV.
  • step (2) Resuspend the nanoparticles (100 mg) prepared in step (2) in 9 mL PBS, and then mix them with 1 mL of the mixed antigen-presenting cell membrane component (2 mg) prepared in step (4).
  • mice were prepared by selecting 6-8 week old female C57BL/6 as model mice. On day 0, each mouse was subcutaneously inoculated with 1.5 ⁇ 10 5 B16F10 cells, and on days 5, 8, 11, 16, and 21, mice were subcutaneously injected with 500 ⁇ g nanovaccine 1 or 500 ⁇ g. Nanovaccine 2 or 100 ⁇ PBS. The mouse tumor volume and survival period were monitored as above.
  • the nanovaccine of the present invention has a therapeutic effect on cancer.
  • the nanovaccine 1 loaded with cancer cells and mixed antigen-presenting cell membrane components on the surface is more effective than the nanovaccine 2 loaded with only mixed antigen-presenting cell membrane components on the surface.
  • the nanovaccine uses CD32 monoclonal antibody as the active target. In practical applications, any target with the ability to target target cells, such as mannose, mannan, CD205 monoclonal antibody, CD19 monoclonal antibody, etc., can also be used. head.
  • 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 inactivated and denatured using ultraviolet light and high-temperature heating respectively, and then an appropriate amount of 6M guanidine hydrochloride aqueous solution is used to lyse the breast cancer cells and dissolve the lysate, which is the source of the antigen raw material.
  • the double emulsion method is used to prepare micron particles.
  • the molecular weight of the micron particle skeleton material PLGA is 38KDa-54KDa.
  • the immune adjuvants used are CpG2395, CpG1018 and Poly(I:C).
  • During preparation first use the double emulsion method to prepare micron particles internally loaded with lysate components and adjuvants, then centrifuge 100 mg of micron particles at 9000g for 20 minutes, resuspend in 10 mL of ultrapure water containing 4% trehalose, and dry for 48 hours before use.
  • the average particle size of this micron particle system is about 2.10 ⁇ m, and the surface potential is about -6mV; each 1 mg of PLGA micron particles is loaded with approximately 110 ⁇ g of protein or polypeptide components, including 0.03 mg each of CpG2395, CpG1018, and Poly(I:C).
  • Antigen-presenting cells are mixed antigen-presenting cells derived from B cells and BMDCs from peripheral blood.
  • the preparation method of BMDC is the same as above.
  • Peripheral blood mononuclear cells (PBMC) were isolated from the peripheral blood of mice after killing C57BL/6, and then CD19 + B cells were sorted from PBMC using flow cytometry. Mix BMDC and B cells at a ratio of 1:1 to form mixed antigen-presenting cells.
  • step (2) Mix 1 mg of micron particles loaded with whole cell components of cancer cells prepared in step (2) with 20 million mixed antigen-presenting cells (including 10 million BMDC + 10 million B cells) in 20 mL RPMI1640 complete medium and incubate for 48 hours (37°C, 5% CO 2 ), the incubation system contains IL-2 (500U/mL), IL-7 (500U/mL), IL-15 (500U/mL), and GM-CSF (500U/mL) . After the incubation is completed, centrifuge the incubated cells at 400g for 5 minutes, discard the supernatant, and wash twice with PBS to obtain activated antigen-presenting cells.
  • mixed antigen-presenting cells including 10 million BMDC + 10 million B cells
  • Collect the cell culture 4T1 cells centrifuge at 400g for 5 minutes, discard the supernatant, and resuspend the precipitated cells in PBS.
  • cancer cells and BMDC cells activated in step (3) were used to prepare.
  • 4T1 cells and BMDC were mixed at a ratio of 1:1, and then the mixed cells were washed twice with PBS containing phosphatase inhibitors and protease inhibitors, and then mechanically disrupted by ultrasonic treatment at 10W for 5 minutes. Centrifuge the sample at 2000g for 5 minutes, discard the precipitate, and centrifuge the supernatant at 6000g for 10 minutes, then discard the precipitate. Centrifuge the supernatant at 15000g for 60 minutes, discard the supernatant, collect the precipitate, and rehydrate the precipitate in PBS. After suspension, the mixed cell membrane components of cancer cells and DCs are obtained.
  • Micron Vaccine 1 Resuspend the microparticles (100 mg) prepared in step (2) in 9 mL PBS, then mix with 1 mL of the cancer cells and mixed antigen-presenting cell membrane components (2 mg) prepared in step (4), ultrasonicate at 20W for 5 minutes, and then After centrifugation at 10,000 g for 15 minutes, discard the supernatant and collect the precipitate. Resuspend the precipitate in PBS to obtain Micron Vaccine 1.
  • the particle size of Micron Vaccine 1 is 2.12 ⁇ m and the surface potential is -4mV.
  • Micron Vaccine 2 Resuspend the micron particles (100mg) prepared in step (2) in 9mL PBS, then mix with 1mL mixed cell membrane fraction (2mg) of cancer cells and DCs, sonicate at 20W for 5 minutes, and then centrifuge at 10000g for 15 minutes. Discard the supernatant and collect the precipitate. Resuspend the precipitate in PBS to obtain Micron Vaccine 2.
  • the particle size of Micron Vaccine 2 is 2.12 ⁇ m and the surface potential is -4mV.
  • mice Female BALB/c mice aged 6-8 weeks were selected as model mice to prepare breast cancer tumor-bearing mice. Each mouse in the vaccine group was vaccinated with 800 ⁇ g Micron vaccine 1 or 800 ⁇ g Micron vaccine 2 or 100 ⁇ L PBS. On day 0, each mouse was subcutaneously inoculated with 1 ⁇ 10 6 4T1 cells, and the mouse tumor and survival monitoring methods were the same as above.
  • the tumor growth rate of mice treated with micron vaccine was significantly slower and the survival period was significantly prolonged.
  • the micron vaccine of the present invention has a preventive effect on breast cancer.
  • the effect of Micron Vaccine 1 is better than that of Micron Vaccine 2, which means that the cell membrane components of the micron vaccine loaded with cancer cells and mixed antigen-presenting cells are more effective than the cell membrane components of the micron vaccine loaded with cancer cells and single antigen-presenting cells. Theoretically, the process of activating T cells after the vaccine is taken up by antigen-presenting cells.
  • DC is the most important antigen-presenting cell ( The initial activation of T cells), the DC cell membrane can better guide the micron vaccine to be engulfed by DC. This experiment proves that the cell membrane components of B cells can improve the DC cell membrane components to guide the micron vaccine to be engulfed by DC and subsequently activate T cells for the first time.
  • Example 8 Nanovaccine with membrane components loaded on the surface of calcified nanoparticles for cancer prevention
  • This example illustrates the use of calcified nanoparticle surface-loaded film components to prepare nanovaccines to prevent cancer.
  • other biomineralization technologies cross-linking, gelation and other modified particles can also be used.
  • mouse melanoma tumor tissue was lysed and dissolved with 10% sodium deoxycholate aqueous solution (containing 8M arginine), the tumor tissue lysate was loaded on nanoparticles, and membrane components were loaded on the surface of the nanoparticles. Preparation of nanovaccines for cancer prevention.
  • Collect mouse B16F10 melanoma tumor tissue cut the tumor tissue into small pieces, lyse the tumor tissue with 10% sodium deoxycholate aqueous solution (containing 8M arginine), and dissolve the whole cell component of the tumor tissue.
  • the nanoparticles are biocalcified after loading whole cell components of cancer cells inside and on the surface of the nanoparticles.
  • nanoparticle 1 is prepared by a solvent evaporation method.
  • the molecular weight of the nanoparticle preparation material PLGA used is 7KDa-17KDa.
  • the immune adjuvants CpG2006 and Poly(I:C) are loaded inside the nanoparticles.
  • the preparation method is as follows.
  • the double emulsion method is first used to load the lysate components inside the nanoparticles, then 100mg PLGA nanoparticles are centrifuged at 13000g for 20 minutes and resuspended in 18mL PBS, and then 2mL of tumor dissolved in 8M urea is added. Tissue lysis solution (60 mg/mL) was incubated at room temperature for 10 minutes, centrifuged at 12,000 g for 20 minutes, and the precipitate was collected. The 100 mg PLGA nanoparticles were then resuspended in 20 mL DMEM medium, and then 200 ⁇ L of CaCl 2 (1 mM) was added and reacted at 37°C for two hours.
  • Nanoparticle 1 the average particle size is about 240nm, each 1 mg of PLGA nanoparticles is loaded with approximately 140 ⁇ g of protein or peptide components, 0.03 mg of CpG2006 and Poly(I:C) each.
  • the preparation materials and methods of blank nanoparticle 2 are the same as nanoparticle 1, but only the immune adjuvant is loaded and not the cancer cell lysate component.
  • the average particle size is about 240nm, and each 1 mg PLGA nanoparticle is loaded with approximately 140 ⁇ g of protein or peptide components. , CpG2006 and Poly(I:C) 0.03mg each.
  • This example uses DC2.4 cell line and BMDM as antigen-presenting cells.
  • the preparation method of BMDM is as follows: C57 mice are anesthetized and killed by dislocation. The mice are disinfected with 75% ethanol. Then use scissors to cut a small opening on the back of the mouse. Tear the skin directly to the calf joint of the mouse and remove it. Mouse foot joints and skin. Use scissors to remove the hind limbs along the greater trochanter at the root of the mouse's thigh, remove the muscle tissue and place it in a petri dish containing 75% ethanol to soak for 5 minutes. Replace the petri dish with a new one with 75% ethanol and move it to a clean bench.
  • Macrophage colony-stimulating factor stimulates bone marrow cells to differentiate into mononuclear macrophages at a concentration of 40ng/mL. After culturing for 8 days, the morphological changes of macrophages were observed under a light microscope. After 8 days, the cells were digested and collected, and incubated with anti-mouse F4/80 antibody and anti-mouse CD11b antibody for 30 minutes at 4°C in the dark, and flow cytometry was used to identify the proportion of successfully induced macrophages.
  • M-CSF Macrophage colony-stimulating factor
  • the nanoparticles 1 (1000 ⁇ g) prepared in step (2) were incubated with DC2.4 (5 million cells) and BMDM cells (5 million cells) in 15 mL high-glucose DMEM complete medium for 48 hours (37°C, 5% CO 2 ); the incubation system contains GM-CSF (2000U/mL), IL-2 (500U/mL), IL-7 (200U/mL), IL-12 (200U/mL) and CD40 antibody (20ng/mL).
  • the incubated cells were collected, centrifuged at 400g for 5 minutes, resuspended in PBS and washed twice. That is, mixed antigen-presenting cells are obtained.
  • Lactobacillus rhamnosus was cultured in LB medium containing 30 ⁇ M doxorubicin, and then the cultured samples were collected and centrifuged at 5000g for 30 minutes. The supernatant was collected after discarding the pellet and ultrasonicated at 15W for 10 minutes. , centrifuge the supernatant at 16000g for 90 minutes, and resuspend the pellet in PBS to obtain the collected bacterial outer vesicle component 1.
  • B16F10 cells were cultured in high-glucose DMEM complete medium for 6 days.
  • the medium contained 30 ⁇ M doxorubicin, and the medium was changed every 2 days.
  • Collect the cell culture B16F10 cells discard the supernatant after centrifugation at 400g for 5 minutes, and use PBS to resuspend the precipitated cells.
  • Ultrasonicate the cancer cells at 20W for 2 minutes to mechanically disrupt the cells. Centrifuge the sample at 2000g for 5 minutes and discard. After precipitation, centrifuge the supernatant at 6000g for 10 minutes, then discard the precipitate. Centrifuge the supernatant at 15000g for 60 minutes, then discard the supernatant to collect the precipitate. Resuspend the precipitate in PBS to obtain the cancer cell membrane component 1. .
  • Or culture B16F10 cells in high-glucose DMEM complete medium for 6 days, and change the medium every 2 days. Collect the cell culture B16F10 cells, discard the supernatant after centrifugation at 400g for 5 minutes, and resuspend the precipitated cells in PBS. Ultrasonicate the cancer cells at 20W for 2 minutes to mechanically disrupt the cells. Centrifuge the sample at 2000g for 5 minutes and discard. After precipitation, centrifuge the supernatant at 6000g for 10 minutes, then discard the precipitate. Centrifuge the supernatant at 15000g for 60 minutes, then discard the supernatant to collect the precipitate. Resuspend the precipitate in PBS to obtain the cancer cell membrane fraction 2. .
  • Nanoparticle 1 (100 mg) prepared in step (2) in 9 mL PBS, and then mix it with 1 mL of the mixed antigen-presenting cell membrane component (5 mg) prepared in step (3) and the cancer cell membrane component 1 prepared in step (4).
  • 5mg) and bacterial external vesicle component 1 were mixed, sonicated at 20W for 5 minutes, and then repeatedly co-extruded using a 0.45 ⁇ m filter membrane, and then centrifuged at 13000g for 30 minutes and then discarded the supernatant to collect the precipitate.
  • the particle size of Nano Vaccine is 260nm.
  • Nanoparticle 2 (100 mg) prepared in step (2) in 9 mL PBS, and then mix it with 1 mL of the mixed antigen-presenting cell membrane component (5 mg) prepared in step (3) and the cancer cell membrane component 1 prepared in step (4).
  • 5mg) and bacterial external vesicle component 1 were mixed, sonicated at 20W for 5 minutes, and then repeatedly co-extruded using a 0.45 ⁇ m filter membrane, and then centrifuged at 13000g for 30 minutes and then discarded the supernatant to collect the precipitate.
  • the particle size of Nano Vaccine is 260nm.
  • Nanoparticle 1 100 mg
  • step (2) Resuspend the nanoparticle 1 (100 mg) prepared in step (2) in 9 mL PBS, and then mix it with 1 mL of the mixed antigen-presenting cell membrane component (5 mg) prepared in step (3) and the cancer cell membrane component 2 prepared in step (4).
  • 5mg and bacterial external vesicle component 2 were mixed, sonicated at 20W for 5 minutes, and then repeatedly co-extruded using a 0.45 ⁇ m filter membrane, and then centrifuged at 13000g for 30 minutes and then discarded the supernatant to collect the precipitate.
  • the particle size of Nano Vaccine is 260nm.
  • mice Select 6-8 week old female C57BL/6 as model mice to prepare melanoma tumor-bearing mice. On days -35, -28, -21, -14 and -14 before the mice are inoculated with cancer cells, On day -7, each mouse was vaccinated with 800 ⁇ g Nano Vaccine 1, or 800 ⁇ g Nano Vaccine 2, or 800 ⁇ g Nano Vaccine 3, or 100 ⁇ L PBS. On day 0, each recipient mouse was inoculated subcutaneously with 1.5 ⁇ 10 5 B16F10 cells on the lower right side of the back. The methods for monitoring mouse tumor volume and survival were as above.
  • Nano Vaccine 1 is more effective than Nano Vaccine 2 and Nano Vaccine 3, indicating that loading the whole cell components of cancer cells inside the Nano Vaccine is beneficial to improving the efficacy of the vaccine.
  • the cell membrane prepared by using doxorubicin-pretreated cancer cells and bacteria Components can also improve the efficacy of nanovaccines.
  • the supernatant part is the water-soluble antigen; the precipitate part uses 10% sodium deoxycholate aqueous solution to dissolve the non-water-soluble antigen.
  • the water-soluble antigen and the non-water-soluble antigen dissolved in sodium deoxycholate are miscible at a mass ratio of 1:1, which is the source of the antigen raw material for preparing the nanoparticle system.
  • the nanoparticles were prepared using the double emulsion method.
  • the preparation material of nanoparticle 1 is PLGA with a molecular weight of 24KDa-38KDa.
  • the immune adjuvants used are poly(I:C), CpG1018 and CpG2216.
  • the substance that increases lysosomal immune escape is KALA polypeptide (WEAKLAKALAKALAKHLAKALAKALKACEA), and the adjuvants, KALA peptide is contained inside the nanoparticles.
  • the preparation method is as mentioned above. During the preparation process, the double emulsion method is first used to load the lysis solution components, adjuvants, and KALA peptides inside the nanoparticles.
  • each 1 mg of PLGA nanoparticles is loaded with approximately 100 ⁇ g of protein or peptide components, and each 1 mg of PLGA nanoparticles is loaded with poly(I:C), CpG1018 and CpG2216 immune
  • Each adjuvant contains 0.02 mg, and the loaded KALA polypeptide is 0.05 mg.
  • nanoparticle 2 The preparation materials and preparation method of nanoparticle 2 are the same as above. Its particle size is about 250nm, its surface potential is about -5mV, it does not load KALA polypeptide, and it loads equal amounts of adjuvants and cell lysis components.
  • nanoparticle 3 The preparation materials and preparation methods of nanoparticle 3 are the same as above. Its particle size is about 250nm and its surface potential is about -5mV. Each 1mg PLGA nanoparticle is loaded with approximately 100 ⁇ g of protein and polypeptide components. Each 1mg PLGA nanoparticle is loaded with poly(I :C)0.02mg, loaded CpG1018 is 0.04mg, loaded KALA polypeptide is 0.05mg.
  • the antigen-presenting cells are BMDCs, and their preparation method is the same as above.
  • the incubation system contains IL-2 (500 U/mL) , IL-7 (500U/mL), IL-15 (500U/mL), GM-CSF (500U/mL).
  • the incubated cells were centrifuged at 400g for 5 minutes, the supernatant was discarded, and then washed twice with PBS containing phosphatase inhibitors and protease inhibitors, and then ultrasonicated at 15W for 2 minutes to mechanically disrupt the cells. Centrifuge the sample at 3000g for 5 minutes. After discarding the precipitate, centrifuge the supernatant at 8000g for five minutes. Then discard the precipitate. Use a 0.22 ⁇ m filter to repeatedly co-extrude the supernatant. Centrifuge the extrudate at 15000g for 60 minutes. Discard the supernatant, collect the precipitate, and resuspend the precipitate in PBS to obtain the antigen-presenting cell membrane fraction.
  • step (2) Resuspend the nanoparticles (100 mg) prepared in step (2) in 9 mL PBS, then mix with 1 mL of the cancer cell membrane component (2 mg) prepared in step (3), ultrasonicate at 4°C for 3 minutes at low power (20W), and then After centrifugation at 12000 g for 60 minutes, collect and discard the supernatant to collect the precipitate. Resuspend the precipitate in PBS to obtain the nano vaccine.
  • the vaccine prepared from nanoparticle 1 is nanovaccine 1, with a particle size of 260 nanometers, and a surface charge of -5mV;
  • the vaccine prepared from nanoparticles 2 is nanovaccine 2, with a particle size of 260 nanometers, and a surface charge of -5mV;
  • nanoparticle 3 is prepared
  • the vaccine is Nano Vaccine 3, with a particle size of 260 nanometers and a surface charge of -5mV.
  • nano vaccine 4 is obtained, with a particle size of 160 nanometers and a surface charge of -5mV.
  • Nano vaccines are used to treat cancer
  • Melanoma tumor-bearing mice were prepared by selecting 6-8 week old female C57BL/6 as model mice. On day 0, 1.5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated into the lower right side of the back of each mouse. On days 6, 10, 15 and 20 after melanoma inoculation, 60 ⁇ g of one of nanovaccines 1-4 or 100 ⁇ g of PBS were administered subcutaneously. In the experiment, the mouse tumor volume and survival period were monitored as above.
  • Nanovaccine prepared from cell membrane components is presented 4.
  • the nanovaccine 1 prepared by adding nanoparticles that increase lysosome escape substances is better than the nanovaccine 2 prepared by not adding nanoparticles that increase lysosome escape.
  • nanovaccine 1 prepared using nanoparticles using two kinds of CpG and Poly(I:C) as mixed adjuvants is better than that of nanoparticles prepared using only one type of CpG and Poly(I:C) mixed adjuvants.
  • Vaccine 3 This shows that internal loading of whole cell components of cancer cells is crucial for preparing cancer cell membrane components into nano-vaccines.
  • the nanovaccine according to the present invention has good therapeutic effect on cancer.
  • KALA polypeptide is used as a lysosome escape substance loaded inside nanoparticles or microparticles.
  • any substance that increases lysosome escape can be used, such as polypeptides, amino acids, organic polymer substances, and proton sponges. Effect of inorganic substances, etc.
  • Example 10 Nanovaccines with membrane components loaded on the surface of nanoparticles are used to prevent cancer
  • Mouse Pan02 pancreatic cancer cells were collected, the cancer cells were lysed with 10% sodium dodecyl sulfate aqueous solution, and then the whole cell components of the cancer cells were dissolved.
  • the nanoparticles are prepared by a solvent evaporation method.
  • the molecular weight of the nanoparticle preparation material PLGA used is 7KDa-17KDa, and the immune adjuvants CpG2395 and Poly(I:C) are used.
  • the immune adjuvant is loaded inside the nanoparticles together with the cancer cell lysate component, 8M urea or Triton-solubilized bacterial membrane component.
  • the mass ratio of the cancer cell lysate component to the bacterial membrane component solubilized by 8M urea or Triton is 1:1.
  • the preparation method is as follows.
  • the double emulsion method is first used to load the lysate component inside the nanoparticles, and then 100mg PLGA nanoparticles are centrifuged at 13000g for 20 minutes and then reused with 10mL of an aqueous solution containing 2% sucrose and 2% mannitol. After suspension, freeze-dry for 48 hours.
  • the nanoparticles loaded with immune adjuvants, cancer cell lysate components, and 8M urea-dissolved bacterial membrane components are nanoparticles 1, with an average particle size of about 240nm.
  • Each 1 mg PLGA nanoparticle is loaded with approximately 140 ⁇ g of protein or peptide components, CpG2006 and Poly(I:C) 0.03 mg each.
  • the nanoparticles loaded with immune adjuvants, cancer cell lysate components, and Triton-dissolved bacterial membrane components are nanoparticles 2, with an average particle size of about 240 nm.
  • Each 1 mg PLGA nanoparticle is loaded with approximately 140 ⁇ g of protein or peptide components, and CpG2006 and Poly(I:C) 0.03 mg each.
  • This example uses DC2.4 cell line and BMDM as antigen-presenting cells.
  • the preparation method of BMDM is the same as in Example 8.
  • the nanoparticles 1 (1000 ⁇ g) prepared in step (3) were incubated with DC2.4 (5 million cells) and BMDM cells (5 million cells) in 15 mL high-glucose DMEM complete medium for 48 hours (37°C, 5% CO 2 ); the incubation system contains GM-CSF (2000U/mL), IL-2 (500U/mL), IL-7 (200U/mL), IL-12 (200U/mL) and CD40 antibody (20ng/mL).
  • the incubated cells were collected, centrifuged at 400g for 5 minutes, resuspended in PBS and washed twice. That is, mixed antigen-presenting cells are obtained.
  • Bifidobacterium breve was cultured in LB medium containing 10 nM sunitinib. The cultured samples were then collected and centrifuged at 5000g for 30 minutes. The supernatant was collected after discarding the pellet and ultrasonicated at 15W for 10 minutes. , centrifuge the supernatant at 16000g for 90 minutes, and resuspend the pellet in PBS to obtain the collected bacterial outer vesicle component 1.
  • B16F10 cells were cultured in high-glucose DMEM complete medium for 6 days.
  • the medium contained 10 nM sunitinib, and the medium was changed every 2 days.
  • Collect the cell culture B16F10 cells discard the supernatant after centrifugation at 400g for 5 minutes, and resuspend the precipitated cells in PBS.
  • Ultrasonicate the cancer cells at 20W for 2 minutes to mechanically disrupt the cells. Centrifuge the sample at 2000g for 5 minutes and discard. After precipitation, centrifuge the supernatant at 6000g for 10 minutes, then discard the precipitate. Centrifuge the supernatant at 15000g for 60 minutes, then discard the supernatant to collect the precipitate. Resuspend the precipitate in PBS to obtain the cancer cell membrane component 1. .
  • Or culture B16F10 cells in high-glucose DMEM complete medium for 6 days, and change the medium every 2 days. Collect the cell culture B16F10 cells, discard the supernatant after centrifugation at 400g for 5 minutes, and resuspend the precipitated cells in PBS. Ultrasonicate the cancer cells at 20W for 2 minutes to mechanically disrupt the cells. Centrifuge the sample at 2000g for 5 minutes and discard. After precipitation, centrifuge the supernatant at 6000g for 10 minutes, then discard the precipitate. Centrifuge the supernatant at 15000g for 60 minutes, then discard the supernatant to collect the precipitate. Resuspend the precipitate in PBS to obtain the cancer cell membrane fraction 2. .
  • Nanoparticle 1 (100 mg) prepared in step (3) in 9 mL PBS, and then mix it with 1 mL of the mixed antigen-presenting cell membrane component (5 mg) prepared in step (4) and the cancer cell membrane component 1 prepared in step (5).
  • 5mg) and bacterial external vesicle component 1 were mixed, sonicated at 20W for 5 minutes, and then repeatedly co-extruded using a 0.45 ⁇ m filter membrane, and then centrifuged at 13000g for 30 minutes and then discarded the supernatant to collect the precipitate. Resuspend the precipitate in PBS to obtain Nano Vaccine 1.
  • the particle size of Nano Vaccine is 260nm.
  • Nanoparticle 2 (100 mg) prepared in step (3) in 9 mL PBS, and then mix it with 1 mL of the mixed antigen-presenting cell membrane component (5 mg) prepared in step (4) and the cancer cell membrane component 1 prepared in step (5).
  • 5mg) and bacterial external vesicle component 1 were mixed, sonicated at 20W for 5 minutes, and then repeatedly co-extruded using a 0.45 ⁇ m filter membrane, and then centrifuged at 13000g for 30 minutes and then discarded the supernatant to collect the precipitate.
  • the particle size of Nano Vaccine is 260nm.
  • Nanoparticle 1 100 mg prepared in step (3) in 9 mL PBS, and then mix it with 1 mL of the mixed antigen-presenting cell membrane component (5 mg) prepared in step (4) and the cancer cell membrane component 2 prepared in step (5).
  • 5mg and bacterial external vesicle component 2 were mixed, sonicated at 20W for 5 minutes, and then repeatedly co-extruded using a 0.45 ⁇ m filter membrane, and then centrifuged at 13000g for 30 minutes and then discarded the supernatant to collect the precipitate.
  • the particle size of Nano Vaccine is 260nm.
  • mice Female C57BL/6 6-8 weeks old were selected as model mice to prepare Pan02 pancreatic cancer tumor-bearing mice.
  • the mice were inoculated with cancer cells on days -35, -28, -21, -14 and On day -7, each mouse was vaccinated with 800 ⁇ g nanovaccine 1, or 800 ⁇ g nanovaccine 2, or 800 ⁇ g nanovaccine 3, or 100 ⁇ L PBS.
  • each recipient mouse On day 0, each recipient mouse was inoculated subcutaneously with 2 ⁇ 10 6 Pan02 cells on the lower right side of the back. The methods for monitoring mouse tumor volume and survival were the same as above.
  • Nano Vaccine 1 is more effective than Nano Vaccine 2 and Nano Vaccine 3, indicating that the bacterial membrane components lysed by the specific lysate are loaded inside the Nano Vaccine, which is beneficial to improving the efficacy of the vaccine.
  • the cancer cells and bacteria pretreated with sunitinib The prepared cell membrane components can also improve the efficacy of nano vaccines.

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Abstract

提供一种负载癌细胞全细胞组分和混合膜组分的疫苗的制备方法及其应用,制备方法包括以下步骤:S1、获取癌细胞的膜组分;S2、激活抗原提呈细胞,获取膜组分;S3、获取细菌的膜组分;S4、将S1和S2的产物,和/或S1和S3的产物与第二粒子共作用,使膜组分负载于第二粒子上,得到疫苗。可实现源于肿瘤组织或癌细胞的疫苗负载广谱多样的癌细胞抗原的同时表面负载混合膜组分,赋予了疫苗适当的仿生膜特性。提供的方法能够制备负载广谱癌细胞抗原表位的癌症疫苗,可用于各类癌症的预防和治疗。

Description

负载癌细胞全细胞组分和混合膜组分的疫苗的制备方法及其应用 技术领域
本发明涉及免疫治疗领域,尤其涉及一种负载癌细胞全细胞组分和混合膜组分的疫苗的制备方法及其应用。
背景技术
癌症免疫治疗是癌症最主要的治疗方法之一,其中癌症疫苗是癌症免疫治疗的重要方法之一。目前,采用癌细胞膜包被的纳米粒子作为癌症疫苗用于肿瘤的预防和治疗已非常广泛,如Zhu J Y等(Preferential cancer cell self-recognition and tumor self-targeting by coating nanoparticles with homotypic cancer cell membranes)提供了一种包被特异性来源于同源肿瘤的癌细胞膜的纳米颗粒,具体地,制备了一种磁性氧化铁纳米粒(Fe 3O 4MNPs)平台,用来自多种癌细胞系的CMBMNPs(细胞膜仿生修饰纳米粒)研究其同源靶向能力,实验结果表明,癌细胞膜仿生修饰的Fe 3O 4MNPs可以在体外对源癌细胞系实现高度特异性的自我识别,并且对同源肿瘤具有出色的靶向能力。甚至当存在异型肿瘤竞争时,该NPs仍然选择性地靶向同源肿瘤。除此之外,有研究公开了癌细胞膜包被明胶纳米粒子(PDTC@GNPs)用于肿瘤治疗、MIA-PaCa-2胰腺癌细胞膜包裹纳米金颗粒用于胰腺癌治疗等多种癌细胞膜包被纳米颗粒的癌症疫苗。但由于其负载的抗原种类有限,以及负载的膜种类有限,并且由于传统的裸磁性纳米颗粒的杀菌药物负载容量有限制,或有些情况下纳米抗菌材料也会使细菌细胞内部产生超氧阴离子自由基、羟基自由基、过氧化氢和单线态分子氧等活性氧物质,这些物质的过度累积会造成细菌细胞的凋亡,因此,目前尚未有同时负载多种细胞膜组分、负载癌细胞全细胞抗原且包载抗肿瘤细菌的癌症疫苗。
发明内容
为解决上述技术问题,本发明提供了一种内部负载肿瘤组织和/或癌细胞全细胞组分,表面负载癌细胞细胞膜和/或细胞外囊泡膜的膜组分,以及激活后的抗原提呈细胞细胞膜组分和/或来源于细菌的膜组分的纳米疫苗或微米疫苗,注射进入体内后由于仿生模拟了膜结构,因而稳定性更好也更容易激活癌细胞特异性免疫反应,从而能更好地发挥预防癌症或治疗癌症的功能。
本发明提供了一种负载癌细胞全细胞组分和混合膜组分的疫苗的制备方法,包括以下步骤:
S1、从癌细胞中获取癌细胞的细胞膜组分和/或癌细胞细胞外囊泡膜组分;
S2、将抗原提呈细胞与第一粒子共孵育以激活抗原提呈细胞,获取激活后的抗原提呈 细胞的细胞膜组分;其中,第一粒子负载癌症相关抗原;
S3、从细菌中获取细菌的细胞膜组分和/或细菌细胞外囊泡膜组分;
S4、将第一混合膜组分和/或第二混合膜组分与第二粒子共作用,使混合膜组分负载于第二粒子上,得到负载癌细胞全细胞组分和混合膜组分的疫苗;其中,第二粒子负载癌细胞全细胞组分,第一混合膜组分为S1产物和S2产物的混合物,第二混合膜组分为S1产物和S3产物的混合物;
其中,癌细胞全细胞组分包括癌细胞和/或肿瘤组织经水裂解得到的水溶性组分和非水溶性组分,非水溶性组分经溶解剂溶解后负载于第二粒子上;或癌细胞全细胞组分包括癌细胞和/或肿瘤组织经含有溶解剂的溶解液裂解后溶解得到的可溶组分。
进一步地,在步骤S1中,获取膜组分前还包括将癌细胞进行预处理的步骤,预处理为将癌细胞在含有阿霉素、替尼类药物、氯喹或氮杂胞苷的培养基中培养。
进一步地,替尼类药物包括吉非替尼、伊马替尼、甲磺酸伊马替尼、尼罗替尼、舒尼替尼、拉帕替尼等。
进一步地,在步骤S2中,癌症相关抗原为多肽抗原或癌细胞全细胞组分。
进一步地,在步骤S3中,获取膜组分前还包括将细菌进行预处理的步骤,预处理为将细菌在含有阿霉素、替尼类药物、氯喹或氮杂胞苷的培养基中培养。
进一步地,第二粒子还负载有细菌组分,细菌组分通过用含有溶解剂的溶解液裂解细菌或细菌外囊泡,后将裂解产物用溶解液溶解得到。
进一步地,从癌细胞、抗原提呈细胞、细菌中获取膜组分的方式包括超声、均质化、匀浆、高速搅拌、高压破坏、高剪切力破坏、溶胀、化学物质、皱缩等。
进一步地,第二粒子与细胞膜组分共作用的方式包括共孵育、超声、搅拌、均质化、共挤出、超滤、透析、匀浆等。
进一步地,细菌包括卡介苗、益生菌、溶瘤细菌等。包括但不限于卡介苗、大肠杆菌、长双歧杆菌、短双歧杆菌、乳双歧杆菌、鼠李糖乳杆菌、嗜酸乳杆菌、格氏乳杆菌、罗伊氏乳杆菌等。
进一步地,负载癌细胞全细胞组分和细菌组分的第二粒子由以下步骤制备得到:
(1)将肿瘤组织和/或癌细胞经含有溶解剂的溶解液裂解,后使用溶解液溶解;或者使用溶胀、反复冻融和/或超声裂解肿瘤组织和/或癌细胞后先收集裂解物中的水溶性组分,再使用含有溶解剂的溶解液溶解裂解物中的非水溶性组分;
(2)将细菌经含有溶解剂的溶解液裂解,得到细菌裂解物后使用溶解液溶解;或者使用溶胀、反复冻融和/或超声裂解细菌后先收集细菌的水溶性组分,再使用含有溶解剂的溶解液溶解细菌中的非水溶性组分;
(3)将(1)和/或(2)负载于纳米粒子或微米粒子内部和/或表面。
进一步地,溶解剂可为尿素、盐酸胍、脱氧胆酸盐、十二烷基硫酸盐、甘油、蛋白质降解酶、白蛋白、卵磷脂、无机盐、Triton、吐温、氨基酸、糖苷、胆碱等。
进一步地,抗原提呈细胞为树突状细胞或含有树突状细胞(DC)的混合抗原提呈细胞,即混合抗原提呈细胞中还可含有B细胞、巨噬细胞中的一种或两种。
进一步地,制备第一粒子或第二粒子的材料可以为天然高分子材料、有机合成高分子材料、无机材料等任何可以植被纳米粒子或微米粒子的材料。
有机合成高分子材料选自聚乳酸-羟基乙酸共聚物、聚乳酸、聚乙醇酸、聚乙二醇、聚己内酯、泊洛沙姆、聚乙烯醇、聚乙烯吡咯烷酮、聚乙烯亚胺、聚三亚甲基碳酸酯、聚酸酐、聚对二氧六环酮、聚对二氧环己酮、聚甲基丙烯酸甲酯、PLGA-PEG、PLA-PEG、PGA-PEG、聚氨基酸、合成多肽和合成脂质中的至少一种;天然高分子材料选自卵磷脂、胆固醇、海藻酸盐、白蛋白、胶原蛋白、明胶、细胞膜、淀粉、糖类和多肽中的至少一种;无机材料选自三氧化二铁、四氧化三铁、碳酸钙和磷酸钙中的至少一种。
进一步地,第一粒子和/或第二粒子还负载有免疫增强佐剂。免疫增强佐剂包括但不限于微生物来源的免疫增强剂、人或动物免疫系统的产物、固有免疫激动剂、适应性免疫激动剂、化学合成药物、真菌多糖类、中药及其他类中的至少一类;免疫增强佐剂包括但不限于模式识别受体激动剂、卡介苗(BCG)、锰相关佐剂、卡介苗细胞壁骨架、卡介苗甲醇提取残余物、卡介苗胞壁酰二肽、草分枝杆菌、多抗甲素、矿物油、病毒样颗粒、免疫增强的再造流感病毒小体、霍乱肠毒素、皂苷及其衍生物、Resiquimod、胸腺素、新生牛肝活性肽、米喹莫特、多糖、姜黄素、免疫佐剂CpG、免疫佐剂poly(I:C)、免疫佐剂poly ICLC、短小棒状杆菌苗、溶血性链球菌制剂、辅酶Q10、左旋咪唑、聚胞苷酸、锰佐剂、铝佐剂、钙佐剂、各种细胞因子、白细胞介素、干扰素、聚肌苷酸、聚腺苷酸、明矾、磷酸铝、羊毛脂、角鲨烯、细胞因子、植物油、内毒素、脂质体佐剂、MF59、双链RNA、双链DNA、铝相关佐剂、CAF01、人参、黄芪的有效成分中的至少一种。
优选地,免疫增强佐剂为Toll样受体激动剂;更优选为两种及以上Toll样受体激动剂联用,保证纳米粒子或微米粒子被抗原提呈细胞吞噬后可以更好地激活癌细胞特异性T细胞。
进一步地,两种及以上Toll样受体激动剂联用为poly(I:C)/Poly(ICLC)与CpG-ODN(CpG寡脱氧核苷酸)联用。优选地,CpG-ODN为两种及以上的CpG-ODN。
进一步地,所述佐剂可以负载于第一粒子或第二粒子的内部和/或表面。
进一步地,第一粒子或第二粒子还负载有增强溶酶体逃逸的物质,比如氨基酸、聚氨基酸(如精氨酸、聚精氨酸、赖氨酸、聚赖氨酸、组氨酸、聚组氨酸)、核酸、带正电的多肽(如KALA多肽、RALA多肽、蜂毒肽等)、脂类、糖类、具有质子海绵效应的无机物(如NH 4HCO 3)、鱼精蛋白、组蛋白等。
进一步地,纳米粒子或微米粒子可以采用已有的制备方法制备得到,包括但不仅限于常见的溶剂挥发法、透析法、微流控法、挤出法、热熔法。
进一步地,纳米粒子或微米粒子在制备过程中可以不做修饰处理,也可以采用适当的修饰技术以提高纳米粒子或微米粒子的抗原负载量。修饰技术包括但不限于生物矿化(如 硅化、钙化、镁化)、凝胶化、交联、化学修饰、添加带电物质等。
进一步地,抗原被负载于纳米粒子或微米粒子表面的方式包括但不限于吸附、共价连接、电荷相互作用(如添加带正电的物质、添加带负电的物质)、疏水相互作用、一步或多步的固化、矿化、包裹等。
进一步地,负载于纳米粒子或微米粒子表面的水溶性抗原和/或非水溶性抗原负载后为一层或多层,表面负载多层水溶性抗原和/或非水溶性抗原时,层与层之间为修饰物。
进一步地,第一粒子和第二粒子分别独立地选自纳米粒子或微米粒子,这样能保证粒子被抗原提呈细胞吞噬,而为了提高吞噬效率,粒径大小要在适宜的范围内。纳米粒子的粒径大小为1nm-1000nm,更优选地,粒径大小为30nm-1000nm,最优选地,粒径大小为100nm-600nm;微米粒子的粒径大小为1μm-1000μm,更优选地,粒径大小为1μm-100μm,更优选地,粒径大小为1μm-10μm,最优选地,粒径大小为1μm-5μm。
进一步地,激活抗原提呈细胞的过程中,孵育体系中含有细胞因子和/或抗体;细胞因子选自白介素、肿瘤坏死因子、干扰素、集落刺激因子中的至少一种;抗体包括但不限于αCD-3抗体、αCD-4抗体、αCD-8抗体、αCD-28抗体、αCD-40抗体、αOX-40抗体、αOX-40L抗体。
进一步地,细胞因子包括但不限于白介素2(IL-2)、白介素7(IL-7)、白介素14(IL-14)、白介素4(IL-4)、白介素15(IL-15)、白介素21(IL-21)、白介素17(IL-17)、白介素12(IL-12)、白介素6(IL-6)、白介素33(IL-33)、γ干扰素(IFN-γ)、TNF-α、粒细胞-巨噬细胞集落刺激因子(GM-CSF)、巨噬细胞集落刺激因子(M-CSF)。
进一步地,被激活的抗原提呈细胞、癌细胞或细菌制备成纳米疫苗或微米疫苗之前可以进行适当洗涤,洗涤过称中使用的洗涤液中可含有蛋白酶抑制剂和/或磷酸酶抑制剂。
进一步地,第一粒子或第二粒子还修饰有具有主动靶向功能的靶头,靶头可为甘露糖、甘露聚糖、CD19抗体、CD20抗体、BCMA抗体、CD32抗体、CD11c抗体、CD103抗体、CD44抗体等。
本发明所述的纳米疫苗或微米疫苗在制备用于治疗或预防癌症药物中的应用。
进一步地,抗原提呈细胞来源于自体、同种异体、细胞系或者干细胞分化中的一种或多种。
借由上述方案,本发明至少具有以下优点:
本发明的纳米疫苗或微米疫苗表面除了负载癌细胞的膜组分,还同时负载了其他细胞的膜组分,比如抗原提呈细胞细胞膜或细胞外囊泡膜组分、或细菌细胞膜或细胞外囊泡膜组分,混合膜组分的负载,加之内部全细胞组分和/或细菌的负载,使本发明的疫苗有更多样、更广谱的抗原表位,且具有仿生膜特性,增强了稳定性,同时克服了活细胞疫苗保存和活性保持难的问题。
上述说明仅是本发明技术方案的概述,为了能够更清楚了解本发明的技术手段,并可依照说明书的内容予以实施,以下以本发明的较佳实施例并配合详细附图说明如后。
附图说明
为了使本发明的内容更容易被清楚的理解,下面根据本发明的具体实施例并结合附图,对本发明作进一步详细的说明。
图1为本发明纳米疫苗或微米疫苗的制备过程及应用示意图;其中,a为水溶性抗原和非水溶性抗原分别收集和制备纳米粒子或微米粒子的示意图;b为采用含有溶解剂的溶解液溶解肿瘤组织和/或癌细胞或细菌全细胞组分和制备纳米粒子或微米粒子的示意图;c为制备本发明所述纳米疫苗或微米疫苗示意图。
图2-11分别为实施例1-10中用纳米疫苗或微米疫苗预防或治疗癌症时小鼠肿瘤生长速度和生存期实验结果;图2-4以及6-11中,a代表预防或治疗癌症时的肿瘤生长速度实验结果(n≥8);b代表预防或治疗癌症时的小鼠生存期实验结果(n≥8),每个数据点为平均值±标准误差(mean±SEM);a中肿瘤生长抑制实验的显著性差异采用ANOVA法分析,b中显著性差异采用Kaplan-Meier和log-rank test分析;***表示与PBS空白对照组相比p<0.005,有显著性差异;&&&表示与空白纳米粒/微米粒表面负载癌细胞膜组分后制备的纳米疫苗/微米疫苗对照组相比p<0.005,有显著性差异;δ代表与内部负载癌细胞全细胞组分的纳米粒/微米粒表面负载癌细胞膜组分制备的纳米疫苗/微米疫苗相比p<0.05,有显著性差异;δδ代表与内部负载癌细胞全细胞组分的纳米粒/微米粒表面负载癌细胞膜组分制备的纳米疫苗/微米疫苗相比p<0.01,有显著性差异;μ表示与内部负载癌细胞全细胞组分,同时表面负载癌细胞膜组分和被激活的抗原提呈细胞细胞膜组分的纳米疫苗/微米疫苗组相比p<0.05,有显著性差异;#表示与内部负载癌细胞全细胞组分,同时表面负载癌细胞膜组分、未被激活的抗原提呈细胞细胞膜组分和细菌细胞外囊泡组分的纳米疫苗/微米疫苗组相比p<0.05,有显著性差异;##表示与内部负载癌细胞全细胞组分,同时表面负载癌细胞膜组分、未被激活的抗原提呈细胞细胞膜组分和细菌细胞外囊泡组分的纳米疫苗/微米疫苗组相比p<0.01,有显著性差异;η表示与内部负载癌细胞全细胞组分,同时表面负载癌细胞膜组分、被负载4种抗原多肽的纳米粒子激活的抗原提呈细胞细胞膜组分和细菌细胞外囊泡组分的纳米疫苗/微米疫苗组相比p<0.05,有显著性差异;λλλ表示与内部只负载细菌外囊泡组分和免疫佐剂,同时表面负载混合膜组分的的纳米疫苗比p<0.005,有显著性差异;λλ表示与内部只负载细菌外囊泡组分和免疫佐剂,同时表面负载混合膜组分的的纳米疫苗比p<0.01,有显著性差异;γ表示与内部负载癌细胞全细胞组分和免疫佐剂,同时表面负载混合膜组分的的纳米疫苗比p<0.05,有显著性差异;ω表示与内部负载癌细胞组分和细菌组分,同时表面负载癌细胞膜组分的的纳米疫苗比p<0.05,有显著性差异;ρ表示与内部负载癌细胞组分和细菌组分,表面负载癌细胞膜组分和嗜酸乳杆菌的纳米疫苗/微米疫苗比,p<0.05,有显著性差异;φ表示与内部负载癌细胞组分和8M尿素裂解和溶解的细菌组分,表面负载细菌外囊泡组分的纳米疫苗/微米疫苗比,p<0.05,有显著性差异;ββ表示与内部负载癌细胞组分和吐温80裂解和溶解的细菌组分,表面负载细菌外囊泡组分和癌细胞细胞外囊泡组分的纳米疫苗/微米疫苗比,p<0.05,有显著性差 异;θ表示与内部负载癌细胞全细胞组分,表面只负载抗原提呈细胞细胞膜组分的纳米疫苗/微米疫苗比,p<0.05,有显著性差异;Ω代表与内部负载癌细胞全细胞组分,表面负载癌细胞和DC细胞细胞膜组分的纳米疫苗/微米疫苗组相比p<0.05,有显著性差异;ππ代表与内部只负载佐剂,同时表面负载被激活的抗原提呈细胞膜组分、处理过的癌细胞膜组分和处理过的细菌膜组分的纳米疫苗/微米相比p<0.005,有显著性差异;τ代表与内部负载癌细胞全细胞组分,同时表面负载被激活的抗原提呈细胞膜组分、未处理的癌细胞膜组分和未处理的细菌膜组分的纳米疫苗/微米相比p<0.05,有显著性差异;
Figure PCTCN2022108965-appb-000001
代表与内部负载癌细胞全细胞组分以及1种CpG+Poly(I:C)混合佐剂+溶酶体逃逸物质,表面负载癌细胞膜组分和抗原提呈细胞膜组分的纳米疫苗/微米相比p<0.05,有显著性差异;ξ代表与内部负载癌细胞全细胞组分以及2种CpG+Poly(I:C)混合佐剂,表面负载癌细胞膜组分和抗原提呈细胞膜组分的纳米疫苗/微米疫苗相比p<0.05,有显著性差异;Δ代表与内部负载Triton裂解和溶解的细菌组分和癌细胞全细胞组分,同时表面负载被激活的抗原提呈细胞膜组分、处理过的癌细胞膜组分和处理过的细菌膜组分的纳米疫苗/微米相比p<0.05,有显著性差异。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好地理解本发明并能予以实施,但所举实施例不作为对本发明的限定。
本发明所述的用于预防或治疗癌症的疫苗,其内部负载癌细胞和/或肿瘤组织全细胞组分和/或细菌组分的同时,表面负载混合膜组分。表面负载的混合膜组分包括癌细胞、抗原提呈细胞和/或细菌的细胞膜或细胞外囊泡的膜组分,其制备过程及应用领域如图1所示。
在制备用于内部负载癌细胞和/或肿瘤组织全细胞组分的纳米粒子或微米粒子时,需要先制备全细胞组分。制备全细胞组分时可裂解细胞或组织后先分别收集水溶性抗原和水不溶性抗原并分别制备纳米或微米粒子系统;或者也可以直接采用含有溶解剂的溶解液直接裂解细胞或组织并溶解细胞全细胞组分并制备纳米或微米粒子。本发明所述细胞全细胞组分在裂解前或(和)裂解后既可经过包括但不限于灭活或(和)变性、固化、生物矿化、离子化、化学修饰、核酸酶处理等处理后再制备纳米粒子或微米粒子;也可细胞裂解前或(和)裂解后不经过任何灭活或(和)变性、固化、生物矿化、离子化、化学修饰、核酸酶处理直接制备纳米粒子或微米粒子。本发明部分实施例中,细胞在裂解前经过了灭活或(和)变性处理,在实际使用过程中也可以在细胞裂解后做灭活或(和)变性处理,或者也可以细胞裂解前和裂解后均做灭活或(和)变性处理;本发明部分实施例中细胞裂解前或(和)裂解后的灭活或(和)变性处理方法为紫外照射和高温加热,在实际使用过程中也可以采用包括但不限于放射线辐照、高压、固化、生物矿化、离子化、化学修饰、核酸酶处理、胶原酶处理、冷冻干燥等处理方法。本领域技术人员可以理解,在实际应用过程中技术人员可根据具体情况进行适当调整。
在表面负载混合膜组分时,需要先把癌细胞、抗原提呈细胞和/或细菌的混合细胞使用 超声、搅拌、高压、高剪切力、均质化等方法将细胞机械破坏,然后采用离心和/或依次通过不同孔径的滤膜过滤和/或共挤出和/或超滤和/或透析等方法收集膜组分,然后将膜组分与已经负载肿瘤组织和/或癌细胞全细胞组分和/或细菌组分的纳米粒子或微米粒子共作用,从而将膜组分负载于纳米粒子或微米粒子表面。
如果使用的抗原提呈细胞为激活过的抗原提呈细胞,在使用纳米粒子或微米粒子体外激活抗原提呈细胞时,可以使用细胞因子和/或抗体辅助提高激活效率,抗原提呈细胞可以来源于自体或者同种异体,也可以来自于细胞系或者干细胞。抗原提呈细胞可以是DC细胞、B细胞、巨噬细胞或者上述三者的任意混合物,也可以是其他具有抗原提呈功能的细胞。
如果使用的癌细胞经过适当处理以增加抗原含量,处理方式包括但不限于与阿霉素等化学药物共孵育一段时间、高钙离子环境增加细胞压力等。
在一些实施方案中,使用了溶剂挥发法制备第一和第二粒子。
下面以使用复乳法制备第一和第二粒子为例,采用负载癌细胞全细胞组分和/或细菌组分的纳米粒子或微米粒子激活的抗原提呈细胞制备纳米疫苗或微米疫苗的具体制备方法如下:
步骤1,将第一预定体积的含有第一预定浓度的水相溶液加入第二预定体积的含有第二预定浓度制备粒子原材料的有机相中。
在一些实施例中,水相溶液可含有癌细胞和/或细菌裂解物中的各组分以及免疫增强佐剂;癌细胞和/或细菌裂解物中的各组分在制备时分别为水溶性抗原或是溶于尿素或盐酸胍等溶解剂中的原非水溶性抗原。水相溶液所含有的水溶性抗原的浓度或原非水溶性抗原的浓度,也即第一预定浓度要求蛋白质多肽浓度含量大于1ng/mL,能负载足够癌细胞全细胞组分以激活相关细胞。免疫增强佐剂在初始水相中的浓度为大于0.01ng/mL。
在一些实施例中,水相溶液含有肿瘤组织和/或癌细胞裂解物中的各组分以及免疫增强佐剂;肿瘤组织和/或癌细胞裂解物中的各组分在制备时分别为水溶性抗原或者是溶于尿素或盐酸胍等溶解剂中的原非水溶性抗原。水相溶液所含有的水溶性抗原的浓度或原非水溶性抗原的浓度,也即第一预定浓度要求蛋白质多肽浓度含量大于0.01ng/mL,能负载足够癌细胞全细胞组分以激活相关细胞。免疫增强佐剂在初始水相中的浓度为大于0.01ng/mL。
在一些实施例中,水相溶液含有细菌裂解物中的各组分以及免疫增强佐剂;细菌裂解物中的各组分在制备时分别为水溶性抗原或者是溶于尿素或盐酸胍等溶解剂中的原非水溶性抗原。水相溶液所含有的水溶性抗原的浓度或原非水溶性抗原的浓度,也即第一预定浓度要求蛋白质多肽浓度含量大于0.01ng/mL,能负载足够细菌组分以激活相关细胞。免疫增强佐剂在初始水相中的浓度为大于0.01ng/mL。
在一些实施例中,制备粒子原材料为PLGA或PLA,有机溶剂选用二氯甲烷。另外,在一些实施例中,制备粒子原材料的第二预定浓度的范围为0.5mg/mL-5000mg/mL,优选为100mg/mL。
在本发明中,之所以选择PLGA或修饰的PLGA,是由于该材料为生物可降解材料且已被FDA批准用作药物敷料。研究表明PLGA具有一定的免疫调节功能,因而适合作为纳米粒子或微米粒子制备时的辅料。在实际应用中可根据实际情况选择合适的材料。
实际中,有机相的第二预定体积根据其和水相的第一预定体积的比例进行设定,在本发明中,水相的第一预定体积和有机相的第二预定体积之比的范围为1:1.1-1:5000,优选地为1:10。在具体实施过程中可根据需要对第一预定体积、第二预定体积和第一预定体积与第二预定体积之比进行调整以调整制备的纳米粒或微米粒的尺寸大小。
优选地,水相溶液为裂解物组分溶液时,其中蛋白质和多肽的浓度大于1ng/mL,优选1mg/mL~100mg/mL;水相溶液为裂解物组分/免疫佐剂溶液时,其中蛋白质和多肽的浓度大于1ng/mL,优选1mg/mL~100mg/mL,免疫佐剂的浓度大于0.01ng/mL,优选0.01mg/mL~20mg/mL。有机相溶液中,溶剂为DMSO、乙腈、乙醇、氯仿、甲醇、DMF、异丙醇、二氯甲烷、丙醇、乙酸乙酯等,优选二氯甲烷;有机相的浓度为0.5mg/mL~5000mg/mL,优选为100mg/mL。
步骤2,将步骤1得到的混合液进行大于2秒的超声处理或大于1分钟的搅拌或均质处理或微流控处理。优选地,搅拌为机械搅拌或者磁力搅拌时,搅拌速度大于50rpm,搅拌时间大于1分钟,比如搅拌速度为50rpm~1500rpm,搅拌时间为0.1小时~24小时;超声处理时,超声功率大于5W,时间大于0.1秒,比如2~200秒;均质处理时使用高压/超高压均质机或高剪切均质机,使用高压/超高压均质机时压力大于5psi,比如20psi~100psi,使用高剪切均质机时转速大于100rpm,比如1000rpm~5000rpm;使用微流控处理流速大于0.01mL/min,比如0.1mL/min-100mL/min。超声或者搅拌或者均质处理或者微流控处理进行纳米化和/或微米化,超声时间长短或搅拌速度或均质处理压力及时间能控制制备的微纳粒子大小,过大或过小都会带来粒径大小的变化。
步骤3,将步骤2处理后得到的混合物加入第三预定体积的含有第三预定浓度乳化剂的水溶液中并进行大于2秒的超声处理或大于1分钟的搅拌或进行均质处理或微流控处理。该步骤将步骤2得到的混合物加入到乳化剂水溶液中继续超声或搅拌纳米化或微米化。在本发明中,超声时间大于0.1秒,比如2~200秒,搅拌速度大于50rpm,比如50rpm~500rpm,搅拌时间大于1分钟,比如60~6000秒。优选地,搅拌为机械搅拌或者磁力搅拌时,搅拌速度大于50rpm,搅拌时间大于1分钟,比如搅拌速度为50rpm~1500rpm,搅拌时间为0.5小时~5小时;超声处理时,超声功率为50W~500W,时间大于0.1秒,比如2~200秒;均质处理时使用高压/超高压均质机或高剪切均质机,使用高压/超高压均质机时压力大于20psi,比如20psi~100psi,使用高剪切均质机时转速大于1000rpm,比如1000rpm~5000rpm;使用微流控处理流速大于0.01mL/min,比如0.1mL/min-100mL/min。超声或者搅拌或者均质处理或者微流控处理进行纳米化或微米化,超声时间长短或搅拌速度或均质处理压力及时间能控制制备的纳米或微米粒子大小,过大或过小都会带来粒径大小的变化。
在一些实施例中,乳化剂水溶液为聚乙烯醇(PVA)水溶液,第三预定体积为5mL, 第三预定浓度为20mg/mL。第三预定体积根据其与第二预定体积的比例进行调整。在本发明中,第二预定体积与第三预定体积之的范围为1:1.1-1:1000进行设定,优选地可以为2:5。在具体实施过程中为了控制纳米粒子或微米粒子的尺寸,可以对第二预定体积和第三预定体积之比进行调整。同样地,本步骤的超声时间或搅拌时间、乳化剂水溶液的体积以及浓度的取值根据,均为了得到尺寸大小合适的纳米粒或微米粒。
步骤4,将步骤3处理后得到的液体加入第四预定体积的第四预定浓度的乳化剂水溶液中,并进行搅拌直至满足预定搅拌条件。
本步骤中,乳化剂水溶液为PVA溶液或其他溶液。
第四预定浓度为5mg/mL,第四预定浓度的选择,以得到尺寸大小合适的纳米粒或微米粒为依据。第四预定体积的选择依据第三预定体积与第四预定体积之比决定。在本发明中,第三预定体积与第三预定体积之比为范围为1:1.5-1:2000,优选地为1:10。在具体实施过程中为了控制纳米粒子或微米粒子的尺寸可以对第三预定体积和第四预定体积之比进行调整。
在本发明中,本步骤的预定搅拌条件为直至有机溶剂挥发完成,也即步骤1中的二氯甲烷挥发完成。
步骤5,将步骤4处理满足预定搅拌条件的混合液在以大于100RPM的转速进行大于1分钟的离心后,去除上清液,并将剩下的沉淀物重新混悬于第五预定体积的第五预定浓度的含有冻干保护剂的水溶液中或者第六预定体积的PBS(或生理盐水)中。
在本发明一些实施方案中,步骤5所得沉淀重新混悬于第六预定体积的PBS(或生理盐水)中时不需要冻干,可直接进行后续纳米粒子或微米粒子与膜组分共作用的相关实验。
在本发明一些实施方案中,步骤5所得沉淀重新混悬于含有冻干保护剂的水溶液中时需进行冷冻干燥,再冷冻干燥以后再进行后续实验。
在本发明中,所述冻干保护剂选用海藻糖(Trehalose)。
在本发明中,该步骤的冻干保护剂的第五预定浓度为质量百分比4%,之所以如此设定,是为了在后续进行冷冻干燥中不影响冻干效果。
步骤6,将步骤5得到的含有冻干保护剂的混悬液进行冷冻干燥处理后,将冻干物质备用。
步骤7,将癌细胞、抗原提呈细胞或者细菌或者细胞外囊泡采用低功率超声、机械搅拌、均质化、高剪切力、高压、溶胀等方法机械破坏细胞,并收集破坏后的细胞或囊泡的膜组分。
步骤8,将步骤7制备的膜组分与前述制备的纳米粒子和/或微米粒子共作用一定时间。制备纳米粒子和/微米粒子的肿瘤组织和/或癌细胞与癌细胞、抗原提呈细胞可以来自于自体或者同种异体。
步骤9,收集共作用后的纳米粒子或微米粒子,采用离心、超滤或透析等方法纯化纳米粒子或微米粒子,即为纳米疫苗或微米疫苗,可以直接使用、冷冻备用或者冷冻干燥后 制备成冻干粉备用。
在另一些实施方案中,使用复乳法制备负载抗原的纳米粒子或微米粒子后制备纳米疫苗或微米疫苗的具体制备方法如下:
步骤1~4同上。
步骤5,将步骤4处理满足预定搅拌条件的混合液在以大于100RPM的转速进行大于1分钟的离心后,去除上清液,并将剩下的沉淀物重新混悬于第五预定体积的第五预定浓度的含有癌细胞全细胞组分中水溶性和/或非水溶性抗原的溶液中,或者将剩下的沉淀物重新混悬于第五预定体积的第五预定浓度的含有癌细胞全细胞组分中水溶性和/或非水溶性抗原与佐剂混合的溶液中。
步骤6,将步骤5处理满足预定搅拌条件的混合液在以大于100RPM的转速进行大于1分钟的离心后,去除上清液,并将剩下的沉淀物重新混悬于第六预定体积的固化处理试剂或矿化处理试剂,作用一定时间后离心洗涤,然后加入第七预定提交的含有带正电或者带负电的物质并作用一定时间。
在本发明一些实施方案中,步骤6所得沉淀重新混悬于第七预定体积的带电物质后可不需要冻干,可直接进行后续纳米粒子或微米粒子与膜组分共作用的相关实验。
在本发明一些实施方案中,步骤6所得沉淀重新混悬于含有干燥保护剂的水溶液中后进行室温真空干燥或者冷冻真空干燥,在干燥以后再进行后续实验。
在本发明中,所述冻干保护剂选用海藻糖(Trehalose),或者甘露醇与蔗糖的混合溶液。在本发明中,该步骤的干燥保护剂的浓度为质量百分比4%,之所以如此设定,是为了在后续进行干燥中不影响干燥效果。
步骤7,将步骤6得到的含有干燥保护剂的混悬液进行干燥处理后,将干燥后的物质备用。
在本发明中,步骤5-步骤7的修饰和抗原负载步骤可重复多次以提高抗原的负载量。而且在添加带正电或带负电的物质时可以多次添加带同种电荷的或者也可以交替添加带不同电荷的物质。
步骤8,将癌细胞、抗原提呈细胞或者细菌或者细胞外囊泡采用低功率超声、机械搅拌、均质化、高剪切力、高压、溶胀等方法机械破坏细胞,并收集破坏后的细胞或囊泡的膜组分。
步骤9,将步骤8制备的膜组分与前述制备的纳米粒子和/或微米粒子共作用一定时间。制备纳米粒子和/微米粒子的肿瘤组织和/或癌细胞与癌细胞、抗原提呈细胞可以来自于自体或者同种异体。
步骤10,收集共作用后的纳米粒子或微米粒子,采用离心、超滤或透析等方法纯化纳米粒子或微米粒子,即为纳米疫苗或微米疫苗,可以直接使用、冷冻备用或者冷冻干燥后制备成冻干粉备用。
实施例1 纳米疫苗用于黑色素瘤的预防
本实施例以小鼠黑色素瘤为癌症模型来说明如何使用纳米疫苗预防黑色素瘤。本实施例中,裂解B16F10黑色素瘤肿瘤组织以制备肿瘤组织的水溶性抗原和非水溶性抗原,然后,以有机高分子材料PLGA为纳米粒骨架材料,以Polyinosinic-polycytidylic acid(poly(I:C))为免疫佐剂采用溶剂挥发法制备负载有肿瘤组织的水溶性抗原和非水溶性抗原的纳米粒子,然后在纳米粒子表面负载癌细胞细胞膜组分和/或被激活的抗原提呈细胞细胞膜组分和细菌细胞外囊泡组分后用于预防癌症。
(1)肿瘤组织的裂解及各组分的收集
在每只C57BL/6小鼠背部皮下接种1.5×10 5个B16F10细胞,在肿瘤长到体积分别为约1000mm 3时处死小鼠并摘取肿瘤组织。将肿瘤组织切块后研磨,通过细胞过滤网加入适量超纯水并反复冻融5次,并伴有超声以破坏裂解细胞。待细胞裂解后,将裂解物以5000g的转速离心5分钟并取上清液即为可溶于纯水的水溶性抗原;在所得沉淀部分中加入8M尿素水溶液溶解沉淀部分即可将不溶于纯水的非水溶性抗原转化为在8M尿素水溶液中可溶。将水溶性抗原和非水溶性抗原按质量比1:1混合,即为制备纳米粒子的抗原原料来源。
(2)内部负载全细胞组分的纳米粒子的制备
本实施例中纳米粒子及作为对照的空白纳米粒采用溶剂挥发法中的复乳法制备。所采用的纳米粒子制备材料PLGA分子量为7KDa-17KDa,所采用的免疫佐剂为poly(I:C)且poly(I:C)包载于纳米粒子内。制备方法如前所述,在制备过程中首先采用复乳法在纳米粒子内部负载全细胞裂解物组分和佐剂,然后将100mg纳米粒子在10000g离心20分钟,并使用10mL含4%海藻糖的超纯水重悬后冷冻干燥48h。该纳米粒子1平均粒径为260nm左右,表面电位为-9mV左右;每1mg PLGA纳米粒子1约负载100μg蛋白质或多肽组分,每1mgPLGA纳米粒1所使用的poly(I:C)免疫佐剂为0.02mg。空白纳米粒制备材料和制备方法相同,粒粒径为260nm左右,负载等量佐剂但是不负载任何裂解物组分。
(3)癌细胞细胞膜组分的制备
收集B16F10癌细胞,然后使用生理盐水洗涤细胞两遍,将细胞重悬在生理盐水中后在7.5W超声20分钟。然后将样品在2000g离心20分钟并收集上清液,将上清液在7000g离心20分钟后收集上清液,然后在15000g离心120分钟后收集弃去上清液收集沉淀,将沉淀在PBS中重悬后备用。
(4)骨髓来源的树突状细胞(BMDC)的制备及激活
本实施例以从小鼠骨髓细胞制备树突状细胞为例来说明如何制备BMDC。首先,取1只6-8周龄C57小鼠颈椎脱臼处死,手术取出后腿的胫骨和股骨放入PBS中,用剪刀和镊子将骨周围的肌肉组织剔除干净。用剪刀剪去骨头两端,再用注射器抽取PBS溶液,针头分别从骨头两端插入骨髓腔,反复冲洗骨髓到培养皿中。收集骨髓溶液,400g离心3min后加入1mL红细胞裂解液裂红。加入3mL RPMI 1640(10%FBS)培养基终止裂解,400g离心3min,弃上清。将细胞放置10mm培养皿中培养,使用RPMI 1640(10%FBS)培养基,同时加入重组小鼠GM-CSF(20ng/mL),37度,5%CO 2培养7天。第3天轻轻摇晃培养瓶, 补充同样体积含有GM-CSF(20ng/mL)RPMI 1640(10%FBS)培养基。第6天,对培养基进行半量换液处理。第7天,收集少量悬浮及半贴壁细胞,通过流式检测,当CD86 +CD80 +细胞在CD11c +细胞中的比例为15-20%之间,诱导培养的BMDC即可被用来做下一步实验。
将负载来源于肿瘤组织的癌细胞全细胞组分的纳米粒子(800μg)与BMDC(1000万个)在15mL RPMI1640完全培养基中共孵育48小时(37℃,5%CO 2),孵育体系中含有粒细胞-巨噬细胞集落刺激因子(GM-CSF,1000U/mL)、IL-2(500U/mL)、IL-7(1000U/mL)和IL-15(500U/mL)。然后收集被激活后的DC并在400g离心5分钟,然后使用含有蛋白酶抑制剂的4℃磷酸盐缓冲溶液(PBS)洗涤细胞两遍,将细胞重悬在PBS水中后在4℃低功率(22.5W)超声1分钟。然后将样品在3000g离心15分钟并收集上清液,将上清液在8000g离心15分钟后收集上清液,然后在16000g离心90分钟后收集弃去上清液收集沉淀,将沉淀在PBS中重悬后使用膜过滤器过滤样品即得基于抗原提呈细胞细胞膜组分的纳米粒子,粒径为130纳米。
(5)细菌细胞外囊泡(Outer membrane vesicles,OMV)的制备
收集培养后的大肠杆菌(BL21),在3000g离心10分钟,去除沉淀后将上清液在13000g离心90分钟,将沉淀在PBS中重悬后即为收集到的细菌细胞外囊泡(3mg/mL)。
(6)纳米疫苗的制备
将50mg步骤(2)制备的空白纳米粒子重悬于9mLPBS中,然后与1mL的3mg步骤(3)制备的癌细胞细胞膜组分混合后在室温共孵育15分钟,然后反复通过0.45μm的挤出膜共挤出,尔后在13000g离心25分钟,弃去上清液后使用PBS重悬沉淀即为纳米疫苗1,粒径为270nm。
或者将50mg步骤(2)制备的负载全细胞组分的纳米粒子重悬于9mLPBS中,然后与1mL的3mg步骤(3)制备的癌细胞细胞膜组分混合后在室温共孵育15分钟,然后反复通过0.45μm的挤出膜共挤出,尔后在13000g离心25分钟,弃去上清液后使用PBS重悬沉淀即为纳米疫苗2,粒径为270nm。
或者将1mL的1.5mg步骤(3)制备的癌细胞细胞膜组分与1.5mg步骤(4)制备的基于抗原提呈细胞细胞膜组分的纳米粒子混合后共孵育10分钟后反复通过0.22μm的挤出膜共挤出。然后将100mg步骤(2)制备的负载全细胞组分的纳米粒子重悬于9mLPBS中,然后与前述混合膜组分混合后在室温共孵育15分钟,然后反复通过0.45μm的挤出膜共挤出,尔后在13000g离心25分钟,弃去上清液后使用PBS重悬沉淀即为纳米疫苗3,粒径为270nm。
或者将1mL的1mg步骤(3)制备的癌细胞细胞膜组分与1mg步骤(4)制备的基于抗原提呈细胞细胞膜组分的纳米粒子以及1mg步骤(5)制备的细菌细胞外囊泡混合后共孵育10分钟后反复通过0.22μm的挤出膜共挤出。然后将100mg步骤(2)制备的负载全细胞组分的纳米粒子重悬于9mLPBS中,然后与前述混合膜组分混合后在室温共孵育15分钟,然后反复通过0.45μm的挤出膜共挤出,尔后在13000g离心25分钟,弃去上清液后使用PBS重悬沉淀即为纳米疫苗4,粒径为270nm。
(7)纳米疫苗用于癌症的预防
选取6-8周的雌性C57BL/6为模型小鼠制备黑色素瘤荷瘤小鼠。在小鼠接种癌细胞之前第-42天、第-35天、第-28天、第-21天、第-14天和第-7天分别给每只小鼠皮下注射1mg纳米疫苗1、或者注射1mg纳米疫苗2、或者注射1mg纳米疫苗3,或者注射1mg纳米疫苗4,或者注射100μLPBS。在第0天,给每只小鼠背部右下方皮下接种1.5×10 5个B16F10细胞。监测小鼠肿瘤生长速度和小鼠生存期。在实验中,从第3天开始每3天记录一次小鼠肿瘤体积的大小。肿瘤体积采用公式v=0.52×a×b 2计算,其中v为肿瘤体积,a为肿瘤长度,b为肿瘤宽度。出于动物实验伦理,在小鼠生存期试验中当小鼠肿瘤体积超过2000mm 3即视为小鼠死亡并将小鼠安乐死。
(8)实验结果
如图2所示,PBS对照组的小鼠其肿瘤生长速度很快,生存期很短;接种纳米疫苗组的小鼠其肿瘤生长速度都明显变慢。而且,纳米疫苗2、纳米疫苗3和纳米疫苗4的效果好于纳米疫苗1;纳米疫苗3和纳米疫苗4的效果好于纳米疫苗2;纳米疫苗4的效果好于纳米疫苗3。这说明,癌细胞细胞膜组分、被负载癌细胞全细胞组分的纳米粒子激活的抗原提呈细胞的膜组分以及细菌细胞外囊泡的膜组分加入均可以提高纳米疫苗的疗效。综上所述,本发明所述纳米疫苗对黑色素瘤具有良好的预防效果。
本实施例中,使用了超声的方法制备癌细胞膜和抗原提呈细胞细胞膜组分,在实际应用中也可以使用搅拌、高压、高剪切力、均质化等方法将细胞机械破坏,然后采用离心和/或依次通过不同孔径的滤膜过滤和/或超滤和/或透析等方法收集膜组分。本实施例使用了癌细胞膜组分,癌细胞与抗原提呈细胞的混合细胞膜,或者癌细胞及抗原提呈细胞与细菌细胞外囊泡的混合细胞膜,在实际应用中还可以使用癌细胞细胞外囊泡组分与抗原提呈细胞和细菌的混合膜组分,或者任意上述几类细胞的细胞外囊泡的混合膜组分。本实施例采用了共孵育和共挤出的方法将纳米粒子与膜组分共作用,在实际应用中也可以使用搅拌、均质化、超声、超滤、透析和匀浆中的一种或多种。本实施例使用了负载全细胞组分的纳米粒子与膜组分共作用制备纳米疫苗,在实际应用中也可以使用负载全细胞组分的微米粒子与膜组分共作用制备微米疫苗。
实施例2 纳米疫苗用于黑色素瘤的治疗
本实施例以小鼠黑色素瘤为癌症模型来说明如何使用纳米疫苗治疗黑色素瘤。本实施例中,裂解B16F10黑色素瘤肿瘤组织以制备肿瘤组织的水溶性抗原和非水溶性抗原,然后,以有PLGA为纳米粒骨架材料,以同为Toll样受体激动剂的poly(I:C)、CpG BW006和CpG2395为混合免疫佐剂采用溶剂挥发法将肿瘤组织的水溶性抗原和非水溶性抗原负载于纳米粒子内,然后将癌细胞和抗原提呈细胞的细胞膜组分负载于纳米粒子表面,即为黑色素瘤疫苗。
(1)肿瘤组织的裂解及各组分的收集
在每只C57BL/6小鼠背部皮下接种1.5×10 5个B16F10细胞,在肿瘤长到体积分别为约 1000mm 3时处死小鼠并摘取肿瘤组织。将肿瘤组织切块后研磨,通过细胞过滤网加入适量纯水并反复冻融5次,并可伴有超声以破坏裂解细胞。待细胞裂解后,将裂解物以5000g的转速离心5分钟并取上清液即为可溶于纯水的水溶性抗原;在所得沉淀部分中加入8M尿素溶解沉淀部分即可将不溶于纯水的非水溶性抗原转化为在8M尿素水溶液中可溶。以上即为制备纳米粒子系统的抗原原料来源。
(2)纳米粒子系统的制备
本实施例中纳米粒采用溶剂挥发法制备。在制备时负载癌细胞全细胞组分中水溶性抗原的纳米粒子和负载癌细胞全细胞组分中非水溶性抗原的纳米粒子分别制备,然后使用时一起使用。纳米粒子1制备材料PLGA分子量为24Da-38KDa,所采用的免疫佐剂为poly(I:C)、CpG BW006和CpG2395且佐剂包载于纳米粒子内部。制备方法如前所述,在制备过程中首先采用复乳法在纳米粒子内部负载抗原和佐剂,在内部负载抗原(裂解组分)后,将100mg纳米粒子在10000g离心20分钟,并使用10mL含4%海藻糖的超纯水重悬后冷冻干燥48h。该纳米粒子平均粒径为320nm左右;每1mg PLGA纳米粒子1约负载100μg蛋白质和多肽组分,负载poly(I:C)、CpG BW006和CpG2395免疫佐剂各0.025mg。
对照多肽纳米粒子制备材料和制备方法同上,所负载的四种多肽新生抗原为B16-M20(Tubb3,FRRKAFLHWYTGEAMDEMEFTEAESNM),B16-M24(Dag1,TAVITPPTTTTKKARVSTPKPATPSTD),B16-M46(Actn4,NHSGLVTFQAFIDVMSRETTDTDTADQ)和TRP2:180-188(SVYDFFVWL)。每1mgPLGA纳米粒负载多肽组分100μg,负载poly(I:C)、CpG BW006和CpG2395各0.025mg,平均粒径为320nm左右。
(3)抗原提呈细胞及其膜组分的制备
本实施例使用BMDC和B细胞的混合抗原提呈细胞。BMDC的制备方法同实施例1。B细胞的分离方法如下:处死C57BL/6小鼠后摘取小鼠脾脏,制备小鼠脾细胞单细胞悬液,使用磁珠分选法分离脾细胞中活细胞中(使用活死细胞染料标记死细胞以去除死细胞)的CD19 +B细胞。然后将BMDC和B细胞按照数量比1:1混合后作为混合抗原提呈细胞使用。
将1mg负载癌细胞全细胞组分的纳米粒子1(其中负载水溶性组分的纳米粒子500μg+负载非水溶性组分的纳米粒子500μg)或者将1mg多肽纳米粒子与2000万个混合抗原提呈细胞(其中DC1000万个+B细胞1000万个)在20mLRPMI1640完全培养基中混合后共孵育48小时(37℃,5%CO 2),孵育体系中含有IL-2(1000U/mL)、IL-7(1000U/mL)、IL-12(200U/mL)、GM-CSF(500U/mL)和白蛋白(50ng/mL)。孵育完成后,将孵育后的细胞在400g离心5分钟后弃去上清液后再使用PBS洗涤两遍即得被激活的抗原提呈细胞。
本实施例中分别使用未被任何纳米粒激活的抗原提呈细胞或者纳米粒激活过的抗原提呈细胞分别制备膜组分。制备膜组分时,首先收集未被激活的混合抗原提呈细胞(2000万个)或者收集已经被纳米粒子激活后的混合抗原提呈细胞(2000万个),并在400g离心5分钟,然后使用含有蛋白酶抑制剂的4℃磷酸盐缓冲溶液(PBS)洗涤相应混合抗原提呈 细胞两遍,将细胞重悬在PBS水中后在4℃低功率(12W)超声3分钟。然后将样品在3000g离心15分钟并收集上清液,将上清液在8000g离心15分钟后收集上清液,将上清液使用0.22μm的膜过滤器反复过滤样品后在16000g离心90分钟后收集弃去上清液收集沉淀,将沉淀在PBS中重悬后即得抗原提呈细胞的细胞膜组分。
(4)细菌细胞外囊泡(Outer membrane vesicles,OMV)的制备
收集培养后的长双歧杆菌(BL21),在3000g离心10分钟,去除沉淀后将上清液在15000g离心90分钟,将沉淀在PBS中重悬后即为收集到的细菌细胞外囊泡(3mg/mL)。
(5)癌细胞细胞膜组分的制备
收集B16F10癌细胞,然后使用生理盐水洗涤细胞两遍,将细胞重悬在生理盐水中后在7.5W超声20分钟。然后将样品在2000g离心20分钟并收集上清液,将上清液在7000g离心20分钟后收集上清液,然后在15000g离心120分钟后收集弃去上清液收集沉淀,将沉淀在PBS中重悬后备用。
(6)混合膜组分的制备
将步骤(3)收集的未被激活的混合抗原提呈细胞膜组分或者已经被纳米粒子激活的混合抗原提呈细胞膜组分、步骤(4)收集的细菌细胞外囊泡组分和步骤(5)收集的癌细胞细胞膜组分按质量比2:1:1混合,然后在室温下1500RPM搅拌2分钟后使用膜挤出器反复共挤出即得三者的混合膜组分。
(7)纳米疫苗的制备
将100mg步骤(2)制备的纳米粒1(50mg负载水溶性组分的纳米粒子+50mg负载非水溶性组分的纳米粒子)与10mg步骤(6)制备的混合膜组分混合后共孵育20分钟,然后使用0.45μm的滤膜反复过滤,将滤液在13000g离心20分钟,弃去上清液后使用4%海藻糖水溶液重悬沉淀,冷冻干燥48小时后所得冻干粉即为纳米疫苗。其中,使用内部负载全细胞组分的纳米粒子1激活的混合抗原提呈细胞膜组分与癌细胞和细菌细胞外囊泡混合制备的纳米疫苗1粒径为340nm;使用内部负载四种多肽抗原的纳米粒子激活的混合抗原提呈细胞膜组分与癌细胞和细菌细胞外囊泡混合制备的纳米疫苗2粒径为340nm;使用未被激活的混合抗原提呈细胞膜组分与癌细胞和细菌细胞外囊泡混合制备的纳米疫苗3粒径为340nm。
(8)纳米疫苗用于癌症的治疗
选取6-8周的雌性C57BL/6为模型小鼠制备黑色素瘤荷瘤小鼠,在第0天,给每只受体小鼠背部右下方皮下接种1.5×10 5个B16F10细胞。在小鼠接种癌细胞前后5天、第8天、第11天、第15天和第20天给每只小鼠分别接种1mg纳米疫苗1或者纳米疫苗2或者纳米疫苗3或者100μL的PBS。小鼠肿瘤生长速度和小鼠生存期监测方法同上。
(9)实验结果
如图3所示,PBS对照组小鼠肿瘤生长速度都很快,小鼠生存期很短。纳米疫苗组小鼠的肿瘤生长速度都明显变慢,小鼠生存期都明显延长。其中纳米疫苗1和纳米疫苗2效果好于纳米疫苗3;纳米疫苗1效果好于纳米疫苗2。这说明,被负载抗原的纳米粒子激 活的混合抗原提呈细胞膜组分加入到混合膜组分中后效果好于未被任何纳米粒子激活的混合抗原提呈细胞膜组分;而且内部负载癌细胞全细胞组分纳米粒子激活混合抗原提呈细胞膜组分加入混合膜组分中后效果好于内部负载多肽抗原的纳米粒子。
实施例3 纳米疫苗用于黑色素瘤的治疗
本实施例以小鼠黑色素瘤为癌症模型来说明如何使用纳米疫苗治疗癌症。本实施例中,首先裂解B16F10黑色素瘤肿瘤组织和癌细胞以制备肿瘤组织和癌细胞的水溶性抗原混合物(质量比1:1)和非水溶性抗原混合物(质量比1:1),并将水溶性抗原混合物和非水溶性抗原混合物按质量比1:1混合。然后,以PLGA为纳米粒骨架材料,以Poly(I:C)、CpG2006和CpG2395为佐剂制备内部负载裂解物组分的纳米粒子,然后将纳米粒子与癌细胞和激活的抗原提呈细胞的混合细胞膜共作用一定时间,制备即得纳米疫苗用于治疗癌症。
(1)肿瘤组织和癌细胞的裂解及各组分的收集
收集肿瘤组织时先在每只C57BL/6小鼠背部皮下接种1.5×10 5个B16F10细胞,在肿瘤长到体积分别为约1000mm 3时处死小鼠并摘取肿瘤组织,将肿瘤组织切块后研磨,通过细胞过滤网加入适量纯水并反复冻融5次,并可伴有超声以破坏裂解所得样品;收集培养的B16F10癌细胞系时,先离心去除培养基后使用PBS洗涤两次并离心收集癌细胞,将癌细胞在超纯水中重悬,反复冻融3次,并伴有超声破坏裂解癌细胞。待肿瘤组织或癌细胞裂解后,将裂解物以5000g的转速离心5分钟并取上清液即为可溶于纯水的水溶性抗原;在所得沉淀部分中加入8M尿素溶解沉淀部分即可将不溶于纯水的非水溶性抗原转化为在8M尿素水溶液中可溶。将肿瘤组织的水溶性抗原和癌细胞的水溶性抗原按质量比1:1混合;肿瘤组织的非水溶性抗原和癌细胞的非水溶性抗原按质量比1:1混合。将水溶性抗原混合物和非水溶性抗原混合物按质量比1:1混合,即为制备纳米粒子的抗原原料来源。
(2)细菌细胞外囊泡(Outer membrane vesicles,OMV)的制备
收集培养后的大肠杆菌(BL21),然后在4℃下5000×g离心10min,得到的上清液(200mL)经0.45μm EPS过滤器(微孔)过滤,然后用50K超滤管浓缩至50mL。浓缩液用0.22μm EPS膜(微孔)进一步过滤后使用150,000×g在4℃超速离心1小时从滤液中收集OMV,弃去上清液后使用10%脱氧胆酸钠水溶液溶解所得沉淀使其在水溶液中完全溶解以能被负载于纳米粒子内部。
(3)纳米粒子的制备
本实施例中纳米粒采用溶剂挥发法制备。纳米粒子1制备材料PLGA分子量为24Da-38KDa,所采用的免疫佐剂为poly(I:C)、CpG2006和CpG2395且佐剂包载于纳米粒子内部;在制备纳米粒子时,第一内水相中所含有的全细胞裂解物组分和细菌细胞外囊泡组分质量比为3:1。制备方法如前所述,在制备过程中首先采用复乳法在纳米粒子内部负载步骤(1)制备的含有全细胞抗原的裂解物组分、步骤(2)制备的细菌细胞外囊泡组分和佐剂,然后将100mgPLGA纳米粒子在10000g离心20分钟,并使用10mL含4%海藻糖的超纯水重悬后冷冻干燥48h。该纳米粒子1平均粒径为350nm左右;每1mg PLGA纳米粒子1约负载 100μg蛋白质和多肽组分,负载poly(I:C)、CpG2006和CpG2395免疫佐剂各0.025mg。
纳米粒子2制备材料和制备方法相同,只是内部只负载佐剂和癌细胞全细胞裂解物组分,但是不负载任何细菌细胞外囊泡组分,纳米粒子2平均粒径为350nm左右,每1mg PLGA纳米粒子2约负载100μg蛋白质和多肽组分,负载poly(I:C)、CpG2006和CpG2395免疫佐剂各0.025mg。
纳米粒子3制备材料和制备方法相同,只是内部只负载佐剂和细菌细胞外囊泡组分,但是不负载任何癌细胞裂解液组分,纳米粒子3平均粒径为350nm左右,每1mg PLGA纳米粒子1约负载100μg蛋白质和多肽组分,负载poly(I:C)、CpG2006和CpG2395免疫佐剂各0.025mg。
(4)抗原提呈细胞及其膜组分的制备
本实施例使用BMDC和B细胞的混合抗原提呈细胞。BMDC的制备方法同实施例1。B细胞的分离方法如下:处死C57BL/6小鼠后摘取小鼠脾脏,制备小鼠脾细胞单细胞悬液,使用磁珠分选法分离脾细胞中活细胞中(使用活死细胞染料标记死细胞以去除死细胞)的CD19 +B细胞。然后将BMDC和B细胞按照数量比1:1混合后作为混合抗原提呈细胞使用。
将1mg负载癌细胞全细胞组分的纳米粒子1与2000万个混合抗原提呈细胞(其中DC1000万个+B细胞1000万个)在20mL RPMI1640完全培养基中混合后共孵育48小时(37℃,5%CO 2),孵育体系中含有IL-2(1000U/mL)、IL-7(1000U/mL)、IL-12(200U/mL)、GM-CSF(500U/mL)和白蛋白(50ng/mL)。孵育完成后,将孵育后的细胞在400g离心5分钟后弃去上清液后再使用PBS洗涤两遍即得被激活的抗原提呈细胞。
制备膜组分时,首先收集已经被纳米粒子激活后的混合抗原提呈细胞(2000万个),并在400g离心5分钟,然后使用含有蛋白酶抑制剂的4℃磷酸盐缓冲溶液(PBS)洗涤相应混合抗原提呈细胞两遍,将细胞重悬在PBS水中后在4℃低功率(12W)超声3分钟。然后将样品在3000g离心15分钟并收集上清液,将上清液在8000g离心15分钟后收集上清液,将上清液使用0.22μm的膜过滤器反复过滤样品后在16000g离心90分钟后收集弃去上清液收集沉淀,将沉淀在PBS中重悬后即得抗原提呈细胞的细胞膜组分。
(5)癌细胞细胞膜组分的制备
收集B16F10癌细胞,在400g离心5分钟后弃去上清液,并使用PBS将沉淀细胞重悬,然后使用含有0.0759M蔗糖和0.225M甘露醇的30mM pH 7.0Tris-HCl缓冲液中400g离心5min清洗三次,然后使用含有磷酸酶抑制剂和蛋白酶抑制剂的PBS清洗两次,然后高压均质化(5mPa)处理1分钟机械破坏细胞。然后将样品依次过孔径为30μm、10μm、5μm、2μm、0.45μm的膜过滤后,将滤液在18000g离心30分钟后弃去上清液收集沉淀,将沉淀在PBS中重悬后备用。
(6)混合膜组分的制备
将步骤(4)收集被纳米粒子激活的混合抗原提呈细胞膜组分和步骤(5)收集的癌细胞细胞膜组分按质量比1:1混合,然后在室温下1500RPM搅拌3分钟后使用膜挤出器反复共 挤出即得二者的混合膜组分。
(7)纳米疫苗的制备
将100mg步骤(3)制备的纳米粒1或者纳米粒2或者纳米粒3重悬于9mLPBS中,然后与1mL的5mg步骤(6)制备的混合膜组分混合后,在1500rpm机械搅拌10分钟,然后使用反复通过0.45μm的膜共挤出,收集挤出液后在13000g离心25分钟后弃去上清液后使用PBS重悬沉淀即为纳米疫苗。其中,使用纳米粒子1制备的为纳米疫苗1,粒径为370nm;使用纳米粒子2制备的为纳米疫苗2,粒径为370nm;使用纳米粒子3制备的为纳米疫苗3,粒径为370nm。
(8)纳米疫苗用于癌症的治疗
选取6-8周的雌性C57BL/6为模型小鼠制备黑色素瘤荷瘤小鼠。在第0天给每只小鼠背部右下方皮下接种1.5×10 5个B16F10细胞。在接种黑色素瘤后第4天、第7天、第10天、第15天、第20天和第25天分别每只小鼠皮下注射500μg纳米疫苗1或者纳米疫苗2或者纳米疫苗3或者100μL PBS。在实验中,从第3天开始每3天记录一次小鼠肿瘤体积的大,小鼠肿瘤体积和生存期监测方法同上。
(9)实验结果
如图4中a和b所示,PBS对照组的小鼠肿瘤生长速度都很快,小鼠生存期很短。纳米疫苗1、纳米疫苗2和纳米疫苗3都可以显著抑制肿瘤生长和延长小鼠生存期。其中,纳米疫苗1效果好于纳米疫苗2和纳米疫苗3。这说明,内部负载细菌细胞外囊泡组分、内部负载癌细胞全细胞组分都有利于提高表面负载混合膜组分的癌症纳米疫苗的效果。
实施例4 纳米疫苗用于黑色素瘤肺转移的预防
本实施例以小鼠黑色素瘤肺模型来说明使用纳米疫苗预防癌症转移。本实施例中,首先裂解B16F10黑色素瘤肿瘤组织以制备肿瘤组织的水溶性抗原和非水溶性抗原;然后,制备负载有肿瘤组织的水溶性抗原和非水溶性抗原的纳米粒子系统。在本实施例中采用了硅化和添加带电物质的方法来增加抗原的负载量,且只进行了一轮矿化处理。本实施例中,先使用纳米粒子激活抗原提呈细胞,然后使用抗原提呈细胞制备纳米疫苗并用于预防癌症转移。
(1)肿瘤组织的裂解及各组分的收集
在每只C57BL/6小鼠背部皮下接种1.5×10 5个B16F10细胞,在肿瘤长到体积分别为约1000mm 3时处死小鼠并摘取肿瘤组织。将肿瘤组织切块后研磨,加入胶原酶在RPMI 1640培养基中孵育30min,然后通过细胞过滤网加入适量纯水并反复冻融5次,并可伴有超声以破坏裂解细胞。待细胞裂解后,将裂解物以5000g的转速离心5分钟并取上清液即为可溶于纯水的水溶性抗原;在所得沉淀部分中加入10%的脱氧胆酸钠溶解沉淀部分即可将不溶于纯水的非水溶性抗原转化为在10%脱氧胆酸钠水溶液中可溶,将水溶性抗原和非水溶性抗原按质量比2:1混合,即为制备粒子的抗原原料来源。
(2)细菌膜组分的制备
收集培养后的长双歧杆菌后在5000g离心30分钟,使用PBS离心洗涤两次后重悬于PBS中,然后在4℃使用20W超声处理5分钟,然后依次通过20μm、10μm、5μm、1μm、0.45μm的滤膜过滤挤出,去除沉淀后将上清液在13000g离心90分钟,将沉淀在PBS中重悬后即为收集到的长双歧杆菌细菌膜组分。
收集培养后的嗜酸乳杆菌后在5000g离心30分钟,使用PBS离心洗涤两次后重悬于PBS中,然后在4℃使用20W超声处理5分钟,然后依次通过20μm、10μm、5μm、1μm、0.45μm的滤膜过滤挤出,去除沉淀后将上清液在13000g离心90分钟,将沉淀在PBS中重悬后即为收集到的嗜酸乳杆菌细菌膜组分。
(3)纳米粒子的制备
本实施例中纳米粒子及作为对照的空白纳米粒采用溶剂挥发法制备,并进行了适当的修饰改进,在纳米粒子制备过程中采用低温硅化技术和添加带电物质两种修饰方法提高抗原的负载量。为了将细菌细胞膜组分负载于纳米粒子,先将细菌膜组分使用8M尿素溶解,然后再将溶解后的细菌细胞膜组分与肿瘤组织全细胞组分混合。制备纳米粒子时所使用的肿瘤组织全细胞组分和细菌膜组分(长双歧杆菌或者嗜酸乳杆菌)质量比为1:1。纳米粒子的制备材料PLGA分子量为24KDa-38KDa,所采用的免疫佐剂为poly(I:C)、CpG1018和CpG2395。制备方法如前所述,在制备过程中首先采用复乳法在纳米粒子内部负载肿瘤组织裂解物组分、细菌膜(长双歧杆菌或者嗜酸乳杆菌)组分和佐剂,然后将100mg纳米粒子在10000g离心20分钟,然后使用7mL PBS重悬纳米粒子并与3mL含有细胞裂解物(60mg/mL)的PBS溶液混合,尔后在10000g离心20分钟,然后采用10mL硅酸盐溶液(含150mM NaCl、80mM原硅酸四甲酯和1.0mM HCl,pH 3.0)重悬,并在室温固定10min,尔后在-80℃固定24h,使用超纯水离心洗涤后使用3mL含鱼精蛋白(5mg/mL)和聚赖氨酸(10mg/mL)的PBS重悬并作用10min,然后10000g离心20min洗涤,采用10mL含有细胞裂解物(50mg/mL)的PBS溶液重悬并作用10min,然后在10000g离心20分钟并使用10mL含4%海藻糖的超纯水重悬后冷冻干燥48h;在粒子使用前将其用7mLPBS重悬然后加入3mL含佐剂的癌组织裂解液组分(蛋白质浓度50mg/mL)并室温作用10min,得到内外都负载裂解物的经冷冻硅化和添加阳离子物质的修饰的纳米粒子。其中负载肿瘤组织裂解物组分和长双歧杆菌的纳米粒子为纳米粒子1,平均粒径为350nm左右,每1mg PLGA纳米粒子约负载300μg蛋白质或多肽组分,每1mgPLGA纳米粒1所负载的poly(I:C)、CpG1018和CpG2395各0.02mg。负载肿瘤组织裂解物组分和嗜酸乳杆菌的纳米粒子为纳米粒子2,平均粒径为350nm左右,每1mg PLGA纳米粒子约负载300μg蛋白质或多肽组分,每1mgPLGA纳米粒2所负载的poly(I:C)、CpG1018和CpG2395各0.02mg。
(4)癌细胞和细菌细胞膜组分的制备
收集细胞培养的B16F10黑色素瘤细胞,在400g离心5分钟后弃去上清液,然后使用含有蛋白酶抑制剂的4℃磷酸盐缓冲溶液(PBS)洗涤混合两遍,将细胞重悬在PBS中。将1000万个B16F10细胞在4℃低功率(15W)超声1分钟,然后将样品在3000g离心15分钟并收集 上清液,将上清液在8000g离心15分钟后收集上清液,将所得上清液与步骤(2)制备的3mg细菌(长双歧杆菌或者嗜酸乳杆菌)膜组分混合后在室温共孵育10分钟,然后使用0.22μm的滤膜反复共挤出,收集挤出液后将其在16000g离心90分钟后收集弃去上清液收集沉淀,将沉淀在PBS中重悬后即得混合细胞细胞膜组分。
作为对照的膜组分只使用癌细胞细胞膜组分。收集细胞培养的B16F10黑色素瘤细胞,在400g离心5分钟后弃去上清液,然后使用含有蛋白酶抑制剂的4℃磷酸盐缓冲溶液(PBS)洗涤混合两遍,将细胞重悬在PBS中。然后将癌细胞在4℃低功率(15W)超声1分钟,然后将样品在3000g离心15分钟并收集上清液,将上清液在8000g离心15分钟后收集上清液,然后使用0.22μm的滤膜反复挤出,收集挤出液后将其在16000g离心90分钟后收集弃去上清液收集沉淀,将沉淀在PBS中重悬后即得癌细胞细胞膜组分。
(5)纳米疫苗的制备
将100mg步骤(3)制备的纳米粒1重悬于9mLPBS中,然后与10mL的20mg步骤(4)制备的癌细胞与长双歧杆菌细菌膜组分混合;将100mg步骤(3)制备的纳米粒子2重悬于9mLPBS中,然后与10mL的20mg步骤(4)制备的癌细胞与嗜酸乳杆菌细菌膜组分混合;或者将100mg步骤(3)制备的纳米粒子1重悬于9mLPBS中,然后与10mL的20mg步骤(4)制备的癌细胞细胞膜膜组分混合。将上述纳米粒子与膜组分混合物使用匀浆机在1500rpm处理5分钟,然后使用0.45μm的滤膜反复共挤出,收集挤出液后将其在13000g离心20分钟,弃去上清液后将沉淀使用PBS重悬沉淀即为纳米疫苗。其中,使用癌细胞和长双歧杆菌混合膜组分制备的纳米疫苗为纳米疫苗1,粒径为370nm;使用癌细胞和嗜酸乳杆菌混合膜组分制备的纳米疫苗为纳米疫苗2,粒径为370nm;使用癌细胞膜组分制备的纳米疫苗为纳米疫苗3,粒径为370nm。
(6)纳米疫苗用于癌症转移的预防
选取6-8周的雌性C57BL/6为模型小鼠制备黑色素瘤荷瘤小鼠。在小鼠癌症造模前第-35天、第-28天、第-21天、第-14天和第-7天每只小鼠皮下注射200μg纳米疫苗1或者纳米疫苗2或者纳米疫苗3或者100μL PBS。在第0天给每只小鼠静脉注射接种1×10 5个B16F10细胞,第16天处死小鼠,观察记录小鼠肺部黑色素瘤癌灶数量。
(7)实验结果
如图5所示,PBS对照组小鼠的癌灶较多且癌灶较大,而纳米疫苗处理的小鼠癌症数量都明显减少。而且,纳米疫苗1和纳米疫苗2对癌症肺转移的预防效果明显优于纳米疫苗3;而且纳米疫苗1对癌症肺转移的预防效果明显优于纳米疫苗2。
实施例5 纳米疫苗用于治疗结肠癌
本实施例首先裂解结肠癌肿瘤组织以制备水溶性抗原混合物(质量比1:1)和非水溶性抗原(质量比1:1)混合物,并将水溶性抗原混合物和非水溶性抗原混合物按质量比1:1混合。然后,以PLA为纳米粒骨架材料,以CpGSL03和Poly ICLC为免疫佐剂制备纳米粒子,并在该纳米粒子表面负载癌细胞细胞外囊泡和细菌外囊泡后制备得到纳米疫苗,用于治疗 结肠癌。
(1)肿瘤组织和癌细胞的裂解及各组分的收集
在每只C57BL/6小鼠背部皮下接种2×10 6个MC38细胞在肿瘤长到体积分别为约1000mm 3时处死小鼠并摘取肿瘤组织。将肿瘤组织切块后研磨,通过细胞过滤网加入适量纯水并反复冻融5次,并可伴有超声以破坏裂解细胞。待细胞裂解后,将裂解物以大于5000g的转速离心5分钟并取上清液即为可溶于纯水的水溶性抗原;在所得沉淀部分中加入8M尿素水溶液溶解沉淀部分即可将不溶于纯水的非水溶性抗原转化为水溶液中可溶。将来自结肠癌肿瘤组织的和肺癌癌细胞的的水溶性抗原按质量比1:1混合;该混合物为制备纳米粒子的原料来源。
(2)细菌膜组分的制备
选取活的卡介苗并在5000g离心30分钟,然后使用PBS离心洗涤两遍后重悬于PBS中,在4℃下使用20W超声处理5分钟,然后依次通过20μm、10μm、5μm、1μm、0.45μm的滤膜过滤挤出,去除沉淀后将上清液在13000g离心90分钟,将沉淀在PBS中重悬后即为收集到的细菌膜组分,然后使用8M尿素水溶液裂解和溶解细菌膜组分。
或者选取活的卡介苗并在5000g离心30分钟,然后使用PBS离心洗涤两遍后重悬于PBS中,在4℃下使用20W超声处理5分钟,然后依次通过20μm、10μm、5μm、1μm、0.45μm的滤膜过滤挤出,去除沉淀后将上清液在13000g离心90分钟,将沉淀在PBS中重悬后即为收集到的细菌膜组分,然后使用吐温80水溶液裂解和溶解细菌膜组分。
(3)纳米粒子的制备
本实施例中纳米粒采用溶剂挥发法制备,将肿瘤组织裂解物组分、步骤(2)所裂解和溶解的细菌膜组分以及免疫佐剂负载于纳米粒子中。所采用的纳米粒子制备材料PLA分子量为20KDa,所采用的免疫佐剂为CpG SL03和Poly ICLC,且佐剂分布于纳米粒子内部。制备方法如前所述,在制备过程中首先采用复乳法在纳米粒子内部负载裂解物混合物和佐剂,在内部负载裂解物和佐剂后,将100mg纳米粒子在10000g离心20分钟,并使用10mL含4%海藻糖的超纯水重悬后冷冻干燥48h。使用8M尿素溶解的细菌膜组分制备的纳米粒子为纳米粒子1,该纳米粒子平均粒径为280nm左右,每1mg PLGA纳米粒子约负载90μg蛋白质或多肽组分,每1mgPLGA纳米粒含有CpGSL03和Poly ICLC免疫佐剂各0.03mg;使用吐温80溶解的细菌膜组分制备的纳米粒子为纳米粒子2,该纳米粒子平均粒径为280nm左右,每1mg PLGA纳米粒子约负载90μg蛋白质或多肽组分,每1mgPLGA纳米粒含有CpGSL03和Poly ICLC免疫佐剂各0.03mg。
(4)细菌外囊泡(OMV)和癌细胞外囊泡的制备
将活的卡介苗在LB培养基中培养,然后收集培养后的样品在5000g离心30分钟,弃去沉淀后收集上清液并在15W超声处理10分钟,将上清液在16000g离心90分钟,将沉淀在PBS中重悬后即为收集到的细菌外囊泡组分。
将MC38细胞在含有50ng/mL的阿霉素的DMEM高糖培养基中培养6天,期间每2天换液 一次,收集培养的MC38细胞使用含有蛋白酶抑制剂的4℃磷酸盐缓冲溶液(PBS)洗涤两遍,在400g离心5分钟,去除沉淀后将上清液在15W超声处理10分钟,然后在15000g离心90分钟,将沉淀在PBS中重悬后即为收集到的癌细胞外囊泡膜组分。
(5)纳米疫苗的制备
将100mg步骤(3)制备的纳米粒1重悬于9mLPBS中,与1mL的步骤(4)制备的癌细胞细胞外囊泡(5mg)与细菌外囊泡(5mg)混合,然后在4℃的10W超声作用2分钟,然后使用0.45μm的滤膜反复共挤出,将挤出液在13000g离心30分钟,弃去上清液后将沉淀使用PBS重悬沉淀即得纳米疫苗1,粒径为300nm。
将100mg步骤(3)制备的纳米粒2重悬于9mLPBS中,与1mL的步骤(4)制备的癌细胞细胞外囊泡(5mg)与细菌外囊泡(5mg)混合,然后在4℃的10W超声作用2分钟,然后使用0.45μm的滤膜反复共挤出,将挤出液在13000g离心30分钟,弃去上清液后将沉淀使用PBS重悬沉淀即得纳米疫苗2,粒径为300nm。
将100mg步骤(3)制备的纳米粒1重悬于9mLPBS中,然后与1mL的步骤(4)制备的细菌外囊泡(10mg)混合,然后在4℃的10W超声作用10分钟,然后在13000g离心20分钟,弃去上清液后将沉淀使用PBS重悬沉淀即得纳米疫苗3,粒径为300nm。
(6)纳米疫苗用于癌症的治疗
选取6-8周的雌性C57BL/6为模型小鼠制备结肠癌荷瘤小鼠。在第0天给每只小鼠皮下接种2×10 6个MC38细胞,在第4、第7天、第10天、第15天和第20天分别给每只小鼠皮下注射800μg纳米疫苗1或者800μg纳米疫苗2或者800μg纳米疫苗3或100μLPBS。肿瘤生长和小鼠生存期监测方法同上。
(7)实验结果
如图6所示,对照组小鼠的肿瘤都很快长大,而经三种纳米疫苗处理的小鼠肿瘤生长速度都明显变慢或肿瘤消失。而且,纳米疫苗1好于纳米疫苗2和纳米疫苗3。由此可见,癌细胞细胞外囊泡膜组分的加入有利于疫苗的功效,而且,不同裂解液裂解得到的细菌膜组分负载于纳米疫苗内部制备的纳米疫苗效果有差异。
实施例6 纳米疫苗用于治疗黑色素瘤
本实施例以黑色素瘤为癌症模型来说明如何使用内部负载黑色素瘤肿瘤组织全细胞组分,表面负载肺癌癌细胞和抗原提呈细胞膜组分的纳米疫苗治疗黑色素瘤。本实施例中,首先裂解B16F10黑色素瘤肿瘤组织中的水溶性组分和非水溶性组分。然后以PLGA为纳米粒骨架材料,以锰颗粒和CpG2395为免疫佐剂制备纳米粒子,然后在纳米粒表面负载膜组分后用于癌症治疗。
(1)肿瘤组织的裂解及各组分的收集
在每只C57BL/6小鼠背部皮下接种1.5×10 5个B16F10细胞,在肿瘤长到体积约1000mm 3时处死小鼠并摘取肿瘤组织。将肿瘤组织切成小块后磨碎通过细胞筛网过滤得到肿瘤组织单细胞悬液,加入超纯水后将肿瘤组织单细胞悬液反复冻融5次,并辅以超声裂解。在裂 解液中加入核酸酶在37℃作用10分钟,然后将含有核酸酶的裂解液在95℃加热5分钟。尔后在5000g离心5分钟,上清液即为水溶性组分,将沉淀使用10%的辛基葡萄糖苷溶解后即为溶解后的原非水溶性组分。将来自黑色素瘤肿瘤组织的水溶性组分和溶解于辛基葡萄糖苷水溶液中的原非水溶性组分按质量比3:1混合即为制备纳米粒子的抗原来源。
(2)纳米粒子的制备
本实施例中纳米粒子采用复乳法制备。所采用的纳米粒子制备材料PLGA分子量为24KDa-38KDa,所采用的免疫佐剂为STING激动剂和Toll样受体激动剂的混合佐剂:锰胶体颗粒(STING激动剂)和CpG2395(Toll样受体激动剂)。先制备锰佐剂,然后将锰佐剂与癌细胞全细胞组分中的水溶性组分或非水溶性组分混合后作为第一水相采用复乳法制备内部负载裂解物组分和佐剂的纳米粒。在制备锰佐剂时,先将1mL 0.3M的Na 3PO 4溶液加入到9mL生理盐水中,后加入2mL 0.3M的MnCl 2溶液,放置过夜后,即得到Mn 2OHPO 4胶体锰佐剂,锰佐剂粒径约为13nm。然后将锰佐剂与癌细胞全细胞组分的水溶性组分(60mg/mL)或非水溶性组分(60mg/mL)按1:3体积比混合后采用复乳法将抗原和锰佐剂负载到纳米粒内部。在内部负载裂解组分和佐剂后,将100mg纳米粒子在10000g离心20分钟,使用10mL含4%海藻糖的超纯水重悬后冷冻干燥48h后备用。该纳米粒子平均粒径为370nm左右,纳米粒子表面电位为-5mV左右;每1mg PLGA纳米粒子约负载120μg蛋白质和多肽组分,每1mg PLGA纳米粒使用CpG2395佐剂为0.04mg。
(3)抗原提呈细胞的制备及激活
抗原提呈细胞为来源于外周血的B细胞和BMDC的混合抗原提呈细胞。BMDC制备方法同上。处死C57BL/6后收集小鼠外周血,从外周血中分离外周血单核细胞(PBMC),然后使用流式细胞术从PBMC中分选出CD19 +B细胞。将BMDC和B细胞按照数量比1:1混合即为混合抗原提呈细胞。
将1mg步骤(2)制备的负载癌细胞全细胞组分的纳米粒子与2000万个混合抗原提呈细胞(其中BMDC1000万个+B细胞1000万个)在20mL RPMI1640完全培养基中混合后共孵育48小时(37℃,5%CO 2),孵育体系中含有IL-21(500U/mL)、IL-2(500U/mL)、IL-7(500U/mL)和CD80抗体(10ng/mL)。孵育完成后,将孵育后的细胞在400g离心5分钟后弃去上清液后再使用PBS洗涤两遍即得被激活的抗原提呈细胞。
(4)癌细胞和抗原提呈细胞膜组分的制备
收集细胞培养的B16F10黑色素瘤细胞,在400g离心5分钟后弃去上清液,并使用PBS将沉淀细胞重悬,将黑色素瘤细胞和激活的混合抗原提呈细胞按照数量比2:1混合,然后使用含有磷酸酶抑制剂和蛋白酶抑制剂的PBS清洗两次,然后在15W超声处理2分钟机械破坏细胞,将样品在3000g离心5分钟,弃去沉淀后将上清液在8000g离心五分钟,然后弃去沉淀将上清液在15000g离心60分钟后弃去上清液收集沉淀,将沉淀在PBS中重悬后即得混合细胞细胞膜组分。
作为对照膜组分,只使用激活的混合抗原提呈细胞制备。将混合抗原提呈细胞用含有 磷酸酶抑制剂和蛋白酶抑制剂的PBS清洗两次,然后在15W超声处理2分钟机械破坏细胞。将样品在3000g离心5分钟,弃去沉淀后将上清液在8000g离心五分钟,然后弃去沉淀将上清液在15000g离心60分钟后弃去上清液收集沉淀,将沉淀在PBS中重悬后即得混合抗原提呈细胞细胞膜组分。
(5)纳米疫苗的制备
将步骤(2)制备的纳米粒(100mg)在9mLPBS中重悬,然后与1mL步骤(4)制备的癌细胞与抗原提呈细胞的混合细胞膜组分(2mg)混合,在上述混合物中加入DSPE-PEG-CD32单抗(0.1mg)并在50W超声处理2分钟,然后使用0.45μm的滤膜反复共挤出,将挤出液在12000g离心20分钟后弃去上清液收集沉淀,将沉淀在PBS中重悬后即得纳米疫苗1,纳米疫苗粒径为390纳米,表面电位为-4mV。
将步骤(2)制备的纳米粒(100mg)在9mLPBS中重悬,然后与1mL步骤(4)制备的混合抗原提呈细胞膜组分(2mg)混合,在上述混合物中加入DSPE-PEG-CD32单抗(0.1mg)并在50W超声处理2分钟,然后使用0.45μm的滤膜反复共挤出,将挤出液在12000g离心20分钟后弃去上清液收集沉淀,将沉淀在PBS中重悬后即得纳米疫苗2,纳米疫苗粒径为390纳米,表面电位为-4mV。
(6)纳米疫苗用于癌症的治疗
选取6-8周的雌性C57BL/6为模型小鼠制备黑色素瘤荷瘤小鼠。在第0天给每只小鼠皮下接种1.5×10 5个B16F10细胞,在第5、第8天、第11天、第16天和第21天分别给小鼠皮下注射500μg纳米疫苗1或者500μg纳米疫苗2或者100μLPBS。小鼠肿瘤体积和生存期监测同上。
(7)实验结果
如图7所示,PBS对照组小鼠的肿瘤都很快长大,与对照组相比,纳米疫苗处理的小鼠肿瘤速度都明显变慢,而且,生存期延长。综上所述,本发明所述纳米疫苗对癌症具有治疗效果。而且,表面负载癌细胞和混合抗原提呈细胞细胞膜组分的纳米疫苗1效果好于表面只负载混合抗原提呈细胞细胞膜组分的纳米疫苗2。本实施例纳米疫苗中使用CD32单克隆抗体作为主动靶向的靶头,在实际应用中也可以使用甘露糖、甘露聚糖、CD205单抗、CD19单抗等任何具有靶向靶细胞能力的靶头。
实施例7 微米疫苗用于乳腺癌的预防
(1)癌细胞的裂解
将培养的4T1细胞在400g离心5分钟,然后用PBS洗涤两遍后重悬于超纯水中。所得癌细胞分别采用紫外线和高温加热进行灭活和变性处理,然后采用适量6M盐酸胍水溶液裂解乳腺癌细胞并溶解裂解物即为抗原原料来源。
(2)微米粒子系统的制备
本实施例中制备微米粒子采用复乳法,微米粒子骨架材料PLGA分子量为38KDa-54KDa,所采用的免疫佐剂为CpG2395、CpG1018和Poly(I:C)。制备时先采用复乳法制备内部负载裂解物组分和佐剂的微米粒子,然后将100mg微米粒子在9000g离心20分 钟,使用10mL含4%海藻糖的超纯水重悬后干燥48h后备用。该微米粒子系统平均粒径为2.10μm左右,表面电位为-6mV左右;每1mg PLGA微米粒子约负载110μg蛋白质或多肽组分,含CpG2395、CpG1018和Poly(I:C)各0.03mg。
(3)抗原提呈细胞的制备及激活
抗原提呈细胞为来源于外周血的B细胞和BMDC的混合抗原提呈细胞。BMDC制备方法同上。处死C57BL/6后收集小鼠外周血,从外周血中分离外周血单核细胞(PBMC),然后使用流式细胞术从PBMC中分选出CD19 +B细胞。将BMDC和B细胞按照数量比1:1混合即为混合抗原提呈细胞。
将1mg步骤(2)制备的负载癌细胞全细胞组分的微米粒子与2000万个混合抗原提呈细胞(其中BMDC1000万个+B细胞1000万个)在20mLRPMI1640完全培养基中混合后共孵育48小时(37℃,5%CO 2),孵育体系中含有IL-2(500U/mL)、IL-7(500U/mL)、IL-15(500U/mL)、GM-CSF(500U/mL)。孵育完成后,将孵育后的细胞在400g离心5分钟后弃去上清液后再使用PBS洗涤两遍即得被激活的抗原提呈细胞。
或者将1mg步骤(2)制备的负载癌细胞全细胞组分的微米粒子与2000万个BMDC在20mL RPMI1640完全培养基中混合后共孵育48小时(37℃,5%CO 2),孵育体系中含有IL-2(500U/mL)、IL-7(500U/mL)、IL-15(500U/mL)、GM-CSF(500U/mL)。孵育完成后,将孵育后的细胞在400g离心5分钟后弃去上清液后再使用PBS洗涤两遍即得被激活的抗原提呈细胞。
(4)癌细胞和抗原提呈细胞膜组分的制备
收集细胞培养4T1细胞,在400g离心5分钟后弃去上清液,并使用PBS将沉淀细胞重悬,将乳腺癌细胞和步骤(3)激活的混合抗原提呈细胞按照数量比1:1混合,然后使用含有蛋白酶抑制剂的PBS清洗两次,然后在10W超声处理5分钟机械破坏细胞,将样品在2000g离心5分钟,弃去沉淀后将上清液在6000g离心10分钟,然后弃去沉淀将上清液在15000g离心60分钟后弃去上清液收集沉淀,将沉淀在PBS中重悬后即得混合细胞细胞膜组分。
作为对照膜组分,使用癌细胞和步骤(3)激活的BMDC细胞制备。4T1细胞和BMDC按数量比1:1混合,然后将混合后的细胞用含有磷酸酶抑制剂和蛋白酶抑制剂的PBS清洗两次,然后在10W超声处理5分钟机械破坏细胞。将样品在2000g离心5分钟,弃去沉淀后将上清液在6000g离心10分钟,然后弃去沉淀将上清液在15000g离心60分钟后弃去上清液收集沉淀,将沉淀在PBS中重悬后即得癌细胞和DC的混合细胞细胞膜组分。
(5)微米疫苗的制备
将步骤(2)制备的微米粒子(100mg)在9mLPBS中重悬,然后与1mL步骤(4)制备的癌细胞和混合抗原提呈细胞膜组分(2mg)混合,在20W超声处理5分钟,然后在10000g离心15分钟后弃去上清液收集沉淀,将沉淀在PBS中重悬后即得微米疫苗1,微米疫苗1粒径为2.12μm,表面电位为-4mV。
将步骤(2)制备的微米粒子(100mg)在9mL PBS中重悬,然后与1mL癌细胞与DC 的混合细胞膜组分(2mg)混合,在20W超声处理5分钟,然后在10000g离心15分钟后弃去上清液收集沉淀,将沉淀在PBS中重悬后即得微米疫苗2,微米疫苗2粒径为2.12μm,表面电位为-4mV。
(6)微米疫苗用于癌症的预防
选取6-8周的雌性BALB/c为模型小鼠制备乳腺癌荷瘤小鼠。在小鼠接种癌细胞前第-35天、第-28天、第-21天、第-14天和第-7天分别给疫苗组每只小鼠接种800μg微米疫苗1或者800μg微米疫苗2或者100μLPBS。在第0天给每只小鼠皮下注射接种1×10 6个4T1细胞,小鼠肿瘤和生存期监测方法同上。
(7)实验结果
如图8所示,PBS对照组相比,微米疫苗处理的小鼠肿瘤生长速度明显变慢且生存期明显延长。由此可见,本发明所述的微米疫苗对乳腺癌具有预防效果。而且,微米疫苗1效果好于微米疫苗2,说明微米疫苗表面负载癌细胞与混合抗原提呈细胞的细胞膜组分效果好于微米疫苗表面负载癌细胞与单一抗原提呈细胞的细胞膜组分。理论上疫苗被抗原提呈细胞摄取后激活T细胞的过程DC是最重要的抗原提呈细胞(
Figure PCTCN2022108965-appb-000002
T细胞的初次激活),DC细胞膜能更好地引导微米疫苗被DC吞噬,本实验证明B细胞的细胞膜组分能够提高DC细胞膜组分引导微米疫苗被DC吞噬以及后续初次激活T细胞。
实施例8 钙化纳米粒子表面负载膜组分的纳米疫苗用于预防癌症
本实施例说明使用钙化的纳米粒子表面负载膜组分制备纳米疫苗预防癌症,在实际使用时也可以使用其他生物矿化技术、交联、凝胶化等修饰粒子。本实施例中,将小鼠黑色素瘤肿瘤组织以10%脱氧胆酸钠水溶液(含8M精氨酸)裂解后溶解,将肿瘤组织裂解物负载于纳米粒子,并在纳米粒子表面负载膜组分制备纳米疫苗用于癌症的预防。
(1)肿瘤组织和癌细胞的裂解
收集小鼠B16F10黑色素瘤肿瘤组织,将肿瘤组织切成小块后采用10%脱氧胆酸钠水溶液(含8M精氨酸)裂解肿瘤组织后溶解肿瘤组织全细胞组组分。
(2)纳米粒子的制备
本实施例在纳米粒子内部和表面负载癌细胞全细胞组分后生物钙化纳米粒子。本实施例中纳米粒子1采用溶剂挥发法制备,所采用的纳米粒子制备材料PLGA分子量为7KDa-17KDa,所采用免疫佐剂CpG2006和Poly(I:C),负载于纳米粒子内部。制备方法如下所述,在制备过程中首先采用复乳法在纳米粒子内部负载裂解物组分,然后将100mg PLGA纳米粒子在13000g离心20min后使用18mLPBS重悬,然后加入2mL溶解于8M尿素的肿瘤组织裂解液(60mg/mL),在室温作用10分钟后在12000g离心20分钟后收集沉淀。然后将该100mg PLGA纳米粒子重悬于20mL DMEM培养基中,然后加入200μL of CaCl 2(1mM)并在37℃反应两小时。然后在10000g离心20分钟后收集沉淀,并采用超纯水重悬后离心洗涤两遍。纳米粒子1,平均粒径为240nm左右,每1mg PLGA纳米粒子约负载140μg蛋白质或多肽组分,CpG2006和Poly(I:C)各0.03mg。
空白纳米粒子2的制备材料和制备方法同纳米粒子1,但是只负载免疫佐剂而不负载癌细胞裂解物组分,平均粒径为240nm左右,每1mgPLGA纳米粒子约负载140μg蛋白质或多肽组分,CpG2006和Poly(I:C)各0.03mg。
(3)抗原提呈细胞膜组分的制备
本实施例使用DC2.4细胞系和BMDM作为抗原提呈细胞。BMDM制备方法如下:将C57小鼠麻醉后脱臼处死,将小鼠使用75%乙醇的消毒,然后用剪刀在小鼠背部剪开一小口,用手直接撕开皮肤至小鼠小腿关节处,去除小鼠足关节以及皮肤。用剪刀沿着小鼠大腿根部大转子将后肢拆下来,去掉肌肉组织后放置在含有75%乙醇的培养皿内浸泡5min,更换新的75%乙醇的培养皿移入超净台中。将乙醇浸泡的腿骨移入冷的PBS浸泡,洗去胫骨、股骨表面的乙醇,此过程可重复3次。将清洗好的股骨、胫骨分开,并用剪刀分别将股骨、胫骨两端剪断,使用1mL注射器吸取冷的诱导培养基将骨髓从股骨、胫骨中吹出,反复吹洗3次,直至腿骨内看不到明显的红色为止。用5mL移液枪将含有骨髓细胞的培养基反复吹打,使细胞团块分散,然后使用70μm细胞滤器将细胞过筛,转移至15mL离心管内,1500rpm/min离心5min,弃上清,加入红细胞裂解液重悬静置5min后1500rpm/min离心5min,弃上清用冷的配置好的骨髓巨噬细胞诱导培养基(含有15%L929培养基的DMEM高糖培养基)重悬,铺板。将细胞培养过夜,以去除贴壁较快的其他杂细胞如纤维细胞等等。收集未贴壁细胞按实验设计安排种入皿或细胞培养板内。巨噬细胞集落刺激因子(M-CSF)以40ng/mL浓度刺激使骨髓细胞向单核巨噬细胞分化。培养8天,光镜下观察巨噬细胞形态变化。8天后消化收集细胞,用抗小鼠F4/80抗体和抗小鼠CD11b抗体,4℃避光孵育30min后,使用流式细胞术鉴定所诱导成功的巨噬细胞的比例。
将步骤(2)制备的纳米粒子1(1000μg)与DC2.4(500万个)及BMDM细胞(500万个)在15mL高糖DMEM完全培养基中共孵育48小时(37℃,5%CO 2);孵育体系中含有GM-CSF(2000U/mL)、IL-2(500U/mL)、IL-7(200U/mL)、IL-12(200U/mL)和CD40抗体(20ng/mL)。收集孵育后的细胞,将孵育后的细胞在400g离心5分钟,使用PBS重悬并洗涤两遍。即得混合抗原提呈细胞。
将混合抗原提呈细胞在20W超声处理2分钟机械破坏细胞,将样品在2000g离心5分钟,弃去沉淀后将上清液在6000g离心10分钟,然后弃去沉淀将上清液在15000g离心60分钟后弃去上清液收集沉淀,将沉淀在PBS中重悬后即得混合抗原提呈细胞的细胞膜组分。
(4)细菌外囊泡(OMV)和癌细胞外囊泡的制备
将鼠李糖乳杆菌在LB培养基中培养,培养基中含有30μM的阿霉素,然后收集培养后的样品在5000g离心30分钟,弃去沉淀后收集上清液并在15W超声处理10分钟,将上清液在16000g离心90分钟,将沉淀在PBS中重悬后即为收集到的细菌外囊泡组分1。
将B16F10细胞在高糖DMEM完全培养基中培养6天,培养基中含有30μM的阿霉素,每2天换液一次。收集细胞培养B16F10细胞,在400g离心5分钟后弃去上清液,并使用PBS将沉淀细胞重悬,将癌细胞在20W超声处理2分钟机械破坏细胞,将样品在2000g离心5分钟, 弃去沉淀后将上清液在6000g离心10分钟,然后弃去沉淀将上清液在15000g离心60分钟后弃去上清液收集沉淀,将沉淀在PBS中重悬后即得癌细胞细胞膜组分1。
或者将鼠李糖乳杆菌在LB培养基中培养,然后收集培养后的样品在5000g离心30分钟,弃去沉淀后收集上清液并在15W超声处理10分钟,将上清液在16000g离心90分钟,将沉淀在PBS中重悬后即为收集到的细菌外囊泡组分2。
或者将B16F10细胞在高糖DMEM完全培养基中培养6天,每2天换液一次。收集细胞培养B16F10细胞,在400g离心5分钟后弃去上清液,并使用PBS将沉淀细胞重悬,将癌细胞在20W超声处理2分钟机械破坏细胞,将样品在2000g离心5分钟,弃去沉淀后将上清液在6000g离心10分钟,然后弃去沉淀将上清液在15000g离心60分钟后弃去上清液收集沉淀,将沉淀在PBS中重悬后即得癌细胞细胞膜组分2。
(5)纳米疫苗的制备
将步骤(2)制备的纳米粒子1(100mg)在9mLPBS中重悬,然后与1mL步骤(3)制备的混合抗原提呈细胞膜组分(5mg)、步骤(4)制备的癌细胞膜组分1(5mg)和细菌外囊泡组分1(5mg)混合,在20W超声处理5分钟,然后使用0.45μm的滤膜反复共挤出,然后在13000g离心30分钟后弃去上清液收集沉淀,将沉淀在PBS中重悬后即得纳米疫苗1,纳米疫苗粒径为260nm。
将步骤(2)制备的纳米粒子2(100mg)在9mLPBS中重悬,然后与1mL步骤(3)制备的混合抗原提呈细胞膜组分(5mg)、步骤(4)制备的癌细胞膜组分1(5mg)和细菌外囊泡组分1(5mg)混合,在20W超声处理5分钟,然后使用0.45μm的滤膜反复共挤出,然后在13000g离心30分钟后弃去上清液收集沉淀,将沉淀在PBS中重悬后即得纳米疫苗2,纳米疫苗粒径为260nm。
将步骤(2)制备的纳米粒子1(100mg)在9mLPBS中重悬,然后与1mL步骤(3)制备的混合抗原提呈细胞膜组分(5mg)、步骤(4)制备的癌细胞膜组分2(5mg)和细菌外囊泡组分2(5mg)混合,在20W超声处理5分钟,然后使用0.45μm的滤膜反复共挤出,然后在13000g离心30分钟后弃去上清液收集沉淀,将沉淀在PBS中重悬后即得纳米疫苗3,纳米疫苗粒径为260nm。
(6)纳米疫苗癌症的预防
选取6-8周的雌性C57BL/6为模型小鼠制备黑色素瘤荷瘤小鼠,在小鼠接种癌细胞前第-35天、第-28天、第-21天、第-14天和第-7天每只小鼠分别接种800μg纳米疫苗1、或800μg纳米疫苗2、或800μg纳米疫苗3、或者100μL PBS。在第0天,给每只受体小鼠背部右下方皮下接种1.5×10 5个B16F10细胞。小鼠肿瘤体积和生存期监测方法同上。
(7)实验结果
如图9所示,PBS组小鼠肿瘤生长很快并且小鼠很快死亡。3种纳米疫苗都可以显著减缓小鼠肿瘤生长速度和延长小鼠生存期。而且纳米疫苗1效果好于纳米疫苗2和纳米疫苗3,说明将癌细胞全细胞组分负载于纳米疫苗内部有利于提高疫苗功效,而且,使用阿 霉素预处理的癌细胞和细菌所制备细胞膜组分也能提高纳米疫苗的疗效。
实施例9 纳米疫苗用于黑色素瘤的治疗
(1)肿瘤组织和癌细胞的裂解及各组分的收集
收集肿瘤组织时先在每只C57BL/6小鼠背部皮下接种1.5×10 5个B16F10细胞,在肿瘤长到体积分别为约1000mm 3时处死小鼠并摘取肿瘤组织,将肿瘤组织切块后研磨,通过细胞过滤网后制备单细胞悬液,加入超纯水后反复冻融并伴有超声裂解上述细胞,然后加入核酸酶作用5分钟,再在95℃作用10分钟灭活核酸酶。尔后在8000g离心3分钟,上清液部分即为水溶性抗原;沉淀部分使用10%脱氧胆酸钠水溶液溶解非水溶性抗原。将水溶性抗原和脱氧胆酸钠溶解后的非水溶性抗原按质量比1:1混溶即为制备纳米粒子系统的抗原原料来源。
(2)纳米粒子的制备
本实施例中纳米粒采用复乳法制备。纳米粒子1制备材料为PLGA分子量为24KDa-38KDa,所采用的免疫佐剂为poly(I:C)、CpG1018和CpG2216,增加溶酶体免疫逃逸的物质为KALA多肽(WEAKLAKALAKALAKHLAKALAKALKACEA),且佐剂、KALA多肽包载于纳米粒子内部。制备方法如前所述,在制备过程中首先采用复乳法在纳米粒子内部负载裂解液组分、佐剂、KALA多肽,在然后将100mg纳米粒子在12000g离心25分钟,并使用10mL含4%海藻糖的超纯水重悬后冷冻干燥48h。该纳米粒子平均粒径为250nm左右,表面电位为-5mV左右;每1mg PLGA纳米粒子约负载100μg蛋白质或多肽组分,每1mg PLGA纳米粒所负载的poly(I:C)、CpG1018和CpG2216免疫佐剂各0.02mg,负载KALA多肽0.05mg。
纳米粒子2的制备材料和制备方法同上,其粒径为250nm左右,表面电位为-5mV左右,不负载KALA多肽,负载等量佐剂和细胞裂解组分。
纳米粒子3的制备材料和制备方法同上,其粒径为250nm左右,表面电位为-5mV左右;每1mg PLGA纳米粒子约负载100μg蛋白质和多肽组分,每1mg PLGA纳米粒所负载的poly(I:C)0.02mg,负载CpG1018为0.04mg,负载KALA多肽0.05mg。
(3)癌细胞细胞膜组分及抗原提呈细胞细胞膜组分的制备
收集细胞培养B16F10细胞,在400g离心5分钟后弃去上清液,并使用PBS将沉淀细胞重悬,然后在20W超声处理2分钟机械破坏细胞,将样品在2000g离心5分钟,弃去沉淀后将上清液在6000g离心10分钟,然后弃去沉淀将上清液在15000g离心60分钟后弃去上清液收集沉淀,将沉淀在PBS中重悬后即得癌细胞细胞膜组分。
抗原提呈细胞为BMDC,其制备方法同上。将2mg步骤(2)制备的纳米粒子1与3000万个BMDC在20mLRPMI1640完全培养基中混合后共孵育48小时(37℃,5%CO 2),孵育体系中含有IL-2(500U/mL)、IL-7(500U/mL)、IL-15(500U/mL)、GM-CSF(500U/mL)。孵育完成后,将孵育后的细胞在400g离心5分钟后弃去上清液后再使用含有磷酸酶抑制剂和蛋白酶抑制剂的PBS清洗两次,然后在15W超声处理2分钟机械破坏细胞,将样品在3000g 离心5分钟,弃去沉淀后将上清液在8000g离心五分钟,然后弃去沉淀将上清液使用0.22μm的滤膜反复共挤出,将挤出液在15000g离心60分钟后弃去上清液收集沉淀,将沉淀在PBS中重悬后即得抗原提呈细胞细胞膜组分。
将癌细胞细胞膜组分和抗原提呈细胞细胞膜组分按质量比3:1混合后使用0.22μm的滤膜反复共挤出,将挤出液在15000g离心60分钟后弃去上清液收集沉淀,将沉淀在PBS中重悬后即得癌细胞和抗原提呈细胞的混合细胞膜组分。
(4)纳米疫苗的制备
将步骤(2)制备的纳米粒子(100mg)在9mLPBS中重悬,然后与1mL步骤(3)制备的癌细胞细胞膜组分(2mg)混合,在4℃低功率(20W)超声3分钟,然后在12000g离心60分钟后收集弃去上清液收集沉淀,将沉淀在PBS中重悬后即得纳米疫苗。纳米粒子1制备的疫苗为纳米疫苗1,粒径为260纳米,表面电荷为-5mV;纳米粒子2制备的疫苗为纳米疫苗2,粒径为260纳米,表面电荷为-5mV;纳米粒子3制备的疫苗为纳米疫苗3,粒径为260纳米,表面电荷为-5mV。
将步骤(3)制备的癌细胞细胞膜组分(2mg)在4℃低功率(20W)超声3分钟,然后在12000g离心60分钟后收集弃去上清液收集沉淀,将沉淀在PBS中重悬后即得纳米疫苗4,粒径为160纳米,表面电荷为-5mV。
(5)纳米疫苗用于治疗癌症
选取6-8周的雌性C57BL/6为模型小鼠制备黑色素瘤荷瘤小鼠。在第0天给每只小鼠背部右下方皮下接种1.5×10 5个B16F10细胞。在接种黑色素瘤后第6天、第10天、第15天和第20天分别皮下60μg纳米疫苗1-4之一或者100μLPBS。在实验中,小鼠肿瘤体积和生存期监测方法同上。
(6)实验结果
如图10所示,PBS对照组的肿瘤很快都长大。与对照组相比,纳米疫苗处理的小鼠肿瘤生长速度明显变慢生存期明显延长。而且,内部负载癌细胞全细胞组分的纳米粒子表面负载癌细胞细胞膜组分和抗原提呈细胞细胞膜组分的纳米疫苗1、纳米疫苗2和纳米疫苗3都明显好于由癌细胞细胞膜和抗原提呈细胞细胞膜组分制备的纳米疫苗4。而且,加入增加溶酶体逃逸物质的纳米粒子制备的纳米疫苗1好于未加入溶酶体逃逸的纳米粒子制备的纳米疫苗2。而且,使用两种CpG和Poly(I:C)作为混合佐剂的纳米粒子制备的纳米疫苗1治疗效果好于只使用一种CpG和Poly(I:C)混合佐剂的纳米粒子制备的纳米疫苗3。这说明,内部负载癌细胞全细胞组分对于将癌细胞细胞膜组分制备成纳米疫苗至关重要。综上所述,本发明所述的纳米疫苗对癌症具有良好的治疗效果。本实施例使用了KALA多肽作为溶酶体逃逸物质负载于纳米粒子或微米粒子内部,在实际使用中可以使用任何增加溶酶体逃逸的物质,比如多肽、氨基酸、有机高分子物质、具有质子海绵效应的无机物等。
实施例10 纳米粒子表面负载膜组分的纳米疫苗用于预防癌症
(1)癌细胞的裂解
收集小鼠Pan02胰腺癌细胞,用10%十二烷基硫酸钠水溶液裂解癌细胞后溶解癌细胞全细胞组分。
(2)细菌膜组分的制备
将培养后的短双歧杆菌在5000g离心30分钟,然后使用PBS离心洗涤两遍后重悬于PBS中,在4℃下使用20W超声处理5分钟,然后依次通过20μm、10μm、5μm、1μm、0.45μm的滤膜过滤挤出,去除沉淀后将上清液在13000g离心90分钟,将沉淀在PBS中重悬后即为收集到的细菌膜组分,然后使用8M尿素水溶液裂解和溶解细菌膜组分。
将培养后的短双歧杆菌在5000g离心30分钟,然后使用PBS离心洗涤两遍后重悬于PBS中,在4℃下使用20W超声处理5分钟,然后依次通过20μm、10μm、5μm、1μm、0.45μm的滤膜过滤挤出,去除沉淀后将上清液在13000g离心90分钟,将沉淀在PBS中重悬后即为收集到的细菌膜组分,然后使用1%的Triton110水溶液裂解和溶解细菌膜组分。
(3)纳米粒子的制备
本实施例中纳米粒子采用溶剂挥发法制备,所采用的纳米粒子制备材料PLGA分子量为7KDa-17KDa,所采用免疫佐剂CpG2395和Poly(I:C)。免疫佐剂与癌细胞裂解物组分、8M尿素或者Triton溶解的细菌膜组分一起负载于纳米粒子内部。癌细胞裂解物组分与8M尿素或者Triton增溶的细菌膜组分质量比为1:1。制备方法如下所述,在制备过程中首先采用复乳法在纳米粒子内部负载裂解物组分,然后将100mg PLGA纳米粒子在13000g离心20min后使用10mL含有2%蔗糖和2%甘露醇的水溶液重悬后冷冻干燥48小时。负载免疫佐剂与癌细胞裂解物组分、8M尿素溶解的细菌膜组分的纳米粒子为纳米粒子1,平均粒径为240nm左右,每1mg PLGA纳米粒子约负载140μg蛋白质或多肽组分,CpG2006和Poly(I:C)各0.03mg。负载免疫佐剂与癌细胞裂解物组分、Triton溶解的细菌膜组分的纳米粒子为纳米粒子2,平均粒径为240nm左右,每1mg PLGA纳米粒子约负载140μg蛋白质或多肽组分,CpG2006和Poly(I:C)各0.03mg。
(4)抗原提呈细胞膜组分的制备
本实施例使用DC2.4细胞系和BMDM作为抗原提呈细胞。BMDM制备方法同实施例8。
将步骤(3)制备的纳米粒子1(1000μg)与DC2.4(500万个)及BMDM细胞(500万个)在15mL高糖DMEM完全培养基中共孵育48小时(37℃,5%CO 2);孵育体系中含有GM-CSF(2000U/mL)、IL-2(500U/mL)、IL-7(200U/mL)、IL-12(200U/mL)和CD40抗体(20ng/mL)。收集孵育后的细胞,将孵育后的细胞在400g离心5分钟,使用PBS重悬并洗涤两遍。即得混合抗原提呈细胞。
将混合抗原提呈细胞在20W超声处理2分钟机械破坏细胞,将样品在2000g离心5分钟,弃去沉淀后将上清液在6000g离心10分钟,然后弃去沉淀将上清液在15000g离心60分钟后弃去上清液收集沉淀,将沉淀在PBS中重悬后即得混合抗原提呈细胞的细胞膜组分。
(5)细菌外囊泡(OMV)和癌细胞外囊泡的制备
将短双歧杆菌在LB培养基中培养,培养基中含有10nM的舒尼替尼,然后收集培养后 的样品在5000g离心30分钟,弃去沉淀后收集上清液并在15W超声处理10分钟,将上清液在16000g离心90分钟,将沉淀在PBS中重悬后即为收集到的细菌外囊泡组分1。
将B16F10细胞在高糖DMEM完全培养基中培养6天,培养基中含有10nM的舒尼替尼,每2天换液一次。收集细胞培养B16F10细胞,在400g离心5分钟后弃去上清液,并使用PBS将沉淀细胞重悬,将癌细胞在20W超声处理2分钟机械破坏细胞,将样品在2000g离心5分钟,弃去沉淀后将上清液在6000g离心10分钟,然后弃去沉淀将上清液在15000g离心60分钟后弃去上清液收集沉淀,将沉淀在PBS中重悬后即得癌细胞细胞膜组分1。
或者将短双歧杆菌在LB培养基中培养,然后收集培养后的样品在5000g离心30分钟,弃去沉淀后收集上清液并在15W超声处理10分钟,将上清液在16000g离心90分钟,将沉淀在PBS中重悬后即为收集到的细菌外囊泡组分2。
或者将B16F10细胞在高糖DMEM完全培养基中培养6天,每2天换液一次。收集细胞培养B16F10细胞,在400g离心5分钟后弃去上清液,并使用PBS将沉淀细胞重悬,将癌细胞在20W超声处理2分钟机械破坏细胞,将样品在2000g离心5分钟,弃去沉淀后将上清液在6000g离心10分钟,然后弃去沉淀将上清液在15000g离心60分钟后弃去上清液收集沉淀,将沉淀在PBS中重悬后即得癌细胞细胞膜组分2。
(6)纳米疫苗的制备
将步骤(3)制备的纳米粒子1(100mg)在9mLPBS中重悬,然后与1mL步骤(4)制备的混合抗原提呈细胞膜组分(5mg)、步骤(5)制备的癌细胞膜组分1(5mg)和细菌外囊泡组分1(5mg)混合,在20W超声处理5分钟,然后使用0.45μm的滤膜反复共挤出,然后在13000g离心30分钟后弃去上清液收集沉淀,将沉淀在PBS中重悬后即得纳米疫苗1,纳米疫苗粒径为260nm。
将步骤(3)制备的纳米粒子2(100mg)在9mLPBS中重悬,然后与1mL步骤(4)制备的混合抗原提呈细胞膜组分(5mg)、步骤(5)制备的癌细胞膜组分1(5mg)和细菌外囊泡组分1(5mg)混合,在20W超声处理5分钟,然后使用0.45μm的滤膜反复共挤出,然后在13000g离心30分钟后弃去上清液收集沉淀,将沉淀在PBS中重悬后即得纳米疫苗2,纳米疫苗粒径为260nm。
将步骤(3)制备的纳米粒子1(100mg)在9mLPBS中重悬,然后与1mL步骤(4)制备的混合抗原提呈细胞膜组分(5mg)、步骤(5)制备的癌细胞膜组分2(5mg)和细菌外囊泡组分2(5mg)混合,在20W超声处理5分钟,然后使用0.45μm的滤膜反复共挤出,然后在13000g离心30分钟后弃去上清液收集沉淀,将沉淀在PBS中重悬后即得纳米疫苗3,纳米疫苗粒径为260nm。
(7)纳米疫苗癌症的预防
选取6-8周的雌性C57BL/6为模型小鼠制备Pan02胰腺癌荷瘤小鼠,在小鼠接种癌细胞前第-35天、第-28天、第-21天、第-14天和第-7天每只小鼠分别接种800μg纳米疫苗1、或800μg纳米疫苗2、或800μg纳米疫苗3、或者100μL PBS。在第0天,给每只受体小鼠背部右下方 皮下接种2×10 6个Pan02细胞。小鼠肿瘤体积和生存期监测方法同上。
(8)实验结果
如图11所示,PBS组小鼠肿瘤生长很快并且小鼠很快死亡。3种纳米疫苗都可以显著减缓小鼠肿瘤生长速度和延长小鼠生存期。而且纳米疫苗1效果好于纳米疫苗2和纳米疫苗3,说明特定裂解液裂解的细菌膜组分负载于纳米疫苗内部有利于提高疫苗功效,而且,使用舒尼替尼预处理的癌细胞和细菌所制备细胞膜组分也能提高纳米疫苗的疗效。
显然,上述实施例仅仅是为清楚地说明所作的举例,并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。

Claims (10)

  1. 一种负载癌细胞全细胞组分和混合膜组分的疫苗的制备方法,其特征在于,包括以下步骤:
    S1、从癌细胞中获取癌细胞的细胞膜组分和/或癌细胞细胞外囊泡膜组分;
    S2、将抗原提呈细胞与第一粒子共孵育以激活所述抗原提呈细胞,获取激活后的抗原提呈细胞的细胞膜组分;其中,第一粒子负载癌症相关抗原;
    S3、从细菌中获取细菌的细胞膜组分和/或细菌细胞外囊泡膜组分;
    S4、将第一混合膜组分和/或第二混合膜组分与第二粒子共作用,使混合膜组分负载于所述第二粒子上,得到负载癌细胞全细胞组分和混合膜组分的疫苗;其中,第二粒子负载癌细胞全细胞组分,第一混合膜组分为S1产物和S2产物的混合物,第二混合膜组分为S1产物和S3产物的混合物;
    所述癌细胞全细胞组分包括癌细胞和/或肿瘤组织经水裂解得到的水溶性组分和非水溶性组分,所述非水溶性组分经溶解剂溶解后负载于所述第二粒子上;或所述癌细胞全细胞组分包括癌细胞和/或肿瘤组织经含有溶解剂的溶解液裂解并溶解后得到的可溶组分。
  2. 根据权利要求1所述的制备方法,其特征在于:在步骤S1中,获取膜组分前还包括将癌细胞进行预处理的步骤,所述预处理为将癌细胞在含有阿霉素、替尼类药物、氯喹或氮杂胞苷的培养基中培养。
  3. 根据权利要求1所述的制备方法,其特征在于:在步骤S2中,所述癌症相关抗原为多肽抗原或癌细胞全细胞组分。
  4. 根据权利要求1所述的制备方法,其特征在于:在步骤S3中,获取膜组分前还包括将细菌进行预处理的步骤,所述预处理为将细菌在含有阿霉素、替尼类药物、氯喹或氮杂胞苷的培养基中培养。
  5. 根据权利要求1所述的制备方法,其特征在于:所述第二粒子还负载有细菌组分,所述细菌组分通过用含有溶解剂的溶解液裂解细菌或细菌外囊泡,后将裂解产物用溶解液溶解得到。
  6. 根据权利要求1所述的制备方法,其特征在于:所述细菌选自卡介苗、益生菌和溶瘤细菌中的一种或多种。
  7. 根据权利要求1所述的制备方法,其特征在于:所述溶解剂选自尿素、盐酸胍、脱氧胆酸盐、十二烷基硫酸盐、甘油、蛋白质降解酶、白蛋白、卵磷脂、无机盐、Triton、吐温、氨基酸、糖苷和胆碱的水溶液中的一种或多种。
  8. 根据权利要求1所述的制备方法,其特征在于:所述第一粒子或第二粒子负载有免疫佐剂和/或增加溶酶体逃逸物质。
  9. 权利要求1-8任一项所述的制备方法制备得到的疫苗。
  10. 权利要求9所述的疫苗在制备用于治疗或预防癌症药物中的应用。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110090298A (zh) * 2019-05-09 2019-08-06 武汉大学 一种细胞膜肿瘤疫苗及制备方法与应用
CN113440605A (zh) * 2020-03-26 2021-09-28 苏州大学 一种全细胞组分的输送系统及其应用
CN113663060A (zh) * 2020-04-30 2021-11-19 中国科学院上海药物研究所 全细胞肿瘤纳米疫苗、其制备方法和用途

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110090298A (zh) * 2019-05-09 2019-08-06 武汉大学 一种细胞膜肿瘤疫苗及制备方法与应用
CN113440605A (zh) * 2020-03-26 2021-09-28 苏州大学 一种全细胞组分的输送系统及其应用
CN113663060A (zh) * 2020-04-30 2021-11-19 中国科学院上海药物研究所 全细胞肿瘤纳米疫苗、其制备方法和用途

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CHEN LONG, QIN HAO, ZHAO RUIFANG, ZHAO XIAO, LIN LIANGRU, CHEN YANG, LIN YIXUAN, LI YAO, QIN YUTING, LI YIYE, LIU SHAOLI, CHENG KE: "Bacterial cytoplasmic membranes synergistically enhance the antitumor activity of autologous cancer vaccines", SCIENCE TRANSLATIONAL MEDICINE, vol. 13, no. 601, 7 July 2021 (2021-07-07), pages eabc2816, XP093121940, ISSN: 1946-6234, DOI: 10.1126/scitranslmed.abc2816 *
CHENG SHANSHAN, XU CONG, JIN YUE, LI YU, ZHONG CHENG, MA JUN, YANG JIANI, ZHANG NAN, LI YUAN, WANG CHAO, YANG ZHIYOU, WANG YU: "Artificial Mini Dendritic Cells Boost T Cell–Based Immunotherapy for Ovarian Cancer", ADVANCED SCIENCE, vol. 7, no. 7, 1 April 2020 (2020-04-01), XP093113661, ISSN: 2198-3844, DOI: 10.1002/advs.201903301 *
WANG DONGDONG, LIU CONGHUI, YOU SHIQUAN, ZHANG KAI, LI MENG, CAO YU, WANG CHANGTAO, DONG HAIFENG, ZHANG XUEJI: "Bacterial Vesicle-Cancer Cell Hybrid Membrane-Coated Nanoparticles for Tumor Specific Immune Activation and Photothermal Therapy", APPLIED MATERIALS & INTERFACES, AMERICAN CHEMICAL SOCIETY, US, vol. 12, no. 37, 16 September 2020 (2020-09-16), US , pages 41138 - 41147, XP093121938, ISSN: 1944-8244, DOI: 10.1021/acsami.0c13169 *
ZENG, YINGPING ET AL.: "Cell membrane coated-nanoparticles for cancer immunotherapy", ACTA PHARM SIN B, vol. 12, no. 8, 28 February 2022 (2022-02-28), pages 3233 - 3254, XP093119283, DOI: 10.1016/j.apsb.2022.02.023 *

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