WO2024016688A1 - 基于激活的抗原提呈细胞的核酸递送粒子、核酸递送系统及制备方法 - Google Patents

基于激活的抗原提呈细胞的核酸递送粒子、核酸递送系统及制备方法 Download PDF

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WO2024016688A1
WO2024016688A1 PCT/CN2023/081489 CN2023081489W WO2024016688A1 WO 2024016688 A1 WO2024016688 A1 WO 2024016688A1 CN 2023081489 W CN2023081489 W CN 2023081489W WO 2024016688 A1 WO2024016688 A1 WO 2024016688A1
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antigen
nucleic acid
loaded
nanoparticles
acid delivery
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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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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules 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
    • A61K9/5005—Wall or coating material
    • A61K9/5063—Compounds of unknown constitution, e.g. material from plants or animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules 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
    • A61K9/5089—Processes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules 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
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5176—Compounds of unknown constitution, e.g. material from plants or animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules 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
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5192—Processes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5154—Antigen presenting cells [APCs], e.g. dendritic cells or macrophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present disclosure relates to the field of immunotherapy, and in particular to a nucleic acid delivery particle, a nucleic acid delivery system and a preparation method based on activated antigen-presenting cells.
  • mRNA Messenger RNA
  • mRNA is a type of single-stranded ribonucleic acid that is transcribed from one strand of DNA as a template and carries genetic information that can guide protein synthesis. Theoretically, mRNA can guide the synthesis of any encoded protein, so it has broad application potential.
  • Current research on mRNA focuses on vaccination, protein replacement therapy and the treatment of genetic diseases.
  • mRNA vaccines are currently being used to prevent COVID-19. Technological advances in RNA biology, chemistry, stability, and delivery systems have accelerated the development of mRNA vaccines and the observation of effective, durable, and safe immune responses in animal models.
  • mRNA vaccines can activate the body's cellular immunity and humoral immunity at the same time, producing a stronger immune response.
  • the intracellular delivery of mRNA is more difficult than that of oligonucleotides, because the molecular weight of mRNA is large, extremely unstable, and easily degraded by RNase. Therefore, a suitable delivery carrier is extremely critical, which can not only protect the mRNA but also facilitate the successful entry of the mRNA into the cell. function within.
  • Current research methods for delivering mRNA mainly include RNA copolymers, modified RNA, viral delivery vectors, polymer delivery vectors, lipid delivery vectors, etc. Among them, the most mature one is lipid nanoparticles (LNP).
  • LNP delivery of mRAN there are also some problems with LNP delivery of mRAN, such as the inability to effectively prepare stable lyophilized formulations, so they can only be stored at -70°C or -20°C for a few months, which requires harsh transportation and storage conditions.
  • the surface of LNP is positively charged, and it is easy to adsorb proteins and cells after injection into the human body, so it has certain toxic side effects.
  • the use of PEG can cause allergic reactions to a certain extent.
  • the expression efficiency and efficacy of LNP-delivered mRNA in vivo also need to be further improved. Therefore, there is an urgent need to develop new delivery technologies to better deliver nucleic acid drugs such as mRNA.
  • the present disclosure provides a nucleic acid delivery particle prepared by using antigen-presenting cells activated by nanoparticles (NP) or microparticles (MP) loaded with antigen components, and a nucleic acid delivery system containing nucleic acid delivery particles. and preparation method thereof.
  • the present disclosure loads nucleic acids inside nanoparticles or microparticles, and then uses biofilm components derived from activated antigen-presenting cells and/or their secreted extracellular vesicles to be loaded on the surface of nanoparticles or microparticles to prepare nucleic acids. Deliver particles.
  • the surface flexibility of the nucleic acid delivery particles in the present disclosure is effectively improved, and problems such as particle homing to lymph nodes, particle targeting to antigen-presenting cells, and lysosome escape after particles enter cells are solved during nucleic acid delivery, thereby improving the efficiency of the delivery system to deliver nucleic acid drugs.
  • the efficiency increases the efficacy of nucleic acid drugs.
  • the first object of the present disclosure is to provide a nucleic acid delivery system, which has a skeleton structure formed of particle materials, the interior of the skeleton structure is loaded with nucleic acid, and the surface of the skeleton structure is loaded with biofilm components;
  • the biofilm component contains extracellular vesicles derived from activated antigen-presenting cells and/or secreted by the activated antigen-presenting cells; the activated antigen-presenting cells are composed of antigen-presenting cells and Antigen delivery particles are obtained through the interaction of antigen delivery particles.
  • the antigen delivery particles have a skeleton structure formed by particle materials, and antigen components are loaded inside and/or on the surface of the skeleton structure.
  • the nucleic acid delivery particles and the antigen delivery particles are independently selected from nanoparticles or microparticles.
  • the particle size of the nucleic acid delivery particles or antigen delivery particles is nanometer or micrometer, which can ensure that the particles are engulfed by antigen-presenting cells.
  • the particle size of the nanoparticles is 1nm-1000nm, more preferably, the particle size is 30nm-1000nm, more preferably, the particle size is 50nm-600nm; more preferably, the particle size is 50-500nm; More preferably, the particle size is 100-400 nm.
  • the particle diameter of the nanoparticles is 10nm, 50nm, 100nm, 200nm, 210nm, 220nm, 230nm, 240nm, 250nm, 260nm, 270nm, 280nm, 290nm, 300nm, 310nm, 320nm, 330nm, 340nm, 350nm, 4 00nm, 500nm and so on.
  • the particle size of the micron particles 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, more preferably, the particle size is 1 ⁇ m-5 ⁇ m, More preferably, it is 1-10 ⁇ m; more preferably, it is 1-2 ⁇ m.
  • the particle diameters of microparticles are 1 ⁇ m, 1.1 ⁇ m, 1.2 ⁇ m, 1.3 ⁇ m, 1.4 ⁇ m, 1.5 ⁇ m, 1.6 ⁇ m, 1.7 ⁇ m, 1.8 ⁇ m, 1.9 ⁇ m, 2 ⁇ m, 2.5 ⁇ m, 3 ⁇ m, 5 ⁇ m, 10 ⁇ m, etc. wait.
  • the nucleic acid delivery particles have a negative surface charge.
  • Nucleic acids described in this disclosure include DNA and RNA.
  • RNA described in this disclosure includes but is not limited to mRNA, siRNA, lcncRNA, etc.
  • the surface potential of nucleic acid delivery particles is -150mV to -1mv. More preferably, it is -50mv to -1mv; more preferably, it is -20mv to -1mv.
  • the antigenic component of the present disclosure includes at least one of the following:
  • the immunogenic protein and/or polypeptide is derived from (1) below, and at least one of (2)-(3) optionally present;
  • Whole cell lysate wherein the whole cell lysate components are derived from tumor tissue and/or tumor cells;
  • Extracellular vesicle lysate wherein the extracellular vesicles are secreted by bacteria or tumor cells.
  • the whole cell lysate includes one or both of the following: lysates of water-soluble antigens and lysates of non-water-soluble antigens.
  • the mass ratio of the mixture of the lysate of the non-water-soluble antigen and the lysate of the water-soluble antigen is (0.1-10): (0.1-10) ; Preferably (0.5-2): (0.5-2).
  • the mass ratio of the dissolved product of the non-water-soluble antigen and the water-soluble antigen is 1:1, 0.5:1, 0.8:1, 1:1.2, 1:1.5, 1:2, 2:1, 3:1, 4:1, 5:1, 1:3, 1:4, 1:5 and so on.
  • immunogenic proteins and/or polypeptides are derived from whole cell lysates and extracellular vesicles Lysates.
  • the extracellular vesicle lysate is selected from the group consisting of extracellular vesicle lysates of cancer cells and/or extracellular vesicle lysates of bacteria.
  • the mass ratio of the whole cell lysate to the extracellular vesicle lysate is (0.1-10): (0.1-10); preferably (0.5-2): (0.5-2).
  • the mass ratio of the whole cell lysate to the extracellular vesicle lysate is 1:1, 0.5:1, 0.8:1, 1:1.2, 1:1.5, 1:2, 2:1 , 3:1, 4:1, 5:1, 1:3, 1:4, 1:5 and so on.
  • immunogenic proteins and/or polypeptides are derived from whole cell lysates and bacterial lysates.
  • the mass ratio of the whole cell lysate to the bacterial lysate is (0.1-10): (0.1-10); preferably (0.5-2): (0.5-2).
  • the mass ratio of the whole cell lysate to the bacterial lysate is 1:1, 0.5:1, 0.8:1, 1:1.2, 1:1.5, 1:2, 2:1, 3: 1, 4:1, 5:1, 1:3, 1:4, 1:5 and so on.
  • the antigen delivery particles of the present disclosure are also loaded with at least one of the following:
  • a positively charged substance selected from the group consisting of positively charged amino acids, positively charged polypeptides, positively charged lipids, positively charged proteins, positively charged polymers, and/or positively charged of inorganic matter.
  • nucleic acid delivery particles are obtained by the co-action of nucleic acid delivery precursor particles and biofilm components;
  • nucleic acid delivery precursor particles and/or the nucleic acid delivery particles are also loaded with at least one of the following:
  • a positively charged substance selected from the group consisting of positively charged amino acids, positively charged polypeptides, positively charged lipids, positively charged proteins, positively charged polymers, and/or positively charged of inorganic matter.
  • the mass ratio (mg: ⁇ g: ⁇ g) of the particle material, nucleic acid and biofilm components is 1: (1-100): (10-300).
  • the nucleic acid delivery particle further includes an immune adjuvant and a charged substance, and the mass ratio of the particle material, nucleic acid, immune adjuvant, positively charged substance and the biofilm component (mg: ⁇ g: ⁇ g: ⁇ g: ⁇ g) is 1: (1-100): (1-200): (10-500): (10-300).
  • the nucleic acid delivery particle loaded biofilm component further contains at least one component as follows:
  • Cancer cell membrane components derived from whole cell lysate wherein the whole cell lysate component is derived from tumor tissue and/or tumor cells;
  • bacteria include, but are not limited to, Bacillus Calmette-Guerin, Escherichia coli, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium lactis, Lactobacillus acidophilus, Lactobacillus formati, Lactobacillus reuteri, Lactobacillus rhamnosus wait.
  • immune-enhancing adjuvants include, but are not limited to, pattern recognition receptor agonists, BCG, BCG cell wall scaffolds, BCG methanol extraction residues, BCG muramyl dipeptide, Mycobacterium phlei, polyantigen, mineral Oil, virus-like particles, immune-enhanced reconstituted influenza virions, cholera enterotoxin, saponins and their derivatives, Resiquimod, thymosin, neonatal bovine liver active peptide, miquimod, polysaccharide, curcumin, 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 adjuvant, calcium adjuvant, STING agonist, cytokine, interleukin , interferon, poly
  • the immune-enhancing adjuvant is selected from two or more Toll-like receptor agonists.
  • the immune adjuvant includes at least one of a Toll-like receptor 3 agonist and a Toll-like receptor 9 agonist.
  • immune-enhancing adjuvants include (1) Poly(I:C) and/or Poly(ICLC); (2) CpG-ODN; wherein, CpG-ODN is type A CpG-ODN, type B CpG-ODN and At least one type of CpG-ODN; preferably at least two types, and at least one of them is type B CpG-ODN or type C CpG-ODN.
  • CpG or “CpG-ODN” (CpG oligonucleotide, CpG oligodeoxynucleotide) is a synthetic oligomeric oligonucleotide containing unmethylated cytosine-guanine dinucleotide (CpG). Deoxyribonucleotide (ODN).
  • CpG-ODN Deoxyribonucleotide
  • Different types of CpG-ODN have different structural characteristics and immune effects, and are generally divided into three categories: A, B, and C.
  • CpG-ODNs include, but are not limited to: CpG 1018 (Class B), CpG 7909 (Class B), CpG 2006 (Class B), CpG-BW006 (Class B), CpG 2395 (Class C), CpG SL01, CpG 1585 (Category A), CpG 2216 (Category A), CpG SL03, CpG 2395 (Category C), CpG M362 (Category C), CpG 2336 (Category A).
  • the positively charged substance includes, but is not limited to, at least one of the following: positively charged amino acids, positively charged polypeptides, positively charged lipids, positively charged proteins, positively charged High molecular polymers and positively charged inorganic substances.
  • positively charged polypeptides include, but are not limited to, arginine-containing polypeptides, histidine-containing polypeptides, and/or KALA polypeptides of histidine and/or lysine, RALA polypeptides, melittin, and the like.
  • positively charged amino acids include, but are not limited to, arginine, histidine, lysine, and the like.
  • positively charged polymers include, but are not limited to, polyarginine, polylysine, polyhistidine, and the like.
  • positively charged lipids include, but are not limited to, DOTAP and the like.
  • positively charged proteins include, but are not limited to, protamine, histones, and the like.
  • positively charged inorganic substances include, but are not limited to, NH 4 HCO 3 , aluminum hydroxide, and the like.
  • the positively charged substance includes melittin, RALA polypeptide, KALA polypeptide, R8 polypeptide, arginine, histidine, lysine, polyarginine, polylysine, poly Any one or any combination of histidine and NH 4 HCO 3 .
  • the antigen delivery particles and/or nucleic acid delivery particles are also loaded with target molecules.
  • the target molecules include at least one of the following: mannose, mannan, CD19 antibody, CD20 antibody, BCMA antibody, CD32 antibody, CD11c antibody, CD103 antibody, CD44 antibody, etc.
  • the water-insoluble antigen, the bacterial lysate or the extracellular vesicle lysate are independently dissolved in a dissolving solution containing at least one of the following dissolving agents: urea, guanidine hydrochloride, deoxygenated Cholate, lauryl sulfate, glycerol, protein degrading enzyme, albumin, lecithin, Triton, Tween, amino acids, glycosides and choline; more preferably, the solute includes at least one of the following: urea, Sodium deoxycholate, octylglucoside and arginine.
  • the bacterial lysate and/or extracellular vesicle lysate is obtained by lysing bacteria and/or extracellular vesicles with a lysis solution containing a lysis agent; the lysis agent is selected from the group consisting of urea, guanidine hydrochloride, and deoxychol.
  • a lysis agent is selected from the group consisting of urea, guanidine hydrochloride, and deoxychol.
  • the antigen-presenting cells include at least one of B cells, dendritic cells (DC) and macrophages, preferably two or more, and more preferably a combination of three types of cells.
  • B cells B cells
  • DC dendritic cells
  • macrophages preferably two or more, and more preferably a combination of three types of cells.
  • the particle material forming the nucleic acid delivery particles is formed of natural polymer materials and/or synthetic polymer materials.
  • the antigen delivery particles are formed of natural polymer materials and/or synthetic polymer materials.
  • organic synthetic polymer materials include, but are not limited to, PLGA, PLA, PGA, PEG, PCL, Poloxamer, PVA, PVP, PEI, PTMC, polyanhydrides, PDON, PPDO, PMMA, polyamino acids, synthetic polypeptides, etc.
  • natural polymer materials include, but are not limited to, lecithin, cholesterol, alginate, albumin, collagen, gelatin, cell membrane components, starch, sugars, polypeptides, etc.
  • inorganic materials include, but are not limited to, ferric oxide, ferric tetroxide, carbonates, phosphates, and the like.
  • nucleic acid delivery particles nucleic acid delivery precursor particles and antigen delivery particles described in the present disclosure are common arbitrary shapes, including but not limited to spherical, ellipsoidal, barrel, polygonal, rod-shaped, sheet-shaped, linear, and worm-shaped. , square, triangle, butterfly, disc, vesicle, etc.
  • the activated antigen-presenting cells are obtained by co-incubation of antigen-presenting cells and antigen delivery particles; wherein the surface and/or interior of the antigen delivery particles are loaded with antigen components; wherein the antigen delivery particles Load whole cell antigen components and/or nucleic acids of cancer cells and/or tumor tissues; wherein the whole cell antigen components include water-soluble antigen components and non-water-soluble antigen components.
  • the co-incubation system of antigen-presenting cells and antigen-delivery particles may include cytokines and/or antibodies.
  • 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), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 17 (IL-17), IL-12, interleukin 12 (IL-12), interleukin 6 (IL-6) , macrophage colony-stimulating factor (M-CSF), interleukin 33 (IL-33), gamma interferon (IFN- ⁇ ), and TNF- ⁇ .
  • IL-2 interleukin 2
  • IL-7 interleukin 7
  • IL-14 interleukin 14
  • IL-4 interleukin 4
  • IL-15 interleukin 15
  • IL-21 Interleukin 21
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • antibodies include, but are not limited to, CD80 antibodies, CD86 antibodies, ⁇ CD-3 antibodies, ⁇ CD-4 antibodies, ⁇ CD-8 antibodies, ⁇ CD-28 antibodies, ⁇ CD-40 antibodies, ⁇ OX-40 antibodies, and ⁇ OX-40L antibodies.
  • the incubation system also includes at least one of the following:
  • Whole cell lysate wherein the whole cell lysate components are derived from tumor tissue and/or tumor cells;
  • Extracellular vesicle lysate wherein the extracellular vesicles are secreted by bacteria or tumor cells.
  • nucleic acids are loaded into nanoparticles or microparticles, and antigen delivery particles loaded with antigen components are used to specifically activate antigen-presenting cells, and then the antigen-presenting cells derived from and/or secreted by the activated antigen-presenting cells are used. extracellular
  • the biofilm components of the vesicles are loaded on the surface of nanoparticles or microparticles, forming nucleic acid delivery particles with nucleic acids loaded inside and biofilm components loaded on the surface.
  • the nucleic acid delivery examples provided in this disclosure can effectively home to lymph nodes, target antigen-presenting cells, and can improve the delivery efficiency and drug efficacy of nucleic acid drugs.
  • Nucleic acid delivery particles can be stored at 4°C for a long time after freeze-drying without affecting drug efficacy.
  • the second object of the present disclosure is to provide a nucleic acid delivery system, which includes the nucleic acid delivery particles provided by the present disclosure. Furthermore, the nucleic acid delivery particles contained in the nucleic acid delivery system can be the same or different, and can achieve in vivo delivery of one or more nucleic acid drugs. It has the advantages of high delivery efficiency and high safety, and can exert significant drug prevention or treatment effects.
  • the third object of the present disclosure is to provide a pharmaceutical composition, which includes the nucleic acid delivery particles or nucleic acid delivery system provided by the present disclosure.
  • compositions further include one or more pharmaceutically acceptable carriers.
  • the pharmaceutical composition provided by the present disclosure can achieve efficient nucleic acid delivery through nucleic acid delivery particles or nucleic acid delivery systems, and exert significant disease prevention or treatment effects.
  • the fourth object of the present disclosure is to provide a nucleic acid vaccine, which includes the nucleic acid delivery particles or nucleic acid delivery system provided by the present disclosure.
  • nucleic acid delivery particles or nucleic acid delivery system in the present disclosure can target antigen-presenting cells, effectively activate the body's immune response, and effectively exert the preventive or therapeutic effects of nucleic acid vaccines.
  • the fifth object of the present disclosure is to provide the use of the above-mentioned nucleic acid delivery particles, nucleic acid delivery systems or pharmaceutical compositions in at least one of the following (1)-(3):
  • the disease is cancer or tumor
  • the cancer or tumor is a solid tumor or hematological tumor, including but not limited to squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, glioma , hepatocellular carcinoma (HCC), Hodgkin lymphoma, non-Hodgkin lymphoma, T-cell lymphoma, acute myeloid leukemia (AML), various myeloma, gastrointestinal (tract) cancer, kidney cancer, ovarian cancer Cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colon cancer, rectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, polymorphic Glioblastoma, cervical cancer, brain cancer, stomach cancer, bladder cancer, liver cancer, breast cancer, colon cancer and head and neck cancer.
  • HCC hepatocellular carcinoma
  • HCC hepatocellular carcinoma
  • the antigen-presenting cells used to form biofilm components and the antigen components are derived from subjects, cell lines, or transformed from stem cells. Further, the antigen-presenting cells and the subject from which the antigen component originates are from the same individual or allogeneic species.
  • the antigenic component is derived from cells or tissues associated with the disease. Further, the antigen component is derived from the whole cell antigen component of cancer cells and/or tumor tissue.
  • the sixth object of the present disclosure is to provide a method for preparing the above-mentioned nucleic acid delivery particles, which includes the following steps:
  • S1 co-incubate the antigen-presenting cells with the antigen-delivery particles to obtain activated antigen-presenting cells; wherein the surface and/or interior of the antigen-delivery particles are loaded with antigen components;
  • biofilm components on the surface of the nucleic acid delivery precursor particles to obtain nucleic acid delivery particles; wherein the biofilm components are derived from the activated antigen-presenting cells, or are derived from the activated antigen-presenting cells. Extracellular vesicles secreted by antigen-presenting cells.
  • loading a biofilm component onto the surface of the nucleic acid delivery precursor particle includes:
  • step S31 the activated antigen-presenting cells are mechanically destroyed and subjected to membrane filtration or gradient centrifugation to collect biofilm components.
  • the mechanical destruction includes but is not limited to one or more of ultrasonic, homogenization, extrusion, homogenization, high-speed stirring, high-pressure destruction, high-shear force destruction, swelling, chemical substances, and shrinkage. .
  • the co-action includes, but is not limited to, one or more of co-incubation, co-extrusion, sonication, stirring, dialysis, ultrafiltration, homogenization and homogenization.
  • the preparation steps of the antigen delivery particles include:
  • the steps of preparing the antigenic component include:
  • S12 Dissolve the precipitate in a solution containing a dissolving agent, and then combine it with the water-soluble antigen component to obtain the antigen component derived from the target cells and/or target tissue.
  • the target cells are cancer cells
  • the target tissue is tumor tissue, one or more types of cancer cells, and one or more types of tumor tissues.
  • the step of lysing the target cells and/or target tissue in water or a lysis solution without a dissolving agent includes: freezing the current cells and/or target tissue at -20°C to -273°C, adding water or not. The solution containing the dissolving agent is then subjected to repeated freeze-thaw lysis.
  • the step of preparing the antigen component includes: using a dissolving solution containing a dissolving agent to lyse and simultaneously dissolve the water-soluble components and non-water-soluble components in the target cells and/or target tissues to obtain the antigen. components.
  • the target cells are cancer cells
  • the target tissue is tumor tissue
  • the cancer cells are one or more than one type
  • the tumor The tumor tissue is one type or more than one type.
  • solubilizing agents include, but are not limited to, urea, guanidine hydrochloride, deoxycholate, dodecyl sulfate (such as SDS), glycerol, protein degrading enzymes, albumin, lecithin, inorganic salts (0.1-2000mg/ mL), Triton, Tween, at least one of peptides, amino acids, glycosides, and choline.
  • This disclosure breaks through the limitations of existing nucleic acid delivery technology, enabling particles to be loaded with nucleic acid drugs and at the same time loaded with whole cell antigen epitopes and/or antigen epitopes of proteins or polypeptides expressed by nucleic acids, as well as antigen presentation derived from activation.
  • the biofilm components of cells or the biofilm components of extracellular vesicles secreted by them can more effectively exert the functions of nucleic acid drugs.
  • the present disclosure has at least the following advantages:
  • Nucleic acid drugs such as mRNA are currently mainly delivered using lipid nanoparticle (LNP) technology.
  • LNPs are positively charged nanoparticles and can easily adsorb negatively charged proteins or cells, making them highly cytotoxic.
  • PEG lipid nanoparticle
  • the present disclosure provides a technology that uses nanoscale or micron-scale particles to deliver nucleic acid drugs, which can not only efficiently load nucleic acid drugs, but also be able to home in lymph nodes and target antigen-presenting cells in lymph nodes, thereby reducing toxic and side effects while improving
  • the nucleic acid delivery particles in the present disclosure can be used to deliver nucleic acid drugs for the prevention and treatment of diseases such as cancer.
  • the surface of the nucleic acid delivery particles described in the present disclosure is negatively charged, so the toxic side effects caused by the adsorption of negatively charged proteins or polypeptides by the delivery system are reduced.
  • the nucleic acid delivery particles described in the present disclosure can be freeze-dried and stored for long-term use, overcoming the current problems of LNP being unable to be freeze-dried and stored for a long time.
  • Figure 1 is a schematic diagram of the preparation process and application of nucleic acid delivery particles in the present disclosure
  • a is a schematic diagram of the process of collecting water-soluble antigen components and non-water-soluble antigen components and preparing antigen delivery particles
  • b is a dissolution process containing a dissolving agent
  • c is the use of the antigen delivery particles prepared in a or b to activate antigen-presenting cells, and transfer the biofilm derived from the activated antigen-presenting cells
  • Figure 2 is the experimental results of the analysis of the expression of the delivered nucleic acid after delivery of nucleic acid by different particle delivery systems in Example 1.
  • Figures 3-14 are respectively the experimental results of mouse tumor growth rate and survival time when nucleic acid vaccines (nano vaccines or micron vaccines) are used to prevent or treat cancer in Example 2-13; in Figure 3-14, a is the prevention or treatment of cancer.
  • Figure 3c and d use flow cytometry to analyze the ratio of CD8 + IFN- ⁇ + T cells to CD8 + T cells and the ratio of CD4 + IFN- ⁇ + T cells to CD4 + T cells in mouse splenocytes after co-incubation; in
  • && means p ⁇ 0.01, there is a significant difference compared with the control group of nanoparticles and/or microparticles that only carry nucleic acid internally but no membrane components on the surface; ⁇ means there is a significant difference compared with the blank control nanoparticles/micron P ⁇ 0.005, there is a significant difference compared to the nanovaccine/microvaccine group prepared by the interaction between the biofilm components of antigen-presenting cells activated by particles + free lysate and nucleic acid delivery precursor particles; ⁇ indicates that it is different from the blank control p ⁇ 0.05, there is a significant difference between the nanoparticles/microparticles + the membrane components of the antigen-presenting cells activated by free lysate and the nucleic acid delivery precursor particles, p ⁇ 0.05; ⁇ represents p ⁇ 0.005, there is a significant difference compared with the nanovaccine/microvaccine group prepared by co-acting the membrane components of antigen-presenting cells activated with polypeptide antigen delivery particles and
  • ⁇ represents the difference compared with the use of two type A CpG and Poly ICLC/ p ⁇ 0.05, there is a significant difference between the nanovaccine/microvaccine group prepared by using poly(I:C) as a mixed adjuvant for antigen delivery particles to activate antigen presenting cells and nucleic acid delivery precursor particles; ⁇ represents Compared with the nanovaccine/microvaccine group prepared by cooperating the membrane components of antigen-presenting cells activated by antigen particle delivery with class A CpG and class B CpG as mixed adjuvants and nucleic acid delivery precursor particles p ⁇ 0.005, there is a significant difference; ⁇ represents the nanovaccine prepared by cooperating with the membrane components of antigen-presenting cells activated by antigen delivery particles using only Poly(I:C) as an adjuvant and nucleic acid delivery precursor particles/ Compared with the micron
  • ⁇ represents the membrane component of antigen-presenting cells activated by antigen delivery particles that do not load lysosomal escape substances.
  • ⁇ represents the activation of antigen delivery particles loaded with only one CpG+Poly(I:C) mixed adjuvant p ⁇ 0.05, there is a significant difference compared to the nucleic acid vaccine prepared by co-operating the membrane components of antigen-presenting cells with nucleic acid delivery precursor particles; ⁇ represents the antigen presentation activated by antigen-loaded antigen delivery particles without adjuvant.
  • ⁇ represents the antigen delivery activated by antigen delivery particles loaded with cancer cell whole cell components.
  • p ⁇ 0.05 there is a significant difference between the nanovaccine/microvaccine prepared by the co-action of the membrane components of cells and nucleic acid delivery precursor particles; ⁇ represents the activation of antigen delivery particles with only two types of CpG as adjuvants.
  • ⁇ represents the difference between the nanovaccine/micron vaccine and the lipid nanoparticle (LNP) vaccine loaded with mRNA.
  • eta means that compared with the vaccine that is loaded with biofilm components on the surface and internally loaded with nucleic acid and two types of A CpG and polyIC mixed adjuvants, p ⁇ 0.05, there is a significant difference; It represents that there is a significant difference at p ⁇ 0.005 between the vaccine with biofilm components loaded on the surface and nucleic acid loaded internally and a mixed adjuvant of class B CpG and polyIC at p ⁇ 0.005; ⁇ represents the biofilm component loaded on the surface while nucleic acid loaded internally with the vaccine.
  • ⁇ Represents a vaccine with cells activated by antigen delivery particles loaded on the surface.
  • Membrane component p ⁇ 0.05 compared to the vaccine group that only loads adjuvants and polypeptides but not nucleic acid components (except adjuvants) internally;
  • p ⁇ 0.01 compared with the vaccine group that only loaded adjuvants and polypeptides but not nucleic acid components (except adjuvants)
  • ⁇ represents the biofilm component with cells activated by antigen delivery particles loaded on the surface , at the same time, p ⁇ 0.005 compared to the vaccine group that only loaded adjuvants and polypeptides but not nucleic acid components (except adjuvants).
  • treatment refers to contacting (e.g., administering) a nucleic acid delivery particle, a nucleic acid delivery system, a nucleic acid vaccine, a nucleic acid-loaded drug, or a pharmaceutical composition to a subject after suffering from a disease, such that the subject is treated better than if he or she were not exposed.
  • Reducing symptoms of the disease does not necessarily mean that symptoms of the disease must be completely suppressed.
  • Suffering from a disease means that the body has symptoms of the disease.
  • prevention refers to contacting (e.g., administering) the nucleic acid delivery particles, nucleic acid delivery systems, nucleic acid vaccines, nucleic acid-loaded drugs, and pharmaceutical compositions of the present disclosure to a subject before suffering from a disease, thereby preventing contact with the subject. Reducing the symptoms of a disease does not mean that it is necessary to completely suppress the disease.
  • pharmaceutically acceptable excipients or “pharmaceutically acceptable carriers” refers to auxiliary materials widely used in the field of pharmaceutical production.
  • the main purpose of using excipients is to provide a pharmaceutical composition that is safe to use, stable in nature and/or has specific functionality, and also to provide a method so that after the drug is administered to the subject, the active ingredient can be used in the desired manner. rate dissolution, or promote effective absorption of the active ingredient in the subject to whom it is administered.
  • Pharmaceutically acceptable excipients may be inert fillers or functional ingredients that provide a certain function for the pharmaceutical composition (such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredients in the composition).
  • Non-limiting examples of pharmaceutically acceptable excipients include, but are not limited to, binders, suspending agents, emulsifiers, diluents (or fillers), granulating agents, adhesives, disintegrants, lubricants, and anti-adhesive agents. , glidants, wetting agents, gelling agents, absorption delaying agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, sweeteners, etc.
  • compositions of the present disclosure may be prepared using any method known to those skilled in the art. For example, conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding and/or lyophilizing processes.
  • the methods of administration can be varied or adapted in any applicable manner to meet the needs of the nature of the drug, convenience of the patient and medical staff, and other relevant factors.
  • mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., , mice and rats).
  • domestic animals e.g., cattle, sheep, cats, dogs, and horses
  • primates e.g., humans and non-human primates such as monkeys
  • rabbits e.g., mice and rats.
  • mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., , mice and rats).
  • domestic animals e.g., cattle, sheep, cats, dogs, and horses
  • primates e.g., humans and non-human primates such as monkeys
  • rabbits e.g., mice and rats.
  • tumor and cancer are used interchangeably herein to encompass both solid and liquid tumors.
  • neoplastic refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer cancerous cells and tissues.
  • cancer cancerous cells and tissues.
  • cancer cancerous cells and tissues.
  • the nucleic acid delivery system (vaccine system) described in the present disclosure is loaded with nucleic acid inside and cell membrane components or extracellular vesicle membrane components of antigen-presenting cells activated by particles are loaded on the surface.
  • the antigen-presenting cells used to prepare nanoparticles or microparticles are first activated by nanoparticles and/or microparticles (antigen delivery particles) loaded with tumor tissue and/or cancer cell whole cell antigens and/or nucleic acids or mixtures thereof; Then, the membrane components of the activated antigen-presenting cells are loaded on the surface of the internally loaded particles (nucleic acid delivery precursor particles) to prepare a particle system (nucleic acid delivery particles) for preventing or treating cancer.
  • Its preparation process and application fields are as follows: As shown in Figure 1.
  • nanoparticles or microparticles that activate antigen-presenting cells
  • cells or tissues can be lysed, water-soluble antigens and water-insoluble antigens can be collected separately and nano or microparticle systems (antigen delivery particles) can be prepared respectively;
  • a lysing solution containing a dissolving agent can be used to directly lyse cells or tissues and dissolve whole cell antigens of cancer cells to prepare nano or micro particle systems (antigen delivery particles).
  • the cancer cell and/or tumor tissue whole cell antigens of the present disclosure may undergo a process including but not limited to inactivation or (and) denaturation, solidification, biomineralization, ionization, chemical modification, Nanoparticles or microparticles can be prepared after protein and/or polypeptide separation and purification, nucleic acid separation and purification, protease endolysis or degradation, nuclease treatment, etc.; or without any inactivation or ( and) denaturation, solidification, biomineralization, ionization, chemical modification, protease endolysis or degradation, and nuclease treatment to directly prepare nanoparticles or microparticles.
  • tumor tissue cells have been inactivated or (and) denatured before lysis. In actual use, they can also be inactivated or (and) denatured after cell lysis, or cells can also be lysed. Inactivation or (and) denaturation treatment is performed before and after lysis; in some embodiments of the present disclosure, the inactivation or (and) denaturation treatment method before or (and) after cell lysis is ultraviolet irradiation and high-temperature heating.
  • the process may also include but is not limited to radiation irradiation, high pressure, protein and/or peptide separation and purification, nucleic acid separation and purification, solidification, biomineralization, ionization, chemical modification, nuclease treatment, protease endolysis or degradation, collagen Enzyme treatment, freeze-drying and other processing methods.
  • radiation irradiation high pressure, protein and/or peptide separation and purification
  • nucleic acid separation and purification solidification, biomineralization, ionization, chemical modification, nuclease treatment, protease endolysis or degradation, collagen Enzyme treatment, freeze-drying and other processing methods.
  • the antigen-presenting cells When preparing activated antigen-presenting cells into nanoparticles or microparticles, the antigen-presenting cells are first mechanically destroyed, then centrifuged and/or filtered with a filter membrane of a certain pore size, and then combined with the loaded whole cell component and/or Or nanoparticles or microparticles of nucleic acids work together.
  • Activated antigen-presenting cells contain a certain cell membrane structure after mechanical destruction.
  • Nucleic acid delivery particles are formed after activated antigen-presenting cells are mechanically damaged and interact with nucleic acid delivery precursor particles. Wherein, biofilm components derived from activated antigen-presenting cells are located on the outer layer of the nucleic acid delivery particles.
  • the antigen-presenting cells used to prepare biofilm components can be derived from autologous or allogeneic sources, or 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.
  • nucleic acid delivery particles can also simultaneously load biofilm components derived from cancer cells, and /or biofilm components derived from extracellular vesicles secreted by cancer cells, and/or bacteria, and/or biofilm components derived from bacteria or extracellular vesicles secreted by them.
  • the system when using antigen delivery particles loaded with antigen components to activate antigen-presenting cells, the system can contain cytokines and/or antibodies to improve activation efficiency. .
  • antigen-presenting cells are first activated using antigen delivery particles loaded with whole cell components derived from cancer cells, and then the biofilm components of the antigen-presenting cells are loaded on the surface of nanoparticles or microparticles to prepare nucleic acids. Deliver particles.
  • any method for preparing nanoparticles and microparticles known to those in the art can be used to prepare the nucleic acid delivery particles described in the present disclosure, including but not limited to solvent evaporation method, dialysis method, microfluidic method, homogeneous emulsification method, and dispersion method. , precipitation method, etc.
  • this disclosure takes the solvent evaporation method as an example and provides the following exemplary preparation methods:
  • Step 1 Mix the initial aqueous phase and the organic phase, specifically adding the first predetermined volume of the aqueous phase solution containing the first predetermined concentration of the antigen component to the second predetermined volume of the organic phase containing the second predetermined concentration of the raw material for preparing particles. middle.
  • the aqueous solution may contain at least one of the following i) to iii): i) components in cancer cell lysates, ii) components in tumor tissue lysates, iii) encoding specific Nucleic acids of proteins or polypeptides, and immune-enhancing adjuvants.
  • Each component in the lysate is a water-soluble antigen during preparation, or an original non-water-soluble antigen dissolved in a solution containing a dissolving agent such as urea or guanidine hydrochloride.
  • the first predetermined concentration is the concentration of the nucleic acid contained in the aqueous phase solution, or the concentration of the water-soluble antigen and/or the concentration of the original non-water-soluble antigen contained in the aqueous phase solution.
  • the first predetermined concentration requires that the protein polypeptide concentration is greater than 1ng/mL, or the concentration of nucleic acid encoding a specific protein or polypeptide is greater than 0.01ng/mL, so that sufficient nucleic acid or cancer cell whole cell antigen can be loaded to activate relevant cells.
  • the concentration of the immune-enhancing adjuvant in the initial aqueous phase is greater than 0.01ng/mL.
  • the second predetermined concentration of raw materials for preparing particles ranges from 0.5 mg/mL to 5000 mg/mL, and is selected as 100 mg/mL.
  • the second predetermined volume of the organic phase is set according to its ratio to the first predetermined volume of the aqueous phase.
  • the ratio of the first predetermined volume of the aqueous phase to the second predetermined volume of the organic phase is within a range. 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 proteins and polypeptides is greater than 1 ng/mL, preferably 1 mg/mL to 100 mg/mL. In some embodiments, when the aqueous phase solution is a solution containing a lysate component and an immune adjuvant, the concentration of the protein and polypeptide is greater than 1 ng/mL, preferably 1 mg/mL to 100 mg/mL, and the concentration of the immune adjuvant is greater than 0.01 ng/mL, preferably 0.01 mg/mL to 20 mg/mL.
  • the solvent in the organic phase solution, is DMSO, acetonitrile, ethanol, chloroform, methanol, DMF, isopropyl alcohol, dichloromethane, propanol, ethyl acetate, etc., preferably dichloromethane;
  • the concentration of the organic phase It is 0.5mg/mL ⁇ 5000mg/mL, preferably 100mg/mL.
  • the concentration of the nucleic acid is greater than 0.01ng/mL, preferably 1 ⁇ g/mL to 1 mg/mL. In some embodiments, when the aqueous solution is a solution containing nucleic acid and an immune adjuvant, the concentration of the nucleic acid is greater than 1 ng/mL, preferably 1 ⁇ g/mL ⁇ 1 mg/mL, and the concentration of the immune adjuvant is greater than 0.01 ng/mL, Preferably, it is 0.01mg/mL ⁇ 20mg/mL.
  • the solvent in the organic phase solution, is DMSO, acetonitrile, ethanol, chloroform, methanol, DMF, isopropyl alcohol, dichloromethane, propanol, ethyl acetate, etc., preferably dichloromethane;
  • the concentration of the organic phase It is 0.5mg/mL ⁇ 5000mg/mL, preferably 100mg/mL.
  • Step 2 Subject the mixed solution obtained in Step 1 to any of the following treatments: i) ultrasonic treatment for more than 2 seconds; ii) stirring for more than 1 minute; iii) homogenization treatment; iv) microfluidic treatment.
  • 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
  • use a high-pressure/ultra-high-pressure homogenizer or high-shear homogenizer for homogenization processing use a high-pressure/ultra-high-pressure homogenizer or high-shear homogenizer for homogenization processing.
  • 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 is 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 nanoparticles or microparticles, whether they are too large or too large. Small will bring about 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 any of the following treatments: i) ultrasonic treatment for more than 2 seconds; ii) ultrasonic treatment for more than 1 minute. Stirring; iii) Homogenization treatment; iv) Microfluidic treatment.
  • the mixture obtained in step 2 is added to the aqueous emulsifier solution and continued to be ultrasonic or stirred or homogenized or mixed to achieve nanonization or micronization.
  • 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 50rpm to 1500rpm
  • the stirring time is 0.5 to 5 hours
  • 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 nanoparticles or microparticles. Too large or too small will result. bring about 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, stirring time or homogenization time, the volume and concentration of the emulsifier aqueous solution in this step are all determined in order to obtain 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 fourth predetermined concentration is selected to obtain nanoparticles or microparticles of suitable size. Based on rice grains.
  • the selection of the fourth predetermined volume is determined based on the ratio of the third predetermined volume to the fourth predetermined volume. In the present disclosure, 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. In the specific implementation process, 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 lyophilized protective agent or a sixth predetermined volume of PBS (or physiological saline); or use ultrafiltration centrifugation or use a dialysis method that can remove specific molecular weight substances to remove free PVA and other substances at the same time The solution in the system is replaced with a fifth predetermined volume of aqueous solution containing a fifth predetermined concentration of lyoprotectant or a sixth predetermined volume of PBS (or physiological saline).
  • 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 Resuspend a sixth predetermined volume of the nanoparticle-containing suspension obtained in Step 5 in PBS (or physiological saline) or use a sixth predetermined volume of PBS (or physiological saline) to resuspend the nanoparticle-containing suspension obtained in Step 6
  • PBS or physiological saline
  • the freeze-dried substance containing nanoparticles or microparticles and a lyoprotectant is used directly; or the above sample is mixed with a seventh predetermined volume of water-soluble antigen or the dissolved original non-water-soluble antigen and used.
  • the volume ratio of the sixth predetermined volume to the seventh predetermined volume is 1:10000 to 10000:1, the preferred volume ratio is 1:100 to 100:1, and the optimal volume ratio is 1:30 to 30:1 .
  • the nucleic acid delivery precursor particles forming the nucleic acid delivery particles are loaded with 0.1-500 ⁇ g of nucleic acid per 1 mg of particle material. In some embodiments, or the nucleic acid delivery precursor particle is also loaded with an immune-enhancing adjuvant, wherein 1-400 ⁇ g of immune-enhancing adjuvant is loaded per 1 mg of particle material. In some embodiments, the nucleic acid delivery precursor particles are also loaded with positively charged molecules, wherein 1-800 ⁇ g of positively charged molecules are loaded per 1 mg of particle material.
  • Step 8 Incubate the antigen-presenting cells with the antigen delivery particles prepared above for a certain period of time.
  • the tumor tissue and/or cancer cells and the antigen-presenting cells used to prepare the antigen delivery particles can be from the same individual or allogeneic.
  • each (500,000-50 million) of the antigen-presenting cells is co-incubated with (10 ⁇ g-1500 ⁇ g) of the antigen delivery particles.
  • Step 9 Collect the co-incubated antigen-presenting cells, perform mechanical destruction such as ultrasound, homogenization, mechanical stirring, etc., and then centrifuge the sample and/or use a filter membrane with a certain pore size to filter and/or co-extrusion. A biofilm component of activated antigen-presenting cells and/or a biofilm component of extracellular vesicles is obtained.
  • Step 10 Load nucleic acids such as mRNA or DNA into nanoparticles or microparticles using the method of steps 1-7 to prepare nucleic acid delivery precursor particles.
  • Step 11 combine the biofilm component obtained in step 9 (a biofilm component containing activated antigen-presenting cells, which may also contain a biofilm component of cancer cells and/or a biofilm component of bacteria) with step 10
  • Prepared nucleic acid-loaded nucleic acid The delivery precursor particles work together to prepare nanoparticles or microparticles that are internally loaded with nucleic acid and surface loaded with membrane components of activated antigen-presenting cells and/or membrane components of extracellular vesicles secreted by them.
  • the nucleic acid delivery particles are loaded with 0.1-500 ⁇ g of nucleic acid per 1 mg of particle material. In some embodiments, the nucleic acid delivery delivery particles are also loaded with an immune-enhancing adjuvant, wherein 0.1-400 ⁇ g of immune-enhancing adjuvant is loaded per 1 mg of particle material. In some embodiments, the nucleic acid delivery delivery particles are also loaded with positively charged molecules, wherein 1-800 ⁇ g of positively charged molecules are loaded per 1 mg of particle material.
  • Step 12 Use the particle delivery system (vaccine system) prepared in step 11 to prevent or treat cancer and other diseases.
  • the experimental techniques and experimental methods used in this example are all conventional technical methods unless otherwise specified.
  • the experimental methods without specifying specific conditions in the following examples usually follow conventional conditions or conditions recommended by the manufacturer.
  • the materials, reagents, etc. used in the examples can be obtained through regular commercial channels unless otherwise specified.
  • Example 1 In vitro expression efficiency after phagocytosis of nanoparticles loaded with mRNA and antigen-presenting cell membrane components
  • This example evaluates the expression efficiency of the nucleic acid loaded after the nucleic acid delivery nanoparticles (ie, nanovaccines) loaded with mRNA internally and loaded with membrane components of activated antigen-presenting cells on the surface are phagocytosed by cells.
  • the nucleic acid delivery nanoparticles ie, nanovaccines
  • mRNA RNA
  • membrane components of activated antigen-presenting cells on the surface are phagocytosed by cells.
  • 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, and Polyinosinic-polycytidylic acid (poly(I:C )) uses a solvent evaporation method to prepare antigen delivery nanoparticles loaded with water-soluble antigens and non-water-soluble antigens of tumor tissue for immune adjuvants, and then uses the antigen delivery nanoparticles to activate antigen-presenting cells and mechanically destroy the antigen-presenting cells. After centrifugation, the cell membrane components of the activated antigen-presenting cells are obtained. At the same time, the mRNA encoding Enhanced Green Fluorescent Protein (EGFP) is loaded into the nucleic acid delivery precursor nanoparticles.
  • EGFP Enhanced Green Fluorescent Protein
  • nucleic acid delivery particles After the membrane components of the activated antigen-presenting cells are combined with the nucleic acid delivery precursor particles loaded with mRNA, nucleic acid delivery particles are obtained that are loaded with nucleic acids internally and at the same time loaded with the membrane components of the activated antigen-presenting cells on the surface. Then, the expression level of the nucleic acid loaded by the nucleic acid delivery particle system after it is phagocytosed by cells is detected.
  • the antigen delivery nanoparticles 1 are prepared by the double emulsion method in the solvent evaporation method. During preparation, nanoparticles loaded with water-soluble antigens in whole cell antigens of cancer cells and nanoparticles loaded with non-water-soluble antigens in whole cell antigens of cancer cells are prepared separately, and then used together.
  • the molecular weight of PLGA, the material used to prepare the antigen delivery nanoparticles, is 7KDa-17KDa.
  • the immune adjuvants used are poly(I:C) and poly(I:C) and are loaded inside the nanoparticles. The preparation method is as mentioned above.
  • the double emulsion method is first used to load cellular antigen components and adjuvants inside the antigen delivery nanoparticles, and then 100 mg of the antigen delivery nanoparticles is centrifuged at 10,000g for 20 minutes, and 10 mL containing 4% seaweed is used. The sugar was resuspended in ultrapure water and freeze-dried for 48 h.
  • the average particle size of the nanoparticles 1 is about 240nm, and each 1 mg of PLGA nanoparticles is loaded with approximately 100 ⁇ g of protein or peptide components.
  • the poly(I:C) immune adjuvant used per 1 mg of PLGA nanoparticles is 0.02 mg.
  • nucleic acid delivery precursor particles (nanoparticle 2) loaded with EGFP-mRNA
  • the mRNA loaded in the nucleic acid delivery precursor nanoparticles 2 loaded with mRNA can be EGFP; the nucleic acid delivery precursor nanoparticles are also loaded with immune adjuvants poly(I:C) and R8 (RRRRRRRR) polypeptides.
  • the preparation materials and preparation method of nucleic acid delivery precursor particles loaded with mRNA are the same as step (2). First, the mRNA, poly(I:C) and R8 polypeptide are mixed in water, and then the above mixture is loaded inside the nanoparticles using the double emulsion method.
  • each 1 mg PLGA nanoparticle is loaded with approximately 10 ⁇ g of mRNA components.
  • Each 1 mg PLGA nanoparticle is loaded with 20 ⁇ g of poly(I:C) immune adjuvant and 40 ⁇ g of R8 polypeptide.
  • 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.
  • 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) complete medium, with recombinant mouse GM-CSF (20 ng/mL) added, and cultured at 37°C and 5% CO2 for 7 days. On the third day, shake the culture bottle gently and add the same volume of RPMI 1640 (10% FBS) medium containing GM-CSF (20ng/mL).
  • Antigen delivery nanoparticles 1 loaded with whole cell components of cancer cells derived from tumor tissues were mixed with BMDC (10 million) in 15 mL Incubate in RPMI1640 complete medium for 48 hours (37°C, 5% CO 2 ); the incubation system contains a combination of cytokines 1: IL-2 (500U/mL), IL-7 (500U/mL), IL-15 (500U/mL) or containing cytokine components 2: IL-4 (500U/mL), IL-33 (500U /mL), IL10 (500U/mL).
  • IL-2 500U/mL
  • IL-7 500U/mL
  • IL-15 500U/mL
  • cytokine components 2 IL-4 (500U/mL), IL-33 (500U /mL), IL10 (500U/mL).
  • the nucleic acid delivery nanoparticle 3 also known as nanovaccine 3, has a particle size of 270nm and a surface potential of -6mV.
  • Each 1 mg of PLGA nanoparticles is loaded with approximately 10 ⁇ g of mRNA components, each 1 mg of PLGA nanoparticles is loaded with 20 ⁇ g of poly(I:C) immune adjuvant, and 40 ⁇ g of R8 polypeptide; each 1 mg of PLGA nanoparticles is loaded with approximately 50 ⁇ g of cell membrane components.
  • nucleic acid delivery nanoparticles 4 also known as nanovaccines 4 have a particle size of 270nm and a surface potential of -6mV.
  • Each 1 mg of PLGA nanoparticles is loaded with approximately 10 ⁇ g of mRNA components, each 1 mg of PLGA nanoparticles is loaded with 20 ⁇ g of poly(I:C) immune adjuvant, and 40 ⁇ g of R8 polypeptide; each 1 mg of PLGA nanoparticles is loaded with approximately 50 ⁇ g of cell membrane components.
  • nucleic acid delivery nanoparticles 5, also known as nanovaccines 5, have a particle size of 270nm and a surface potential of -6mV.
  • Each 1 mg of PLGA nanoparticles is loaded with approximately 10 ⁇ g of mRNA components, each 1 mg of PLGA nanoparticles is loaded with 20 ⁇ g of poly(I:C) immune adjuvant, and 40 ⁇ g of R8 polypeptide; each 1 mg of PLGA nanoparticles is loaded with approximately 50 ⁇ g of cell membrane components.
  • Nanovaccine 5 was tested immediately after preparation or after storage at 4°C for 18 months.
  • This example uses microfluidic method to prepare LNP.
  • the particle size of LNP is 95 nanometers, the surface potential is 8mV, and the load of mRNA per 1 mg of LNP is 50 ⁇ g.
  • This LNP is the LNP vaccine, and the LNP vaccine is currently manufactured and available for use.
  • Antigen-presenting cell DC2.4 cells were seeded in a 24-well plate in RPMI complete medium (containing 10% FBS) at a density of 100,000 cells per well, and cultured overnight at 37°C (5% CO 2 ). Add 25ng free EGFP-mRNA, or 0.5 ⁇ g of the LNP vaccine just prepared in step (7), or 1.25 ⁇ g of the nanovaccine prepared in step (6) (newly prepared nanovaccine 3, just prepared) to the above cells. Nano vaccine 4, newly prepared nano vaccine 5, or nano vaccine that has been stored for a long time 5), and then incubate for 24 hours. The above-mentioned cells were then collected and flow cytometry was used to analyze the proportion of cells containing EGFP fluorescent signals to all cells and the mean fluorescence signal intensity (MFI) of EGFP-positive cells.
  • MFI mean fluorescence signal intensity
  • Naked mRNA, LNP, Nanovaccine 3(Fresh), Nanovaccine 4(Fresh), Nanovaccine 5(Fresh), and Nanovaccine 5(Long term storage) in Figure 2 correspond to naked mRNA, LNP vaccine, and just prepared Nano vaccines 3.
  • nano vaccine 5 Whether it is freeze-dried for short-term storage or long-term storage of nano vaccine 5, its effect is significantly better than nano vaccine 3, nano vaccine 4 and LNP vaccine; moreover, nano vaccine 5 is well prepared. Long-term storage does not affect its efficacy. This shows that the nanovaccine disclosed in this disclosure can be stored at 4°C for a long time after freeze-drying without affecting its efficacy; and that adding cytokine combination 1 during the activation of antigen-presenting cells is more effective than cytokine component 2; it is loaded with cancer Antigen Delivery Nanoparticles of Whole Cell Components The effect of nucleic acid delivery nanoparticles prepared by activated antigen-presenting cells is much better than that of nucleic acid delivery nanoparticles prepared by unactivated antigen-presenting cells. In summary, the nucleic acid particle delivery system described in the present disclosure can deliver mRNA very efficiently, and the particle delivery system can be stored at 4°C for a long time after freeze-drying.
  • Example 2 Nanoparticles loaded with mRNA and antigen-presenting cell membrane components are used for the prevention of melanoma
  • This example uses a cancer model to illustrate how to prevent disease using nucleic acid delivery nanoparticles (nano-vaccines) loaded internally with mRNA and surface-loaded with membrane components of activated antigen-presenting cells.
  • nucleic acid delivery nanoparticles nano-vaccines
  • 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 framework material
  • poly(I:C) was used as the immune adjuvant
  • a solvent evaporation method was used.
  • an antigen delivery nanoparticle system loaded with water-soluble antigens and non-water-soluble antigens of tumor tissue, and then use the antigen delivery nanoparticles to activate antigen-presenting cells and deliver the anti-
  • the original presenting cells are mechanically destroyed and centrifuged to obtain the membrane fraction of the activated antigen-presenting cells.
  • the mRNA encoding four melanoma neoantigen polypeptides was loaded into the nanoparticles.
  • nucleic acid delivery particles are obtained that are loaded with nucleic acids internally and at the same time loaded with the membrane components of the activated antigen-presenting cells on the surface. Then, the nanoparticles are used Particles prevent melanoma.
  • the antigen delivery nanoparticles 1 are prepared by the double emulsion method in the solvent evaporation method. During preparation, nanoparticles loaded with water-soluble antigens in whole cell antigens of cancer cells and nanoparticles loaded with non-water-soluble antigens in whole cell antigens of cancer cells are prepared separately, and then used together.
  • the molecular weight of PLGA, the material used to prepare the nanoparticles, is 7KDa-17KDa.
  • the immune adjuvants used are poly(I:C) and poly(I:C) and are loaded inside the nanoparticles. The preparation method is as mentioned above.
  • the double emulsion method is first used to load cell antigen components and adjuvants inside the nanoparticles, and then 100 mg of nanoparticles are centrifuged at 10,000 g for 20 minutes, and 10 mL of ultrapure solution containing 4% trehalose is used. Resuspend in water and freeze-dry for 48 hours.
  • the average particle size of the nanoparticles 1 is about 240nm, and each 1 mg of PLGA nanoparticles is loaded with approximately 100 ⁇ g of protein or peptide components.
  • the poly(I:C) immune adjuvant used per 1 mg of PLGA nanoparticles is 0.02 mg.
  • the mRNA loaded on the nucleic acid delivery precursor nanoparticles 2 can encode four polypeptide neoantigens B16-M20 (Tubb3, FRRKAFLHWYTGEAMDEMEFTEEAESNM), B16-M24 (Dag1, TAVITPPTTTTKKARVSTPKPPAPSTD), and B16-M46 (Actn4, NHSGLVTFQAFIDVMSRETTDTDTADQ) ) and TRP2:180-188(SVYDFFVWL); nucleic acid delivery precursor nanoparticles are simultaneously loaded with immune adjuvants poly(I:C) and R8(RRRRRRRR) polypeptides.
  • the preparation materials and preparation method of mRNA-loaded nucleic acid delivery precursor particles are the same as step (2).
  • the mRNA, poly(I:C) and R8 polypeptide are mixed in water, and then the above mixture is loaded on the nanoparticles using the double emulsion method 2 Internally, 100 mg of PLGA nanoparticles 2 were then 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 is about 250nm.
  • Each 1 mg of PLGA nanoparticles is loaded with approximately 10 ⁇ g of mRNA component.
  • Each 1 mg of PLGA nanoparticles is loaded with 20 ⁇ g of poly(I:C) immune adjuvant and 40 ⁇ g of R8 polypeptide.
  • 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.
  • Use scissors to cut off both ends of the bone, and then use a syringe to draw the PBS solution.
  • the needles are Insert into the medullary cavity from both ends of the bone and repeatedly rinse the bone marrow into the culture dish. Collect the bone marrow solution, centrifuge at 400g for 3 minutes, and then add 1 mL of red blood cell lysis solution to lyse the red blood.
  • Antigen delivery nanoparticles 1 loaded with whole cell components of cancer cells derived from tumor tissues were mixed with BMDC (10 million) in 15 mL Incubate in RPMI1640 complete medium for 48 hours (37°C, 5% CO 2 ); the incubation system contains cytokine combination 1: IL-2 (500U/mL), IL-7 (500U/mL), IL-15 (500U /mL) or contains cytokine component 2: IL-4 (500U/mL), IL-33 (500U/mL), IL10 (500U/mL).
  • IL-2 500U/mL
  • IL-7 500U/mL
  • IL-15 500U /mL
  • cytokine component 2 IL-4 (500U/mL), IL-33 (500U/mL), IL10 (500U/mL).
  • the nucleic acid delivery nanoparticle 3 also known as nanovaccine 3, has a particle size of 270nm and a surface potential of -6mV.
  • Each 1 mg of PLGA nanoparticles is loaded with approximately 10 ⁇ g of mRNA components, each 1 mg of PLGA nanoparticles is loaded with 20 ⁇ g of poly(I:C) immune adjuvant, and 40 ⁇ g of R8 polypeptide; each 1 mg of PLGA nanoparticles is loaded with approximately 50 ⁇ g of cell membrane components.
  • the nano vaccine is used directly after preparation.
  • nucleic acid delivery nanoparticles 4 also known as nanovaccines 4, with a particle size of 270nm and a surface potential of -6mV.
  • Each 1 mg of PLGA nanoparticles is loaded with approximately 10 ⁇ g of mRNA components, each 1 mg of PLGA nanoparticles is loaded with 20 ⁇ g of poly(I:C) immune adjuvant, and 40 ⁇ g of R8 polypeptide; each 1 mg of PLGA nanoparticles is loaded with approximately 50 ⁇ g of cell membrane components.
  • the nano vaccine is used directly after preparation.
  • nucleic acid delivery nanoparticles 5, also known as nanovaccines 5, have a particle size of 270nm and a surface potential of -6mV.
  • Each 1 mg of PLGA nanoparticles is loaded with approximately 10 ⁇ g of mRNA components, each 1 mg of PLGA nanoparticles is loaded with 20 ⁇ g of poly(I:C) immune adjuvant, and 40 ⁇ g of R8 polypeptide; each 1 mg of PLGA nanoparticles is loaded with approximately 50 ⁇ g of cell membrane components.
  • the nano-vaccine can be used directly after preparation or stored at 4°C for 18 months before use.
  • This example uses microfluidic method to prepare LNP.
  • mice Select 6-8 week old female C57BL/6 as model mice to prepare melanoma tumor-bearing mice.
  • the mice will be inoculated on days -35, -28, -21, -14 and - On 7 days, mice were subcutaneously inoculated with 1 mg of nano vaccine (Nano vaccine 3, or Nano vaccine 4, or Nano vaccine 5) that had just been freeze-dried, or 1 mg of Nano vaccine 5 that had been freeze-dried and stored at 4°C for 18 months. Either inoculate 0.5 mg of the current LNP vaccine or inoculate 100 ⁇ L PBS. On day 0, 1.5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated into the lower right corner of the back of each mouse. Monitor mouse tumor growth rate and mouse survival time.
  • 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 - before the mice are inoculated with tumors. On the 7th day, mice were subcutaneously inoculated with 1 mg of nanovaccine that had just been freeze-dried (Nanovaccine 3, or Nanovaccine 4, or Nanovaccine 5), or 1mg of freeze-dried nanovaccine was injected into the mice. Store nanovaccine 3 at 4°C for 18 months, or inoculate 0.5 mg of the current LNP vaccine or inoculate 100 ⁇ L PBS.
  • mice were sacrificed on day 0, the mouse splenocytes were removed, and a single cell suspension of mouse splenocytes was prepared, and then 2 million mouse splenocytes were mixed with 400 ⁇ g antigen delivery nanoparticles 1 (200 ⁇ g loaded water-soluble nanoparticles 1 + 200 ⁇ g loaded non-water-soluble nanoparticles 1) + 20 ⁇ L mixture of mouse tumor tissue lysate (80mg/mL, with equal mass of water-soluble and non-water-soluble components) prepared in step (1) in DMEM high sugar Incubate in complete medium at 37°C (5% CO2 ) for 48 hours.
  • antigen delivery nanoparticles 1 200 ⁇ g loaded water-soluble nanoparticles 1 + 200 ⁇ g loaded non-water-soluble nanoparticles 1 + 20 ⁇ L mixture of mouse tumor tissue lysate (80mg/mL, with equal mass of water-soluble and non-water-soluble components) prepared in step (1) in DMEM high sugar Incubate in complete medium at 37°C (5%
  • the incubated cells were collected and labeled with anti-mouse CD3 antibody, anti-mouse CD4 antibody and anti-mouse CD8 antibody with fluorescent probes, and then fixed with 2% paraformaldehyde, and then disrupted with After the membrane solution breaks the cell membrane, the IFN- ⁇ antibody with a fluorescent probe is used for labeling.
  • Flow cytometry was then used to analyze the proportion of CD8 + IFN- ⁇ + T cells among CD8 + T cells and the proportion of CD4 + IFN- ⁇ + T cells among CD4 + T cells.
  • the whole cell antigen of cancer cells loaded with antigen delivery nanoparticles can be degraded into antigen epitopes after being engulfed by antigen-presenting cells and presented to the surface of the antigen-presenting cell membrane.
  • Nanoparticles prepared by antigen-presenting cells are loaded with the above-mentioned degradation epitopes.
  • the presented antigenic epitope can be recognized by cancer cell-specific T cells and activate cancer cell-specific T cells, which secrete killer cytokines after activation.
  • IFN- ⁇ is the most important cytokine secreted by antigen-specific T cells that are activated after recognizing antigens.
  • CD8 + IFN- ⁇ + T cells and CD4 + IFN- ⁇ + T cells analyzed by flow cytometry are cancer cell-specific T cells that can recognize and kill cancer cells.
  • Nano Vaccine 3 Whether it is freeze-dried for short-term storage or long-term storage, its effect is significantly better than Nano Vaccine 3, Nano Vaccine 4 and LNP Vaccine; Moreover, after Nano Vaccine 5 is prepared, Long-term storage does not affect its efficacy.
  • the nanovaccine disclosed in this disclosure can be stored at 4°C for a long time after freeze-drying without affecting its efficacy; and that adding cytokine combination 1 during the activation of antigen-presenting cells is more effective than cytokine component 2; it is loaded with cancer
  • the effect of nanoparticles prepared by antigen-presenting cells activated by nanoparticles of whole cell components is much better than that of nanoparticles prepared by unactivated antigen-presenting cells.
  • the nanovaccine described in the present disclosure has a good preventive effect on melanoma.
  • the antigen-presenting cells activated by the antigen-delivery nanoparticles loaded with cancer cell whole-cell components will degrade and present the whole-cell antigens in the cancer cell whole-cell components loaded with the engulfed nanoparticles, which are presented to Cancer cell antigen epitopes on the cell membrane surface have been bound to major histocompatibility complex (MHC) molecules.
  • MHC major histocompatibility complex
  • the cell membrane components in the above antigen-presenting cells will be loaded on the surface of the nanoparticles or microparticles, forming new nanoparticles or microparticles. , namely nano vaccine or micro vaccine.
  • the nano-vaccine or micro-vaccine prepared by the above method is internally loaded with mRNA encoding specific antigens, and at the same time, the surface is loaded with complexes of MHC molecules and various cancer cell antigen epitopes, so its ability to activate the body's cancer cell-specific immune response will be stronger It is also broader-spectrum and therefore more effective in preventing or treating cancer.
  • Nano vaccine 5 has the highest proportion of activated cancer cell-specific T cells. Whether it is short-term storage after freeze-drying or long-term storage, the effect of nano vaccine 5 is significantly better than that of nano vaccine 3, nano vaccine 4 and LNP vaccine; and, Long-term storage of Nano Vaccine 5 after preparation will not affect its efficacy. This result is consistent with the efficacy results, indicating that the nanovaccine works by activating more cancer cell-specific T cells.
  • This example uses mouse melanoma as a cancer model to illustrate how to use nanoparticle-activated antigen-presenting cells to prepare mRNA-loaded nanoparticles for preventing 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 framework material
  • poly(I:C) and CpG1018 were used as immune adjuvants and solvents were used.
  • An antigen delivery particle system loaded with water-soluble antigens and non-water-soluble antigens of tumor tissue is prepared by evaporation method, and then the antigen delivery particles are used to activate antigen-presenting cells, and the membrane components of the activated antigen-presenting cells are loaded for nucleic acid delivery On the surface of the precursor particles, nucleic acid delivery particles are prepared to prevent cancer.
  • 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.
  • Mixing water-soluble components and non-water-soluble components at a mass ratio of 1:1 is the source of the antigen raw material for preparing the antigen delivery nanoparticle system.
  • the antigen delivery nanoparticles and the blank nanoparticles and peptide nanoparticles used as controls were prepared using a solvent evaporation method.
  • the preparation material for antigen delivery nanoparticles 1 loaded with whole cell components is PLGA with a molecular weight of 7Da-17KDa.
  • the immune adjuvants used are poly(I:C) and CpG1018 and the adjuvants are encapsulated 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.
  • each 1 mg of PLGA nanoparticles is loaded with approximately 100 ⁇ g of protein and peptide components, and each 1 mg of PLGA nanoparticles uses 0.02 mg of poly(I:C) and CpG1018 immune adjuvants.
  • polypeptide neoantigens B16-M20 (Tubb3, FRRKAFLHWYTGEAMDEMEFTEAESNM), B16-M24 (Dag1, TAVITPPTTTTKKARVSTPKPATPSTD), B16-M46 (Actn4, NHSGLVTFQAFIDVMSRETTDTDTADQ) and TRP2:180-188 (SVYDFFVWL) were loaded with equal mass.
  • Peptide nanoparticle 2 is used as a control nanoparticle. Its preparation materials and preparation methods are the same as nanoparticle 1.
  • the particle size of control nanoparticle 2 is about 220 nm, and it is loaded with 100 ⁇ g of peptide component and an equal amount of adjuvant.
  • the preparation materials and methods of blank nanoparticle 3 are the same as those of nanoparticle 1.
  • the particle size is about 220 nm. It only carries the same amount of immune adjuvant but does not load any antigen component.
  • the mRNA loaded on the nucleic acid delivery precursor nanoparticles 4 loaded with mRNA can encode four polypeptides.
  • Neoantigens B16-M20 (Tubb3, FRRKAFLHWYTGEAMDEMEFTEAESNM), B16-M24 (Dag1, TAVITPPTTTTKKARVSTPKPATPSTD), B16-M46 (Actn4, NHSGLVTFQAFIDVMSRETTDTDTADQ) and TRP2:180-188 (SVYDFFVWL); the nanoparticles are also loaded with the immune adjuvant poly(I : C) and positively charged melittin (GIGAVLKVLTTGLPALISWIKRKRQQ).
  • the preparation materials and preparation method of the particles loaded with mRNA are the same as step (2).
  • the mRNA, poly(I:C) and melittin are mixed in water, and then the above mixture is loaded inside the nanoparticles using the double emulsion method, and then 100 mg of 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 4 is about 210 nm, and each 1 mg PLGA nanoparticle is loaded with approximately 10 ⁇ g of mRNA component, each 1 mg PLGA nanoparticle is loaded with 10 ⁇ g of poly(I:C) immune adjuvant, and 20 ⁇ g of melittin.
  • Bone marrow-derived dendritic cells (BMDC) and B cells were used as antigen-presenting cells.
  • the preparation of BMDC is the same as in Example 1.
  • the B cell extraction process is as follows: kill the mouse and remove the mouse spleen, then prepare a mouse splenocyte single cell suspension, and then use magnetic bead sorting to sort CD19 + B cells from the splenocyte single cell suspension. Mix BMDC and B cells at a ratio of 1:1 and use them as mixed antigen-presenting cells.
  • the antigen delivery nanoparticle 1 and 20 million BMDCs were incubated in 15 mL RPMI1640 complete medium for 48 hours (37°C, 5% CO 2 ); the incubation system contained a combination of cytokines: IL-15 (500U/mL), IL- 7(500U/mL), IL-21(1000U/mL).
  • the nanoparticles prepared by using the mixed antigen-presenting cell membrane components activated by blank nanoparticles 3 and the nucleic acid delivery precursor nanoparticles 4 are nanovaccines 7, with a particle size of 220nm and a surface potential of -6mV; each 1 mg PLGA nanoparticles are loaded with approximately 10 ⁇ g of mRNA components, each 1 mg of PLGA nanoparticles is loaded with 10 ⁇ g of poly(I:C) immune adjuvant, and 20 ⁇ g of melittin; each 1 mg of PLGA nanoparticles is loaded with approximately 100 ⁇ g of cell membrane components.
  • the nanoparticles prepared by using mixed antigen-presenting cell membrane components activated by peptide nanoparticles 2 and nucleic acid delivery precursor nanoparticles 4 are nanovaccine 6, with a particle size of 220nm and a surface potential of -6mV; per 1mg PLGA nanometer
  • the particles are loaded with approximately 10 ⁇ g of mRNA components, each 1 mg of PLGA nanoparticles is loaded with 10 ⁇ g of poly(I:C) immune adjuvant, and 20 ⁇ g of melittin; each 1 mg of PLGA nanoparticles is loaded with approximately 100 ⁇ g of cell membrane components.
  • the nanoparticles prepared by using the mixed antigen-presenting cell membrane components activated by nanoparticle 1 and the nucleic acid delivery precursor nanoparticle 4 are nanovaccine 5, with a particle size of 220nm and a surface potential of -6mV; per 1 mg of PLGA nanoparticles
  • the particles are loaded with approximately 10 ⁇ g of mRNA components, each 1 mg of PLGA nanoparticles is loaded with 10 ⁇ g of poly(I:C) immune adjuvant, and 20 ⁇ g of melittin; each 1 mg of PLGA nanoparticles is loaded with approximately 100 ⁇ g of cell membrane components.
  • Nano Vaccine 8 with a particle size of 220nm and a surface potential of -6mV.
  • Each 1 mg of PLGA nanoparticles is loaded with approximately 10 ⁇ g of mRNA components, each 1 mg of PLGA nanoparticles is loaded with 10 ⁇ g of poly(I:C) immune adjuvant, and 20 ⁇ g of melittin; each 1 mg of PLGA nanoparticles is loaded with approximately 100 ⁇ g of cell membrane components.
  • 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 - before the mice are inoculated with tumors. Mice were subcutaneously inoculated with 1 mg of nanovaccine (nanovaccine 5, or nanovaccine 6, or nanovaccine 7, or nanovaccine 8, or nanoparticle 4) or 100 ⁇ L PBS on 7 days. On day 0, 1.5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated into the lower right corner of the back of each mouse. Monitor mouse tumor growth rate and mouse survival time. In the experiment, the size of the mouse tumor volume was recorded every 3 days starting from the 3rd day.
  • Nanovaccine 4 As shown in Figure 4, in Figure 4, PBS control, Nanovaccine 4, Nanovaccine 5, Nanovaccine 6, Nanovaccine 7, and Nanovaccine 8 are the PBS control group, Nanovaccine 4, Nanovaccine 5, Nanovaccine 6, Nanovaccine 7, and Nanovaccine in order. 8.
  • the results showed that the mice in the PBS control group had very fast tumor growth and a short survival period. Compared with the control group, the tumor growth rate of mice in the vaccine group was significantly slower, and some mice had tumors that disappeared and recovered.
  • Nano Vaccine 5 has the best effect, and Nano Vaccine 5 is significantly better than Nano Vaccine 6, Nano Vaccine 7 and Nano Vaccine 8; and Nano Vaccine 5, Nano Vaccine 6 and Nano Vaccine 8 are all significantly better than Nano Particle 4.
  • This example uses mouse melanoma as a cancer model to illustrate how to use the membrane components of antigen-presenting cells activated by antigen-delivery nanoparticles to be loaded on the surface of nucleic acid-loaded nanoparticles as a nanovaccine to treat melanoma.
  • 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 was used as the nanoparticle skeleton material
  • Poly(I:C), CpG7909 and CpG2006 were used as adjuvants to prepare antigen delivery nanoparticles loaded with lysate components, and then the antigen delivery nanoparticles were incubated with antigen-presenting cells for a period of time. After a period of time, the antigen-presenting cells are activated, and the membrane components of the antigen-presenting cells are loaded on the surface of the nucleic acid delivery precursor nanoparticles that have internally loaded nucleic acids to prepare a nano-vaccine for the treatment of melanoma.
  • 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.
  • centrifuge Bifidobacterium longum at 5000g for 30 minutes, then discard the precipitate and collect the supernatant. Filter the supernatant with a 1 ⁇ m filter, ultrasonicate at 4°C for 5 minutes at 20W, and then centrifuge at 16000g for 90 minutes. , resuspend the pellet in PBS to form the collected bacterial outer vesicle membrane components, and then use Tween 80 aqueous solution to lyse and dissolve the bacterial membrane components.
  • the antigen delivery nanoparticle 1 is prepared by the double emulsion method.
  • the molecular weight of the preparation material PLGA is 24KDa-38KDa.
  • the immune adjuvants used are poly(I:C), CpG7909 and CpG2006 and are loaded in the nanoparticles.
  • the preparation method is as mentioned above.
  • the double emulsion method is first used to load the lysis solution components and adjuvants inside the nanoparticles. Then 100 mg of nanoparticles are centrifuged at 10,000g for 20 minutes, and 10 mL of ultrapure solution containing 4% trehalose is used. Resuspend in water and freeze-dry for 48 hours before use.
  • the average particle size of nanoparticles 1 is about 250nm.
  • Each 1mg PLGA nanoparticle 1 is loaded with approximately 130 ⁇ g of protein or peptide components.
  • Each 1mg PLGA nanoparticle 1 is loaded with poly(I:C), CpG7909 and CpG2006 immunoadjuvant. Each dose is 0.005mg.
  • the preparation materials and methods of the antigen delivery nanoparticle 2 are the same as the nanoparticle 1.
  • the nanoparticle 2 is internally loaded with the antigen component prepared in step (1) and the 8M urea-dissolved bacterial outer vesicle membrane component prepared in step (2) at the same time, and the mass ratio of the two is 1:1.
  • the immune adjuvants used were poly(I:C), CpG7909 and CpG2006 and were loaded into nanoparticles.
  • the double emulsion method was first used to load tumor tissue antigen components, bacterial external vesicle components and adjuvants inside the nanoparticles.
  • nanoparticles were centrifuged at 10,000g for 20 minutes, and 10 mL of ultrasonic acid containing 4% trehalose was used. Resuspend in pure water and freeze-dry for 48 hours before use.
  • the average particle size of nanoparticles 2 is about 250nm.
  • Each 1 mg of PLGA nanoparticles 2 is loaded with approximately 130 ⁇ g of protein or peptide components.
  • Each 1 mg of PLGA nanoparticles 2 carries 0.005 mg of poly(I:C), CpG7909 and CpG2006 immune adjuvants. .
  • the preparation materials and preparation method of nanoparticle 3 are the same as nanoparticle 1.
  • the nanoparticle 3 is simultaneously loaded with the antigen component prepared in step (1) and the bacterial outer vesicle membrane component dissolved in Tween 80 prepared in step (2), and the mass ratio of the two is 1:1.
  • the immune adjuvants used were poly(I:C), CpG7909 and CpG2006 and were loaded into nanoparticles.
  • the double emulsion method was first used to load the tumor tissue inside the nanoparticles with lysate components, bacterial external vesicle components and adjuvants.
  • nanoparticles 3 100 mg were centrifuged at 10,000g for 20 minutes, and 10 mL containing 4% trehalose was used. Resuspend in ultrapure water and freeze-dry for 48 hours before use.
  • the average particle size of nanoparticles 3 is about 250nm.
  • Each 1 mg of PLGA nanoparticles 3 is loaded with approximately 30 ⁇ g of protein or peptide components.
  • Each 1 mg of PLGA nanoparticles 3 is loaded with 0.005 mg of poly(I:C), CpG7909 and CpG2006 immune adjuvants. .
  • blank nanoparticle 4 The preparation materials and methods of blank nanoparticle 4 are the same as nanoparticle 1, but blank nanoparticle 4 only carries the same amount of adjuvant and does not load any antigen components in the tumor tissue lysate.
  • the particle size of the nanoparticles 4 is approximately 250 nm.
  • the mRNA loaded on the nucleic acid delivery precursor nanoparticles 5 loaded with mRNA can encode four polypeptide neoantigens B16-M20 (Tubb3, FRRKAFLHWYTGEAAMDEMEFTEAESNM), B16-M24 (Dag1, TAVITPPTTTTKKARVSTPKPPAPSTD), and B16-M46 (Actn4, NHSGLVTFQAFIDVMSRETTDTDTADQ) ) and TRP2:180-188 (SVYDFFVWL); the nanoparticles are simultaneously loaded with immune adjuvants poly(I:C), CpG7909, CpG1018 and melittin (GIGAVLKVLTTGLPALISWIKRKRQQ).
  • the preparation materials and preparation method of the particles loaded with mRNA are the same as step (3).
  • the mRNA, poly(I:C), CpG1018, CpG7909 and melittin are mixed in water, and then the above mixture is loaded on the nanoparticles using the double emulsion method.
  • 100 mg of PLGA nanoparticles were then centrifuged at 10,000 g for 20 min, resuspended in 10 mL of ultrapure water containing 4% trehalose and freeze-dried for 48 h.
  • the average particle size of the nanoparticles 5 is about 210nm.
  • Each 1 mg PLGA nanoparticle is loaded with approximately 2 ⁇ g of mRNA components.
  • Each 1 mg PLGA nanoparticle is loaded with 5 ⁇ g each of poly(I:C), CpG7909 and CpG1018 immune adjuvants, and 40 ⁇ g of melittin.
  • mice After the C57BL/6 mice were sacrificed, the spleens of the mice were removed, a single cell suspension of mouse splenocytes was prepared, and CD19 + B cells in the splenocytes were isolated using magnetic bead sorting.
  • the incubation system contains IL-2 (500 U/mL) and IL-7 (200 U/mL). , IL-12 (200U/mL) and CD80 antibody (10ng/mL).
  • the incubated B cells (10 million cells) were collected by centrifugation at 400g for 5 minutes, then washed three times with PBS, resuspended in PBS water and sonicated at low power (10W) for 15 minutes. Then centrifuge the sample at 500g for 5 minutes and collect the supernatant. The supernatant is filtered through membranes with pore sizes of 30um, 10um, 5um, 0.45um, and 0.22um in sequence. The resulting filtrate sample is centrifuged at 18000g for 60 minutes and then discarded.
  • the supernatant and the pellet were resuspended in PBS and incubated with the nucleic acid delivery precursor nanoparticles 5 for 2 minutes, then ultrasonicated for 1 minute, and then repeatedly co-extruded using a 0.22um filter membrane, and then centrifuged at 13000g for 30 minutes. , and resuspended in 10 mL of ultrapure water containing 2% sucrose and 2% mannitol and freeze-dried for 48 hours for later use.
  • the nanoparticles prepared by using the membrane components of antigen-presenting cells activated by nanoparticles 1 and nanoparticles 5 are nanovaccine 6, with a particle size of 220 nanometers and a surface potential of -10mV; each 1 mg of PLGA nanoparticles is about Loading 10 ⁇ g of mRNA components, each 1 mg of PLGA nanoparticles is loaded with 5 ⁇ g of poly(I:C), CpG7909 and CpG1018 immune adjuvants, and 40 ⁇ g of melittin; each 1 mg of PLGA nanoparticles is loaded with approximately 40 ⁇ g of cell membrane components.
  • nanoparticles prepared by using the membrane components of antigen-presenting cells activated by nanoparticles 2 and nanoparticles 5 to interact with each other are nanovaccine 7, with a particle size of 220 nm and a surface potential of -10mV; each 1 mg of PLGA nanoparticles is loaded with approximately 10 ⁇ g.
  • each 1 mg of PLGA nanoparticles is loaded with 5 ⁇ g each of poly(I:C), CpG7909, and CpG1018 immune adjuvants, and 40 ⁇ g of melittin; each 1 mg of PLGA nanoparticles is loaded with approximately 40 ⁇ g of cell membrane components.
  • nanoparticles prepared by using the membrane components of antigen-presenting cells activated by nanoparticles 3 and nanoparticles 5 to interact with each other are nanovaccine 8, with a particle size of 220 nm and a surface potential of -10mV; each 1 mg of PLGA nanoparticles is loaded with approximately 10 ⁇ g.
  • each 1 mg of PLGA nanoparticles is loaded with 5 ⁇ g each of poly(I:C), CpG7909, and CpG1018 immune adjuvants, and 40 ⁇ g of melittin; each 1 mg of PLGA nanoparticles is loaded with approximately 40 ⁇ g of cell membrane components.
  • the nanoparticles prepared by using the membrane components of antigen-presenting cells activated by nanoparticles 4 and nanoparticles 5 to interact with each other are nanovaccine 9, with a particle size of 220 nm and a surface potential of -10mV; each 1 mg of PLGA nanoparticles is loaded with approximately 10 ⁇ g.
  • each 1 mg of PLGA nanoparticles is loaded with 5 ⁇ g each of poly(I:C), CpG7909, and CpG1018 immune adjuvants, and 40 ⁇ g of melittin; each 1 mg of PLGA nanoparticles is loaded with approximately 40 ⁇ g of cell membrane components.
  • 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, 7th, 10th, 15th, 20th and 25th days after melanoma vaccination, 1 mg of nanovaccine (Vaccine 6, or Vaccine 7, or Vaccine 8, or Vaccine 9) was injected subcutaneously. ) or subcutaneously inject 100 ⁇ L PBS. In the experiment, the size of the mouse tumor volume was recorded every 3 days starting from the 3rd day.
  • PBS control, Nanovaccine 6, Nanovaccine 7, Nanovaccine 8, and Nanovaccine 9 are the PBS control group, Nanovaccine 6, Nanovaccine 7, Nanovaccine 8, and Nanovaccine 9, respectively.
  • the results showed that, as shown in a and b in Figure 5, the tumors of mice in the PBS control group grew rapidly and had a short survival period.
  • the tumor growth rate of mice treated with several nano-vaccines was significantly slowed down, and some mice had tumors disappearing and cured.
  • Nano Vaccine 7 is better than that of Nano Vaccine 6, Nano Vaccine 8 and Nano Vaccine 9, which shows that the membrane of antigen-presenting cells is activated after the bacterial outer vesicle components lysed and dissolved using appropriate methods are loaded into nanoparticles. After the components are loaded onto the surface of the particle vaccine described in the present disclosure, it is beneficial to improve the vaccine effect.
  • the vaccine described in the present disclosure has excellent therapeutic effect on cancer.
  • 6M guanidine hydrochloride was first used to cleave the whole cell antigen of B16F10 melanoma cancer cells. Then, an antigen delivery microparticle system loaded with cancer cell whole cell antigens was prepared using PLGA as the micron particle skeleton material, CpG BW006 (B type), CPG2216 (A type) and Poly ICLC as immune adjuvants. After activating antigen-presenting cells using antigen-delivery microparticles, the membrane components of the antigen-presenting cells are loaded on the surface of nucleic acid-loaded nucleic acid delivery precursor nanoparticles to prepare a nanovaccine for the treatment of cancer.
  • the cultured B16F10 melanoma cancer cell line was collected and centrifuged at 350g for 5 minutes, then the supernatant was discarded and washed twice with PBS, and then the cancer cells were resuspended and lysed with 6M guanidine hydrochloride.
  • the whole cell antigen of the cancer cells was lysed and dissolved in 6M Guanidine hydrochloride is the source of antigen raw material for preparing the antigen delivery microparticle system.
  • the antigen delivery microparticles are prepared using the double emulsion method.
  • the molecular weight of PLGA, the material used to prepare micron particles 1, is 38KDa-54KDa, and the immune adjuvants used are CpG BW006, CpG 2216 and Poly ICLC.
  • Poly ICLC is a toll-like receptor 3 agonist, while various CpGs are Toll-like receptor 9 agonists, and both Toll-like receptor 3 and Toll-like receptor 9 are located in the endosome membrane structure within the cell.
  • the lysate components and immune adjuvant were loaded into the micron particles, and then centrifuged at 10,000g for 15 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and then freeze-dried for 48 hours; before use, the particles were Resuspend in 7 mL of PBS and then add 3 mL of cancer cell lysate component (protein concentration 50 mg/mL) and incubate at room temperature for 10 min to obtain micron particles 1 loaded with lysate both inside and outside.
  • cancer cell lysate component protein concentration 50 mg/mL
  • the average particle size of the microparticles is about 2.50 ⁇ m, and the surface potential is about -22mV; each 1 mg of PLGA microparticles is loaded with approximately 140 ⁇ g of protein or polypeptide components, and the loaded CpG BW006 (Class B), CpG 2216 (Class A) and Poly ICLC 0.02mg each.
  • the preparation materials and preparation methods of Micron Particle 2 are the same, and the loaded immune adjuvants are CpG2336 (Class A), CpG 2216 (Class A) and Poly ICLC.
  • the particle size of the control micron particles 2 is about 2.50 ⁇ m, and the surface potential is about -22 mV.
  • Each 1 mg PLGA micron particle is loaded with approximately 140 ⁇ g of protein or peptide components.
  • Each 1 mg PLGA micron particle is loaded with CpG2336 (Class A) and CpG 2216 (Class A). ) and Poly ICLC immune adjuvant are 0.02mg each.
  • microparticle 3 The preparation materials and preparation methods of microparticle 3 are the same, and the loaded immune adjuvants are CpG BW006 (type B) and CpG 2216 (type A).
  • the adjuvant used in control microparticle 3 is 0.02 mg per 1 mg PLGA microparticle, the particle size is about 2.50 ⁇ m, and the surface potential is about -22 mV.
  • Each 1 mg PLGA microparticle is loaded with approximately 140 ⁇ g of protein or peptide components.
  • Each 1 mg PLGA microparticle contains Loaded CpG BW006 (Category B) and CpG 2216 (Category A) are 0.03mg each.
  • the mRNA loaded in the nucleic acid delivery precursor nanoparticle 1 loaded with mRNA can encode four polypeptide neoantigens B16-M20 (Tubb3, FRRKAFLHWYTGEAMDEMEFTEAESNM), B16-M24 (Dag1, TAVITPPTTTTKKARVSTPKPPATSTD), and B16-M46 (Actn4, NHSGLVTFQAFIDVMSRETTDTDTADQ) ) and TRP2:180-188 (SVYDFFVWL); the nanoparticles are simultaneously loaded with immune adjuvants CpG2395, CpG1018, poly(I:C) and positively charged peptide melittin (GIGAVLKVLTTGLPALISWIKRKRQQ).
  • the preparation materials and preparation method of PLGA nanoparticles loaded with mRNA are the same as in Example 3.
  • mRNA, poly(I:C), CpG2395, CpG1018 and melittin are mixed in water, and then the above mixture is loaded on the nanoparticles using the double emulsion method.
  • 100mg PLGA nanoparticles were then centrifuged at 10,000g for 20 minutes, resuspended in 10mL of ultrapure water containing 4% trehalose and freeze-dried for 48h.
  • the average particle size of the nanoparticles 1 is about 210nm.
  • Each 1 mg PLGA nanoparticle is loaded with approximately 1 ⁇ g of mRNA component.
  • Each 1 mg PLGA nanoparticle is loaded with 2 ⁇ g each of poly(I:C), CpG2395 and CpG1018 immune adjuvants, and 10 ⁇ g of polyarginine. .
  • mice After the mice were killed, mouse lymph nodes and spleens were collected. The mouse lymph nodes or spleens were minced and ground, and filtered through a cell mesh to prepare a single cell suspension. After mixing the lymph node single cell suspension and the spleen single cell suspension, flow cytometry was used. Cytometry separates CD19 + B cells and CD11c + DCs. B cells and DCs are mixed at a ratio of 1:1 and used as mixed antigen-presenting cells.
  • the antigen delivery microparticles (100 ⁇ g) loaded with cancer cell whole cell components and the mixed antigen-presenting cells (20 million) prepared in step (4) were incubated in 20 mL of high-sugar DMEM complete medium for 48 hours (37°C, 5 %CO 2 ), the incubation system contained GM-CSF (500U/mL), IL-2 (500U/mL), IL-15 (200U/mL) and CD86 antibody (10ng/mL).
  • the incubated DC and B cells were collected by centrifugation 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 (PBS). 22.5W) Ultrasonic 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.
  • PBS 4°C phosphate buffer solution
  • nanoparticles obtained by using the membrane components of antigen-presenting cells activated by microparticles 1 and nanoparticles 1 are nanoparticles 2, with a particle size of 220 nm and a surface potential of -7mV; each 1mg PLGA nanoparticle is loaded with approximately 1 ⁇ g of mRNA.
  • Each 1 mg of PLGA nanoparticles is loaded with 2 ⁇ g each of poly(I:C), CpG2395, and CpG1018 immune adjuvants, and 10 ⁇ g of polyarginine; each 1 mg of PLGA nanoparticles is loaded with approximately 200 ⁇ g of cell membrane components.
  • the nanoparticles obtained by using the membrane components of antigen-presenting cells activated by microparticles 2 and nanoparticles 1 to interact with each other are nanoparticles 3, with a particle size of 220 nanometers and a surface potential of -7mV; each 1 mg of PLGA nanoparticles loads approximately 1 ⁇ g of mRNA.
  • Components, per 1 mg PLGA nanoparticles 2 ⁇ g each of poly(I:C), CpG2395 and CpG1018 immune adjuvants are loaded, 10 ⁇ g polyarginine is loaded, and approximately 200 ⁇ g of cell membrane components are loaded per 1 mg of PLGA nanoparticles.
  • the nanoparticles obtained by using the membrane components of antigen-presenting cells activated by microparticles 3 and nanoparticles 1 to interact with each other are nanoparticles 4, with a particle size of 220 nm and a surface potential of -7mV; each 1 mg of PLGA nanoparticles loads approximately 1 ⁇ g of mRNA.
  • Each 1 mg of PLGA nanoparticles is loaded with 2 ⁇ g of poly(I:C), CpG2395, and CpG1018 immune adjuvants, 10 ⁇ g of polyarginine, and approximately 200 ⁇ g of cell membrane components per 1 mg of PLGA nanoparticles.
  • Melanoma tumor-bearing mice were prepared by selecting 6-8-week-old female C57BL/6 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, 7th, 10th, 15th, 20th and 25th days after melanoma vaccination, 0.8mg of Nanovaccine (Nanovaccine 2, or Nanovaccine 3, or Nanovaccine 4) was injected subcutaneously. ) or subcutaneously inject 100 ⁇ L PBS. The methods for monitoring tumor growth rate and survival period in mice are the same as above.
  • PBS control, Nanovaccine 2, Nanovaccine 3, and Nanovaccine 4 are the PBS control group, Nanovaccine 2, Nanovaccine 3, and Nanovaccine 4 in order.
  • the results showed that, as shown in Figure 6, the tumors of the mice in the control group all grew, while the tumor growth rate of the nanovaccine-treated mice was significantly slower and the survival period was significantly prolonged.
  • the effect of the nanovaccine prepared by cooperating with antigen-presenting cells activated by antigen-delivering microparticles loaded with CpG adjuvant and Poly ICLC mixed adjuvant and nucleic acid-loaded nucleic acid delivery precursor nanoparticles is better than that of the nanovaccine loaded with two CpG mixed adjuvants.
  • a vaccine prepared by the co-action of antigen-presenting cells activated by antigen-delivery microparticles and nucleic acid-loaded nucleic acid delivery precursor nanoparticles is better than A nanovaccine prepared by using antigen-presenting cells activated by antigen-delivery microparticles loaded with two types of Class A CpG and PolyICLC mixed adjuvants and nucleic acid-loaded nucleic acid delivery precursor nanoparticles.
  • the particle vaccine prepared by the co-operation of antigen-delivery microparticles loaded with mixed adjuvants of two different toll-like receptors activated antigen-presenting cells and nucleic acid-loaded nucleic acid delivery precursor particles is more effective and contains class B CpG.
  • the mixed CpG and Toll-like receptor 3 agonist are used as mixed adjuvants for antigen delivery micron particles to activate antigen-presenting cells and nucleic acid-loaded nucleic acid delivery precursor particles to prepare nucleic acid delivery particle vaccines that are more effective.
  • 8M urea was first used to lyse B16F10 melanoma tumor tissue and dissolve the tumor tissue lysate components. Then, PLGA was used as the microparticle framework material, and Poly(I:C), CpG2006 (B type) and CpGSL01 (B type) were used as immune adjuvants to prepare antigen delivery micron particles loaded with cancer cell whole cell antigens. After the micron particles activate the antigen-presenting cells, the activated antigen-presenting cells interact with the nucleic acid-loaded nucleic acid delivery precursor micron particles to prepare a nucleic acid-delivery micron vaccine for cancer prevention.
  • 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 8M urea through a cell filter to lyse the cells, dissolve the whole cell lysate, and use protein extraction and separation technology. technology to separate and extract total protein from whole cell lysates. The total protein obtained by separation and extraction is the source of antigen raw materials for preparing nanoparticle systems.
  • the antigen delivery microparticles are prepared by solvent evaporation method.
  • the molecular weight of PLGA, the preparation material used for microparticle 1, is 38KDa-54KDa.
  • the immune adjuvants used are Poly(I:C), CpG2006 and CpGSL01, and the lysate antigen component and adjuvant are loaded inside the microparticles.
  • the preparation method is as described above. After loading the lysate antigen component and adjuvant inside the microparticles, 100mg of the microparticles are centrifuged at 12000g for 20 minutes, resuspended in 10mL of ultrapure water containing 4% trehalose and then freeze-dried for 48h.
  • the average particle size of the microparticles is about 1.0 ⁇ m, and the surface potential of the microparticles is about -13mV; each 1 mg of PLGA microparticles is loaded with approximately 80 ⁇ g of protein or polypeptide components, and each 1 mg of PLGA microparticles is loaded with Poly(I:C), CpG2006 and CpGSL01 0.02mg each.
  • the preparation materials and preparation methods of the antigen delivery microparticles are the same as above.
  • the particle size is about 1.0 ⁇ m. They are loaded with an equal amount of lysate antigen components.
  • the loaded immune adjuvant is Poly(I:C). Each 1 mg of PLGA is loaded with Poly(I: C)0.06mg.
  • Antigen delivery micron particles 3 have a particle size of about 1.0 ⁇ m and are loaded with an equal amount of lysate antigen components.
  • the loaded immune adjuvants are Poly(I:C), CpG1585 (Class A) and CpG2216 (Class A). Each 1 mg of PLGA is loaded Poly(I:C), CpG1585 (Class A) and CpG2216 (Class A) 0.02mg each.
  • the mRNA loaded on the nucleic acid delivery precursor microparticles 4 can encode four polypeptide neoantigens B16-M20 (Tubb3, FRRKAFLHWYTGEAMDEMEFTEEAESNM), B16-M24 (Dag1, TAVITPPTTTTKKARVSTPKPPAPSTD), and B16-M46 (Actn4, NHSGLVTFQAFIDVMSRETTDTDTADQ) ) and TRP2:180-188 (SVYDFFVWL); the micron particles are simultaneously loaded with immune adjuvants poly(I:C), CpG (mixed CpG of class B and class C) and RALA polypeptide (WEARLARALARALARHLARALARALRACEA).
  • the preparation materials and preparation method of microparticles loaded with mRNA are the same as step (2). First, mix mRNA, poly(I:C), CpGSL03 (Category C), CpG1018 (Category B) and RALA polypeptide in water, and then use double emulsion. The above mixture is loaded inside the microparticles, and then 100mg PLGA microparticles are centrifuged at 9000g for 20 minutes, resuspended in 10mL of ultrapure water containing 4% trehalose and freeze-dried for 48h. The average particle size of the microparticles 4 is about 1.0 ⁇ m. Each 1 mg PLGA micron particle is loaded with approximately 5 ⁇ g of mRNA components. Each 1 mg PLGA micron particle is loaded with 30 ⁇ g each of poly(I:C), CpG SL03 and CpG1018 immune adjuvants, and 200 ⁇ g of RALA polypeptide. .
  • mouse lymph nodes were removed to prepare mouse lymph node single cell suspension, and then flow cytometry was used to sort CD11c + DC and CD19 + B cells from the lymph node cell single cell suspension.
  • Antigen delivery microparticles 1 800 ⁇ g
  • antigen delivery microparticles 2 800 ⁇ g
  • antigen delivery microparticles 3 800 ⁇ g loaded with cancer cell whole cell components were combined with DCs (5 million) and B cells (5 million) Incubate in 20mL high-glucose DMEM complete medium for a total of 72 hours (37°C, 5% CO 2 ); the incubation system contains GM-CSF (500U/mL), IL-2 (500U/mL), IL-7 (200U/mL).
  • step (3) After suspension, mix with the nanoparticles 1 prepared in step (3) and incubate for 10 minutes, then use a 2 ⁇ m filter to repeatedly co-extrude, then centrifuge the extrusion at 13000g for 30 minutes, and use 10 mL of ultrasonic solution containing 4% trehalose. Resuspend in pure water and freeze-dry for 48 hours before use.
  • the nucleic acid delivery microparticles prepared by the interaction of the membrane components of the antigen-presenting cells activated by the antigen delivery microparticles 1 and the nucleic acid delivery precursor microparticles 4 are micron vaccines 5, with a particle size of 1.1 ⁇ m and a surface potential of -7mV; each 1 mg PLGA is loaded with approximately 5 ⁇ g of mRNA components, each 1 mg of PLGA is loaded with 30 ⁇ g each of poly(I:C), CpG SL03 and CpG1018 immune adjuvants, and 200 ⁇ g of RALA polypeptide; each 1 mg of PLGA is loaded with approximately 80 ⁇ g of cell membrane components.
  • the nucleic acid delivery microparticles prepared by the interaction between the membrane components of the antigen-presenting cells activated by the antigen delivery microparticles 2 and the nucleic acid delivery precursor microparticles 4 are micron vaccines 6, with a particle size of 1.1 ⁇ m and a surface potential of -7mV. ;
  • Each 1 mg PLGA is loaded with approximately 5 ⁇ g of mRNA components, each 1 mg of PLGA is loaded with 30 ⁇ g each of poly(I:C), CpG SL03, and CpG1018 immune adjuvants, and 200 ⁇ g of RALA peptide; each 1 mg of PLGA is loaded with approximately 80 ⁇ g of cell membrane components.
  • the nucleic acid delivery microparticles prepared by the co-action of the membrane components of the antigen-presenting cells activated by the antigen delivery microparticles 3 and the nucleic acid delivery precursor microparticles 4 are micron vaccines 7, with a particle size of 1.1 ⁇ m and a surface potential of -7mV. ;
  • Each 1 mg PLGA is loaded with approximately 5 ⁇ g of mRNA components, each 1 mg of PLGA is loaded with 30 ⁇ g each of poly(I:C), CpG SL03, and CpG1018 immune adjuvants, and 200 ⁇ g of RALA peptide; each 1 mg of PLGA is loaded with approximately 80 ⁇ g of cell membrane components.
  • 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 - before the mice are inoculated with tumors. On 7 days, mice were subcutaneously inoculated with 1 mg of freshly freeze-dried Micron vaccine (Micron vaccine 5, or Micron vaccine 6, or Micron vaccine 7), or 100 ⁇ L of PBS. On day 0, 1.5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated into the lower right corner of the back of each mouse. The methods for monitoring tumor growth rate and survival period in mice are the same as above.
  • PBS control, Microvaccine 5, Microvaccine 6, and Microvaccine 7 are the PBS control group, Microvaccine 5, Microvaccine 6, and Microvaccine 7 in order.
  • the results showed that, as shown in Figure 7, the tumors of the mice in the PBS control group grew very quickly, while the tumors of the mice in the micron vaccine treatment group all slowed down significantly, and most of the mice's tumors disappeared after the cancer cells were inoculated.
  • the micron vaccine prepared by cooperating with antigen-presenting cells activated by antigen-delivery microparticles loaded with two types of B-type CpG and Poly(I:C) as a mixed adjuvant and nucleic acid-loaded nucleic acid delivery precursor micron particles has a good effect.
  • Antigen-presenting cells activated by antigen-delivery microparticles loaded with two types of A-type CpG and Poly(I:C) as a mixed adjuvant or loaded with only Poly(I:C) as an adjuvant and nucleic acid-loaded Micron vaccine prepared by combining nucleic acid delivery precursor micron particles.
  • Example 7 Vaccine for treating T lymphoma
  • E.G7-OVA mouse T lymphoma as a cancer model to illustrate how to use nanoparticle-activated antigen-presenting cells and nucleic acid-loaded nanoparticles to prepare a nanovaccine for the treatment of T lymphoma.
  • E.G7-OVA cells are lysed and protease is used to degrade the lysates into peptides in vitro.
  • other enzymes or other methods can also be used to first degrade the proteins in the whole cell components into polypeptides. Then prepare water-soluble components and non-water-soluble components respectively, and mix them in a mass ratio of 1:1.
  • PLA as the nanoparticle skeleton material
  • CpG2395 Category C
  • CpGM362 Category C
  • Poly ICLC immune adjuvants to prepare antigen delivery nanoparticles
  • use the antigen delivery nanoparticles to activate antigen-presenting cells in vitro and then Activated antigen-presenting cells interact with nucleic acid-loaded nucleic acid delivery precursor nanoparticles to prepare nanovaccines.
  • Collect BCG use 10% sodium deoxycholate (containing 1M arginine) aqueous solution to cleave the BCG, and then use 10% sodium deoxycholate (containing 1M arginine) aqueous solution to dissolve the cleavage components for later use.
  • nanoparticle 1 is prepared by solvent evaporation method.
  • the material for preparing the antigen delivery nanoparticle 1, PLA has a molecular weight of 20KDa.
  • the nanoparticles are loaded with cancer cell lysates, bacterial lysates and immune adjuvants inside, and the cancer cell lysate components are loaded on the surface.
  • the immune adjuvants used are CpG2395 (Category C), CpGM362 (Category C) and poly ICLC, and the adjuvants are loaded inside the nanoparticles.
  • the difference between the cancer cell lysate component and the bacterial lysate component used in the preparation of the nanoparticles is The mass ratio is 1:1.
  • the preparation method is as described above.
  • the double emulsion method is first used to load cancer cell lysate antigens, bacterial lysate components and adjuvants inside the nanoparticles. Then 100 mg of nanoparticles are centrifuged at 10,000g for 20 minutes, and 10 mL of nanoparticles containing Resuspend in 4% trehalose ultrapure water and freeze-dry for 48 h. Before use, resuspend 20 mg of nanoparticles in 0.9 mL of PBS, mix with 0.1 mL of sample containing cancer cell lysate antigen component (80 mg/mL), and incubate at room temperature for 5 minutes before use. The average particle size of nanoparticles 1 is about 400nm.
  • Each 1 mg of PLA nanoparticles 1 is loaded with approximately 400 ⁇ g of protein or peptide components.
  • Each 1 mg of PLA nanoparticles is loaded with 0.005 mg each of CpG2395 (Category C), CpGM362 (Category C) and Poly ICLC immune adjuvant. .
  • Nanoparticles 1 Preparation materials and preparation methods Nanoparticles 1, the particle size is about 400nm, each 1mg PLA nanoparticles is loaded with approximately 140 ⁇ g of protein or peptide components, each 1mg PLA is loaded with CpG1585 (type A), CpG2336 (type A) and Poly ICLC 0.005mg each.
  • the mRNA loaded on the mRNA-loaded nanoparticles 3 can encode the OVA antigen; the nanoparticles are also loaded with immune adjuvants poly ICLC, CpG, arginine and lysine.
  • the preparation materials and preparation method of mRNA-loaded nanoparticles are the same as in step (3).
  • PBMC Peripheral blood mononuclear cells
  • BMDC Peripheral blood mononuclear cells
  • the preparation method of BMDC is the same as in Example 1.
  • 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.
  • M-CSF Macrophage colony-stimulating factor
  • Antigen delivery nanoparticle 1 1000 ⁇ g and BMDC (30 million) were incubated in RPMI1640 complete medium for 48 hours (37°C, 5% CO 2 ); the incubation system contained IL-2 (500 U/mL), IL- 7 (500U/mL), IL-15 (500U/mL) and CD80 antibody (10ng/mL).
  • antigen delivery nanoparticles 1 1000 ⁇ g were incubated with BMDC (10 million), BMDM (10 million), and B cells (10 million) in 20 mL RPMI1640 complete medium 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 CD80 antibody (10ng/mL).
  • antigen delivery nanoparticles 2 1000 ⁇ g were incubated with BMDC (10 million), BMDM (10 million), and B cells (10 million) in 20 mL RPMI1640 complete medium 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 CD80 antibody (10ng/mL).
  • BMDCs (30 million) after incubation with antigen delivery nanoparticles 1 were collected by centrifugation at 400 g for 5 min, and then washed twice with 4°C phosphate buffer solution (PBS) containing protease inhibitors, and the cells were resuspended in PBS After soaking in water, process it in a high-pressure homogenizer (5000bar) for 5 minutes. The sample was then centrifuged at 2000 g for 15 minutes and the supernatant was collected. The supernatant was centrifuged at 8000 g for 15 minutes and the supernatant was collected. The supernatant was mixed with 50 mg of the nucleic acid delivery precursor nanoparticle 3 prepared in step (4).
  • PBS 4°C phosphate buffer solution
  • each 1 mg of PLA is loaded with approximately 40 ⁇ g of mRNA components.
  • Each 1 mg of PLA is loaded with 5 ⁇ g each of Poly ICLC, CpG2395 and CpG7909 immune adjuvants, and arginine. and 180 ⁇ g of lysine each; each 1 mg of PLGA is loaded with approximately 280 ⁇ g of membrane components. Store at room temperature for 12 months before use.
  • BMDC 10 million
  • B cells 10 million
  • BMDM 10 million
  • 4°C phosphate buffer solution containing protease inhibitors The cells were washed twice with (PBS), resuspended in PBS water and treated in a high-pressure homogenizer (5000bar) for 5 minutes. Then, the sample was centrifuged at 2000 g for 15 minutes and the supernatant was collected. The supernatant was centrifuged at 8000 g for 15 minutes and the supernatant was collected. The supernatant was mixed with the nucleic acid delivery precursor nanoparticle 3 (50 mg) prepared in step (4).
  • the nanovaccine prepared by using mixed antigen-presenting cells activated by antigen delivery nanoparticles 1 and nanoparticles 3 is nanovaccine 5, with a particle size of 400 nanometers, a surface potential of -7mV, and approximately 40 ⁇ g of mRNA per 1 mg of PLA.
  • Each 1 mg of PLGA is loaded with 5 ⁇ g each of Poly ICLC, CpG2395, and CpG7909 immune adjuvants, and 180 ⁇ g each of arginine and lysine; each 1 mg PLA is loaded with approximately 280 ⁇ g of membrane components; use immediately after preparation or store 12 at room temperature.
  • the nanovaccine prepared by using mixed antigen presenting cells activated by antigen delivery nanoparticles 2 and nanoparticles 3 is nanovaccine 6, with a particle size of 400 nanometers, a surface potential of -7mV, and a load of approximately 1 mg of PLGA 40 ⁇ g of mRNA component, each 1 mg of PLA is loaded with 5 ⁇ g each of Poly ICLC, CpG2395 and CpG7909 immune adjuvants, and 180 ⁇ g each of arginine and lysine; each 1 mg PLA is loaded with approximately 280 ⁇ g of membrane components; store at room temperature for 12 months before use.
  • mice Female C57BL/6 mice aged 6-8 weeks were selected as model mice to prepare tumor-bearing mice. On day 0, each mouse was subcutaneously inoculated with 5 ⁇ 10 5 E.G7-OVA cells, and on days 5, 8, 13, and 20, mice were injected with 100 ⁇ L PBS or 1 mg of nanovaccine ( The nano vaccine 5 that has just been prepared, or the nano vaccine 4 that has been stored for 12 months, or the nano vaccine that has been stored for 12 months 5, or the nano vaccine that has been stored for 12 months 6). The methods for monitoring mouse tumor volume and survival were the same as above.
  • PBS control, Nanovaccine 4 (Long term storage), Nanovaccine 5 (Long term storage), Nanovaccine 6 (Long term storage) and Nanovaccine 5 (Fresh) are the PBS control group, long-term stored nanovaccine 4, long-term stored nanovaccine 5, long-term stored nanovaccine 6, and newly prepared nanovaccine 5.
  • the results showed that, as shown in Figure 8, the tumors of the mice in the PBS control group grew very quickly and the mice had a short survival period. Compared with the control group, the tumor growth rate of mice treated with several nanovaccines was significantly slower and the survival period was significantly prolonged.
  • the nanovaccine 5 prepared by using two types of CpG and Poly ICLC as mixed adjuvant antigen delivery nanoparticles to activate antigen-presenting cells and nucleic acid-loaded nucleic acid delivery precursor nanoparticles is more effective than the two types of A CpG and Poly ICLC act as mixed adjuvants for antigen delivery nanoparticles to activate antigen-presenting cells and nucleic acid-loaded nucleic acid delivery precursor nanoparticles to prepare nanovaccine 6; moreover, using the membrane components of mixed antigen-presenting cells Nanovaccine 5 is more effective than nanovaccine 4 using membrane components of a single antigen-presenting cell. Moreover, the effect of the above-mentioned nanovaccine after being stored at room temperature for 12 months is the same as that immediately after preparation, indicating that the above-mentioned nanovaccine has good stability.
  • Example 8 Vaccine for treatment of melanoma
  • the supernatant part is the water-soluble antigen; the precipitate part uses 8M urea (containing 200mM sodium chloride) aqueous solution to dissolve the non-water-soluble antigen.
  • 8M urea containing 200mM sodium chloride
  • the water-soluble antigen and the non-water-soluble antigen dissolved in 8M urea are miscible at a mass ratio of 1:1, which is the source of the antigen raw material for preparing the nanoparticle system.
  • the antigen delivery nanoparticles were prepared using the double emulsion method.
  • the nanoparticle preparation material used 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 and KALA polypeptide are encapsulated in nanoparticles.
  • the preparation method is as described above. During the preparation process, the double emulsion method is first used to load the lysis solution components, adjuvants, and KALA polypeptides 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 0.04 mg, and the loaded KALA polypeptide is 0.3 mg.
  • the preparation materials and methods of the antigen delivery nanoparticles 2 are the same.
  • the particle size is about 250nm, the surface potential is about -5mV, it does not load KALA polypeptide, and it loads equal amounts of adjuvants and cell lysis antigen components.
  • the preparation materials and preparation methods of the antigen delivery nanoparticles 3 are the same, about 250nm, and the surface potential is about -5mV; each 1 mg of PLGA nanoparticles is loaded with approximately 100 ⁇ g of protein and peptide components, and the poly(I: C)0.04mg, loaded CpG1018 is 0.08mg, loaded KALA polypeptide is 0.3mg.
  • the mRNA loaded on the mRNA-loaded nanoparticles 4 in this embodiment can encode four polypeptide neoantigens B16-M20 (Tubb3, FRRKAFLHWYTGEAMDEMEFTEAESNM), B16-M24 (Dag1, TAVITPPTTTTKKARVSTPKPATPSTD), B16-M46 (Actn4, NHSGLVTFQAFIDVMSRETTDTDTADQ) and TRP2: 180-188 (SVYDFFVWL); the nanoparticles are simultaneously loaded with immune adjuvants poly(I:C), CpG and KALA polypeptide (WEAKLAKALAKALAKHLAKALAKALKACEA), a substance that promotes lysosomal escape.
  • B16-M20 Tubb3, FRRKAFLHWYTGEAMDEMEFTEAESNM
  • B16-M24 Dag1, TAVITPPTTTTKKARVSTPKPATPSTD
  • B16-M46 Actn4, NHSGLVTFQAFIDV
  • the preparation materials and preparation method of mRNA-loaded nanoparticles are the same as step (2).
  • 100 mg of PLGA nanoparticles were then centrifuged at 9000 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 4 is about 210nm, and each 1 mg PLGA nanoparticle is loaded with approximately 100 ⁇ g of mRNA components.
  • Each 1 mg PLGA nanoparticle is loaded with 1 ⁇ g each of poly(I:C), CpG7909, and CpGBW2006 immune adjuvants, and 400 ⁇ g of KALA polypeptide.
  • This example uses BMDC and BMDM as mixed antigen-presenting cells.
  • the preparation methods of BMDC and BMDM are the same as above.
  • nanoparticle 1, or nanoparticle 2, or nanoparticle 3 1000 ⁇ g of antigen delivery nanoparticles (nanoparticle 1, or nanoparticle 2, or nanoparticle 3) were incubated with BMDC (10 million pcs) and BMDM (10 million pcs) in 15 mL high-glucose DMEM complete medium for 48 hours (37°C , 5% CO 2 ); the incubation system contains GM-CSF (200U/mL), M-CSF (200U/mL), IL-2 (500U/mL), IL-7 (200U/mL), IL-12 (200U/mL) and CD80 antibody (10ng/mL).
  • the incubated BMDC and BMDM were collected by centrifugation at 400 g for 5 min, then washed three times using 1200 rpm for 3 min in 30 mM pH 7.0 Tris-HCl buffer containing 0.0759 M sucrose and 0.225 M mannitol, and then incubated with phosphatase inhibitors and protease inhibitors. Ultrasound mechanically destroys antigen-presenting cells in the presence of the agent. After centrifugation, the cell membrane obtained was washed with a solution of 10mM Tris-HCl pH 7.5 and 1mM EDTA. The sample was then filtered through membranes with pore sizes of 30 ⁇ m, 10 ⁇ m, 5 ⁇ m, 2 ⁇ m, and 0.45 ⁇ m.
  • the filtrate was centrifuged at 12,000g for 25 minutes and the supernatant was discarded to collect the precipitate.
  • the precipitate was resuspended in PBS and mixed with the nucleic acid before delivery.
  • the bulk nanoparticles 4 were incubated at room temperature for 15 minutes, and then repeatedly co-extruded using a 0.22 ⁇ m filter.
  • the extrudate was centrifuged at 15,000g for 30 minutes, the supernatant was discarded, and the precipitate was placed in physiological solution containing 4% mannitol. Resuspend in saline and freeze-dry to obtain nucleic acid delivery nanovaccine.
  • the nucleic acid delivery nanoparticles prepared by using the antigen presenting cells activated by the antigen delivery nanoparticles 1 and the nucleic acid delivery precursor nanoparticles 4 are nanovaccines 5, with a particle size of 220 nm and a surface potential of -7mV; per 1 mg PLGA is loaded with approximately 10 ⁇ g of mRNA components, each 1 mg of PLGA is loaded with 1 ⁇ g each of poly(I:C), CpG7909, and CpGBW2006 immune adjuvants, and 400 ⁇ g of KALA polypeptide; each 1 mg of PLGA is loaded with approximately 280 ⁇ g of membrane components.
  • the nucleic acid delivery nanoparticles prepared by using antigen-presenting cells activated by antigen delivery nanoparticles 2 and nucleic acid delivery precursor nanoparticles 4 are nanovaccines 6, with a particle size of 220 nm and a surface potential of -7mV; per 1 mg of PLGA is approximately Load 10 ⁇ g of mRNA components, load 1 ⁇ g each of poly(I:C), CpG7909, and CpGBW2006 immune adjuvants per 1 mg PLGA, and load 400 ⁇ g of KALA polypeptide; approximately 280 ⁇ g of membrane components per 1 mg PLGA.
  • the nucleic acid delivery nanoparticles prepared by using antigen-presenting cells activated by antigen delivery nanoparticles 3 and nucleic acid delivery precursor nanoparticles 4 are nanovaccines 7, with a particle size of 220 nm and a surface potential of -7mV; per 1 mg of PLGA is approximately Load 10 ⁇ g of mRNA components, load 1 ⁇ g each of poly(I:C), CpG7909, and CpGBW2006 immune adjuvants per 1 mg PLGA, and load 400 ⁇ g of KALA polypeptide; approximately 280 ⁇ g of membrane components per 1 mg PLGA.
  • 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 the 4th, 7th, 10th, 15th and 20th days after melanoma inoculation, 100 ⁇ L PBS or 0.8 mg of the corresponding nanovaccine was injected subcutaneously. In the experiment, the mouse tumor volume and survival period were monitored as above.
  • PBS control, Nanovaccine 5, Nanovaccine 6, and Nanovaccine 7 in Figure 9 are the PBS control group, Nanovaccine 5, Nanovaccine 6, and Nanovaccine 7 in order.
  • the results showed that the tumors in the PBS control group grew quickly. Compared with the control group, the tumor growth rate of nanovaccine-treated mice was significantly slower and the survival period was significantly prolonged.
  • the effect of nanovaccine 5 prepared by adding antigen delivery nanoparticles that promote lysosomal escape substances to activate antigen-presenting cells and nucleic acid-loaded nucleic acid delivery precursor nanoparticles is better than that of antigen delivery without adding lysosomal escape.
  • Nanovaccine 6 prepared by the co-action of nanoparticle-activated antigen-presenting cells and nucleic acid-loaded nucleic acid delivery precursor nanoparticles; antigen delivery nanoparticle-activated antigen using two CpGs and Poly(I:C) as mixed adjuvants
  • the therapeutic effect of nanovaccine 5 prepared by the co-action of presenting cells with nucleic acid-loaded nucleic acid delivery precursor nanoparticles is better than the antigen presentation activated by antigen delivery nanoparticles using only one CpG and Poly(I:C) mixed adjuvant.
  • Nanovaccine 7 prepared after the interaction between cells and nucleic acid-loaded nucleic acid delivery precursor nanoparticles. This shows that the addition of lysosomal escape substances and the use of mixed adjuvants are beneficial to improving the efficacy of nanovaccines.
  • the nanovaccine disclosed in the present disclosure has a good therapeutic effect on cancer.
  • the cultured E.G7-OVA mouse T lymphoma 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 8M urea aqueous solution (containing 500mM sodium chloride) is used to lyse the cancer cells and dissolve the lysate components, which are the antigen components for preparing the antigen delivery micron particle system. .
  • the precipitate was lysed using 8M urea aqueous solution (containing 500mM sodium chloride) to dissolve the bacterial outer vesicle component.
  • the double emulsion method was used to prepare the antigen delivery microparticles.
  • the skeleton material of micron particle 1 is PLA, and the molecular weight of PLA is 40KDa.
  • the immune adjuvants used are CpG2006 (Class B), CpG2216 (Class A) and Poly ICLC, and the positively charged substances used are arginine and polyhistidine. Cancer cell lysate components used in the preparation of microparticles and The mass ratio of bacterial outer vesicle components is 1:1.
  • the double emulsion method is first used to prepare antigen delivery micron particles that are internally loaded with cancer cell lysate antigen components, bacterial external vesicle components, adjuvants, arginine and polyhistidine. Then, 100 mg of micron particles are placed in 9000g Centrifuge for 20 minutes, resuspend in 10 mL of ultrapure water containing 4% trehalose, and dry for 48 hours to obtain the antigen delivery microparticles 1. The average particle size is about 4.98 ⁇ m. Each 1 mg of PLGA microparticles 1 is approximately loaded with 100 ⁇ g of protein or peptide group. 0.07mg each of CpG2006, CpG2216 and Poly ICLC is loaded, and 0.05mg each of arginine and polyhistidine is loaded.
  • the preparation materials and methods of the antigen delivery micron particles 2 are the same as those of the micron particles 1.
  • the particle size is about 4.98 ⁇ m, and it is loaded with equal amounts of arginine, polyhistidine, and equal amounts of cancer cell lysate components and bacterial outer vesicle components. , but without any adjuvants.
  • the mRNA loaded on the micron particles 3 loaded with mRNA can encode the OVA antigen; the micron particles are also loaded with the immune adjuvant poly(I:C), two types of CpG (type B and type C) and polyhistidine.
  • the preparation materials and preparation method of micron particles loaded with mRNA are the same as step (2).
  • This example uses BMDC, B cells and BMDM as antigen-presenting cells.
  • the preparation methods of BMDC and BMDM are the same as above.
  • B cells were derived from mouse peripheral blood PBMC, and the preparation method was the same as above.
  • Antigen Delivery Microparticles 1 or Antigen Delivery Microparticles 2 were mixed with 40 million mixed antigen-presenting cells (including 20 million BMDCs, 10 million B cells and 10 million BMDMs) in 15 mL of high-glucose DMEM complete medium Incubate with CCP for 48 hours (37°C, 5% CO 2 ); the incubation system contains IL-2 (500U/mL), IL-7 (200U/mL), IL-15 (200U/mL), and CD40 antibody (20mg /mL).
  • IL-2 500U/mL
  • IL-7 200U/mL
  • IL-15 200U/mL
  • CD40 antibody 20mg /mL
  • Micron Vaccine 4 is prepared by using mixed antigen-presenting cells activated by Micron Particle 1 and Micron Particle 3.
  • the particle size is 5.00 ⁇ m and the surface electricity is The position is -6mV; each 1 mg PLGA is loaded with approximately 100 ⁇ g of mRNA components, each 1 mg PLGA is loaded with 60 ⁇ g each of poly(I:C), CpG2395 and CpG1018 immune adjuvants, and 250 ⁇ g of polyhistidine; each 1 mg PLGA is loaded with approximately 280 ⁇ g of membrane components .
  • Micron vaccine 5 is prepared using mixed antigen-presenting cells activated by micron particles 2 and micron particles 3.
  • the particle size is 5.00 ⁇ m and the surface potential is -6mV.
  • Each 1 mg of PLGA is loaded with approximately 100 ⁇ g of mRNA components, and each 1 mg of PLGA is loaded with poly. (I:C), CpG2395 and CpG1018 immune adjuvants are 60 ⁇ g each, and 250 ⁇ g of polyhistidine is loaded; approximately 280 ⁇ g of membrane components are loaded per 1mg of PLGA.
  • PBS phosphate buffer solution
  • Vaccine 6 the particle size of micron vaccine 6 is 5.00 ⁇ m, and the surface potential is -6mV; each 1mg PLGA is loaded with approximately 100 ⁇ g of mRNA components, and each 1mg of PLGA is loaded with 60 ⁇ g each of poly(I:C), CpG2395 and CpG1018 immune adjuvants. 250 ⁇ g of histidine; approximately 280 ⁇ g of membrane component is loaded per 1 mg of PLGA.
  • mice Female C57BL/6 mice aged 6-8 weeks were selected as model mice to prepare tumor-bearing mice.
  • the mice were injected with 100 ⁇ L PBS or 1 mg of Micron vaccine (Micron vaccine 4, or Micron vaccine 5, respectively) on days -35, -28, -21, -14 and -7 before tumor inoculation. Or micron vaccine 6), each mouse was subcutaneously inoculated with 5 ⁇ 10 5 E.G7-OVA cells on day 0.
  • the methods for monitoring mouse tumor volume and survival were the same as above.
  • PBS control, Microvaccine 4, Microvaccine 5, and Microvaccine 6 in Figure 10 are the PBS control group, Microvaccine 4, Microvaccine 5, and Microvaccine 6 in order.
  • the results showed that compared with the PBS control group, the tumor growth rate of mice treated with Micron vaccine was significantly slower and the survival period of mice was significantly prolonged.
  • micron vaccine 4 prepared by the co-action of the components of the antigen-presenting cells activated by the antigen-delivery microparticles 1 containing substances that increase the lysosomal escape function and mixed adjuvants and the nucleic acid delivery precursor microparticles 3 is better than the micron vaccine 4 containing only Micron vaccine 5 prepared by the co-action of membrane components of antigen-presenting cells activated by antigen-delivery microparticles 2 without mixed adjuvant and nucleic acid delivery precursor micron-particles 3.
  • Micron Vaccine 6 is better than Micron Vaccine 4, indicating that the mixed membrane component composed of membrane components of activated antigen-presenting cells and cancer cell membrane components is more effective. It can be seen that the micron vaccine described in the present disclosure can be used to prevent or treat cancer. Moreover, the use of mixed adjuvants and the loading of cancer cell membrane components on the surface of the vaccine will help improve the vaccine effect.
  • This example uses mouse colon cancer as a cancer model to illustrate how to use antigen-presenting cells activated by antigen-delivery nanoparticles loaded with colon cancer whole-cell antigens to prepare a nanovaccine by cooperating with nucleic acid-loaded nucleic acid delivery precursor nanoparticles.
  • Used to treat colon cancer In this example, 10% octylglucoside aqueous solution is first used to lyse colon cancer tumor tissue and dissolve the lysed components. Then, PLGA is used as the skeleton material, Poly(I:C), CpG2336 and CpG2006 are used as adjuvants, and NH is used. 4 HCO 3 is used to increase lysosomal escape substances, and antigen delivery nanoparticles are prepared.
  • the antigen delivery nanoparticles are used to activate the antigen-presenting cells, and then the antigen-presenting cells are combined with extracellular vesicles from the antigen-presenting cells and nucleic acid-loaded nucleic acids.
  • the nucleic acid delivery precursor nanoparticles work together to prepare a nanovaccine, and the nanovaccine is used to treat cancer.
  • Centrifuge Lactobacillus rhamnosus at 5000g for 30 minutes, then discard the precipitate and collect the supernatant. Filter the supernatant with a 1 ⁇ m filter, ultrasonicate at 4°C for 5 minutes at 20W, and then centrifuge at 16000g for 90 minutes. , resuspend the pellet in PBS to form the collected bacterial outer vesicle membrane components, and then use 10% octylglucoside aqueous solution to lyse and dissolve the bacterial outer vesicle membrane components.
  • the antigen delivery nanoparticles were prepared using the double emulsion method.
  • the nanoparticles are internally loaded with tumor tissue lysate components, bacterial extracellular vesicle components, mRNA, immune adjuvants and substances that increase lysosomal escape.
  • the preparation material of nanoparticles, PLGA has a molecular weight of 7KDa-17KDa.
  • the mRNA used is the mRNA encoding OVA.
  • Poly(I:C) and two CpGs are used as adjuvants, and KALA polypeptide (WEAKLAKALAKALAKHLAKALAKALKACEA) is used to increase lysosomal escape substances.
  • the adjuvant and KALA polypeptide are loaded into the nanoparticles; during preparation, the mass ratio of tumor tissue lysate components: bacterial outer vesicle components: mRNA is 4:4:1.
  • the preparation method is as described above.
  • the nanoparticles are first loaded with tumor tissue lysate components, bacterial extracellular vesicle lysate components, increased lysosomal escape substances and adjuvants, and then 100 mg of nanoparticles are added to 10,000 g.
  • each 1 mg of PLGA nanoparticles is loaded with approximately 90 ⁇ g of protein and peptide components.
  • the amount of mRNA encoding OVA is 10 ⁇ g.
  • Each 1 mg of PLGA nanoparticles is loaded with 0.02 mg of poly(I:C), CpG2336 and CpG2006, and 0.12 mg of KALA polypeptide.
  • the preparation materials and preparation methods of Antigen Delivery Nanoparticle 2 are the same as Nanoparticle 1, but they do not load poly(I:C).
  • the particle size is about 110nm.
  • Each 1 mg PLGA nanoparticle is loaded with approximately 90 ⁇ g of protein and peptide components, and is loaded with OVA-encoded
  • the amount of mRNA is 10 ⁇ g, and each 1 mg of PLGA nanoparticles is loaded with 0.12 mg of KALA peptide and 0.03 mg of CpG2336 and CpG2006.
  • the mRNA loaded on the mRNA-loaded nanoparticles 3 in this embodiment can encode OVA antigen.
  • Nanoparticle 3 is also loaded with the immune adjuvant poly(I:C), two types of B CpG and polylysine.
  • the preparation materials and preparation method of mRNA-loaded nanoparticles are the same as step (2). First, mRNA, poly(I:C), CpG7909 (Class B), CpG1018 (Class B) and poly Lysine is mixed in water, and then the above mixture is loaded inside the nanoparticles using the double emulsion method.
  • 100mg PLGA nanoparticles are centrifuged at 9000g for 20 minutes, resuspended in 10mL of ultrapure water containing 4% trehalose and then freeze-dried. 48h.
  • the average particle size of nanoparticle 3 is about 110nm.
  • Each 1 mg PLGA nanoparticle is loaded with approximately 20 ⁇ g of mRNA components.
  • Each 1 mg PLGA nanoparticle is loaded with 20 ⁇ g each of poly(I:C), CpG7909 and CpG1018 immune adjuvants, and 40 ⁇ g of polylysine.
  • This example uses BMDC and B as antigen-presenting cells.
  • the preparation method of BMDC is the same as in Example 1.
  • B cells were derived from mouse peripheral blood PBMC, and the preparation method was the same as above.
  • Antigen Delivery Nanoparticle 1 or Antigen Delivery Nanoparticle 2 was incubated with BMDC (10 million) and B cells (10 million) in 15 mL of high-glucose DMEM complete medium for 48 hours (37°C, 5% CO 2 ) ;
  • the incubation system contains IL-2 (500U/mL), IL-7 (500U/mL), IL-12 (200U/mL) and GM-CSF (200U/mL).
  • the collected extracellular vesicles of activated antigen-presenting cells and cancer cell extracellular vesicles were mixed, then ultrasonicated at 4°C at low power (20W) for 2 minutes, and then repeatedly co-extruded using a 0.22 ⁇ m filter membrane.
  • the extruded liquid is mixed with the nucleic acid delivery precursor nanoparticles (nanoparticle 3) prepared in step (3), treated with a high-pressure homogenizer (10000bar) for 1 minute, and then repeatedly co-extruded using a 0.22 ⁇ m filter membrane, and then incubated at 15000g After centrifugation for 30 minutes, discard the supernatant and collect the precipitate.
  • the nanovaccine prepared by using the antigen-presenting extracellular vesicle membrane component activated by nanoparticle 1 and nanoparticle 3 is nanovaccine 4, with a particle size of 120 nanometers and a surface potential of -6mV; per 1 mg of PLGA is approximately Load 10 ⁇ g of mRNA component, load 20 ⁇ g each of poly(I:C), CpG7909, and CpG1018 immune adjuvants per 1 mg PLGA, and load 40 ⁇ g polylysine; approximately load 280 ⁇ g membrane components per 1 mg PLGA.
  • the nanovaccine prepared using the antigen-presenting extracellular vesicle membrane component activated by nanoparticle 2 and nanoparticle 3 is nanovaccine 5, with a particle size of 120 nm and a surface potential of -6mV; each 1 mg of PLGA is loaded with approximately 10 ⁇ g
  • each 1 mg of PLGA is loaded with 20 ⁇ g each of poly(I:C), CpG7909, and CpG1018 immune adjuvants, and 40 ⁇ g of polylysine; each 1 mg of PLGA is loaded with approximately 280 ⁇ g of membrane components.
  • Nano vaccines are used to treat cancer
  • PBS control, Nanovaccine 4, and Nanovaccine 5 in Figure 11 are the PBS control group, Nanovaccine 4, and Nanovaccine 5 in order.
  • the results showed that compared with the PBS control group, the tumor growth rate of mice treated with nanovaccine was significantly slower and the survival period of mice was significantly prolonged.
  • the nanovaccine 4 that simultaneously loads the extracellular vesicle membrane components of antigen-presenting cells and the extracellular vesicle membrane components of cancer cells activated by particles 1 containing Poly(I:C) and CpG is loaded on the surface of the internally loaded nucleic acid.
  • the effect is better than the nanovaccine 5 which internally loads nucleic acid and simultaneously loads the extracellular vesicle membrane components of antigen-presenting cells and the extracellular vesicle membrane components of cancer cells activated by particles 2 containing only CpG. It can be seen that the nanovaccine described in the present disclosure has excellent therapeutic effect on cancer.
  • Example 11 Nanoparticles loaded with mRNA and antigen-presenting cell membrane components are used for the prevention of melanoma
  • the mRNA loaded on the mRNA-loaded nanoparticle 1 in this embodiment can encode four polypeptide neoantigens B16-M20 (Tubb3, FRRKAFLHWYTGEAMDEMEFTEAESNM), B16-M24 (Dag1, TAVITPPTTTTKKARVSTPKPATPSTD), B16-M46 (Actn4, NHSGLVTFQAFIDVMSRETTDTDTADQ) and TRP2: 180-188 (SVYDFFVWL); the nanoparticles are simultaneously loaded with immune adjuvants poly(I:C), CpGBW006, CpG2395 and R8 (RRRRRRRR) polypeptides.
  • the preparation materials and preparation methods of the particles loaded with mRNA are as described above.
  • the mRNA, poly(I:C), CpGBW006, CpG2395 and R8 polypeptide are mixed in water, and then the above mixture is loaded inside the nanoparticles using the double emulsion method. Then 100mg PLGA nanoparticles were centrifuged at 10000g for 20 minutes, resuspended in 10mL of ultrapure water containing 4% trehalose and freeze-dried for 48h. The average particle size of the nanoparticles 1 is about 250nm. Each 1 mg PLGA nanoparticle is loaded with approximately 10 ⁇ g of mRNA components. Each 1 mg PLGA nanoparticle is loaded with 20 ⁇ g each of poly(I:C), CpGBW006 and CpG2395, and 40 ⁇ g of R8 polypeptide.
  • B cells and BMDC are mixed in a quantity ratio of 1:1 to form the mixed antigen-presenting cells used in this embodiment.
  • IL-2 500U/mL
  • IL-7 500U/mL
  • IL-15 IL-15
  • Collect mixed antigen-presenting cells (20 million) by centrifugation at 400g for 5 minutes, then wash the cells twice with normal saline, resuspend the cells in normal saline and sonicate at 4°C and 7.5W for 20 minutes to destroy the cells and prepare samples containing cell membrane components. The sample was then centrifuged at 2000g for 20 minutes and the supernatant was collected. The supernatant was centrifuged at 7000g for 20 minutes and the supernatant was collected.
  • the supernatant was incubated with 40 mg of nanoparticle 1 prepared in step (1) for 10 minutes, and then Repeated co-extrusion using a 0.45 ⁇ m filter membrane, centrifuge the extrudate at 15,000 g for 120 minutes, collect the supernatant, and collect the precipitate.
  • the precipitate is resuspended in PBS to obtain Nano Vaccine 2, with a particle size of 270 nm; each 1 mg PLGA is loaded with approximately 10 ⁇ g of mRNA components, each 1 mg PLGA is loaded with 20 ⁇ g each of poly(I:C), CpGBW006, and CpG2395, and 40 ⁇ g of R8 polypeptide is loaded; each 1 mg PLGA loads approximately 90 ⁇ g of membrane components.
  • This example uses microfluidic method to prepare LNP.
  • 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 - before the mice are inoculated with tumors. On the 7th day, mice were subcutaneously inoculated with 0.5 mg Nano Vaccine 2, or 0.5 mg LNP Vaccine or 100 ⁇ L PBS. On day 0, 1.5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated into the lower right corner of the back of each mouse. The methods for monitoring tumor growth rate and survival period in mice are the same as above.
  • the PBS control, LNP vaccine, and Nanovaccine 2 in Figure 12 are the PBS control group, LNP vaccine, and Nanovaccine 2 in order.
  • the results showed that the tumors of the mice in the PBS control group grew rapidly and their survival period was very short. The tumor growth rate of mice treated with the vaccine was significantly slower, and the mice's survival period was longer.
  • Nano Vaccine 2 is more effective than LNP vaccine. This shows that the nanovaccine described in the present disclosure is very effective in preventing or treating cancer and is better than the LNP vaccine.
  • the cultured E.G7-OVA mouse T lymphoma 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 8M urea aqueous solution (containing 500mM sodium chloride) is used to lyse the cancer cells and dissolve the lysate components, which are the antigen components for preparing the antigen delivery micron particle system. .
  • the precipitate was lysed using 8M urea aqueous solution (containing 500mM sodium chloride) to dissolve the bacterial outer vesicle component.
  • the double emulsion method is used to prepare the antigen delivery microparticles 1.
  • the skeleton material of micron particle 1 is PLA, and the molecular weight of PLA is 40KDa.
  • the immune adjuvants used are CpG2006 (Class B), CpG2216 (Class A) and Poly ICLC, and the positively charged substances used are arginine and histidine.
  • the mass ratio of cancer cell lysate components and bacterial external vesicle components used in the preparation of micron particles is 1:1.
  • the double emulsion method is first used to prepare antigen delivery micron particles that are internally loaded with cancer cell lysate antigen components, bacterial external vesicle components, adjuvants, arginine and histidine, and then 100 mg of the micron particles are centrifuged at 9000g. For 20 minutes, use 10 mL of ultrapure water containing 4% trehalose to resuspend and dry for 48 hours to obtain the antigen delivery microparticles 1. The average particle size is about 2.45 ⁇ m. Each 1 mg of PLGA microparticles 1 is approximately loaded with 100 ⁇ g of protein or peptide components. , loading CpG2006, CpG2216 and Poly ICLC 0.02mg each, loading arginine and histidine 0.05mg each.
  • the mRNA loaded on the micron particles 2 loaded with mRNA can encode the OVA antigen; the micron particles are also loaded with the immune adjuvant poly(I:C), two types of CpG (type B and type C) and KALA polypeptide (WEAKLAKALAKALAKHLAKALAKALKACEA) .
  • the preparation materials and preparation method of micron particles loaded with mRNA are the same as step (2). First, mix mRNA, poly(I:C), CpG2395 (Category C), CpG1018 (B class) and KALA polypeptide in water, and then use double emulsion.
  • microparticles The above mixture is loaded inside the microparticles, and then 100mg PLA microparticles are centrifuged at 9000g for 20 minutes, resuspended in 10mL of ultrapure water containing 4% trehalose and freeze-dried for 48h.
  • the average particle size of micron particles 2 is about 2.45 ⁇ m.
  • Each 1 mg PLA micron particle is loaded with approximately 20 ⁇ g of mRNA components.
  • Each 1 mg PLGA micron particle is loaded with 20 ⁇ g each of poly(I:C), CpG2395 and CpG1018 immune adjuvants, and 150 ⁇ g of KALA polypeptide.
  • the mRNA loaded on the micron particles 3 loaded with mRNA can encode the OVA antigen; the micron particles 3 are also loaded with the immune adjuvant poly(I:C), two types of A CpG and KALA polypeptide (WEAKLAKALAKALAKHLAKALAKALKACEA).
  • the preparation materials and preparation method of microparticles loaded with mRNA are the same as step (2). First, mix mRNA, poly(I:C), CpG1585 (Class A), CpG2336 (Class A) and KALA polypeptide in water, and then use double emulsion.
  • microparticles The above mixture is loaded inside the microparticles, and then 100mg PLA microparticles are centrifuged at 9000g for 20 minutes, resuspended in 10mL of ultrapure water containing 4% trehalose and freeze-dried for 48h.
  • the average particle size of micron particles 3 is about 2.45 ⁇ m.
  • Each 1 mg PLA micron particle is loaded with approximately 20 ⁇ g of mRNA components.
  • Each 1 mg PLGA micron particle is loaded with 20 ⁇ g each of poly(I:C), CpG1585, and CpG2336 immune adjuvants, and 150 ⁇ g of KALA polypeptide.
  • the mRNA loaded on the micron particles 4 loaded with mRNA can encode the OVA antigen; the micron particles are also loaded with the immune adjuvant poly(I:C), a CpG (type B) and KALA polypeptide (WEAKLAKALAKALAKHLAKALAKALKACEA).
  • the preparation materials and preparation method of microparticles loaded with mRNA are the same as step (2).
  • control blank microparticles 5 that do not load any mRNA are simultaneously loaded with the immune adjuvant poly(I:C), two types of CpG (type B and type C) and KALA polypeptide (WEAKLAKALAKALAKHLAKALAKALKACEA).
  • the preparation materials and preparation method of micron particles 5 are the same as step (2). First, poly(I:C), CpG2395 (Category C), CpG1018 (B category) and KALA polypeptide are mixed in water, and then the above mixture is mixed using the double emulsion method.
  • microparticles Loaded inside the microparticles, 100mg PLA microparticles were then centrifuged at 9000g for 20 minutes, resuspended in 10mL of ultrapure water containing 4% trehalose and freeze-dried for 48h.
  • the average particle size of micron particles 2 is about 2.45 ⁇ m.
  • Each 1 mg PLA micron particle carries 20 ⁇ g each of poly(I:C), CpG2395 and CpG1018 immune adjuvants, and 150 ⁇ g KALA polypeptide.
  • This example uses BMDC, B cells and BMDM as antigen-presenting cells.
  • the preparation methods of BMDC and BMDM are the same as above.
  • B cells were derived from mouse peripheral blood PBMC, and the preparation method was the same as above.
  • antigen delivery microparticles 1 1000 ⁇ g of antigen delivery microparticles 1 were incubated with 40 million mixed antigen-presenting cells (containing 20 million BMDC, 10 million B cells and 10 million BMDM) in 15 mL of high-glucose DMEM complete medium for 48 hours (37 °C, 5% CO 2 ); the incubation system contains IL-2 (500U/mL), IL-7 (200U/mL), IL-15 (200U/mL) and CD40 antibody (20mg/mL).
  • mixed antigen-presenting cells containing 20 million BMDC, 10 million B cells and 10 million BMDM
  • high-glucose DMEM complete medium for 48 hours (37 °C, 5% CO 2 ); the incubation system contains IL-2 (500U/mL), IL-7 (200U/mL), IL-15 (200U/mL) and CD40 antibody (20mg/mL).
  • PBS phosphate buffer solution
  • micron vaccine 6 has a particle size of 2.50 ⁇ m and a surface potential of -6mV.
  • Each 1 mg of PLGA is loaded with approximately 20 ⁇ g of mRNA components, each 1 mg of PLGA is loaded with 20 ⁇ g each of poly(I:C), CpG2395, and CpG1018 immune adjuvants, and 150 ⁇ g of KALA polypeptide; each 1 mg of PLGA is loaded with approximately 190 ⁇ g of membrane components.
  • PBS phosphate buffer solution
  • Micron Vaccine 7 The particle size of Micron Vaccine 7 is 2.50 ⁇ m, the surface potential is -10mV.
  • Each 1 mg of PLGA is loaded with approximately 20 ⁇ g of mRNA components, each 1 mg of PLGA is loaded with 20 ⁇ g of poly(I:C), CpG1585, and CpG2336 immune adjuvants, and 150 ⁇ g of KALA polypeptide; each 1 mg of PLGA is loaded with approximately 190 ⁇ g of membrane components.
  • PBS phosphate buffer solution
  • Each 1 mg of PLGA is loaded with approximately 20 ⁇ g of mRNA components, each 1 mg of PLGA is loaded with 20 ⁇ g of poly(I:C), 40 ⁇ g of CpG1018 immune adjuvant, and 150 ⁇ g of KALA polypeptide; each 1 mg of PLGA is loaded with approximately 190 ⁇ g of membrane components.
  • PBS phosphate buffer solution
  • Each 1 mg of PLGA is loaded with 20 ⁇ g each of poly(I:C), CpG2395, and CpG1018 immune adjuvants, and 150 ⁇ g of KALA polypeptide; each 1 mg of PLGA is loaded with approximately 190 ⁇ g of membrane components.
  • mice Female C57BL/6 mice aged 6-8 weeks were selected as model mice to prepare tumor-bearing mice. On days -35, -28, -21, -14, and -7 before mice were inoculated with tumors, the mice were injected with 100 ⁇ L PBS or 1 mg of Micron vaccine (Micron vaccine 5, Micron vaccine 6, or Micron vaccine 7), each mouse was subcutaneously inoculated with 5 ⁇ 10 5 E.G7-OVA cells on day 0. The methods for monitoring mouse tumor volume and survival were the same as above.
  • Microvaccine 6, Microvaccine 7, Microvaccine 8, Microvaccine 9 is the PBS control group, Microvaccine 6, Microvaccine 7, Microvaccine 8 and Microvaccine 9 in order.
  • the results showed that compared with the PBS control group, the tumor growth rate of mice treated with micron vaccine was significantly slower and the survival period of mice was significantly prolonged.
  • Micron Vaccine 6, Micron Vaccine 7 and Micron Vaccine 8 are all better than Micron Vaccine 9, indicating that loading nucleic acid inside the vaccine can significantly improve the efficacy of the vaccine.
  • Micron Vaccine 6 is more effective than Micron Vaccine 7 and Micron Vaccine 8, indicating that the nucleic acid delivery particles (vaccines) containing two types of CpG (Class B and C) and poly(I:C) mixed adjuvants are more effective than Micron Vaccine 7 and Micron Vaccine 8.
  • the effect of nucleic acid delivery micron particles (vaccine) containing two type A CpG and poly(I:C) mixed adjuvant is also better than that of nucleic acid delivery micron particles (vaccine) containing one type B type CpG and poly(I:C).
  • This example uses mouse colon cancer as a cancer model to illustrate how to use antigen-presenting cells activated by antigen-delivery nanoparticles loaded with colon cancer whole-cell antigens to prepare a nanovaccine by cooperating with nucleic acid-loaded nucleic acid delivery precursor nanoparticles.
  • Used to treat colon cancer In this example, 10% octylglucoside aqueous solution is first used to lyse colon cancer tumor tissue and dissolve the lysed components. Then, PLGA is used as the skeleton material, Poly(I:C), CpG2336 and CpG2006 are used as adjuvants, and NH is used. 4 HCO 3 is used to increase lysosomal escape substances, and antigen delivery nanoparticles are prepared.
  • the antigen delivery nanoparticles are used to activate the antigen-presenting cells, and then the antigen-presenting cells are combined with extracellular vesicles from the antigen-presenting cells and nucleic acid-loaded nucleic acids.
  • the nucleic acid delivery precursor nanoparticles work together to prepare a nanovaccine, and the nanovaccine is used to treat cancer.
  • Centrifuge Lactobacillus rhamnosus at 5000g for 30 minutes, then discard the precipitate and collect the supernatant. Filter the supernatant with a 1 ⁇ m filter, ultrasonicate at 4°C for 5 minutes at 20W, and then centrifuge at 16000g for 90 minutes. , resuspend the pellet in PBS to form the collected bacterial outer vesicle membrane components, and then use 10% octylglucoside aqueous solution to lyse and dissolve the bacterial outer vesicle membrane components.
  • the antigen delivery nanoparticles 1 are prepared by the double emulsion method.
  • Nanoparticle 1 is internally loaded with tumor tissue lysate components, bacterial extracellular vesicle components, mRNA, immune adjuvants and substances that increase lysosomal escape.
  • the preparation material of nanoparticles, PLGA has a molecular weight of 7KDa-17KDa.
  • the mRNA used is the mRNA encoding OVA.
  • Poly(I:C) and two CpGs are used as adjuvants, and KALA polypeptide (WEAKLAKALAKALAKHLAKALAKALKACEA) is used to increase lysosomal escape substances.
  • the adjuvant and KALA polypeptide are loaded into the nanoparticles; during preparation, the mass ratio of tumor tissue lysate components: bacterial outer vesicle components: mRNA is 4:4:1.
  • the preparation method is as mentioned above.
  • the nanoparticles are first loaded with tumor tissue lysate components, bacterial extracellular vesicle lysate components, increased lysosomal escape substances and adjuvants.
  • 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 260 nm, and each 1 mg of PLGA nanoparticles is loaded with approximately 90 ⁇ g.
  • 10 ⁇ g of mRNA encoding OVA is loaded, 0.02 mg each of poly(I:C), CpG2336, and CpG2006 are loaded per 1 mg of PLGA nanoparticles, and 0.12 mg of KALA polypeptide is loaded.
  • the mRNA loaded on the mRNA-loaded nanoparticles 2 in this embodiment can encode OVA antigen.
  • Nanoparticle 2 is also loaded with immune adjuvant poly(I:C), two types of B CpG and KALA polypeptide (WEAKLAKALAKALAKHLAKALAKALKACEA).
  • the preparation materials and preparation method of mRNA-loaded nanoparticles are the same as step (2). First, mix mRNA, poly(I:C), CpG7909 (Class B), CpG1018 (Class B) and KALA polypeptide in water, and then use double emulsion.
  • the above mixture is loaded inside the nanoparticles, and then 100mg PLGA nanoparticles are centrifuged at 9000g for 20 minutes, resuspended in 10mL of ultrapure water containing 4% trehalose and freeze-dried for 48h.
  • the average particle size of nanoparticle 2 is about 210nm.
  • Each 1 mg PLGA nanoparticle is loaded with approximately 20 ⁇ g of mRNA components.
  • Each 1 mg PLGA nanoparticle is loaded with 20 ⁇ g each of poly(I:C), CpG7909 and CpG1018 immune adjuvants, and 140 ⁇ g of KALA polypeptide.
  • the mRNA loaded on the mRNA-loaded nanoparticles 3 in this embodiment can encode OVA antigen.
  • Nanoparticle 3 is also loaded with immune adjuvant poly(I:C), two types of A CpG and KALA polypeptide (WEAKLAKALAKALAKHLAKALAKALKACEA).
  • the preparation materials and preparation method of mRNA-loaded nanoparticles are the same as step (2). First, mix mRNA, poly(I:C), CpG1585 (Class A), CpG2336 (Class A) and KALA polypeptide in water, and then use double emulsion.
  • the above mixture is loaded inside the nanoparticles, and then 100mg PLGA nanoparticles are centrifuged at 9000g for 20 minutes, resuspended in 10mL of ultrapure water containing 4% trehalose and freeze-dried for 48h.
  • the average particle size of nanoparticle 3 is about 210nm.
  • Each 1 mg PLGA nanoparticle is loaded with approximately 20 ⁇ g of mRNA components.
  • Each 1 mg PLGA nanoparticle is loaded with 20 ⁇ g each of poly(I:C), CpG1585 and CpG2336 immune adjuvants, and 140 ⁇ g of KALA polypeptide.
  • the mRNA loaded on the mRNA-loaded nanoparticles 4 in this embodiment can encode OVA antigen.
  • Nanoparticle 4 is loaded with immune adjuvant poly(I:C) and KALA polypeptide (WEAKLAKALAKALAKHLAKALAKALKACEA) at the same time.
  • the preparation materials and preparation method of mRNA-loaded nanoparticles are the same as step (2).
  • the mRNA loaded on the mRNA-loaded nanoparticles 5 in this embodiment can encode OVA antigen.
  • Nanoparticle 5 is simultaneously loaded with two types of immune adjuvants, Class B CpG and KALA polypeptide (WEAKLAKALAKALAKHLAKALAKALKACEA).
  • Preparation materials and materials for mRNA-loaded nanoparticles The preparation method is the same as step (2). First, mix mRNA, CpG7909 (Class B), CpG1018 (Class B) and KALA polypeptide in water, then use the double emulsion method to load the above mixture inside the nanoparticles, and then add 100mg PLGA nanoparticles.
  • Each 1 mg PLGA nanoparticle is loaded with approximately 20 ⁇ g of mRNA components.
  • Each 1 mg PLGA nanoparticle is loaded with 30 ⁇ g each of CpG7909 and CpG1018 immune adjuvants, and 140 ⁇ g of KALA polypeptide.
  • the mRNA loaded on the mRNA-loaded nanoparticles 6 in this embodiment can encode OVA antigen.
  • Nanoparticle 6 is also loaded with the immune adjuvant poly(I:C) and two types of B CpG.
  • the preparation materials and preparation method of mRNA-loaded nanoparticles are the same as step (2).
  • This example uses BMDC and B as antigen-presenting cells.
  • the preparation method of BMDC is the same as in Example 1.
  • B cells were derived from mouse peripheral blood PBMC, and the preparation method was the same as above.
  • the collected extracellular vesicles of activated antigen-presenting cells and cancer cell extracellular vesicles were mixed, then ultrasonicated at 4°C at low power (20W) for 2 minutes, and then repeatedly co-extruded using a 0.22 ⁇ m filter membrane.
  • nanoparticle 2 nanoparticle 3, or nanoparticle 4, or nanoparticle 5, or nanoparticle 6
  • step (3) Mix the extruded liquid with the nucleic acid delivery precursor nanoparticles (nanoparticle 2, nanoparticle 3, or nanoparticle 4, or nanoparticle 5, or nanoparticle 6) prepared in step (3) and then use a high-pressure homogenizer (10000 bar) Treat for 1 minute, then use 0.22 ⁇ m filter membrane to repeatedly co-extrude, then centrifuge at 15000g for 30 minutes, discard the supernatant to collect the precipitate, resuspend the precipitate in 4% trehalose aqueous solution and freeze-dry for 48 hours. Get the nano vaccine.
  • a high-pressure homogenizer 10000 bar
  • the nanovaccine prepared using the antigen-presenting extracellular vesicle membrane component activated by nanoparticle 1 and nanoparticle 2 is nanovaccine 7, with a particle size of 220 nanometers and a surface potential of -6mV; per 1 mg of PLGA is approximately Load 20 ⁇ g of mRNA component, and load poly(I:C), CpG7909 and 20 ⁇ g each of CpG1018 immune adjuvant, loaded with 140 ⁇ g of KALA peptide; approximately 120 ⁇ g of membrane component per 1 mg of PLGA.
  • the nanovaccine prepared using the antigen-presenting extracellular vesicle membrane component activated by nanoparticle 1 and nanoparticle 3 is nanovaccine 8, with a particle size of 220 nm and a surface potential of -6mV; approximately 20 ⁇ g is loaded per 1 mg of PLGA.
  • each 1 mg of PLGA is loaded with 20 ⁇ g each of poly(I:C), CpG1585, and CpG2336 immune adjuvants, and 140 ⁇ g of KALA polypeptide; each 1 mg of PLGA is loaded with approximately 120 ⁇ g of membrane components.
  • the nanovaccine prepared by using the antigen-presenting extracellular vesicle membrane component activated by nanoparticle 1 and nanoparticle 4 is nanovaccine 9, with a particle size of 220 nm and a surface potential of -6mV; approximately 20 ⁇ g is loaded per 1 mg of PLGA.
  • each 1 mg PLGA is loaded with 60 ⁇ g of ppoly(I:C) and 140 ⁇ g of KALA polypeptide; each 1 mg of PLGA is loaded with approximately 120 ⁇ g of membrane components.
  • the nanovaccine prepared using the antigen-presenting extracellular vesicle membrane component activated by nanoparticle 1 and nanoparticle 5 is nanovaccine 10, with a particle size of 220 nm and a surface potential of -6mV; approximately 20 ⁇ g is loaded per 1 mg of PLGA.
  • each 1 mg of PLGA is loaded with 30 ⁇ g of CpG7909 and CpG1018 immune adjuvants, and 140 ⁇ g of KALA peptide; each 1 mg of PLGA is loaded with approximately 120 ⁇ g of membrane components.
  • the nanovaccine prepared using the antigen-presenting extracellular vesicle membrane component activated by nanoparticle 1 and nanoparticle 6 is nanovaccine 11, with a particle size of 220 nm and a surface potential of -6mV; approximately 20 ⁇ g is loaded per 1 mg of PLGA.
  • each 1 mg of PLGA is loaded with 20 ⁇ g each of poly(I:C), CpG7909, and CpG1018 immune adjuvants, but not with KALA polypeptide; each 1 mg of PLGA is loaded with approximately 120 ⁇ g of membrane components.
  • Nano vaccines are used to treat cancer
  • PBS control, Nanovaccine 7, Nanovaccine 8, Nanovaccine 9, Nanovaccine 10, and Nanovaccine 11 in Figure 14 are the PBS control group, Nanovaccine 7, Nanovaccine 8, Nanovaccine 9, Nanovaccine 10, and Nanovaccine in order 11.
  • the results showed that compared with the PBS control group, the tumor growth rate of mice treated with nanovaccine was significantly slower and the survival period of mice was significantly prolonged.
  • the therapeutic effect of Nano Vaccine 7 is better than that of Nano Vaccine 8, Nano Vaccine 9, Nano Vaccine 10 and Nano Vaccine 11. It can be seen that the nanovaccine described in the present disclosure has excellent therapeutic effect on cancer.
  • No target is used in the nanoparticles, microparticles, nanovaccines or microvaccines in the embodiments of the present disclosure.
  • mannose, CD32 antibodies, mannans, CD205 antibodies, CD19 antibodies, etc. can also be added as needed.
  • Any target with the ability to target target cells is attached to the surface of nanoparticles, microparticles, nano-vaccines or micro-vaccines.
  • calcification, silicide and other technologies are not used to treat nanoparticles or microparticles.
  • calcification, silicide or other biomineralization technologies, cross-linking, gelation, etc. can be used to treat or modify particles.
  • the nucleic acid delivered in the embodiments of the present disclosure is mRNA, but in practical applications it can also be DNA or other RNA.
  • amino acid sequences involved in this disclosure are as follows:
  • KALA peptide WEAKLAKALAKALAKHLAKALAKALKACEA (SEQ ID NO:7)
  • RALA peptide WEARLARALARALARHLARALARALRACEA (SEQ ID NO:8)

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Abstract

一种基于激活的抗原提呈细胞膜组分的核酸递送粒子、核酸递送系统及其制备方法及应用，使用负载全细胞组分的纳米粒子或微米粒子激活抗原提呈细胞后将抗原提呈细胞的膜组分负载于已经内部负载mRNA的核酸递送前体粒子后，得到所述核酸递送粒子。所述核酸递送粒子克服了mRNA递送过程中面临的无法在4摄氏度度或室温下长期储存、递送效率低等问题，能够高效的将mRNA递送到抗原提呈细胞并激活癌细胞特异性免疫反应，可用于癌症等疾病的预防和治疗。

Description

基于激活的抗原提呈细胞的核酸递送粒子、核酸递送系统及制备方法 技术领域
本公开涉及免疫治疗领域，尤其涉及一种基于激活的抗原提呈细胞的核酸递送粒子、核酸递送系统及制备方法。
背景技术
信使RNA(Messenger RNA，mRNA)是由DNA的一条链作为模板转录而来的、携带遗传信息能指导蛋白质合成的一类单链核糖核酸。理论上mRNA可以指导合成任意编码的蛋白质，因而具有广泛的应用潜力。目前对mRNA的研究主要集中在疫苗接种、蛋白质替代疗法和遗传疾病的治疗上。而且，mRNA疫苗目前已被应用于预防COVID-19。RNA生物学、化学、稳定性和递送系统的技术进步加速了mRNA疫苗的开发并且在动物模型中观察到有效、持久和安全的免疫反应。相比于传统疫苗，mRNA疫苗能够同时激活机体细胞免疫和体液免疫，产生较强的免疫反应。然而，mRNA的胞内递送较寡核苷酸困难，因为mRNA分子量较大且极其不稳定，容易被RNase降解，因此合适的递送载体就极为关键，其不仅能保护mRNA也有利于mRNA成功进入胞内发挥作用。目前研究用于递送mRNA的手段主要有RNA共聚物、修饰的RNA、病毒递送载体、聚合物递送载体、脂质递送载体等，其中发展最成熟的是脂质纳米颗粒(Lipid nanoparticle,LNP)。但是，LNP递送mRAN也存在一些问题，比如无法有效制备稳定的冻干制剂，因而只能在-70℃或者-20℃储存几个月时间，因而需要的运输和储存条件比较苛刻。而且，LNP表面带正电荷，注射进入人体后容易吸附蛋白质和细胞，因而具有一定的毒副作用，而且，PEG的使用使得其会在一定程度引起过敏反应。而且，LNP递送的mRNA体内的表达效率和疗效也有待进一步提升。因而，目前亟需开发新型递送技术以更好实现mRNA等核酸药物的递送。
发明内容
为解决上述技术问题，本公开提供了一种使用负载抗原组分的纳米粒子(NP)或微米粒子(MP)激活过的抗原提呈细胞制备的核酸递送粒子、包含核酸递送粒子的核酸递送系统及其制备方法。本公开将核酸负载于纳米粒子或微米粒子内部，然后使用来源于激活的抗原提呈细胞和/或其分泌的细胞外囊泡的生物膜组分负载于纳米粒子或微米粒子表面，制备得到核酸递送粒子。本公开中核酸递送粒子的表面柔性有效提高，并解决了核酸递送时粒子归巢淋巴结、粒子靶向抗原提呈细胞以及粒子进入细胞后的溶酶体逃逸等问题，提高了递送系统递送核酸药物的效率，增加了核酸药物的功效。
本公开的第一个目的是提供一种核酸递送系统，其具有由粒子材料形成的骨架结构，所述骨架结构的内部负载有核酸，所述骨架结构的表面负载有生物膜组分；
其中，所述生物膜组分含有来源于激活的抗原提呈细胞和/或由所述激活的抗原提呈细胞分泌的细胞外囊泡；所述激活的抗原提呈细胞由抗原提呈细胞与抗原递送粒子共作用后得到，所述抗原递送粒子具有由粒子材料形成的骨架结构，以及负载于所述骨架结构的内部和/或表面负载有抗原组分。
在一些实施方案中，所述核酸递送粒子和所述抗原递送粒子彼此独立地选自纳米粒子或微米粒子。核酸递送粒子或抗原递送粒子的粒径大小为纳米级或微米级，这样能保证粒子被抗原提呈细胞吞噬。
进一步地，纳米粒子的粒径大小为1nm-1000nm，更优选地，粒径大小为30nm-1000nm，更优选地，粒径大小为50nm-600nm；更优选地，粒径大小为50-500nm；更优选地，粒径大小为100-400nm。示例性地，纳米粒子的粒径为10nm、50nm、100nm、200nm、210nm、220nm、230nm、240nm、250nm、260nm、270nm、280nm、290nm、300nm、310nm、320nm、330nm、340nm、350nm、400nm、500nm等等。
进一步地，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，更优选地，粒径大小为1μm-5μm，更优选为1-10μm；更优选为1-2μm。示例性地，微米粒子的粒径为1μm、1.1μm、1.2μm、1.3μm、1.4μm、1.5μm、1.6μm、1.7μm、1.8μm、1.9μm、2μm、2.5μm、3μm、5μm、10μm等等。
在一些实施方案中，核酸递送粒子表面带有负电荷。
本公开所述核酸包括DNA和RNA。
本公开所述RNA包括但不限于mRNA、SiRNA、lcncRNA等。
核酸递送粒子的表面电位为-150mV到-1mv。更优选为-50mv到-1mv；更优选为-20mv到-1mv。
在一些实施方案中，本公开所述抗原组分包含如下的至少一种：
(i)具有免疫原性的蛋白和/或多肽；
(ii)编码抗原的核酸。
在一些实施方案中，所述具有免疫原性的蛋白和/或多肽来源于如下(1)，和任选存在的(2)-(3)中的至少一种；
(1)全细胞裂解物；其中，所述全细胞裂解物组分来源于肿瘤组织和/或肿瘤细胞；
(2)细菌裂解物；
(3)细胞外囊泡裂解物；其中，所述细胞外囊泡由细菌或肿瘤细胞分泌。
进一步地，所述全细胞裂解物包括如下的一种或两种：水溶性抗原的裂解物和非水溶性抗原的溶解物。在一些优选的实施方案中，所述全细胞裂解物中，所述非水溶性抗原的溶解物与所述水溶性抗原的裂解物混合的质量比为(0.1-10)：(0.1-10)；优选为(0.5-2)：(0.5-2)。示例性地，非水溶性抗原的溶解物与所述水溶性抗原混合的质量比为1:1、0.5:1、0.8:1、1:1.2、1:1.5、1:2、2:1、3:1、4:1、5:1、1:3、1:4、1:5等等。
在一些实施方案中，具有免疫原性的蛋白和/或多肽来源于全细胞裂解物和细胞外囊泡 裂解物。进一步地，所述细胞外囊泡裂解物选自癌细胞的细胞外囊泡裂解物和/或细菌的细胞外囊泡裂解物。所述全细胞裂解物与所述细胞外囊泡裂解物的质量比为(0.1-10)：(0.1-10)；优选为(0.5-2)：(0.5-2)。示例性地，所述全细胞裂解物与所述细胞外囊泡裂解物的质量比为1:1、0.5:1、0.8:1、1:1.2、1:1.5、1:2、2:1、3:1、4:1、5:1、1:3、1:4、1:5等等。
在一些实施方案中，具有免疫原性的蛋白和/或多肽来源于全细胞裂解物和细菌裂解物。进一步地，所述全细胞裂解物与所述细菌裂解物的质量比为(0.1-10)：(0.1-10)；优选为(0.5-2)：(0.5-2)。示例性地，所述全细胞裂解物与所述细菌裂解物的质量比为1:1、0.5:1、0.8:1、1:1.2、1:1.5、1:2、2:1、3:1、4:1、5:1、1:3、1:4、1:5等等。
在一些实施方案中，本公开所述抗原递送粒子还负载如下至少一种：
(iii)免疫佐剂；
(iv)带正电荷的物质，其选自带正电荷的氨基酸、带正电荷的多肽、带正电荷的脂质、带正电荷的蛋白质、带正电荷的聚合物，和/或带正电荷的无机物。
进一步的，所述核酸递送粒子由核酸递送前体粒子与生物膜组分共作用得到；
进一步地，所述核酸递送前体粒子和/或所述核酸递送粒子还负载如下至少一种：
(iii)免疫佐剂；
(iv)带正电荷的物质，其选自带正电荷的氨基酸、带正电荷的多肽、带正电荷的脂质、带正电荷的蛋白质、带正电荷的聚合物，和/或带正电荷的无机物。
在一些实施方案中，所述核酸递送粒子中，所述粒子材料、核酸与生物膜组分的质量比(mg:μg:μg)为1：(1-100)：(10-300)。
进一步地，所述核酸递送粒子进一步包括免疫佐剂和带电荷的物质，所述粒子材料、核酸、免疫佐剂、带正电荷的物质与所述生物膜组分的质量比(mg:μg:μg:μg:μg)为1：(1-100)：(1-200)：(10-500)：(10-300)。
在一些实施方案中，核酸递送粒子负载的生物膜组分进一步含有至少一种如下所示的组分：
(a)来源于全细胞裂解物的癌细胞膜组分；其中，所述全细胞裂解物组分来源于肿瘤组织和/或肿瘤细胞；
(b)来源与细胞外囊泡裂解物的细胞外囊泡膜组分，所述细胞外囊泡由细菌或肿瘤细胞分泌；
(c)来源于细菌裂解物的细菌膜组分。
在本公开中，细菌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、嗜酸乳杆菌、格式乳杆菌、罗伊氏乳杆菌、鼠李糖乳杆菌等。
在本公开中，免疫增强佐剂包括但不限于模式识别受体激动剂、卡介苗、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、STING激动剂、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、植物油、内毒素佐剂、脂质体佐剂、MF59、双链RNA、双链DNA、单链DNA、CAF01、人参有效成分、黄芪有效成分等。
进一步地，免疫增强佐剂选自两种或两种以上Toll样受体激动剂。示例性地，免疫佐剂包括Toll样受体3激动剂和Toll样受体9激动剂中的至少一种。
进一步的，免疫增强佐剂包括(1)Poly(I:C)和/或Poly(ICLC)；(2)CpG-ODN；其中，CpG-ODN为A类CpG-ODN、B类CpG-ODN和C类CpG-ODN中的至少一种；优选为至少两种，且至少其中一种为B类CpG-ODN或C类CpG-ODN。
本公开的上下文中，“CpG”或“CpG-ODN”(CpG oligonucleotide,CpG寡脱氧核苷酸)，是合成的含有非甲基化的胞嘧啶鸟嘌呤二核苷酸(CpG)的寡聚脱氧核苷酸(ODN)。不同类型的CpG-ODN的结构特征与免疫效应各有不同，一般分为A、B、C三类。
在一些实施方案中，CpG-ODN包括但不限于：CpG 1018(B类)、CpG 7909(B类)、CpG 2006(B类)、CpG-BW006(B类)、CpG 2395(C类)、CpG SL01、CpG 1585(A类)、CpG 2216(A类)、CpG SL03、CpG 2395(C类)、CpG M362(C类)、CpG 2336(A类)。
在本公开中，所述带正电荷的物质包括但不限于如下至少一种：带正电荷的氨基酸、带正电荷的多肽、带正电荷的脂质、带正电荷的蛋白质、带正电荷的高分子聚合物、带正电荷的无机物。示例性地，带正电荷的多肽包括但不限于含有精氨酸的多肽、含有组氨酸的多肽和/或组氨酸和/或赖氨酸的KALA多肽、RALA多肽、蜂毒肽等。
示例性地，带正电荷的氨基酸包括但不限于精氨酸、组氨酸、赖氨酸等。
示例性地，带正电荷的高分子聚合物包括但不限于聚精氨酸、聚赖氨酸、聚组氨酸等。
示例性地，带正电荷的脂质包括但不限于DOTAP等。
示例性地，带正电荷的蛋白质包括但不限于鱼精蛋白、组蛋白等。
示例性地，带正电荷的无机物包括但不限于NH4HCO3、氢氧化铝等。
在一些实施方案中，所述带正电荷的物质包括蜂毒肽、RALA多肽、KALA多肽、R8多肽、精氨酸、组氨酸、赖氨酸、聚精氨酸、聚赖氨酸、聚组氨酸和NH4HCO3中的任意一种或任意组合。
在一些实施方案中，抗原递送粒子和/或核酸递送粒子还负载有靶分子。所述靶分子包括如下至少一种：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。
在本公开中，所述非水溶性抗原、所述细菌裂解物或所述细胞外囊泡裂解物彼此独立地溶解于包含如下至少一种溶解剂的的溶解液中：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、Triton、吐温、氨基酸、糖苷和胆碱；更优选地，所述溶质包括如下至少一种：尿素、脱氧胆酸钠、辛基葡萄糖苷和精氨酸。
在本公开中，所述细菌裂解物和/或细胞外囊泡裂解物经含有裂解剂的裂解液裂解细菌和/或细胞外囊泡得到；所述裂解剂选自尿素、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、Triton、吐温、多肽、氨基酸、糖苷和胆碱的水溶液中的一种或多种。
在本公开中，所述抗原提呈细胞包括B细胞、树突状细胞(DC)和巨噬细胞中的至少一种，优选为两种及以上，更优选为三种细胞的组合。
在本公开中，形成所述核酸递送粒子的粒子材料由天然高分子材料和/或合成高分子材料形成。
在本公开中，形成所述抗原递送粒子由天然高分子材料和/或合成高分子材料形成。
示例性地，有机合成高分子材料包括但不限于PLGA、PLA、PGA、PEG、PCL、Poloxamer、PVA、PVP、PEI、PTMC、聚酸酐、PDON、PPDO、PMMA、聚氨基酸、合成多肽等。
示例性地，天然高分子材料包括但不限于卵磷脂、胆固醇、海藻酸盐、白蛋白、胶原蛋白、明胶、细胞膜成分、淀粉、糖类、多肽等。
示例性地，无机材料包括但不限于三氧化二铁、四氧化三铁、碳酸盐、磷酸盐等。
本公开所述的核酸递送粒子、核酸递送前体粒子和抗原递送粒子的形状为常见的任意形状，包括但不限于球形、椭球形、桶形、多角形、棒状、片状、线形、蠕虫形、方形、三角形、蝶形、圆盘形、囊泡形等。
在本公开中，所述激活的抗原提呈细胞由抗原提呈细胞与抗原递送粒子共孵育得到；其中，抗原递送粒子的表面和/或内部负载有抗原组分；其中，所述抗原递送粒子负载癌细胞和/或肿瘤组织的全细胞抗原组分和/或核酸；其中，全细胞抗原组分包含水溶性抗原组分和非水溶性抗原组分。
在本公开，所述抗原提呈细胞与抗原递送粒子的共孵育体系中可以包含细胞因子和/或抗体。
示例性地，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)、粒细胞－巨噬细胞集落刺激因子(GM-CSF)、白介素17(IL-17)、IL-12、白介素12(IL-12)、白介素6(IL-6)、巨噬细胞集落刺激因子(M-CSF)、白介素33(IL-33)、γ干扰素(IFN-γ)、TNF-α。
示例性地，抗体包括但不限于CD80抗体、CD86抗体、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。
在本公开中，所述孵育体系中还包括如下至少一种：
(1)全细胞裂解物；其中，所述全细胞裂解物组分来源于肿瘤组织和/或肿瘤细胞；
(2)细菌裂解物；
(3)细胞外囊泡裂解物；其中，所述细胞外囊泡由细菌或肿瘤细胞分泌。
本公开中，将核酸负载于纳米粒子或微米粒子中，同时使用负载抗原组分的抗原递送粒子先特异性激活抗原提呈细胞，再将来源于激活的抗原提呈细胞和/或由其分泌的细胞外 囊泡的生物膜组分负载于纳米粒子或微米粒子的表面，形成内部负载核酸、表面负载生物膜组分的核酸递送粒子。本公开提供的核酸递送例子可以有效归巢淋巴结，靶向抗原提呈细胞，能够提高核酸药物的递送效率及药物功效。核酸递送粒子在冷冻干燥后可在4℃下长期保存，不影响起药物功效。
本公开的第二个目的在于提供一种核酸递送系统，其包括本公开提供的核酸递送粒子。进一步地，核酸递送系统包含的核酸递送粒子可以相同或不同，能够实现对一种或多种核酸药物的体内递送，具有递送效率高、安全性高等优势，可发挥显著的药物预防或者治疗功效。
本公开的第三个目的在于提供了一种药物组合物，其包括本公开提供的核酸递送粒子或核酸递送系统。
在一些实施方案中，药物组合物还包括一种或多种药学上可接受的载体。
本公开提供的药物组合物，可通过核酸递送粒子或核酸递送系统实现高效的核酸递送，发挥显著的疾病预防或治疗效果。
本公开的第四个目的在于提供一种核酸疫苗，其包括本公开提供的核酸递送粒子或核酸递送系统。
本公开中的核酸递送粒子或核酸递送系统可以靶向抗原提呈细胞，有效激活机体的免疫应答反应，高效发挥核酸疫苗的预防或治疗效果。
本公开的第五个目的在于提供上述核酸递送粒子、核酸递送系统或药物组合物在如下(1)-(3)至少一项中的用途：
(1)预防或治疗疾病，或制备用于预防或治疗疾病的药物；
(2)在受试者中诱导免疫应答，或制备用于在受试者中诱导免疫应答的药物；
(3)作为或用于制备核酸疫苗。
在一些实施方案中，所述疾病为癌症或肿瘤；
在一些实施方案中，所述癌症或肿瘤为实体肿瘤或者血液瘤，所述实体肿瘤或血液瘤包括但不限于鳞状细胞癌、骨髓瘤、小细胞肺癌、非小细胞肺癌、神经胶质瘤、肝细胞癌(HCC)、霍奇金淋巴瘤、非霍奇金淋巴瘤、T细胞淋巴瘤、急性髓性白血病(AML)、多种骨髓瘤、胃肠(道)癌、肾癌、卵巢癌、肝脏癌、淋巴母细胞白血病、淋巴细胞白血病、结肠癌、直肠癌、子宫内膜癌、肾癌、前列腺癌、甲状腺癌、黑色素瘤、软骨肉瘤、神经母细胞瘤、胰腺癌、多形性胶质母细胞瘤、宫颈癌、脑癌、胃癌、膀胱癌、肝癌、乳腺癌、结肠癌和头颈癌。
本公开中，用于形成生物膜组分的抗原提呈细胞，与抗原组分来源于受试者、细胞系或由干细胞转化形成。进一步地，抗原提呈细胞与抗原组分来源的受试者为同一个体或同种异体。
本公开中，抗原组分来源于所述疾病相关的细胞或组织。进一步地，抗原组分来源于癌细胞和/或肿瘤组织的全细胞抗原组分。
本公开的第六个目的在于提供上述核酸递送粒子的制备方法，其包括如下步骤：
S1，将抗原提呈细胞与抗原递送粒子共孵育，得到激活的抗原提呈细胞；其中，抗原递送粒子的表面和/或内部负载有抗原组分；
S2，制备内部负载核酸的核酸递送前体粒子；
S3，将生物膜组分负载于所述核酸递送前体粒子的表面，得到核酸递送粒子；其中，所述生物膜组分来源于所述激活的抗原提呈细胞，或来源于由所述激活的抗原提呈细胞分泌的细胞外囊泡。
在一些实施方案中，所述将生物膜组分负载于所述核酸递送前体粒子的表面包括：
S31，将激活的抗原提呈细胞经过机械破坏，收集生物膜组分；
S32，将所述收集生物膜组分与核酸递送前体粒子共作用，得到核酸递送粒子。
进一步地，在步骤S31中，将激活的抗原提呈细胞经过机械破坏，经膜过滤或梯度离心，收集生物膜组分。
示例性地，所述机械破坏包括但不限于超声、均质化、挤出、匀浆、高速搅拌、高压破坏、高剪切力破坏、溶胀、化学物质、皱缩中的一种或多种。
示例性地，所述共作用包括但不限于共孵育、共挤出、超声、搅拌、透析、超滤、均质化和匀浆中的一种或多种。
在一些实施方案中，所述抗原递送粒子的制备步骤包括：
分别制备负载水溶性抗原的递送粒子，以及负载非水溶性抗原的溶解物的递送粒子，混合两种递送粒子，得到所述抗原递送粒子；或者，混合所述水溶性抗原和非水溶性抗原的溶解物，得到混合后的抗原组分；利用所述混合后的抗原组分，制备所述抗原递送粒子；或者，使用含有溶解剂的溶解液同时溶解水溶性抗原和非水溶性抗原，并利用所述混合抗原组分制备所述抗原递送粒子。
在一些实施方案中，所述抗原组分的制备步骤包括：
S11，在水或不含溶解剂的溶解液中裂解目标细胞和/或目标组织，离心；得到的上清液为水溶性抗原组分，沉淀为非水溶性抗原组分；
S12，将所述沉淀溶解于含有溶解剂的溶解液中，然后与水溶性抗原组分合并，得到来源于目标细胞和/或目标组织的抗原组分。
示例性地，目标细胞为癌细胞，目标组织为肿瘤组织，癌细胞为一种或一种以上，肿瘤组织为一种或者一种以上。
进一步地，所述在水或不含溶解剂的溶解液中裂解目标细胞和/或目标组织的步骤包括：将目前细胞和/或目标组织在-20℃～-273℃下冷冻，加水或不含溶解剂的溶液后进行反复冻融裂解。
在一些实施方案中，所述抗原组分的制备步骤包括：使用含有溶解剂的溶解液裂解并同时溶解目标细胞和/或目标组织织中的水溶性组分和非水溶性组分，得到抗原组分。
示例性地，目标细胞为癌细胞，目标组织为肿瘤组织，癌细胞为一种或一种以上，肿 瘤组织为一种或者一种以上。
示例性地，溶解剂包括但不限于尿素、盐酸胍、脱氧胆酸盐、十二烷基硫酸盐(如SDS)、甘油、蛋白质降解酶、白蛋白、卵磷脂、无机盐(0.1-2000mg/mL)、Triton、吐温、多肽、氨基酸、糖苷、胆碱中的至少一种。
本公开突破现有核酸递送技术的限制，使粒子上负载核酸药物的同时负载有细胞全细胞抗原表位和/或核酸所表达的蛋白质或多肽的抗原表位，以及来源于激活的抗原提呈细胞的生物膜组分或其分泌的细胞外囊泡的生物膜组分，能够更有效的发挥核酸药物的功能。
借由上述方案，本公开至少具有以下优点：
mRNA等核酸药物目前主要使用脂质纳米粒(LNP)技术递送，但是LNP为带正电荷的纳米粒子，极易吸附带负电的蛋白质或者细胞，因而细胞毒性较大。而且，PEG等物质的引入使得LNP易引起过敏反应。本公开提供了一种使用纳米级或微米级粒子递送核酸药物的技术，既能高效负载核酸药物，也能归巢淋巴结和靶向淋巴结内的抗原提呈细胞，从而在降低毒副作用的同时提高了核酸药物的疗效，本公开中的核酸递送粒子可以用于预防和治疗癌症等疾病的核酸药物的递送。而且，本公开所述核酸递送粒子表面带负电，所以降低了递送系统吸附带负电的蛋白质或多肽所引发的毒副作用。而且，本公开所述的核酸递送粒子可以冷冻干燥后长期储存备用，克服了LNP目前无法冷冻干燥以及长期储存的问题。
上述说明仅是本公开技术方案的概述，为了能够更清楚了解本公开的技术手段，并可依照说明书的内容予以实施，以下以本公开的较佳实施例并配合详细附图说明如后。
附图说明
为了使本公开的内容更容易被清楚的理解，下面根据本公开的具体实施例并结合附图，对本公开作进一步详细的说明。
图1为本公开中核酸递送粒子的制备过程及应用示意图；其中，a为收集水溶性抗原组分和非水溶性抗原组分与制备抗原递送粒子的过程示意图；b为采用含有溶解剂的溶解解液直接裂解后溶解癌细胞全细胞抗原和制备抗原递送粒子的过程示意图；c为使用a或b中制备的抗原递送粒子激活抗原提呈细胞，并将来源于激活的抗原提呈细胞生物膜组分或其分泌的细胞外囊泡的生物膜组分与与负载核酸的核酸递送前体粒子共作用后制备得到纳米疫苗或微米疫苗用于预防或治疗癌症的示意图。
图2为实施例1中不同粒子递送体系递送核酸后所递送的核酸表达情况的分析的实验结果。
图3-14分别为实施例2-13中使用核酸疫苗(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3-14中，a为预防或治疗癌症时的肿瘤生长速度实验结果(n≥8)；b为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)；图3c和d为使用流式细胞术分析共孵育后小鼠脾细胞中CD8+IFN-γ+T细胞占CD8+T细胞的比例以及CD4+IFN-γ+T细胞占CD4+T细胞的比例； 其中
图3-12中，a图中肿瘤生长抑制实验的显著性差异采用ANOVA法分析，b图中显著性差异采用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PBS空白对照组相比p＜0.01，有显著性差异；ΔΔΔ表示与负载核酸的LNP疫苗处理组相比p＜0.005，有显著性差异；εε代表与使用特定细胞因子组分加入抗原递送粒子(纳米粒/微米粒)激活抗原提呈细胞共孵育体系，并将其生物膜组分与核酸递送前体粒子(纳米粒/微米粒)共作用后所制备的核酸递送粒子(纳米粒/微米粒)相比p＜0.01，有显著性差异；ε表示与使用特定细胞因子组分加入抗原递送粒子(纳米粒/微米粒)激活抗原提呈细胞共孵育体系，并将其生物膜组分与核酸递送前体粒子(纳米粒/微米粒)共作用后所制备的核酸递送粒子(纳米粒/微米粒)相比p＜0.05，有显著性差异；###表示单独孵育而无任何抗原递送粒子(纳米粒/微米粒)激活的抗原提呈细胞的膜组分与核酸递送前体粒子(纳米粒/微米粒)共作用后所制备的核酸递送粒子(纳米粒/微米粒)相比p＜0.005，有显著性差异；τ代表与抗原递送粒子(纳米粒/微米粒)激活的DC与核酸递送前体粒子(纳米粒/微米粒)共作用后所制备的纳米疫苗/微米疫苗组相比p＜0.05，有显著性差异；&&&表示只是内部负载核酸而表面不负载膜组分的纳米粒子和/或微米粒子对照组相比p＜0.005，有显著性差异；&&表示与只是内部负载核酸而表面不负载膜组分的纳米粒子和/或微米粒子对照组相比p＜0.01，有显著性差异；δδδ表示与空白对照纳米粒子/微米粒子+游离裂解液激活的抗原提呈细胞的生物膜组分与核酸递送前体粒子共作用后所制备的纳米疫苗/微米疫苗组相比p＜0.005，有显著性差异；δ表示与空白对照纳米粒子/微米粒子+游离裂解液激活的抗原提呈细胞的膜组分与核酸递送前体粒子共作用后所制备的纳米疫苗/微米疫苗组相比p＜0.05，有显著性差异；ωωω代表与多肽抗原递送粒子粒激活的抗原提呈细胞的膜组分与核酸递送前体粒子共作用后所制备的纳米疫苗/微米疫苗组相比p＜0.005，有显著性差异；ωω代表与多肽抗原递送粒子激活的抗原提呈细胞的膜组分与核酸递送前体粒子共作用后所制备的纳米疫苗/微米疫苗组相比p＜0.01，有显著性差异；ω代表与多肽抗原递送粒子激活的抗原提呈细胞的膜组分与核酸递送前体粒子共作用后所制备的纳米疫苗/微米疫苗组相比p＜0.05，有显著性差异；χ代表与负载癌细胞全细胞组分和吐温80裂解和溶解的细菌组分的抗原递送粒子的抗原提呈细胞的膜组分与核酸递送前体粒子共作用后所制备的核酸疫苗组相比p＜0.05，有显著性差异；π代表与抗原递送粒子激活的抗原提呈细胞的膜组分与核酸递送前体粒子共作用后所制备的纳米疫苗/微米疫苗组相比p＜0.05，有显著性差异；ππ代表与抗原递送粒子激活的抗原提呈细胞的膜组分与核酸递送前体粒子共作用后所制备的纳米疫苗/微米疫苗组相比p＜0.01，有显著性差异；θ代表与使用两种A类CpG和Poly ICLC/Poly(I:C)作为混合佐剂的抗原递送粒子激活的抗原提呈细胞与核酸递送前体粒子共作用所制备的纳米疫苗/微米疫苗组相比p＜0.05，有显著性差异；γγγ代表与使用A类CpG和B类CpG作为混合佐剂的抗原粒子递送激活的抗原提呈细胞的膜组分与核酸递送前体粒子共作用所制备的纳米疫苗/微米疫苗组相比p ＜0.005，有显著性差异；ΩΩ代表与只使用Poly(I:C)作为佐剂的抗原递送粒子激活的抗原提呈细胞的膜组分与核酸递送前体粒子共作用所制备的纳米疫苗/微米疫苗组相比p＜0.01，有显著性差异；Ο代表与使用两种A类CpG和Poly ICLC/Poly(I:C)作为混合佐剂的抗原递送粒子激活的抗原提呈细胞与核酸递送前体粒子共作用所制备的纳米疫苗/微米疫苗在室温储存12个月后相比p＜0.05，有显著性差异；κ代表与抗原递送粒子激活的DC与核酸递送前体粒子共作用后所制备的纳米疫苗/微米疫苗在室温储存12个月后相比p＜0.05，有显著性差异；β代表与不负载溶酶体逃逸物质的抗原递送粒子激活的抗原提呈细胞的膜组分与核酸递送前体粒子共作用后制备的纳米疫苗/微米疫苗相比p＜0.05，有显著性差异；ξ代表与只负载一种CpG+Poly(I:C)混合佐剂的抗原递送粒子激活的抗原提呈细胞的膜组分与核酸递送前体粒子共作用后制备的核酸疫苗相比p＜0.05，有显著性差异；μμμ代表与负载抗原不负载佐剂的抗原递送粒子激活的抗原提呈细胞的膜组分与核酸递送前体粒子共作用后制备的纳米疫苗/微米疫苗相比p＜0.005，有显著性差异；ρ代表与负载癌细胞全细胞组分的抗原递送粒子激活的抗原提呈细胞的膜组分与核酸递送前体粒子共作用后制备的纳米疫苗/微米疫苗相比p＜0.05，有显著性差异；λλ代表与只负载两类CpG作为佐剂的抗原递送粒子激活的抗原提呈细胞的膜组分与核酸递送前体粒子共作用后制备的纳米疫苗/微米疫苗相比p＜0.01，有显著性差异；ΔΔΔ代表与负载负载mRNA的脂质纳米颗粒(LNP)疫苗相比p＜0.005，有显著性差异；η代表与表面负载生物膜组分同时内部负载核酸与两种A类CpG和polyIC混合佐剂的疫苗相比p＜0.05，有显著性差异；代表与表面负载生物膜组分同时内部负载核酸与一种B类CpG和polyIC混合佐剂的疫苗相比p＜0.005，有显著性差异；∞代表与表面负载生物膜组分同时内部负载核酸与两种A类CpG、polyIC混合佐剂以及阳离子物质的疫苗相比p＜0.05，有显著性差异；代表与表面负载生物膜组分同时内部负载核酸与polyIC和阳离子物质的疫苗相比p＜0.05，有显著性差异；∝代表与表面负载生物膜组分同时内部负载核酸与两种B类CpG和阳离子物质的疫苗相比p＜0.05，有显著性差异；代表与表面负载生物膜组分同时内部负载核酸与两种B类CpG和polyIC混合佐剂的疫苗相比p＜0.05，有显著性差异；■代表与表面负载被抗原递送粒子激活的细胞的生物膜组分，同时内部只负载佐剂和多肽却不负载核酸组分(除佐剂外)的疫苗组相比p＜0.05；■■代表与表面负载被抗原递送粒子激活的细胞的生物膜组分，同时内部只负载佐剂和多肽却不负载核酸组分(除佐剂外)的疫苗组相比p＜0.01；■■■代表与表面负载被抗原递送粒子激活的细胞的生物膜组分，同时内部只负载佐剂和多肽却不负载核酸组分(除佐剂外)的疫苗组相比p＜0.005。
具体实施方式
下面结合附图和具体实施例对本公开作进一步说明，以使本领域的技术人员可以更好地理解本公开并能予以实施，但所举实施例不作为对本公开的限定。
除非有相反陈述，否则在本发明中所使用的术语具有下述含义。
在本发明的权利要求和/或说明书中，词语“一(a)”或“一(an)”或“一(the)”可以指“一个”，但也可以指“一个或多个”、“至少一个”以及“一个或多于一个”。
如在权利要求和说明书中所使用的，词语“包含”、“具有”、“包括”或“含有”是指包括在内的或开放式的，并不排除额外的、未引述的元件或方法步骤。
术语“治疗”是指：在罹患疾病之后，使受试者接触(例如给药)核酸递送粒子、核酸递送系统、核酸疫苗、负载核酸的药物、药物组合物，从而与不接触时相比使该疾病的症状减轻，并不意味着必需完全抑制疾病的症状。罹患疾病是指：身体出现了疾病症状。
术语“预防”是指：在罹患疾病之前，通过使受试者接触(例如给药)本公开的核酸递送粒子、核酸递送系统、核酸疫苗、负载核酸的药物、药物组合物，从而与不接触时相比减轻罹患疾病后的症状，并不意味着必需完全抑制患病。
术语“药学上可接受的辅料”或“药学上可接受的载体”是指在药物生产领域中广泛采用的辅助物料。使用辅料的主要目的在于提供一种使用安全、性质稳定和/或具有特定功能性的药物组合物，还在于提供一种方法，以便在为受试者施用药物之后，活性成分能够以所期望的速率溶出，或者促进活性成分在接受给药的受试者体内得到有效吸收。药学上可接受的辅料可以是具有惰性的填充剂，也可以是为药用组合物提供某种功能(例如稳定组合物的整体pH值或防止组合物中活性成分的降解)的功效成分。药学上可接受的辅料的非限制性实例包括但不限于粘合剂、助悬剂、乳化剂、稀释剂(或填充剂)、成粒剂、胶粘剂、崩解剂、润滑剂、抗粘着剂、助流剂、润湿剂、胶凝剂、吸收延迟剂、溶解抑制剂、增强剂、吸附剂、缓冲剂、螯合剂、防腐剂、着色剂、矫味剂、甜味剂等。
本公开中的药物组合物可以使用本领域技术人员已知的任何方法来制备。例如，常规混合、溶解、造粒、乳化、磨细、包封、包埋和/或冻干工艺。
在本公开中，施用途经能够以任何适用的方式进行变化或调整，以满足药物的性质、患者和医务人员的便利以及其它相关因素的需求。
本公开上下文中使用的术语“个体”、“患者”或“受试者”包括哺乳动物。哺乳动物包括但不限于，家养动物(例如，牛，羊，猫，狗和马)，灵长类动物(例如，人和非人灵长类动物如猴)，兔，以及啮齿类动物(例如，小鼠和大鼠)。
除非另外定义或由背景清楚指示，否则在本公开中的全部技术与科学术语具有如本公开所属领域的普通技术人员通常理解的相同含义。
本公开上下文中使用的术语“个体”、“患者”或“受试者”包括哺乳动物。哺乳动物包括但不限于，家养动物(例如，牛，羊，猫，狗和马)，灵长类动物(例如，人和非人灵长类动物如猴)，兔，以及啮齿类动物(例如，小鼠和大鼠)。
术语“肿瘤”和“癌症”在本文中互换地使用，涵盖实体瘤和液体肿瘤。术语“肿瘤”指所有赘生性(neoplastic)细胞生长和增殖，无论是恶性的还是良性的，及所有癌前(pre-cancerous)和癌性细胞和组织。术语“癌症”、“癌性”和“肿瘤”在本文中提到时并不互相排斥。
本公开所述的核酸递送系统(疫苗系统)，其内部负载核酸，表面负载被粒子激活的抗原提呈细胞的细胞膜组分或细胞外囊泡膜组分。其中，用于制备纳米粒子或微米粒子的抗原提呈细胞先被负载肿瘤组织和/或癌细胞全细胞抗原和/或核酸或其混合物的纳米粒子和/或微米粒子(抗原递送粒子)激活；然后将被激活的抗原提呈细胞的膜组分负载于内部负载mRNA的粒子(核酸递送前体粒子)表面，制备预防或治疗癌症的粒子系统(核酸递送粒子)，其制备过程及应用领域如图1所示。
在制备激活抗原提呈细胞的纳米粒子或微米粒子(抗原递送粒子)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(抗原递送粒子)；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癌细胞全细胞抗原并制备纳米或微米粒子系统(抗原递送粒子)。本公开所述癌细胞和/或肿瘤组织全细胞抗原在裂解前或(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、蛋白质和/或多肽分离提纯、核酸分离提纯、蛋白酶内切或降解、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、蛋白酶内切或降解、核酸酶处理直接制备纳米粒子或微米粒子。本公开部分实施例中，肿瘤组织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；本公开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、蛋白质和/或多肽分离提纯、核酸分离提纯、固化、生物矿化、离子化、化学修饰、核酸酶处理、蛋白酶内切或降解、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。
在将被激活的抗原提呈细胞制备成纳米粒子或微米粒子时，先对抗原提呈细胞进行机械破坏，然后使用离心和/或一定孔径的滤膜过滤，然后与负载全细胞组分和/或核酸的纳米粒子或微米粒子共作用。
被激活的抗原提呈细胞在经过机械破坏后含有一定的细胞膜结构。
激活的抗原提呈细胞在经过机械破坏后，与作为核酸递送前体粒子共作用后所形成核酸递送粒子。其中，来源于激活的抗原提呈细胞的生物膜组分位于核酸递送粒子的外层。
用于制备生物膜组分的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。
核酸递送粒子除了表面负载来源于激活的抗原提呈细胞的生物膜组分和/或其分泌的细胞外囊泡的生物膜组分，还可以同时负载来源于癌细胞的生物膜组分，和/或来源于由癌细胞分泌的细胞外囊泡的生物膜组分，和/或细菌，和/或来源于细菌或其分泌的细胞外囊泡的生物膜组分。
以肿瘤组织，和/或癌细胞的全细胞组分作为抗原组分，在使用负载抗原组分的抗原递送粒子激活抗原提呈细胞时，体系中可含有细胞因子和/或抗体以提高激活效率。
在一些实施方案中，采用负载来源于癌细胞全细胞组分的抗原递送粒子先激活抗原提呈细胞，在将抗原提呈细胞的生物膜组分负载于纳米粒子或微米粒子表面，制备得到核酸递送粒子。
任何本领域人员知晓的纳米粒子、微米粒子制备方法均可以用于制备本公开所述的核酸递送粒子，包括但不限于溶剂挥发法、透析法、微流控法、均质乳化法、分散法、沉淀法等等。
在一些具体的实施方案中，本公开以溶剂挥发法为例，提供如下示例制备方法：
步骤1，混合初水相与有机相，具体为将第一预定体积的含有第一预定浓度的抗原组分的水相溶液加入第二预定体积的含有第二预定浓度的制备粒子原材料的有机相中。
在一些实施例中，水相溶液可含有如下i)～iii)中的至少一种：i)癌细胞裂解物中各组分、ii)肿瘤组织裂解物中的各组分、iii)编码特定蛋白质或多肽的核酸，以及免疫增强佐剂。裂解物中的各组分在制备时分别为水溶性抗原，或是溶于含有尿素或盐酸胍等溶解剂的溶解液中的原非水溶性抗原。第一预定浓度为水相溶液所含有的核酸的浓度，或者是水相溶液中所含有的水溶性抗原的浓度和/或原非水溶性抗原的浓度，第一预定浓度要求蛋白质多肽浓度含量大于1ng/mL，或编码特定蛋白质或多肽的核酸的浓度为大于0.01ng/mL核酸浓度大于0.01ng/mL，以便能负载足够的核酸或者癌细胞全细胞抗原以激活相关细胞。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，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，选为100mg/mL。
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，在本公开中，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。
在一些实施方案中，水相溶液为包含细胞和/或组织裂解物组分的溶液时，其中蛋白质和多肽的浓度大于1ng/mL，优选1mg/mL～100mg/mL。在一些实施方案中，水相溶液为包含裂解物组分与免疫佐剂的溶液时，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。在一些实施方案中，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。
在一些实施方案中，水相溶液为包含核酸的溶液时，其中核酸的浓度大于0.01ng/mL，优选1μg/mL～1mg/mL。在一些实施方案中，水相溶液为包含核酸与免疫佐剂的溶液时，其中核酸的浓度大于1ng/mL，优选1μg/mL～1mg/mL，免疫佐剂的浓度大于0.01ng/mL， 优选0.01mg/mL～20mg/mL。在一些实施方案中，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得到的混合液进行如下任一种处理：i)大于2秒的超声处理；ii)大于1分钟的搅拌；iii)均质处理；iv)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等能控制制备的纳米粒子或微米粒子大小，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如下任一种处理：i)大于2秒的超声处理；ii)大于1分钟的搅拌；iii)进行均质处理；iv)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或均质化或混合以进行纳米化或微米化。在本公开中，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等能控制制备的纳米粒子或微米粒子大小，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。在本公开中，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或均质化时间、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。在本公开中，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。
在本公开中，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；或者使用超滤离心或者使用能够去除特定分子量物质的透析法，去除游离的PVA等物质的同时将体系中的溶液更换为含有第五预定浓度的冻干保护剂的第五预定体积的水溶液或者第六预定体积的PBS(或生理盐水)。
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，将第六预定体积的步骤5中得到的重悬于PBS(或生理盐水)中的含纳米粒的混悬液或者采用第六预定体积的PBS(或生理盐水)重悬步骤6得到的冷冻干燥后的含有纳米粒或微米粒和冻干保护剂的冻干物质直接使用；或者上述样品与第七预定体积的水溶性抗原或者溶解的原非水溶性抗原混合后使用。
在本公开中，第六预定体积与第七预定体积的体积比为1:10000到10000:1，优先体积比为1:100到100:1，最优体积比为1:30到30:1。
在一些实施方案中，所述形成核酸递送粒子的核酸递送前体粒子中，每1mg粒子材料负载0.1-500μg核酸。在一些实施方案中，或所述核酸递送前体粒子还负载免疫增强佐剂，其中，每1mg粒子材料负载1-400μg免疫增强佐剂。在一些实施方案中，所述核酸递送前体粒子还负载带正电性分子，其中，每1mg粒子材料负载1-800μg带正电性分子。
步骤8，将抗原提呈细胞与上述制备的抗原递送粒子共孵育一定时间。制备抗原递送粒子的肿瘤组织和/或癌细胞与抗原提呈细胞可以来自于同一个体或者同种异体。
在一些实施方案中，每(50万-5000万)个所述抗原提呈细胞与(10μg-1500μg)的所述抗原递送粒子进行共孵育。
步骤9，收集共孵育后的抗原提呈细胞，进行超声、均质化、机械搅拌等机械破坏，然后将样品进行离心和/或使用一定孔径的滤膜过滤和/或共挤出等处理，得到被激活的抗原提呈细胞的生物膜组分和/或细胞外囊泡的生物膜组分。
步骤10，将mRNA或DNA等核酸采用步骤1-7的方法负载于纳米粒子或微米粒子中，制备核酸递送前体粒子。
步骤11，将步骤9得到的生物膜组分(含有被激活的抗原提呈细胞的生物膜组分，可同时含有癌细胞的生物膜组分和/或细菌的生物膜组分)与步骤10制备的负载核酸的核酸 递送前体粒子共作用，制备得到内部负载核酸，表面负载被激活的抗原提呈细胞的膜组分和/或其分泌的细胞外囊泡的膜组分的纳米粒子或微米粒子。
在一些实施方案中，所述核酸递送递送粒子中，每1mg粒子材料负载0.1-500μg核酸。在一些实施方案中，所述核酸递送递送粒子还负载免疫增强佐剂，其中，每1mg粒子材料负载0.1-400μg免疫增强佐剂。在一些实施方案中，所述核酸递送递送粒子还负载带正电性分子，其中，每1mg粒子材料负载1-800μg带正电性分子。
步骤12，将步骤11所制备的粒子递送系统(疫苗系统)用于预防或者治疗癌症等疾病。
实施例
本公开的其他目的、特征和优点将从以下详细描述中变得明显。但是，应当理解的是，详细描述和具体实施例(虽然表示本公开的具体实施方式)仅为解释性目的而给出，因为在阅读该详细说明后，在本公开的精神和范围内所作出的各种改变和修饰，对于本领域技术人员来说将变得显而易见。
本实施例中所用到的实验技术与实验方法，如无特殊说明均为常规技术方法，例如下列实施例中未注明具体条件的实验方法，通常按照常规条件或按照制造厂商所建议的条件。实施例中所使用的材料、试剂等，如无特殊说明，均可通过正规商业渠道获得。
实施例1负载mRNA和抗原提呈细胞膜组分的纳米粒子被吞噬后体外表达效率
本实施例评价内部负载mRNA同时表面负载被激活的抗原提呈细胞的膜组分的核酸递送纳米粒子(也即，纳米疫苗)被细胞吞噬后所负载的核酸的表达效率。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抗原递送纳米粒子，然后使用抗原递送纳米粒子激活抗原提呈细胞，并将抗原提呈细胞机械破坏后离心得到被激活的抗原提呈细胞的细胞膜组分。与此同时，将编码增强绿色荧光蛋白(Enhanced Green Fluorescent Protein，EGFP)的mRNA负载于核酸递送前体纳米粒子中。将前述被激活的抗原提呈细胞的膜组分与负载mRNA的核酸递送前体粒子共作用后，即得到内部负载核酸同时表面负载被激活的抗原提呈细胞的膜组分的核酸递送粒子，然后检测该核酸递送粒子系统被细胞吞噬后所负载的核酸表达高低。
(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1.5×105个B16F10细胞，在肿瘤长到体积分别为约1000mm3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 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。以上即为制备抗原递送纳米粒子系统的抗原原料来源。
(2)负载全细胞组分的抗原递送粒子(纳米粒子1)的制备
本实施例中抗原递送纳米粒子1采用溶剂挥发法中的复乳法制备。在制备时负载癌细胞全细胞抗原中水溶性抗原的纳米粒子和负载癌细胞全细胞抗原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。所采用的抗原递送纳米粒子制备材料PLGA分子量为7KDa-17KDa，所采用的免疫佐剂为poly(I:C)且poly(I:C)且负载于纳米粒子内部。制备方法如前所述，在制备过程中首先采用复乳法在抗原递送纳米粒子内部负载细胞抗原组分和佐剂，然后将100mg抗原递送纳米粒子在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240nm左右，每1mg PLGA纳米粒子约负载100μg蛋白质或多肽组分，每1mgPLGA纳米粒所使用的poly(I:C)免疫佐剂为0.02mg。
(3)负载EGFP-mRNA的核酸递送前体粒子(纳米粒子2)的制备
本实施例中负载mRNA的核酸递送前体纳米粒子2所负载的mRNA可以EGFP；核酸递送前体纳米粒子同时负载有免疫佐剂poly(I:C)和R8(RRRRRRRR)多肽。负载mRNA的核酸递送前体粒子的制备材料和制备方法同步骤(2)，首先将mRNA、poly(I:C)和R8多肽在水中混合，然后采用复乳法将上述混合物负载于纳米粒子内部，然后将100mg PLGA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2平均粒径为250nm左右，每1mg PLGA纳米粒子约负载10μg mRNA组分，每1mgPLGA纳米粒负载poly(I:C)免疫佐剂为20μg，负载R8多肽40μg。
(4)骨髓来源的树突状细胞(BMDC)的制备
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6-8周龄的C57BL/6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。将细胞放置在10mm培养皿中培养，使用RPMI 1640(10％FBS)完全培养基，同时加入重组小鼠GM-CSF(20ng/mL)，在37℃，5％CO2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。
(5)抗原提呈细胞的激活
将负载来源于肿瘤组织的癌细胞全细胞组分的抗原递送纳米粒子1(250μg负载水溶性组分的纳米粒子+250μg负载非水溶性组分的纳米粒子)与BMDC(1000万个)在15mL RPMI1640完全培养基中共孵育48小时(37℃，5％CO2)；孵育体系中含有细胞因子组合 1：IL-2(500U/mL)、IL-7(500U/mL)、IL-15(500U/mL)或者含有细胞因子组分2：IL-4(500U/mL)、IL-33(500U/mL)、IL10(500U/mL)。
(6)纳米疫苗的制备
收集步骤(4)制备的未经任何纳米粒子或微米粒子激活的DC(1000万个)，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4℃和7.5W下超声20分钟以破坏细胞并制备含有细胞膜组分的样品。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，将上清液与40mg步骤(3)制备的核酸递送前体纳米粒子2共孵育10分钟，然后使用0.40μm的滤膜反复共挤出，将挤出液在15000g离心120分钟后收集弃去上清液收集沉淀，将沉淀在含2％蔗糖和2％甘露醇的超纯水重悬后冷冻干燥48h备用，即为核酸递送纳米粒子3，也称为纳米疫苗3,粒径为270nm，表面电位为-6mV。每1mg PLGA纳米粒子约负载10μg mRNA组分，每1mgPLGA纳米粒负载poly(I:C)免疫佐剂为20μg，负载R8多肽40μg；每1mg PLGA纳米粒子约负载50μg细胞膜组分。
或者通过在400g离心5分钟收集添加细胞因子组分2的孵育后的DC(1000万个)，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在4℃和7.5W下超声20分钟以破坏细胞并制备含有细胞膜组分的样品。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，将上清液与40mg步骤(3)制备的纳米粒子2共孵育10分钟，然后使用0.40μm的滤膜反复共挤出，将挤出液在15000g离心120分钟后收集弃去上清液收集沉淀，将沉淀在含2％蔗糖和2％甘露醇的超纯水重悬后冷冻干燥48h备用，即为核酸递送纳米粒子4，也称为纳米疫苗4,粒径为270nm，表面电位为-6mV。每1mg PLGA纳米粒子约负载10μg mRNA组分，每1mgPLGA纳米粒负载poly(I:C)免疫佐剂为20μg，负载R8多肽40μg；每1mg PLGA纳米粒子约负载50μg细胞膜组分。
通过在400g离心5分钟收集添加细胞因子组分1的孵育后的DC(1000万个)，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在4℃和7.5W下超声20分钟以破坏细胞并制备含有细胞膜组分的样品。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，将上清液与40mg步骤(3)制备的纳米粒子2共孵育10分钟，然后使用0.40μm的滤膜反复共挤出，将挤出液在15000g离心120分钟后收集弃去上清液收集沉淀，将沉淀在含2％蔗糖和2％甘露醇的超纯水重悬后冷冻干燥48h备用，即为核酸递送纳米粒子5，也称为纳米疫苗5,粒径为270nm，表面电位为-6mV。每1mg PLGA纳米粒子约负载10μg mRNA组分，每1mgPLGA纳米粒负载poly(I:C)免疫佐剂为20μg，负载R8多肽40μg；每1mg PLGA纳米粒子约负载50μg细胞膜组分。纳米疫苗5为制备好后立即测试或者在4℃储存18个月后测试。
(7)负载EGFP-mRNA的脂质纳米粒(Lipid nanoparticles，LNP)的制备
本实施例使用微流控法制备LNP。使用的脂质材料等的摩尔比如下：MC3:PEG2000:DSPC：胆固醇＝50％：1.5％：10％：38.5％；所负载的mRNA同步骤(3)所负载的mRNA。先称取1mM各脂质成分即MC3(0.321mg)、PEG2000(0.037＝mg)、DSPC (0.079mg)、Chol(0.148mg)一起混合后溶于0.586mL无水乙醇中。将0.107mg的EGFP mRNA溶于1mL pH＝4.0的柠檬酸缓冲溶液中。分别将脂质-乙醇溶液和mRNA-柠檬酸缓冲液通过0.22μm滤膜过滤，然后将脂质-乙醇溶液吸入1mL注射器中，将mRNA-柠檬酸缓冲液吸入3mL注射器中(至少吸入1.5mL)，并排出注射器中的空气，将注射器出口和样品导入管连接，并固定在微流控注射泵上。在微流控装置中设置好参数后点击运行(总流速2.4ml/min运行)，观察流出管流速稳定后(一般需要丢弃前200μL液体)，用收集管收集流出的液体即为制备好的LNP。LNP粒径为95纳米,,表面电位为8mV,每1mg LNP负载mRNA为50μg。该LNP即为LNP疫苗，LNP疫苗现制现用。
(8)纳米疫苗递送核酸时的体外细胞表达效率
将抗原提呈细胞DC2.4细胞在RPMI完全培养基(含10％FBS)中以每孔10万个细胞密度接种于24孔板中，在37℃(5％CO2)培养过夜。在上述细胞中分别加入25ng游离EGFP-mRNA、或者0.5μg步骤(7)刚制备好的LNP疫苗、或者1.25μg步骤(6)制备的纳米疫苗(刚制备好的纳米疫苗3，刚制备好的纳米疫苗4，刚制备好的纳米疫苗5，或者已经长期储存的纳米疫苗5)，尔后共孵育24h后。然后收集上述细胞并用流式细胞仪术分析含有EGFP荧光信号的细胞占所有细胞的比例以及EGFP阳性的细胞中的平均荧光信号强度(MFI)。
(9)实验结果
如图2所示，图2中Naked mRNA、LNP、Nanovaccine 3(Fresh)、Nanovaccine 4(Fresh)、Nanovaccine 5(Fresh)、Nanovaccine 5(Long term storage)依次对应裸mRNA、LNP疫苗、刚制备好的纳米疫苗3、刚制备好的纳米疫苗4、刚制备好的纳米疫苗5、已经长期储存的纳米疫苗5。结果显示，裸mRNA于细胞共孵育后细胞内的表达效率几乎为0。纳米疫苗5效果最好表达率最高，不管是冷冻干燥后短期储存的还是长期储存的纳米疫苗5，其效果均显著优于纳米疫苗3、纳米疫苗4和LNP疫苗；而且，纳米疫苗5制备好后长期储存不影响其功效。这说明：本公开所述纳米疫苗冷冻干燥后可以在4℃长期储存，不影响其功效；而且在激活抗原提呈细胞过程中加入细胞因子组合1效果优于细胞因子组分2；被负载癌细胞全细胞组分的抗原递送纳米粒子激活的抗原提呈细胞所制备的核酸递送纳米粒子效果远好于未被激活的抗原提呈细胞制备的核酸递送纳米粒子。综上所述，本公开所述的核酸粒子递送系统可以非常高效的递送mRNA，而且粒子递送系统在冷冻干燥后可以在4℃长期储存。
实施例2负载mRNA和抗原提呈细胞膜组分的纳米粒子用于黑色素瘤的预防
本实施例以癌症模型来说明如何使用内部负载mRNA同时表面负载被激活的抗原提呈细胞的膜组分的核酸递送纳米粒子(纳米疫苗)预防疾病。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PLGA为纳米粒骨架材料，以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抗原递送纳米粒子系统，然后使用抗原递送纳米粒子激活抗原提呈细胞，并将抗 原提呈细胞机械破坏后离心得到被激活的抗原提呈细胞的膜组分。与此同时，将编码四种黑色素瘤新抗原多肽的mRNA负载于纳米粒子中。将前述被激活的抗原提呈细胞的膜组分与负载mRNA的粒子共作用后，即得到内部负载核酸同时表面负载被激活的抗原提呈细胞的膜组分的核酸递送粒子，然后使用该纳米粒子预防黑色素瘤。
(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1.5×105个B16F10细胞，在肿瘤长到体积分别为约1000mm3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)负载全细胞组分的抗原递送粒子(纳米粒子1)的制备
本实施例中抗原递送纳米粒子1采用溶剂挥发法中的复乳法制备。在制备时负载癌细胞全细胞抗原中水溶性抗原的纳米粒子和负载癌细胞全细胞抗原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。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)且负载于纳米粒子内部。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细胞抗原组分和佐剂，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240nm左右，每1mg PLGA纳米粒子约负载100μg蛋白质或多肽组分，每1mgPLGA纳米粒所使用的poly(I:C)免疫佐剂为0.02mg。
(3)负载mRNA的核酸递送前体粒子(纳米粒子2)的制备
本实施例中负载mRNA的核酸递送前体纳米粒子2所负载的mRNA可以编码四种多肽新生抗原B16-M20(Tubb3,FRRKAFLHWYTGEAMDEMEFTEAESNM)，B16-M24(Dag1,TAVITPPTTTTKKARVSTPKPATPSTD)，B16-M46(Actn4,NHSGLVTFQAFIDVMSRETTDTDTADQ)和TRP2:180-188(SVYDFFVWL)；核酸递送前体纳米粒子同时负载有免疫佐剂poly(I:C)和R8(RRRRRRRR)多肽。负载mRNA的核酸递送前体粒子的制备材料和制备方法同步骤(2)，首先将mRNA、poly(I:C)和R8多肽在水中混合，然后采用复乳法将上述混合物负载于纳米粒子2内部，然后将100mg PLGA纳米粒子2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2平均粒径为250nm左右，每1mg PLGA纳米粒子2约负载10μg mRNA组分，每1mgPLGA纳米粒2负载poly(I:C)免疫佐剂为20μg，负载R8多肽40μg。
(4)骨髓来源的树突状细胞(BMDC)的制备
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)完全培养基，同时加入重组小鼠GM-CSF(20ng/mL)，在37℃和5％CO2中培养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。
(5)抗原提呈细胞的激活
将负载来源于肿瘤组织的癌细胞全细胞组分的抗原递送纳米粒子1(250μg负载水溶性组分的纳米粒子+250μg负载非水溶性组分的纳米粒子)与BMDC(1000万个)在15mL RPMI1640完全培养基中共孵育48小时(37℃，5％CO2)；孵育体系中含有细胞因子组合1：IL-2(500U/mL)、IL-7(500U/mL)、IL-15(500U/mL)或者含有细胞因子组分2：IL-4(500U/mL)、IL-33(500U/mL)、IL10(500U/mL)。
(6)纳米疫苗的制备
收集步骤(4)制备的未经任何纳米粒子或微米粒子激活的DC(1000万个)，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4℃和7.5W下超声20分钟以破坏细胞并制备含有细胞膜组分的样品。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，将上清液与40mg步骤(3)制备的核酸递送前体纳米粒子2共孵育10分钟，然后使用0.45μm的滤膜反复共挤出，将挤出液在15000g离心120分钟后收集弃去上清液收集沉淀，将沉淀在含2％蔗糖和2％甘露醇的超纯水重悬后冷冻干燥48h备用，即为核酸递送纳米粒子3，也称为纳米疫苗3,粒径为270nm，表面电位为-6mV。每1mg PLGA纳米粒子约负载10μg mRNA组分，每1mgPLGA纳米粒负载poly(I:C)免疫佐剂为20μg，负载R8多肽40μg；每1mg PLGA纳米粒子约负载50μg细胞膜组分。该纳米疫苗制备后直接使用。
或者通过在400g离心5分钟收集添加细胞因子组分2的孵育后的DC(1000万个)，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在4℃和7.5W下超声20分钟以破坏细胞并制备含有细胞膜组分的样品。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，将上清液与40mg步骤(3)制备的的纳米粒子2共孵育10分钟，然后使用0.45μm的滤膜反复共挤出，将挤出液在15000g离心120分钟后收集弃去上清液收集沉淀，将沉淀在含2％蔗糖和2％甘露醇的超纯水重悬后冷冻干燥48h备用，即为核酸递送纳米粒子4，也称为纳米疫苗4,粒径为270nm，表面电位为-6mV。每1mg PLGA纳米粒子约负载10μg mRNA组分，每1mgPLGA纳米粒负载poly(I:C)免疫佐剂为20μg，负载R8多肽40μg；每1mg PLGA纳米粒子约负载50μg细胞膜组分。该纳米疫苗制备后直接使用。
或者通过在400g离心5分钟收集添加细胞因子组分1的孵育后的DC(1000万个)，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在4℃和7.5W下超声20分钟以破坏细胞并制备含有细胞膜组分的样品。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，将上清液与40mg步骤(3)制备的纳米粒子2共孵育10分钟，然后使用0.45μm的滤膜反复共挤出，将挤出液在15000g离心120分钟后收集弃去上清液收集沉淀，将沉淀在含2％蔗糖和2％甘露醇的超纯水重悬后冷冻干燥48h备用，即为核酸递送纳米粒子5，也称为纳米疫苗5,粒径为270nm，表面电位为-6mV。每1mg PLGA纳米粒子约负载10μg mRNA组分，每1mgPLGA纳米粒负载poly(I:C)免疫佐剂为20μg，负载R8多肽40μg；每1mg PLGA纳米粒子约负载50μg细胞膜组分。该纳米疫苗制备后直接使用或者在4℃储存18个月后使用。
(7)负载mRNA的脂质纳米粒(Lipid nanoparticles，LNP)的制备
本实施例使用微流控法制备LNP。使用的脂质材料等的摩尔比如下：MC3:PEG2000:DSPC：胆固醇＝50％：1.5％：10％：38.5％；所负载的mRNA同步骤(3)所负载的mRNA。先称取1mM各脂质成分即MC3(0.321mg)、PEG2000(0.037＝mg)、DSPC(0.079mg)、Chol(0.148mg)一起混合后溶于0.586mL无水乙醇中。将mRNA溶于1mL pH＝4.0的柠檬酸缓冲溶液中。分别将脂质-乙醇溶液和mRNA-柠檬酸缓冲液通过0.22μm滤膜过滤，然后将脂质-乙醇溶液吸入1mL注射器中，将mRNA-柠檬酸缓冲液吸入3mL注射器中(至少吸入1.5mL)，并排出注射器中的空气，将注射器出口和样品导入管连接，并固定在微流控注射泵上。在微流控装置中设置好参数后点击运行，观察流出管流速稳定后(一般需要丢弃前200μL液体)，用收集管收集流出的液体即为制备好的LNP。LNP粒径为95纳米,表面电位为8mV,每1mg LNP负载mRNA为50μg。该LNP即为LNP疫苗，LNP疫苗现制现用。
(8)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，在小鼠接种肿瘤前第-35天，第-28天，第-21天，第-14天和第-7天分别在小鼠皮下接种1mg刚冷冻干燥后的纳米疫苗(纳米疫苗3，或者纳米疫苗4，或者纳米疫苗5)、或者接种1mg冷冻干燥后在4℃储存18个月的纳米疫苗5、或者接种0.5mg现制的LNP疫苗或者接种100μL PBS。第0天给每只小鼠背部右下方皮下接种1.5×105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×b2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2000mm3即视为小鼠死亡并将小鼠安乐死。
(9)纳米疫苗处理后小鼠体内癌细胞特异性T细胞含量的分析
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，在小鼠接种肿瘤前第-35天，第-28天，第-21天，第-14天和第-7天分别在小鼠皮下接种1mg刚冷冻干燥后的纳米疫苗(纳米疫苗3，或者纳米疫苗4，或者纳米疫苗5)、或者接种1mg冷冻干燥后在 4℃储存18个月的纳米疫苗3、或者接种0.5mg现制的LNP疫苗或者接种100μL PBS。在第0天处死小鼠，摘取小鼠脾细胞，制备小鼠脾细胞单细胞悬液，然后将200万个小鼠脾细胞与400μg抗原递送纳米粒子1(200μg负载水溶性的纳米粒子1+200μg负载非水溶性的纳米粒子1)+20μL步骤(1)制备的小鼠肿瘤组织裂解液(80mg/mL,其中水溶性组分和非水溶性组分等质量)的混合物在DMEM高糖完全培养基中在37℃(5％CO2)共孵育48小时。然后收集孵育后的细胞并用先使用带有荧光探针的抗小鼠CD3抗体、抗小鼠CD4抗体和抗小鼠CD8抗体标记小鼠细胞，然后使用2％多聚甲醛固定细胞后，使用破膜液对细胞破膜后使用带有荧光探针的IFN-γ抗体进行标记。尔后使用流式细胞术分析CD8+T细胞中CD8+IFN-γ+T细胞所占比例以及CD4+T细胞中CD4+IFN-γ+T细胞所占比例。抗原递送纳米粒子所负载的癌细胞全细胞抗原在被抗原提呈细胞吞噬后可被降解成抗原表位被提呈到抗原提呈细胞膜表面，抗原提呈细胞制备的纳米粒子负载有上述降解提呈后的抗原表位，可以被癌细胞特异性T细胞识别并激活癌细胞特异性T细胞，被激活后分泌杀伤性细胞因子。IFN-γ是抗原特异性T细胞识别抗原后被激活所分泌的最主要的细胞因子。使用流式细胞术分析的CD8+IFN-γ+T细胞和CD4+IFN-γ+T细胞即为可以识别和杀伤癌细胞的癌细胞特异性T细胞。
(10)实验结果
结果如图3所示，图3中PBS control、LNP vaccine、Nanovaccine 3(Fresh)、Nanovaccine 4(Fresh)、Nanovaccine 5(Fresh)、Nanovaccine 5(Long term storage)依次为PBS对照组、LNP疫苗、刚制备好的纳米疫苗3、刚制备好的纳米疫苗4、刚制备好的纳米疫苗5、已经长期储存的纳米疫苗5。如图3a和b所示，PBS对照组的小鼠其肿瘤生长速度很快，生存期很短。接受几种疫苗处理的小鼠肿瘤生长速度都明显变慢，小鼠生存期变长。其中，纳米疫苗5效果最好，不管是冷冻干燥后短期储存的还是长期储存的纳米疫苗5，其效果均显著优于纳米疫苗3、纳米疫苗4和LNP疫苗；而且，纳米疫苗5制备好后长期储存不影响其功效。这说明：本公开所述纳米疫苗冷冻干燥后可以在4℃长期储存，不影响其功效；而且在激活抗原提呈细胞过程中加入细胞因子组合1效果优于细胞因子组分2；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所制备的纳米粒子效果远好于未被激活的抗原提呈细胞制备的纳米粒子。综上所述，本公开所述的纳米疫苗对黑色素瘤具有良好的预防效果。被负载癌细胞全细胞组分的抗原递送纳米粒子激活的抗原提呈细胞会降解和提呈所吞噬纳米粒子负载的癌细胞全细胞组分中的全细胞抗原，被抗原提呈细胞提呈到细胞膜表面的癌细胞抗原表位已经与主要组织相容性复合物(MHC)分子结合。将上述抗原提呈细胞经过机械破坏后，抗原提呈细胞的细胞膜组分中含有与MHC结合的抗原表位。通过离心和/或使用一定孔径的滤膜过滤后与纳米粒子或微米粒子共作用，上述抗原提呈细胞中的细胞膜组分会负载于纳米粒子或微米粒子表面，会形成新的纳米粒子或微米粒子，即为纳米疫苗或微米疫苗。上述方法制备的纳米疫苗或微米疫苗内部负载编码特定抗原的mRNA,同时表面负载了MHC分子和各类癌细胞抗原表位的复合物，因而其激活机体癌细胞特异性免疫反应的能力会更强也更广谱，因而预防或治疗癌症的效果也会更佳。
如图3c和d所示，与PBS对照组及LNP疫苗组相比，纳米疫苗可以激活更多的癌细胞特异性T细胞。纳米疫苗5效果激活的癌细胞特异性T细胞比例最高，不管是冷冻干燥后短期储存的还是长期储存的纳米疫苗5，其效果均显著优于纳米疫苗3、纳米疫苗4和LNP疫苗；而且，纳米疫苗5制备好后长期储存不影响其功效。该结果与疗效结果相一致，说明纳米疫苗通过激活更多的癌细胞特异性T细胞发挥作用。
实施例3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粒子激活的抗原提呈细胞制备成的负载mRNA的纳米粒子用于预防黑色素瘤。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PLGA为纳米粒骨架材料，以poly(I:C)和CpG1018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抗原递送粒子系统，然后使用抗原递送粒子激活抗原提呈细胞，并将被激活的抗原提呈细胞的膜组分负载于核酸递送前体粒子表面，制备得到核酸递送粒子预防癌症。
(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1.5×105个B16F10细胞，在肿瘤长到体积分别为约1000mm3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。将水溶性组分和非水溶性组分按质量比1:1混合后即为制备抗原递送纳米粒子系统的抗原原料来源。
(2)负载癌细胞全细胞组分的抗原递送粒子的制备
本实施例中抗原递送纳米粒子及作为对照的空白纳米粒子和多肽纳米粒子采用溶剂挥发法制备。负载全细胞组分的抗原递送纳米粒子1制备材料PLGA分子量为7Da-17KDa，所采用的免疫佐剂为poly(I:C)和CpG1018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，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。该纳米粒子平均粒径为220nm左右；每1mg PLGA纳米粒子约负载100μg蛋白质和多肽组分，每1mgPLGA纳米粒所使用的poly(I:C)和CpG1018免疫佐剂各0.02mg。本实施例中，采用等质量负载四种多肽新生抗原B16-M20(Tubb3,FRRKAFLHWYTGEAMDEMEFTEAESNM)，B16-M24(Dag1,TAVITPPTTTTKKARVSTPKPATPSTD)，B16-M46(Actn4,NHSGLVTFQAFIDVMSRETTDTDTADQ)和TRP2:180-188(SVYDFFVWL)的多肽纳米粒子2作为对照纳米粒子使用，其制备材料和制备方法同纳米粒子1，对照纳米粒2的粒径为220nm左右，负载100μg多肽组分，负载等量佐剂。空白纳米粒3的制备材料和制备方法同纳米粒子1，粒径为220nm左右，只负载等量的免疫佐剂却不负载任何抗原组分。
(3)负载mRNA的核酸递送前体粒子(纳米粒子4)的制备
本实施例中负载mRNA的核酸递送前体纳米粒子4所负载的mRNA可以编码四种多肽 新生抗原B16-M20(Tubb3,FRRKAFLHWYTGEAMDEMEFTEAESNM)，B16-M24(Dag1,TAVITPPTTTTKKARVSTPKPATPSTD)，B16-M46(Actn4,NHSGLVTFQAFIDVMSRETTDTDTADQ)和TRP2:180-188(SVYDFFVWL)；纳米粒子同时负载有免疫佐剂poly(I:C)和带正电荷的蜂毒肽(GIGAVLKVLTTGLPALISWIKRKRQQ)。负载mRNA的粒子的制备材料和制备方法同步骤(2)，首先将mRNA、poly(I:C)和蜂毒肽在水中混合，然后采用复乳法将上述混合物负载于纳米粒子内部，然后将100mg PLGA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4平均粒径为210nm左右，每1mg PLGA纳米粒子约负载10μg mRNA组分，每1mgPLGA纳米粒负载poly(I:C)免疫佐剂为10μg，负载蜂毒肽20μg。
(4)抗原提呈细胞的制备
采用骨髓来源的树突状细胞(BMDC)和B细胞作为抗原提呈细胞。BMDC的制备同实施例1。B细胞提取流程如下：处死小鼠后摘取小鼠脾脏，然后制备小鼠脾细胞单细胞悬液，然后使用磁珠分选法从脾细胞单细胞悬液中分选出CD19+B细胞。将BMDC和B细胞按数量比1:1混合后作为混合抗原提呈细胞使用。
(5)抗原提呈细胞的激活
将抗原递送纳米粒子1(500μg)或多肽纳米粒子2(500μg)或空白纳米粒3(500μg)+游离裂解液与2000万个混合抗原提呈细胞(1000万个BMDC+1000万个B细胞)在15mL RPMI1640完全培养基中共孵育48小时(37℃，5％CO2)；孵育体系中含有细胞因子组合：IL-15(500U/mL)、IL-7(500U/mL)、IL-21(1000U/mL)。
或者抗原递送纳米粒子1与2000万个BMDC在15mL RPMI1640完全培养基中共孵育48小时(37℃，5％CO2)；孵育体系中含有细胞因子组合：IL-15(500U/mL)、IL-7(500U/mL)、IL-21(1000U/mL)。
(6)基于抗原提呈细胞的纳米疫苗的制备
通过在400g离心5分钟收集孵育后的2000万个混合抗原提呈细胞(1000万个BMDC+1000万个B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4℃下使用低功率7.5W超声10分钟以破坏细胞并制备含有细胞膜组分的样品。然后将样品一次通过滤膜孔径为50μm、10μm、5μm、1μm、0.45μm、0.22μm的滤膜过滤，收集所得滤液后与相对应的步骤(3)制备的核酸递送前体纳米粒子4(50mg)共孵育10分钟，然后使用0.22μm的滤膜反复共挤出，将挤出液并在15000g离心60分钟，弃去上清后使用生理盐水重悬所得沉淀即为纳米粒子。其中，使用空白纳米粒子3激活的混合抗原提呈细胞膜组分与核酸递送前体纳米粒子4共作用后所制得的纳米粒子为纳米疫苗7，粒径为220nm,表面电位为-6mV；每1mg PLGA纳米粒子约负载10μg mRNA组分，每1mgPLGA纳米粒负载poly(I:C)免疫佐剂为10μg，负载蜂毒肽20μg；每1mg PLGA纳米粒子约负载100μg细胞膜组分。使用多肽纳米粒子2激活的混合抗原提呈细胞膜组分与核酸递送前体纳米粒子4共作用后所制得的纳米粒子为纳米疫苗6，粒径为220nm,表面电位为-6mV；每1mg PLGA纳米 粒子约负载10μg mRNA组分，每1mgPLGA纳米粒负载poly(I:C)免疫佐剂为10μg，负载蜂毒肽20μg；每1mg PLGA纳米粒子约负载100μg细胞膜组分。使用纳米粒子1激活的混合抗原提呈细胞膜组分与核酸递送前体纳米粒子4共作用后所制得的纳米粒子为纳米疫苗5，粒径为220nm,表面电位为-6mV；每1mg PLGA纳米粒子约负载10μg mRNA组分，每1mgPLGA纳米粒负载poly(I:C)免疫佐剂为10μg，负载蜂毒肽20μg；每1mg PLGA纳米粒子约负载100μg细胞膜组分。
或者通过在400g离心5分钟收集与抗原递送纳米粒子1孵育后的2000万个BMDC，然后使用生理盐水洗涤BMDC两遍，将细胞重悬在生理盐水中后在4℃下使用低功率7.5W超声10分钟以破坏细胞并制备含有细胞膜组分的样品。然后将样品一次通过滤膜孔径为50μm、10μm、5μm、1μm、0.45μm、0.22μm的滤膜过滤，收集所得滤液后与步骤(3)制备的核酸递送前体纳米粒子4(50mg)共孵育10分钟，然后使用0.22μm的滤膜反复共挤出，将挤出液并在15000g离心60分钟，弃去上清后使用含4％海藻糖的超纯水重悬后冷冻干燥48h备用。即为纳米疫苗8，粒径为220nm,表面电位为-6mV。每1mg PLGA纳米粒子约负载10μg mRNA组分，每1mgPLGA纳米粒负载poly(I:C)免疫佐剂为10μg，负载蜂毒肽20μg；每1mg PLGA纳米粒子约负载100μg细胞膜组分。
(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，在小鼠接种肿瘤前第-35天，第-28天，第-21天，第-14天和第-7天分别在小鼠皮下接种1mg纳米疫苗(纳米疫苗5，或者纳米疫苗6，或者纳米疫苗7，或者纳米疫苗8，或者纳米粒子4)或者100μL PBS。第0天给每只小鼠背部右下方皮下接种1.5×105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×b2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2000mm3即视为小鼠死亡并将小鼠安乐死。
(8)实验结果
如图4所示，图4中PBS control、Nanovaccine 4、Nanovaccine 5、Nanovaccine 6、Nanovaccine 7、Nanovaccine 8依次为PBS对照组、纳米疫苗4、纳米疫苗5、纳米疫苗6、纳米疫苗7、纳米疫苗8。结果显示，PBS对照组小鼠肿瘤生长速度都很快，小鼠生存期很短。与对照组相比，疫苗组小鼠的肿瘤生长速度都明显变慢，而且部分小鼠肿瘤消失痊愈。其中，纳米疫苗5效果最好，纳米疫苗5明显好于纳米疫苗6、纳米疫苗7和纳米疫苗8；而且纳米疫苗5、纳米疫苗6和纳米疫苗8的效果都明显好于纳米粒子4。这说明，抗原提呈细胞膜组分的负载有利于提高纳米疫苗的效果；而且，使用负载癌细胞全细胞抗原的抗原递送纳米粒子激活的抗原提呈细胞与负载核酸的核酸递送前体粒子共作用制备的纳米疫苗效果更佳；而且使用DC和B细胞的混合抗原提呈细胞效果好于使用单一抗原提呈细胞DC。
实施例4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抗原递送纳米粒子激活的抗原提呈细胞的膜组分负载于负载核酸的纳米粒子表面作为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)、CpG7909和CpG2006为佐剂制备负载裂解物组分的抗原递送纳米粒子，然后将抗原递送纳米粒子与抗原提呈细胞共孵育一段时间后激活抗原提呈细胞，并将抗原提呈细胞的膜组分负载于已经内部负载核酸的核酸递送前体纳米粒子表面后制备成纳米疫苗，用于治疗黑色素瘤。
(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1.5×105个B16F10细胞，在肿瘤长到体积分别为约1000mm3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组分。
(2)细菌细胞外囊泡(OMV)和癌细胞外囊泡的制备
将长双歧杆菌在5000g离心30分钟，然后弃去沉淀后收集上清液，将上清液使用1μm的滤膜过滤，在4℃下使用20W超声处理5分钟，然后在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膜组分，然后使用8M尿素水溶液裂解和溶解细菌外囊泡膜组分。
或者将长双歧杆菌在5000g离心30分钟，然后弃去沉淀后收集上清液，将上清液使用1μm的滤膜过滤，在4℃下使用20W超声处理5分钟，然后在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膜组分，然后使用吐温80水溶液裂解和溶解细菌膜组分。
(3)抗原递送粒子的制备
本实施例中抗原递送纳米粒子1采用复乳法制备，制备材料PLGA分子量为24KDa-38KDa，所采用的免疫佐剂为poly(I:C)、CpG7909和CpG2006且负载于纳米粒子内。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裂解液组分和佐剂，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备用。纳米粒子1平均粒径为250nm左右，每1mg PLGA纳米粒子1约负载130μg蛋白质或多肽组分，每1mg PLGA纳米粒子1所负载的poly(I:C)、CpG7909和CpG2006免疫佐 剂各0.005mg。
本实施例中抗原递送纳米粒子2制备材料和方法同纳米粒子1。纳米粒子2内部同时负载步骤(1)所制备的抗原组分和步骤(2)所制备的8M尿素溶解的细菌外囊泡膜组分，且二者质量比为1:1。所采用的免疫佐剂为poly(I:C)、CpG7909和CpG2006且负载于纳米粒子内。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抗原组分、细菌外囊泡组分和佐剂，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备用。纳米粒子2平均粒径为250nm左右，每1mg PLGA纳米粒子2约负载130μg蛋白质或多肽组分，每1mg PLGA纳米粒2所负载的poly(I:C)、CpG7909和CpG2006免疫佐剂各0.005mg。
本实施例中纳米粒子3制备材料和制备方法同纳米粒1。纳米粒子3内部同时负载步骤(1)所制备的抗原组分和步骤(2)所制备的吐温80溶解的细菌外囊泡膜组分，且二者质量比为1:1。所采用的免疫佐剂为poly(I:C)、CpG7909和CpG2006且负载于纳米粒子内。在制备过程中首先采用复乳法在纳米粒子内部负肿瘤组织载裂解液组分、细菌外囊泡组分和佐剂，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备用。纳米粒子3平均粒径为250nm左右，每1mg PLGA纳米粒子3约负载30μg蛋白质或多肽组分，每1mg PLGA纳米粒3所负载的poly(I:C)、CpG7909和CpG2006免疫佐剂各0.005mg。
空白纳米粒子4的制备材料和制备方法同纳米粒子1，但是空白纳米粒子4只负载等量的佐剂而不负载任何的肿瘤组织裂解物中的抗原组分。纳米粒子4的粒径为250nm左右。
(4)负载mRNA的核酸递送前体粒子(纳米粒子5)的制备
本实施例中负载mRNA的核酸递送前体纳米粒子5所负载的mRNA可以编码四种多肽新生抗原B16-M20(Tubb3,FRRKAFLHWYTGEAMDEMEFTEAESNM)，B16-M24(Dag1,TAVITPPTTTTKKARVSTPKPATPSTD)，B16-M46(Actn4,NHSGLVTFQAFIDVMSRETTDTDTADQ)和TRP2:180-188(SVYDFFVWL)；纳米粒子同时负载有免疫佐剂poly(I:C)、CpG7909、CpG1018和蜂毒肽(GIGAVLKVLTTGLPALISWIKRKRQQ)。负载mRNA的粒子的制备材料和制备方法同步骤(3)，首先将mRNA、poly(I:C)、CpG1018、CpG7909和蜂毒肽在水中混合，然后采用复乳法将上述混合物负载于纳米粒子内部，然后将100mg PLGA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5平均粒径为210nm左右，每1mg PLGA纳米粒子约负载2μg mRNA组分，每1mgPLGA纳米粒负载poly(I:C)、CpG7909和CpG1018免疫佐剂各5μg，负载蜂毒肽40μg。
(5)B细胞的分离
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的CD19+B细胞。
(6)抗原提呈细胞的激活
将500μg的抗原递送纳米粒子1，或者500μg的抗原递送纳米粒子2，或者500μg的抗原递送纳米粒子3，或者500μg的纳米粒子4+等量的癌细胞裂解物组分和尿素溶解的细菌组分分别与B细胞(1000万个)在15mL RPMI1640完全培养基中共孵育48小时(37℃，5％CO2)，孵育体系中含IL-2(500U/mL)、IL-7(200U/mL)、IL-12(200U/mL)和CD80抗体(10ng/mL)。
(7)纳米疫苗的制备
通过在400g离心5分钟收集孵育后的B细胞(1000万个)，然后使用PBS洗涤细胞三遍，将细胞重悬在PBS水中后在低功率(10W)超声15分钟。然后将样品在500g离心5分钟并收集上清液，将上清液依次过孔径为30um、10um、5um、0.45um、0.22um的膜过滤后，将所得滤液样品在18000g离心60分钟后弃去上清液并将沉淀使用PBS重悬后与核酸递送前体纳米粒子5共孵育2分钟后超声1分钟，然后使用0.22um的滤膜反复共挤出，然后将挤出液在13000g离心30分钟，并使用10mL含2％蔗糖和2％甘露醇的超纯水重悬后冷冻干燥48h备用。其中，使用纳米粒子1激活的抗原提呈细胞的膜组分与纳米粒子5共作用后制备的纳米粒子为纳米疫苗6，粒径为220纳米,表面电位为-10mV；每1mg PLGA纳米粒子约负载10μg mRNA组分，每1mgPLGA纳米粒负载poly(I:C)、CpG7909和CpG1018免疫佐剂各5μg，负载蜂毒肽40μg；每1mg PLGA纳米粒子约负载40μg细胞膜组分。使用纳米粒子2激活的抗原提呈细胞的膜组分与纳米粒子5共作用后制备的纳米粒子为纳米疫苗7，粒径为220纳米,表面电位为-10mV；每1mg PLGA纳米粒子约负载10μg mRNA组分，每1mgPLGA纳米粒负载poly(I:C)、CpG7909和CpG1018免疫佐剂各5μg，负载蜂毒肽40μg；每1mg PLGA纳米粒子约负载40μg细胞膜组分。使用纳米粒子3激活的抗原提呈细胞的膜组分与纳米粒子5共作用后制备的纳米粒子为纳米疫苗8，粒径为220纳米,表面电位为-10mV；每1mg PLGA纳米粒子约负载10μg mRNA组分，每1mgPLGA纳米粒负载poly(I:C)、CpG7909和CpG1018免疫佐剂各5μg，负载蜂毒肽40μg；每1mg PLGA纳米粒子约负载40μg细胞膜组分。使用纳米粒子4激活的抗原提呈细胞的膜组分与纳米粒子5共作用后制备的纳米粒子为纳米疫苗9，粒径为220纳米,表面电位为-10mV；每1mg PLGA纳米粒子约负载10μg mRNA组分，每1mgPLGA纳米粒负载poly(I:C)、CpG7909和CpG1018免疫佐剂各5μg，负载蜂毒肽40μg；每1mg PLGA纳米粒子约负载40μg细胞膜组分。
(8)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1.5×105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天分别皮下注射1mg的纳米疫苗(疫苗6，或者疫苗7，或者疫苗8，或者疫苗9)或者皮下注射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。肿瘤体积采用公式v＝0.52×a×b2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2000mm3即视为小鼠死亡并将小鼠安乐死。
(9)实验结果
图5中PBS control、Nanovaccine 6、Nanovaccine 7、Nanovaccine 8、Nanovaccine 9依次为PBS对照组、纳米疫苗6、纳米疫苗7、纳米疫苗8、纳米疫苗9。结果显示，如图5中a和b所示，PBS对照组小鼠的肿瘤生长速度很快，生存期很短。而几种纳米疫苗处理的小鼠其肿瘤生长速度都明显变慢，而且部分小鼠肿瘤消失痊愈。而且，纳米疫苗7的效果优于纳米疫苗6、纳米疫苗8和纳米疫苗9，这说明使用适当方法裂解和溶解的细菌外囊泡组分负载到纳米粒子后所激活的抗原提呈细胞的膜组分负载到本公开所述粒子疫苗表面后有利于提高疫苗效果。综上所述，本公开所述的疫苗对癌症具有优异的治疗效果。
实施例5疫苗用于治疗癌症
本实施例中，首先使用6M盐酸胍裂解B16F10黑色素瘤癌细胞全细胞抗原。然后，以PLGA为微米粒骨架材料，以CpG BW006(B类)、CPG2216(A类)和Poly ICLC为免疫佐剂制备负载有癌细胞全细胞抗原的抗原递送微米粒子系统。使用抗原递送微米粒子激活抗原提呈细胞后，将抗原提呈细胞的膜组分负载于负载核酸的核酸递送前体纳米粒子表面制备得到纳米疫苗用于治疗癌症。
(1)癌细胞的裂解
将培养的B16F10黑色素瘤癌细胞系收集后在350g离心5分钟，然后弃去上清并用PBS洗涤两遍，然后采用6M盐酸胍重悬和裂解癌细胞，癌细胞全细胞抗原裂解并溶于6M盐酸胍后即为制备抗原递送微米粒子系统的抗原原料来源。
(2)抗原递送粒子(微米粒子)的制备
本实施例中抗原递送微米粒子采用复乳法制备。所采用的微米粒子1制备材料PLGA分子量为38KDa-54KDa，所采用的免疫佐剂为CpG BW006、CpG 2216和Poly ICLC。Poly ICLC是toll样受体3激动剂，而各类CpG是Toll样受体9激动剂，而Toll样受体3和Toll样受体9均位于细胞内的内吞体膜结构中。首先将裂解物组分和免疫佐剂共负载于微米粒子内，然后在10000g离心15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7mL PBS重悬然后加入3mL癌细胞裂解液组分(蛋白质浓度50mg/mL)并室温作用10min，得到内外都负载裂解物的微米粒子1。该微米粒子平均粒径为2.50μm左右，表面电位为-22mV左右；每1mg PLGA微米粒子1约负载140μg蛋白质或多肽组分，负载的CpG BW006(B类)、CpG 2216(A类)和Poly ICLC各0.02mg。
微米粒子2制备材料和制备方法相同，负载的免疫佐剂为CpG2336(A类)、CpG 2216(A类)和Poly ICLC。对照微米粒子2粒径为2.50μm左右，表面电位为-22mV左右，每1mg PLGA微米粒子约负载140μg蛋白质或多肽组分，每1mgPLGA微米粒所负载的CpG2336(A类)、CpG 2216(A类)和Poly ICLC免疫佐剂各为0.02mg。
微米粒子3制备材料和制备方法相同，负载的免疫佐剂为CpG BW006(B类)和CpG 2216(A类)。对照微米粒子3每1mgPLGA微米粒所使用的佐剂为0.02mg，粒径为2.50μm左右，表面电位为-22mV左右，每1mg PLGA微米粒子约负载140μg蛋白质或多肽组分，每1mgPLGA微米粒所负载的CpG BW006(B类)和CpG 2216(A类)各0.03mg。
(3)负载mRNA的核酸递送前体粒子(纳米粒子1)的制备
本实施例中负载mRNA的核酸递送前体纳米粒子1所负载的mRNA可以编码四种多肽新生抗原B16-M20(Tubb3,FRRKAFLHWYTGEAMDEMEFTEAESNM)，B16-M24(Dag1,TAVITPPTTTTKKARVSTPKPATPSTD)，B16-M46(Actn4,NHSGLVTFQAFIDVMSRETTDTDTADQ)和TRP2:180-188(SVYDFFVWL)；纳米粒子同时负载有免疫佐剂CpG2395、CpG1018、poly(I:C)和带正电荷的多肽蜂毒肽(GIGAVLKVLTTGLPALISWIKRKRQQ)。负载mRNA的PLGA纳米粒子的制备材料和制备方法同实施例3，首先将mRNA、poly(I:C)、CpG2395、CpG1018和蜂毒肽在水中混合，然后采用复乳法将上述混合物负载于纳米粒子内部，然后将100mg PLGA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210nm左右，每1mg PLGA纳米粒子约负载1μg mRNA组分，每1mgPLGA纳米粒负载poly(I:C)、CpG2395和CpG1018免疫佐剂各2μg，负载聚精氨酸10μg。
(4)抗原提呈细胞的制备
处死小鼠后收集小鼠淋巴结和脾脏，将小鼠淋巴结或者脾脏切碎研磨分别通过细胞筛网过滤制备单细胞悬液，将淋巴结单细胞悬液和脾脏单细胞悬液混合后，使用流式细胞术从中分选出CD19+B细胞和CD11c+的DC，将B细胞和DC按数量比1:1混合后作为混合抗原提呈细胞使用。
(5)抗原提呈细胞的激活
将负载癌细胞全细胞组分的抗原递送微米粒子(100μg)与步骤(4)制备的混合抗原提呈细胞(2000万个)在20mL高糖DMEM完全培养基中共孵育48小时(37℃，5％CO2)，孵育体系中含有GM-CSF(500U/mL)、IL-2(500U/mL)、IL-15(200U/mL)和CD86抗体(10ng/mL)。
(6)抗原提呈细胞来源的核酸递送纳米粒子的制备
通过在400g离心5分钟收集孵育后的DC和B细胞，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中重悬后与步骤(3)制备的核酸递送前体纳米粒子1混合后共孵育10分钟，然后使用0.22um的滤膜反复共挤出，然后将挤在在13000g离心30分钟，并使用10mL含4％海藻糖的超纯水重悬后冷冻干燥48h备用。使用微米粒子1激活的抗原提呈细胞的膜组分与纳米粒子1共作用得到的纳米粒子为纳米疫苗2，粒径为220纳米，表面电位为-7mV；每1mg PLGA纳米粒子约负载1μg mRNA组分，每1mgPLGA纳米粒负载poly(I:C)、CpG2395和CpG1018免疫佐剂各2μg，负载聚精氨酸10μg；每1mg PLGA纳米粒子约负载200μg细胞膜组分。使用微米粒子2激活的抗原提呈细胞的膜组分与纳米粒子1共作用得到的纳米粒子为纳米疫苗3，粒径为220纳米，表面电位为-7mV；每1mg PLGA纳米粒子约负载1μg mRNA组分，每1mgPLGA纳米粒 负载poly(I:C)、CpG2395和CpG1018免疫佐剂各2μg，负载聚精氨酸10μg，每1mg PLGA纳米粒子约负载200μg细胞膜组分。使用微米粒子3激活的抗原提呈细胞的膜组分与纳米粒子1共作用得到的纳米粒子为纳米疫苗4，粒径为220纳米，表面电位为-7mV；每1mg PLGA纳米粒子约负载1μg mRNA组分，每1mgPLGA纳米粒负载poly(I:C)、CpG2395和CpG1018免疫佐剂各2μg，负载聚精氨酸10μg，每1mg PLGA纳米粒子约负载200μg细胞膜组分。
(7)纳米疫苗癌症的治疗
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1.5×105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天分别皮下注射0.8mg的纳米疫苗(纳米疫苗2，或者纳米疫苗3，或者纳米疫苗4)或者皮下注射100μL PBS。小鼠肿瘤生长速度和生存期监测方法同上。
(8)实验结果
图6中PBS control、Nanovaccine 2、Nanovaccine 3、Nanovaccine 4依次为PBS对照组、纳米疫苗2、纳米疫苗3、纳米疫苗4。结果显示，如图6所示，对照组小鼠的肿瘤都长大，而纳米疫苗处理小鼠肿瘤生长速度都明显变慢且生存期明显延长。而且，负载CpG佐剂和Poly ICLC混合佐剂的抗原递送微米粒子激活的抗原提呈细胞与负载核酸的核酸递送前体纳米粒子共作用制备的纳米疫苗效果优于负载两种CpG混合佐剂的抗原递送微米粒子激活的抗原提呈细胞与负载核酸的核酸递送前体纳米粒子共作用制备的疫苗。而且，负载一种B类CpG、一种A类CpG和Poly ICLC混合佐剂的抗原递送微米粒子激活的抗原提呈细胞与负载核酸的核酸递送前体纳米粒子共作用制备的纳米疫苗效果好于使用负载两种A类CpG和PolyICLC混合佐剂的抗原递送微米粒子激活的抗原提呈细胞和负载核酸的核酸递送前体纳米粒子共作用制备的纳米疫苗。这说明负载两种不同toll样受体的混合佐剂的抗原递送微米粒子激活的抗原提呈细胞与负载核酸的核酸递送前体粒子共作用制备的粒子疫苗效果更好，而且，含有B类CpG的混合CpG与Toll样受体3激动剂作为混合佐剂的抗原递送微米粒子激活的抗原提呈细胞与负载核酸的核酸递送前体粒子共作用制备的核酸递送粒子疫苗效果更好。
实施例6疫苗用于癌症的预防
本实施例中，首先使用8M尿素裂解B16F10黑色素瘤肿瘤组织，并溶解肿瘤组织裂解物组分。然后，以PLGA为微米粒骨架材料，以Poly(I:C)、CpG2006(B类)和CpGSL01(B类)为免疫佐剂制备负载有癌细胞全细胞抗原的抗原递送微米粒子，使用抗原递送微米粒子激活抗原提呈细胞后将被激活的抗原提呈细胞与负载核酸的核酸递送前体微米粒子共作用制备核酸递送微米疫苗用于预防癌症。
(1)肿瘤组织的收集及裂解
在每只C57BL/6小鼠背部皮下接种1.5×105个B16F10细胞，在肿瘤长到体积分别为约1000mm3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8M尿素裂解细胞，并溶解全细胞裂解物，使用蛋白质提取分离技 术从全细胞裂解物中分离提取出总蛋白。分离提取得到的总蛋白即为制备纳米粒子系统的抗原原料来源。
(2)抗原递送粒子(微米粒子)的制备
本实施例中抗原递送微米粒子采用溶剂挥发法制备。微米粒子1所采用的制备材料PLGA分子量为38KDa-54KDa，所采用的免疫佐剂为Poly(I:C)、CpG2006和CpGSL01，且裂解物抗原组分和佐剂负载于微米粒子内部。制备方法如前所述，在微米粒子内部负载裂解物抗原组分和佐剂后，将100mg微米粒子在12000g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，得冻干粉后备用。该微米粒子平均粒径为1.0μm左右，微米粒子表面电位为-13mV左右；每1mg PLGA微米粒子约负载80μg蛋白质或多肽组分，每1mg PLGA微米粒负载Poly(I:C)、CpG2006和CpGSL01各0.02mg。
抗原递送微米粒2子制备材料和制备方法同上，粒径为1.0μm左右，负载等量的裂解物抗原组分，负载免疫佐剂为Poly(I:C)，每1mg PLGA负载Poly(I:C)0.06mg。
抗原递送微米粒子3粒径为1.0μm左右，负载等量的裂解物抗原组分，负载免疫佐剂为Poly(I:C)、CpG1585(A类)和CpG2216(A类)，每1mg PLGA负载Poly(I:C)、CpG1585(A类)和CpG2216(A类)各0.02mg。
(3)负载mRNA的核酸递送前体粒子(微米粒子4)的制备
本实施例中负载mRNA的核酸递送前体微米粒子4所负载的mRNA可以编码四种多肽新生抗原B16-M20(Tubb3,FRRKAFLHWYTGEAMDEMEFTEAESNM)，B16-M24(Dag1,TAVITPPTTTTKKARVSTPKPATPSTD)，B16-M46(Actn4,NHSGLVTFQAFIDVMSRETTDTDTADQ)和TRP2:180-188(SVYDFFVWL)；微米粒子同时负载有免疫佐剂poly(I:C)、CpG(B类和C类混合CpG)和RALA多肽(WEARLARALARALARHLARALARALRACEA)。负载mRNA的微米粒子的制备材料和制备方法同步骤(2)，首先将mRNA、poly(I:C)、CpGSL03(C类)、CpG1018(B类)和RALA多肽在水中混合，然后采用复乳法将上述混合物负载于微米粒子内部，然后将100mg PLGA微米粒子在9000g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。该微米粒子4平均粒径为1.0μm左右，每1mg PLGA微米粒子约负载5μg mRNA组分，每1mgPLGA微米粒子负载poly(I:C)、CpG SL03和CpG1018免疫佐剂各30μg，负载RALA多肽200μg。
(3)DC和B细胞的制备
处死C57BL/6后摘取小鼠淋巴结，制备小鼠淋巴结单细胞悬液，然后使用流式细胞术从淋巴结细胞单细胞悬液中分选出CD11c+DC和CD19+B细胞。
(4)抗原提呈细胞的激活
将负载癌细胞全细胞组分的抗原递送微米粒子1(800μg)、抗原递送微米粒子2(800μg)或抗原递送微米粒子3(800μg)与DC(500万个)和B细胞(500万个)在20mL高糖DMEM完全培养基中共孵育72小时(37℃，5％CO2)；孵育体系中含有GM-CSF(500U/mL)、 IL-2(500U/mL)、IL-7(200U/mL)。
(5)基于抗原提呈细胞的核酸递送微米疫苗的制备
通过在400g离心5分钟收集孵育后的DC和B细胞，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℃使用匀浆机在2000rpm搅拌破坏处理25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15000g离心30分钟后弃去上清液收集沉淀，将沉淀在PBS中重悬后与步骤(3)制备的纳米粒子1混合后共孵育10分钟，然后使用2μm的滤膜反复共挤出，然后将挤在在13000g离心30分钟，并使用10mL含4％海藻糖的超纯水重悬后冷冻干燥48h后备用。其中，由抗原递送微米粒子1激活的抗原提呈细胞的膜组分与核酸递送前体微米粒子4共作用后所制备的核酸递送微米粒子为微米疫苗5，粒径为1.1μm，表面电位为-7mV；每1mg PLGA约负载5μg mRNA组分，每1mgPLGA负载poly(I:C)、CpG SL03和CpG1018免疫佐剂各30μg，负载RALA多肽200μg；每1mg PLGA约负载80μg细胞膜组分。由抗原递送微米粒子2激活的抗原提呈细胞的膜组分与核酸递送前体微米粒子4共作用后所制备的核酸递送微米粒子为微米疫苗6，粒径为1.1μm，表面电位为-7mV；每1mg PLGA约负载5μg mRNA组分，每1mgPLGA负载poly(I:C)、CpG SL03和CpG1018免疫佐剂各30μg，负载RALA多肽200μg；每1mg PLGA约负载80μg细胞膜组分。由抗原递送微米粒子3激活的抗原提呈细胞的膜组分与核酸递送前体微米粒子4共作用后所制备的核酸递送微米粒子为微米疫苗7，粒径为1.1μm，表面电位为-7mV；每1mg PLGA约负载5μg mRNA组分，每1mgPLGA负载poly(I:C)、CpG SL03和CpG1018免疫佐剂各30μg，负载RALA多肽200μg；每1mg PLGA约负载80μg细胞膜组分。
(7)微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，在小鼠接种肿瘤前第-35天，第-28天，第-21天，第-14天和第-7天分别在小鼠皮下接种1mg刚冷冻干燥后的微米疫苗(微米疫苗5，或者微米疫苗6，或者微米疫苗7)、或者接种100μL PBS。第0天给每只小鼠背部右下方皮下接种1.5×105个B16F10细胞。小鼠肿瘤生长速度和生存期监测方法同上。
(8)实验结果
图7中PBS control、Microvaccine 5、Microvaccine 6、Microvaccine 7依次为PBS对照组、微米疫苗5、微米疫苗6、微米疫苗7。结果显示，如图7所示，PBS对照组小鼠的肿瘤都很快长大，而微米疫苗处理组的小鼠肿瘤生长速度都明显变慢，且大部分小鼠癌细胞接种后肿瘤消失。而且，负载两种B类CpG与Poly(I:C)作为混合佐剂的抗原递送微米粒子激活的抗原提呈细胞与负载核酸的核酸递送前体微米粒子共作用后所制备的微米疫苗效果好于负载两种A类CpG与Poly(I:C)作为混合佐剂的抗原递送微米粒子或者只负载Poly(I:C)作为佐剂的抗原递送微米粒子激活的抗原提呈细胞与负载核酸的核酸递送前体微米粒子共作用后所制备的微米疫苗。
实施例7疫苗用于治疗T淋巴瘤
本实施例以E.G7-OVA小鼠T淋巴瘤为癌症模型来说明如何使用纳米粒子激活的抗原提呈细胞与负载核酸的纳米粒子共作用后制备的纳米疫苗用于治疗T淋巴瘤。首先裂解E.G7-OVA细胞并使用蛋白酶在体外先降解裂解物为多肽。在实际应用中也可以使用其它酶或者其它方法先将全细胞组分中的蛋白质降解为多肽。然后再分别制备水溶性组分和非水溶性组分，并按质量比1:1混合。然后，以PLA为纳米粒骨架材料，以CpG2395(C类)、CpGM362(C类)和Poly ICLC为免疫佐剂制备抗原递送纳米粒子，并用该抗原递送纳米粒子体外激活抗原提呈细胞，然后将激活的抗原提呈细胞与负载核酸的核酸递送前体纳米粒子共作用制备纳米疫苗。
(1)癌细胞的裂解及各组分的收集
将培养的E.G7-OVA细胞系收集后在350g离心5分钟，然后弃去上清并用PBS洗涤两遍，然后采用超纯水重悬细胞并反复冻融5次，并可伴有超声以破坏裂解细胞。待细胞裂解后，加入胰蛋白酶(Trypsin，0.5mg/mL)和糜蛋白酶(Chymotrypsin，0.5mg/mL)共孵育30分钟，然后在95℃加热10分钟灭活蛋白酶备用。然后将裂解物以3000g的转速离心6分钟并取上清液即为可溶于纯水的水溶性组分；在所得沉淀部分中加入10％脱氧胆酸钠(含1M精氨酸)水溶液溶解沉淀部分即可将不溶于纯水的非水溶性组分转化为在10％脱氧胆酸钠(含1M精氨酸)水溶液中可溶。然后将水溶性抗原和非水溶性抗原按照质量比1:1混合，该混合物为制备纳米粒子的原料来源。
(2)卡介苗(BCG)的裂解和溶解
收集BCG，使用10％脱氧胆酸钠(含1M精氨酸)水溶液裂解BCG后使用10％脱氧胆酸钠(含1M精氨酸)水溶液溶解裂解组分备用。
(3)抗原递送粒子(纳米粒子)的制备
本实施例中纳米粒子1采用溶剂挥发法制备。抗原递送纳米粒子1制备材料PLA分子量为20KDa，纳米粒子内部负载癌细胞裂解物、细菌裂解物以及免疫佐剂，表面负载癌细胞裂解物组分。所采用的免疫佐剂为CpG2395(C类)、CpGM362(C类)和poly ICLC，且佐剂负载于纳米粒子内部，制备纳米粒子时使用的癌细胞裂解物组分与细菌裂解物组分的质量比为1:1。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癌细胞裂解物抗原、细菌裂解物组分和佐剂，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。使用前将20mg纳米粒重悬于0.9mL PBS中，并于0.1mL含有癌细胞裂解物抗原组分(80mg/mL)的样品室温混合孵育5分钟后即可使用。纳米粒子1平均粒径为400nm左右，每1mg PLA纳米粒子1约负载400μg蛋白质或多肽组分，每1mgPLA纳米粒负载CpG2395(C类)、CpGM362(C类)和Poly ICLC免疫佐剂各0.005mg。
抗原递送纳米粒子2制备材料和制备方法纳米粒子1，粒径为400nm左右，每1mg PLA纳米粒子约负载140μg蛋白质或多肽组分，每1mgPLA负载CpG1585(A类)、CpG2336(A类)和Poly ICLC各0.005mg。
(4)负载mRNA的核酸递送前体粒子(纳米粒子3)的制备
本实施例中负载mRNA的纳米粒子3所负载的mRNA可以编码OVA抗原；纳米粒子同时负载有免疫佐剂poly ICLC、CpG、精氨酸和赖氨酸。负载mRNA的纳米粒子的制备材料和制备方法同步骤(3)，首先将mRNA、poly ICLC、CpG2395(C类)、CpG7909(B类)、精氨酸和赖氨酸在水中混合，然后采用复乳法将上述混合物负载于纳米粒子内部，然后将100mg PLGA纳米粒子在9000g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。纳米粒子3平均粒径为380nm左右，每1mg PLA纳米粒子约负载40μg mRNA组分，每1mgPLA纳米粒子负载poly ICLC、CpG2395和CpG7909免疫佐剂各5μg，负载精氨酸和赖氨酸各180μg。
(5)抗原提呈细胞的制备
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。本实施例中同时使用BMDM作为抗原提呈细胞。BMDC制备方法同实施例1。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即可。
(6)抗原提呈细胞的激活
将抗原递送纳米粒子1(1000μg)与BMDC(3000万个)在RPMI1640完全培养基中共孵育48小时(37℃，5％CO2)；孵育体系中含有IL-2(500U/mL)、IL-7(500U/mL)、IL-15(500U/mL)和CD80抗体(10ng/mL)。
或者将抗原递送纳米粒子1(1000μg)与BMDC(1000万个)、BMDM(1000万个)、B细胞(1000万个)在20mL RPMI1640完全培养基中共孵育48小时(37℃，5％CO2)，孵育体系中含有IL-2(500U/mL)、IL-7(500U/mL)、IL-15(500U/mL)和CD80抗体(10ng/mL)。
或者将抗原递送纳米粒子2(1000μg)与BMDC(1000万个)、BMDM(1000万个)、B细胞(1000万个)在20mL RPMI1640完全培养基中共孵育48小时(37℃，5％CO2)，孵育体系中含有IL-2(500U/mL)、IL-7(500U/mL)、IL-15(500U/mL)和CD80抗体(10ng/mL)。
(7)基于抗原提呈细胞的纳米疫苗的制备
通过在400g离心5分钟收集与抗原递送纳米粒子1孵育后的BMDC(3000万个)，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水中后在高压均质机中(5000bar)中处理5分钟。然后将样品在2000g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，将上清液与50mg步骤(4)所制备的核酸递送前体纳米粒子3在4℃共孵育1小时，然后使用0.45μm的滤膜反复共挤出，将挤出液在13000g离心20分钟后弃去上清液收集沉淀，将沉淀在4％海藻糖水溶液中重悬冷冻干燥48小时后即得纳米疫苗4，粒径为400纳米，表面电位为-7mV，每1mg PLA约负载40μg mRNA组分，每1mgPLA负载Poly ICLC、CpG2395和CpG7909免疫佐剂各5μg，负载精氨酸和赖氨酸各180μg；每1mg PLGA约负载280μg膜组分。在室温储存12个月后使用。
或者通过在400g离心5分钟收集与抗原递送纳米粒子孵育后BMDC(1000万个)、B细胞(1000万个)及BMDM(1000万个)，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水中后在高压均质机中(5000bar)中处理5分钟。然后将样品在2000g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，将上清液与步骤(4)所制备的核酸递送前体纳米粒子3(50mg)在4℃共孵育1小时，然后使用0.45μm的滤膜反复共挤出，将挤出液在13000g离心20分钟后弃去上清液收集沉淀，将沉淀在4％海藻糖水溶液中重悬冷冻干燥48小时后即得纳米疫苗。其中，使用抗原递送纳米粒子1激活的混合抗原提呈细胞与纳米粒子3共作用所制备的纳米疫苗为纳米疫苗5，粒径为400纳米，表面电位为-7mV，每1mg PLA约负载40μg mRNA组分，每1mgPLGA负载Poly ICLC、CpG2395和CpG7909免疫佐剂各5μg，负载精氨酸和赖氨酸各180μg；每1mg PLA约负载280μg膜组分；制备好后立即使用或者在室温储存12个月后使用；使用抗原递送纳米粒子2激活的混合抗原提呈细胞与纳米粒子3共作用所制备的纳米疫苗为纳米疫苗6，粒径为400纳米，表面电位为-7mV，每1mg PLGA约负载40μg mRNA组分，每1mgPLA负载Poly ICLC、CpG2395和CpG7909免疫佐剂各5μg，负载精氨酸和赖氨酸各180μg；每1mg PLA约负载280μg膜组分；在室温储存12个月后使用。
(8)纳米疫苗用于癌症的治疗
选取6-8周的雌性C57BL/6为模型小鼠制备荷瘤小鼠。在第0天给每只小鼠皮下接种5×105个E.G7-OVA细胞，在第5、第8天、第13天和第20天分别给小鼠注射100μLPBS或者1mg的纳米疫苗(刚制备好的纳米疫苗5，或者储存12个月后的纳米疫苗4，或者储存12个月后的纳米疫苗5，或者储存12个月后的纳米疫苗6)。小鼠肿瘤体积及生存期监测方法同上。
(9)实验结果
图8中PBS control、Nanovaccine 4(Long term storage)、Nanovaccine 5(Long term storage)、 Nanovaccine 6(Long term storage)、Nanovaccine 5(Fresh)依次为PBS对照组、长期储存的纳米疫苗4、长期储存的纳米疫苗5、长期储存的纳米疫苗6、刚制备好的纳米疫苗5。结果显示，如图8所示，PBS对照组的小鼠的肿瘤都生长很快小鼠生存期很短。与对照组相比，几种纳米疫苗处理的小鼠肿瘤生长速度都明显变慢且生存期明显延长。而且，使用两种C类CpG与Poly ICLC作为混合佐剂的抗原递送纳米粒子激活的抗原提呈细胞与负载核酸的核酸递送前体纳米粒子共作用制备的纳米疫苗5效果好于两种A类CpG与Poly ICLC作为混合佐剂的抗原递送纳米粒子激活的抗原提呈细胞与负载核酸的核酸递送前体纳米粒子共作用制备的纳米疫苗6；而且，使用混合抗原提呈细胞的膜组分的纳米疫苗5效果好于使用单一抗原提呈细胞的膜组分的纳米疫苗4。而且，上述纳米疫苗在室温储存12个月后效果与刚制备好后相同，说明上述纳米疫苗稳定性良好。
实施例8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1.5×105个B16F10细胞，在肿瘤长到体积分别为约1000mm3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(0.5mg/mL)作用15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8M尿素(含200mM氯化钠)水溶液溶解非水溶性抗原。将水溶性抗原和8M尿素(含200mM氯化钠)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)抗原递送粒子(纳米粒子)的制备
本实施例中抗原递送纳米粒采用复乳法制备。所采用的纳米粒子制备材料为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。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，在内部负载上述组分后将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0.04mg,负载KALA多肽0.3mg。
抗原递送纳米粒子2的制备材料和方法相同，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抗原组分。
抗原递送纳米粒子3的制备材料和制备方法相同，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)0.04mg,负载CpG1018为0.08mg,负载KALA多肽0.3mg。
(3)负载mRNA的核酸递送前体粒子(纳米粒子4)的制备
本实施例中负载mRNA的纳米粒子4所负载的mRNA可以编码四种多肽新生抗原B16-M20(Tubb3,FRRKAFLHWYTGEAMDEMEFTEAESNM)，B16-M24(Dag1,TAVITPPTTTTKKARVSTPKPATPSTD)，B16-M46(Actn4,NHSGLVTFQAFIDVMSRETTDTDTADQ)和TRP2:180-188(SVYDFFVWL)；纳米粒子同时负载有免疫佐剂poly(I:C)、CpG和促进溶酶体逃逸的物质KALA多肽(WEAKLAKALAKALAKHLAKALAKALKACEA)。负载mRNA的纳米粒子的制备材料和制备方法同步骤(2)，首先将mRNA、poly(I:C)、CpG7909、CpGBW2006和KALA多肽在水中混合，然后采用复乳法将上述混合物负载于纳米粒子内部，然后将100mg PLGA纳米粒子在9000g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4平均粒径为210nm左右，每1mg PLGA纳米粒子约负载100μg mRNA组分，每1mgPLGA纳米粒子负载poly(I:C)、CpG7909、CpGBW2006免疫佐剂各1μg，负载KALA多肽400μg。
(4)抗原提呈细胞的制备
本实施例使用BMDC和BMDM作为混合抗原提呈细胞。BMDC和BMDM制备方法同上。
(5)抗原提呈细胞的激活
将1000μg抗原递送纳米粒子(纳米粒子1，或者纳米粒子2，或者纳米粒子3)与BMDC(1000万个)和BMDM(1000万个)在15mL高糖DMEM完全培养基中共孵育48小时(37℃，5％CO2)；孵育体系中含有GM-CSF(200U/mL)、M-CSF(200U/mL)、IL-2(500U/mL)、IL-7(200U/mL)、IL-12(200U/mL)和CD80抗体(10ng/mL)。
(6)基于抗原提呈细胞的纳米疫苗的制备
通过在400g离心5分钟收集孵育后的BMDC和BMDM，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1200rpm 3min离心清洗三次，然后在磷酸酶抑制剂和蛋白酶抑制剂的存在下超声机械破坏抗原提呈细胞。经过离心后所获细胞膜用10mM pH 7.5的Tris-HCl和1mM EDTA的溶液清洗。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12000g离心25分钟后弃去上清液收集沉淀，将沉淀在PBS中重悬后与核酸递送前体纳米粒子4在室温共孵育15分钟，然后使用0.22μm的滤膜反复共挤出，将挤出液在15000g离心30分钟后弃去上清液，将沉淀在含有的4％甘露醇的生理盐水中重悬后冷冻干燥，即得核酸递送纳米疫苗。其中,使用抗原递送纳米粒子1激活的抗原提呈细胞与核酸递送前体纳米粒子4共作用所制备的核酸递送纳米粒子为纳米疫苗5，粒径为220纳米，表面电位为-7mV；每1mg PLGA约负载10μg mRNA组分，每1mgPLGA负载poly(I:C)、CpG7909、CpGBW2006免疫佐剂各1μg，负载KALA多肽400μg；每1mg PLGA约负载280μg膜组分。使用抗原递送纳米粒子2激活的抗原提呈细胞与核酸递送前体纳米粒子4共作用所制备的核酸递送纳米粒子为纳米疫苗6，粒径为220纳米，表面电位为-7mV；每1mg PLGA约负载10μg mRNA组分，每1mgPLGA负载poly(I:C)、CpG7909、CpGBW2006免疫佐剂各1μg，负载KALA多肽400μg；每1mg PLGA约负载280μg膜组分。 使用抗原递送纳米粒子3激活的抗原提呈细胞与核酸递送前体纳米粒子4共作用所制备的核酸递送纳米粒子为纳米疫苗7，粒径为220纳米，表面电位为-7mV；每1mg PLGA约负载10μg mRNA组分，每1mgPLGA负载poly(I:C)、CpG7909、CpGBW2006免疫佐剂各1μg，负载KALA多肽400μg；每1mg PLGA约负载280μg膜组分。
(7)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1.5×105个B16F10细胞。在接种黑色素瘤后第4天、第7天、第10天、第15天和第20天分别皮下注射100μL PBS或者0.8mg相应的纳米疫苗。在实验中，小鼠肿瘤体积和生存期监测方法同上。
(8)实验结果
如图9所示，图9中PBS control、Nanovaccine 5、Nanovaccine 6、Nanovaccine 7依次为PBS对照组、纳米疫苗5、纳米疫苗6、纳米疫苗7。结果显示，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，加入促进溶酶体逃逸物质的抗原递送纳米粒子激活的抗原提呈细胞与负载核酸的核酸递送前体纳米粒子共作用后制备的纳米疫苗5效果好于未加入溶酶体逃逸的抗原递送纳米粒子激活的抗原提呈细胞与负载核酸的核酸递送前体纳米粒子共作用后制备的纳米疫苗6；使用两种CpG和Poly(I:C)作为混合佐剂的抗原递送纳米粒子激活的抗原提呈细胞与负载核酸的核酸递送前体纳米粒子共作用后制备的纳米疫苗5治疗效果好于只使用一种CpG和Poly(I:C)混合佐剂的抗原递送纳米粒子激活的抗原提呈细胞与负载核酸的核酸递送前体纳米粒子共作用后制备的纳米疫苗7。这说明溶酶体逃逸物质的加入以及混合佐剂的使用都有利于提高纳米疫苗的疗效。综上所述，本公开所述的纳米疫苗对癌症具有良好的治疗效果。
实施例9微米疫苗用于癌症的预防
(1)癌细胞及细菌外囊泡组分的制备
将培养的E.G7-OVA小鼠T淋巴瘤细胞在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，然后使用8M尿素水溶液(含500mM氯化钠)裂解癌细胞并溶解裂解物组分，即为制备抗原递送微米粒子系统的抗原组分。
将嗜酸乳杆菌在5000g离心30分钟，然后弃去沉淀后收集上清液，将上清液使用1μm的滤膜过滤，然后在16000g离心90分钟，弃去上清液后所得沉淀即为细菌外囊泡组分，将沉淀使用8M尿素水溶液(含500mM氯化钠)裂解后溶解细菌外囊泡组分。
(2)抗原递送粒子(微米粒子)的制备
本实施例中制备抗原递送微米粒子采用复乳法。微米粒子1骨架材料为PLA，PLA分子量为40KDa。所采用的免疫佐剂为CpG2006(B类)、CpG2216(A类)和Poly ICLC，所采用的带正电荷物质为精氨酸和聚组氨酸。微米粒子制备时所使用的癌细胞裂解物组分和 细菌外囊泡组分质量比为1:1。制备时先采用复乳法制备内部负载癌细胞裂解物抗原组分、细菌外囊泡组分、佐剂、精氨酸和聚组氨酸的抗原递送微米粒子,尔后，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后即得抗原递送微米粒子1，平均粒径为4.98μm左右，每1mg PLGA微米粒子1约负载100μg蛋白质或多肽组分，负载CpG2006，CpG2216和Poly ICLC各0.07mg，负载精氨酸和聚组氨酸各0.05mg。
抗原递送微米粒子2制备材料和制备方法同微米粒子1，粒径为4.98μm左右，负载等量精氨酸、聚组氨酸和等量的癌细胞裂解物组分和细菌外囊泡组分，但是不负载任何佐剂。
(3)负载mRNA的核酸递送前体粒子(微米粒子3)的制备
本实施例中负载mRNA的微米粒子3所负载的mRNA可以编码OVA抗原；微米粒子同时负载有免疫佐剂poly(I:C)、两种CpG(B类和C类)和聚组氨酸。负载mRNA的微米粒子的制备材料和制备方法同步骤(2)，首先将mRNA、poly(I:C)、CpG2395(C类)、CpG1018(B类)和聚组氨酸在水中混合，然后采用复乳法将上述混合物负载于微米粒子内部，然后将100mg PLA微米粒子在9000g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。微米粒子3平均粒径为4.95μm左右，每1mg PLA微米粒子约负载100μg mRNA组分，每1mgPLGA微米粒子负载poly(I:C)、CpG2395和CpG1018免疫佐剂各60μg，负载聚组氨酸250μg。
(4)抗原提呈细胞的制备
本实施例使用BMDC、B细胞以及BMDM作为抗原提呈细胞。BMDC及BMDM制备方法同上。B细胞来自小鼠外周血PBMC，制备方法同上。将BMDC、B细胞和BMDM按数量比2:1:1混和后即为混合抗原提呈细胞。
(5)抗原提呈细胞的激活
将1000μg的抗原递送微米粒子1或抗原递送微米粒子2分别与4000万个混合抗原提呈细胞(含2000万个BMDC，1000万个B细胞及1000万个BMDM)在15mL高糖DMEM完全培养基中共孵育48小时(37℃，5％CO2)；孵育体系中含有IL-2(500U/mL)、IL-7(200U/mL)、IL-15(200U/mL)、和CD40抗体(20mg/mL)。
(6)抗原提呈细胞来源的核酸疫苗的制备
通过在400g离心5分钟收集孵育后的4000万个混合抗原提呈细胞(微米粒子1或者微米粒子2激活)，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(20W)超声2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5000g离心10分钟后收集上清液，将上清液通过0.22μm的膜反复挤出，将挤出液使用高压均质机(10000bar)处理3分钟，然后在16000g离心40分钟后弃去上清液收集沉淀，将沉淀在PBS中重悬后与30mg步骤(3)制备的微米粒子3共孵育10分钟后，使用5μm的滤膜反复共挤出，将挤出液在9000g离心25分钟后弃去上清液收集沉淀部分，将沉淀部分使用4％海藻糖重悬后冷冻干燥48小时后即得微米疫苗。其中，使用微米粒子1激活的混合抗原提呈细胞与微米粒子3共作用制备的为微米疫苗4，粒径为5.00μm，表面电 位为-6mV；每1mg PLGA约负载100μg mRNA组分，每1mgPLGA负载poly(I:C)、CpG2395和CpG1018免疫佐剂各60μg，负载聚组氨酸250μg；每1mg PLGA约负载280μg膜组分。使用微米粒子2激活的混合抗原提呈细胞与微米粒子3共作用制备的为微米疫苗5，粒径为5.00μm，表面电位为-6mV；每1mg PLGA约负载100μg mRNA组分，每1mgPLGA负载poly(I:C)、CpG2395和CpG1018免疫佐剂各60μg，负载聚组氨酸250μg；每1mg PLGA约负载280μg膜组分。
通过在400g离心5分钟收集微米粒子1激活的2000万个混合抗原提呈细胞，与2000万个E.G7-OVA癌细胞混合，然后使用含有蛋白酶抑制剂的4℃磷酸盐缓冲溶液(PBS)洗涤混合细胞两遍，将细胞重悬在PBS水中后在4℃低功率(20W)超声2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5000g离心10分钟后收集上清液，将上清液通过0.22μm的膜反复挤出，将挤出液使用高压均质机(10000bar)处理3分钟，然后在16000g离心40分钟后弃去上清液收集沉淀，将沉淀在PBS中重悬后与30mg步骤(3)制备的核酸递送前体微米粒子3共孵育10分钟后，使用5μm的滤膜反复共挤出，将挤出液在9000g离心25分钟后弃去上清液收集沉淀部分，将沉淀部分使用4％海藻糖重悬后冷冻干燥48小时后即得微米疫苗6，微米疫苗6的粒径为5.00μm，表面电位为-6mV；每1mg PLGA约负载100μg mRNA组分，每1mgPLGA负载poly(I:C)、CpG2395和CpG1018免疫佐剂各60μg，负载聚组氨酸250μg；每1mg PLGA约负载280μg膜组分。
(7)微米疫苗用于癌症的预防
选取6-8周的雌性C57BL/6为模型小鼠制备荷瘤小鼠。在小鼠接种肿瘤前第-35、第-28天、第-21天、第-14天和第-7天分别给小鼠注射100μLPBS或者1mg的微米疫苗(微米疫苗4，或者微米疫苗5，或者微米疫苗6)，在第0天给每只小鼠皮下接种5×105个E.G7-OVA细胞，。小鼠肿瘤体积及生存期监测方法同上。
(8)实验结果
如图10所示，图10中PBS control、Microvaccine 4、Microvaccine 5、Microvaccine 6依次为PBS对照组、微米疫苗4、微米疫苗5、微米疫苗6。结果显示，与PBS对照组相比，微米疫苗处理的小鼠，其肿瘤生长速度明显变慢且小鼠生存期明显延长。而且，微米疫苗4明显好于微米疫苗5；微米疫苗6好于微米疫苗4和微米疫苗5。这说明含有增加溶酶体逃逸功能的物质和混合佐剂的抗原递送微米粒子1激活的抗原提呈细胞的组分与核酸递送前体微米粒子3共作用制备的微米疫苗4效果好于只含有溶酶体逃逸功能的物质而不含有混合佐剂的抗原递送微米粒子2激活的抗原提呈细胞的膜组分与核酸递送前体微米粒子3共作用所制备的微米疫苗5。而且，微米疫苗6好于微米疫苗4，说明被激活的抗原提呈细胞的膜组分与癌细胞膜组分组成的混合膜组分效果更好。由此可见，本公开所述的微米疫苗可被用于预防或者治疗癌症。而且混合佐剂的使用以及癌细胞的膜组分负载于疫苗表面均有助于提高疫苗效果。
实施例10疫苗用于结肠癌的治疗
本实施例以小鼠结肠癌为癌症模型来说明如何使用负载结肠癌全细胞抗原的抗原递送纳米粒子激活的抗原提呈细胞与负载核酸的核酸递送前体纳米粒子共作用后所制备的纳米疫苗用于治疗结肠癌。本实施例中，首先使用10％辛基葡萄糖苷水溶液裂解结肠癌肿瘤组织并溶解裂解组分，然后，以PLGA为骨架材料，以Poly(I:C)、CpG2336和CpG2006为佐剂，以NH4HCO3为增加溶酶体逃逸物质，制备抗原递送纳米粒子，使用该抗原递送纳米粒子激活抗原提呈细胞后将抗原提呈细胞后将来自抗原提呈细胞的细胞外囊泡与负载核酸的核酸递送前体纳米粒子共作用后制备成纳米疫苗，并使用该纳米疫苗治疗癌症。
(1)肿瘤组织裂解物组分及细菌外囊泡组分的制备
收集肿瘤组织时先在每只C57BL/6小鼠背部皮下接种2×106个MC38-OVA结肠癌细胞，在肿瘤长到体积分别为约1000mm3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10％辛基葡萄糖苷水溶液裂解肿瘤组织并溶解裂解后组分。以上即为制备纳米粒子系统的抗原原料来源。
将鼠李糖乳杆菌在5000g离心30分钟，然后弃去沉淀后收集上清液，将上清液使用1μm的滤膜过滤，在4℃下使用20W超声处理5分钟，然后在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膜组分，然后使用10％辛基葡萄糖苷水溶液裂解和溶解细菌外囊泡膜组分。
(2)抗原递送粒子(纳米粒子)系统的制备
本实施例中抗原递送纳米粒子采用复乳法制备。纳米粒子内部负载肿瘤组织裂解物组分、细菌细胞外囊泡组分、mRNA、免疫佐剂和增加溶酶体逃逸物质。纳米粒子的制备材料PLGA分子量为7KDa-17KDa，所使用的mRNA为编码OVA的mRNA,以Poly(I:C)和两种CpG为佐剂，以KALA多肽(WEAKLAKALAKALAKHLAKALAKALKACEA)为增加溶酶体逃逸物质，且佐剂和KALA多肽负载于纳米粒子内；制备时肿瘤组织裂解物组分：细菌外囊泡组分：mRNA的质量比为4:4:1。制备方法如前所述，在制备过程中首先在纳米粒子内部负载肿瘤组织裂解液组分、细菌细胞外囊泡裂解物组分、增加溶酶体逃逸物质和佐剂，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后备用；该纳米粒子1平均粒径为110nm左右，每1mg PLGA纳米粒子约负载90μg蛋白质和多肽组分，负载编码OVA的mRNA为10μg,每1mg PLGA纳米粒负载poly(I:C)、CpG2336和CpG2006各0.02mg，负载KALA多肽0.12mg。
抗原递送纳米粒子2的制备材料和制备方法同纳米粒子1,但是不负载poly(I:C)，粒径为110nm左右，每1mg PLGA纳米粒子约负载90μg蛋白质和多肽组分，负载编码OVA的mRNA为10μg,每1mg PLGA纳米粒负载KALA多肽0.12mg,负载CpG2336和CpG2006各0.03mg。
(3)负载mRNA的核酸递送前体粒子(纳米粒子3)的制备
本实施例中负载mRNA的纳米粒子3所负载的mRNA可以编码OVA抗原。纳米粒子3同时负载有免疫佐剂poly(I:C)、两种B类CpG和聚赖氨酸。负载mRNA的纳米粒子的制备材料和制备方法同步骤(2)，首先将mRNA、poly(I:C)、CpG7909(B类)、CpG1018(B类)和聚 赖氨酸在水中混合，然后采用复乳法将上述混合物负载于纳米粒子内部，然后将100mg PLGA纳米粒子在9000g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。纳米粒子3平均粒径为110nm左右，每1mg PLGA纳米粒子约负载20μg mRNA组分，每1mgPLGA纳米粒子负载poly(I:C)、CpG7909和CpG1018免疫佐剂各20μg，负载聚赖氨酸40μg。
(4)抗原提呈细胞的制备
本实施例使用BMDC和B作为抗原提呈细胞。BMDC制备方法同实施例1。B细胞来自小鼠外周血PBMC，制备方法同上。将BMDC与B细胞按照数量比1:1混合后即为混合抗原提呈细胞。
(5)抗原提呈细胞的激活
将1mg的抗原递送纳米粒子1或者抗原递送纳米粒子2与BMDC(1000万个)及B细胞(1000万个)在15mL高糖DMEM完全培养基中共孵育48小时(37℃，5％CO2)；孵育体系中含有IL-2(500U/mL)、IL-7(500U/mL)、IL-12(200U/mL)和GM-CSF(200U/mL)。
(6)负载抗原提呈细胞的膜组分以及癌细胞细胞外囊泡膜组分的纳米疫苗的制备
收集孵育后的混合抗原提呈细胞2000万个，在400g离心5分钟，弃去沉淀后收集上清液，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中重悬后即得被激活的抗原提呈细胞的细胞外囊泡。
收集培养的MC38-OVA细胞2000万个，在400g离心5分钟，弃去沉淀后收集上清液，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的细胞外囊泡。
将收集的被激活的抗原提呈细胞的细胞外囊泡与癌细胞细胞外囊泡混合，然后在4℃低功率(20W)超声2分钟，然后使用0.22μm的滤膜反复共挤出，将挤出液与步骤(3)制备的核酸递送前体纳米粒子(纳米粒子3)混合后使用高压均质机(10000bar)处理1分钟，然后使用0.22μm的滤膜反复共挤出，然后在15000g离心30分钟后弃去上清液收集沉淀，将沉淀在4％的海藻糖水溶液中重悬后冷冻干燥48小时后即得纳米疫苗。其中，使用纳米粒子1激活的抗原提呈细胞外囊泡膜组分与纳米粒子3共作用所制备的纳米疫苗为纳米疫苗4，粒径为120纳米，表面电位为-6mV；每1mg PLGA约负载10μg mRNA组分，每1mgPLGA负载poly(I:C)、CpG7909和CpG1018免疫佐剂各20μg，负载聚赖氨酸40μg；每1mg PLGA约负载280μg膜组分。使用纳米粒子2激活的抗原提呈细胞外囊泡膜组分与纳米粒子3共作用所制备的纳米疫苗为纳米疫苗5，粒径为120纳米，表面电位为-6mV；每1mg PLGA约负载10μg mRNA组分，每1mgPLGA负载poly(I:C)、CpG7909和CpG1018免疫佐剂各20μg，负载聚赖氨酸40μg；每1mg PLGA约负载280μg膜组分。
(7)纳米疫苗用于治疗癌症
选取6-8周的雌性C57BL/6为模型小鼠制备结肠癌小鼠。在第0天给每只小鼠背部右下方皮下接种2×106个MC38-OVA细胞。在接种结肠癌细胞后第6天、第9天、第12天、第15天、 第20天和第25天分别皮下注射0.4mg纳米疫苗(纳米疫苗4、或者纳米疫苗5)或者100μL的PBS。小鼠肿瘤生长和生存期监测方法同上。
(8)实验结果
如图11所示，图11中PBS control、Nanovaccine 4、Nanovaccine 5依次为PBS对照组、纳米疫苗4、纳米疫苗5。结果显示，与PBS对照组相比，纳米疫苗处理小鼠后其肿瘤生长速度明显变慢且小鼠生存期明显延长。而且，内部负载核酸表面同时负载被含有Poly(I:C)和CpG的粒子1激活的抗原提呈细胞的细胞外囊泡膜组分和癌细胞细胞外囊泡膜组分的纳米疫苗4的效果好于内部负载核酸表面同时负载被只含有CpG的粒子2激活的抗原提呈细胞细胞外囊泡膜组分和癌细胞细胞外囊泡膜组分的纳米疫苗5。由此可见，本公开所述的纳米疫苗对癌症具有优异治疗效果。
实施例11负载mRNA和抗原提呈细胞膜组分的纳米粒子用于黑色素瘤的预防
(1)负载mRNA的纳米粒子1的制备
本实施例中负载mRNA的纳米粒子1所负载的mRNA可以编码四种多肽新生抗原B16-M20(Tubb3,FRRKAFLHWYTGEAMDEMEFTEAESNM)，B16-M24(Dag1,TAVITPPTTTTKKARVSTPKPATPSTD)，B16-M46(Actn4,NHSGLVTFQAFIDVMSRETTDTDTADQ)和TRP2:180-188(SVYDFFVWL)；纳米粒子同时负载有免疫佐剂poly(I:C)、CpGBW006、CpG2395和R8(RRRRRRRR)多肽。负载mRNA的粒子的制备材料和制备方法如前所述，首先将mRNA、poly(I:C)、CpGBW006、CpG2395和R8多肽在水中混合，然后采用复乳法将上述混合物负载于纳米粒子内部，然后将100mg PLGA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250nm左右，每1mg PLGA纳米粒子约负载10μg mRNA组分，每1mgPLGA纳米粒负载poly(I:C)、CpGBW006和CpG2395各20μg，负载R8多肽40μg。
(2)BMDC和B细胞的制备
BMDC和B细胞的制备方法同上，将B细胞和BMDC按数量比1:1混合即为本实施例所使用的混合抗原提呈细胞。
(3)抗原提呈细胞的激活
将1mg纳米粒子1、2000万个混合抗原提呈细胞在15mL RPMI1640完全培养基中共孵育48小时(37℃，5％CO2)；孵育体系中含有细胞因子组合：IL-2(500U/mL)、IL-7(500U/mL)、IL-15(500U/mL)。
(4)纳米疫苗2的制备
通过在400g离心5分钟收集混合抗原提呈细胞(2000万个)，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在4℃和7.5W下超声20分钟以破坏细胞并制备含有细胞膜组分的样品。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，将上清液与40mg步骤(1)制备的纳米粒子1共孵育10分钟，然后使用0.45μm的滤膜反复共挤出，将挤出液在15000g离心120分钟后收集弃去上清液收集沉淀，将 沉淀在PBS中重悬后即得纳米疫苗2，粒径为270nm；每1mg PLGA约负载10μg mRNA组分，每1mgPLGA负载poly(I:C)、CpGBW006和CpG2395各20μg，负载R8多肽40μg；每1mg PLGA约负载90μg膜组分。
(5)负载mRNA的脂质纳米粒(Lipid nanoparticles，LNP)的制备
本实施例使用微流控法制备LNP。使用的脂质材料等的摩尔比如下：MC3:PEG2000:DSPC：胆固醇＝50％：1.5％：10％：38.5％；所负载的mRNA同步骤(1)所负载的mRNA。先称取1mM各脂质成分即MC3(0.321mg)、PEG2000(0.037＝mg)、DSPC(0.079mg)、Chol(0.148mg)一起混合后溶于0.586mL无水乙醇中。将mRNA溶于1mL pH＝4.0的柠檬酸缓冲溶液中。分别将脂质-乙醇溶液和mRNA-柠檬酸缓冲液通过0.22μm滤膜过滤，然后将脂质-乙醇溶液吸入1mL注射器中，将mRNA-柠檬酸缓冲液吸入3mL注射器中(至少吸入1.5mL)，并排出注射器中的空气，将注射器出口和样品导入管连接，并固定在微流控注射泵上。在微流控装置中设置好参数后点击运行，观察流出管流速稳定后(一般需要丢弃前200μL液体)，用收集管收集流出的液体即为制备好的LNP。LNP粒径为95纳米,每1mg LNP负载mRNA为50μg。该LNP即为LNP疫苗。
(6)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，在小鼠接种肿瘤前第-35天，第-28天，第-21天，第-14天和第-7天分别在小鼠皮下接种0.5mg纳米疫苗2、或者0.5mgLNP疫苗或者100μL PBS。第0天给每只小鼠背部右下方皮下接种1.5×105个B16F10细胞。小鼠肿瘤生长速度和生存期监测方法同上。
(7)实验结果
如图12所示，图12中PBS control、LNP vaccine、Nanovaccine 2依次为PBS对照组、LNP疫苗、纳米疫苗2。结果显示，PBS对照组的小鼠其肿瘤生长速度很快，生存期很短。接受疫苗处理的小鼠肿瘤生长速度都明显变慢，小鼠生存期变长。其中，纳米疫苗2效果优于LNP疫苗。这说明本公开所述的纳米疫苗预防或者治疗癌症效果很好，且优于LNP疫苗。
实施例12微米疫苗用于癌症的预防
(1)癌细胞及细菌外囊泡组分的制备
将培养的E.G7-OVA小鼠T淋巴瘤细胞在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，然后使用8M尿素水溶液(含500mM氯化钠)裂解癌细胞并溶解裂解物组分，即为制备抗原递送微米粒子系统的抗原组分。
将嗜酸乳杆菌在5000g离心30分钟，然后弃去沉淀后收集上清液，将上清液使用1μm的滤膜过滤，然后在16000g离心90分钟，弃去上清液后所得沉淀即为细菌外囊泡组分，将沉淀使用8M尿素水溶液(含500mM氯化钠)裂解后溶解细菌外囊泡组分。
(2)抗原递送粒子(微米粒子1)的制备
本实施例中制备抗原递送微米粒子1采用复乳法。微米粒子1骨架材料为PLA，PLA分子量为40KDa。所采用的免疫佐剂为CpG2006(B类)、CpG2216(A类)和Poly ICLC，所采用的带正电荷物质为精氨酸和组氨酸。微米粒子制备时所使用的癌细胞裂解物组分和细菌外囊泡组分质量比为1:1。制备时先采用复乳法制备内部负载癌细胞裂解物抗原组分、细菌外囊泡组分、佐剂、精氨酸和组氨酸的抗原递送微米粒子,尔后，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后即得抗原递送微米粒子1，平均粒径为2.45μm左右，每1mg PLGA微米粒子1约负载100μg蛋白质或多肽组分，负载CpG2006，CpG2216和Poly ICLC各0.02mg，负载精氨酸和组氨酸各0.05mg。
(3)负载mRNA的核酸递送前体粒子(微米粒子2、微米粒子3和微米粒子4及空白微米粒子5)的制备
本实施例中负载mRNA的微米粒子2所负载的mRNA可以编码OVA抗原；微米粒子同时负载有免疫佐剂poly(I:C)、两种CpG(B类和C类)和KALA多肽(WEAKLAKALAKALAKHLAKALAKALKACEA)。负载mRNA的微米粒子的制备材料和制备方法同步骤(2)，首先将mRNA、poly(I:C)、CpG2395(C类)、CpG1018(B类)和KALA多肽在水中混合，然后采用复乳法将上述混合物负载于微米粒子内部，然后将100mg PLA微米粒子在9000g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。微米粒子2平均粒径为2.45μm左右，每1mg PLA微米粒子约负载20μg mRNA组分，每1mgPLGA微米粒子负载poly(I:C)、CpG2395和CpG1018免疫佐剂各20μg，负载KALA多肽150μg。
本实施例中负载mRNA的微米粒子3所负载的mRNA可以编码OVA抗原；微米粒子3同时负载有免疫佐剂poly(I:C)、两种A类CpG和KALA多肽(WEAKLAKALAKALAKHLAKALAKALKACEA)。负载mRNA的微米粒子的制备材料和制备方法同步骤(2)，首先将mRNA、poly(I:C)、CpG1585(A类)、CpG2336(A类)和KALA多肽在水中混合，然后采用复乳法将上述混合物负载于微米粒子内部，然后将100mg PLA微米粒子在9000g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。微米粒子3平均粒径为2.45μm左右，每1mg PLA微米粒子约负载20μg mRNA组分，每1mgPLGA微米粒子负载poly(I:C)、CpG1585、CpG2336免疫佐剂各20μg，负载KALA多肽150μg。
本实施例中负载mRNA的微米粒子4所负载的mRNA可以编码OVA抗原；微米粒子同时负载有免疫佐剂poly(I:C)、一种CpG(B类)和KALA多肽(WEAKLAKALAKALAKHLAKALAKALKACEA)。负载mRNA的微米粒子的制备材料和制备方法同步骤(2)，首先将mRNA、poly(I:C)、CpG1018(B类)和KALA多肽在水中混合，然后采用复乳法将上述混合物负载于微米粒子内部，然后将100mg PLA微米粒子在9000g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。微米粒子4平 均粒径为2.45μm左右，每1mg PLA微米粒子约负载20μg mRNA组分，每1mgPLGA微米粒子负载poly(I:C)20μg、负载CpG1018免疫佐剂40μg，负载KALA多肽150μg。
本实施例中不负载任何mRNA的对照空白微米粒子5同时负载有免疫佐剂poly(I:C)、两种CpG(B类和C类)和KALA多肽(WEAKLAKALAKALAKHLAKALAKALKACEA)。微米粒子5的制备材料和制备方法同步骤(2)，首先将poly(I:C)、CpG2395(C类)、CpG1018(B类)和KALA多肽在水中混合，然后采用复乳法将上述混合物负载于微米粒子内部，然后将100mg PLA微米粒子在9000g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。微米粒子2平均粒径为2.45μm左右，每1mg PLA微米粒子负载poly(I:C)、CpG2395和CpG1018免疫佐剂各20μg，负载KALA多肽150μg。
(4)抗原提呈细胞的制备
本实施例使用BMDC、B细胞以及BMDM作为抗原提呈细胞。BMDC及BMDM制备方法同上。B细胞来自小鼠外周血PBMC，制备方法同上。将BMDC、B细胞和BMDM按数量比2:1:1混和后即为混合抗原提呈细胞。
(5)抗原提呈细胞的激活
将1000μg的抗原递送微米粒子1分别与4000万个混合抗原提呈细胞(含2000万个BMDC，1000万个B细胞及1000万个BMDM)在15mL高糖DMEM完全培养基中共孵育48小时(37℃，5％CO2)；孵育体系中含有IL-2(500U/mL)、IL-7(200U/mL)、IL-15(200U/mL)和CD40抗体(20mg/mL)。
(6)抗原提呈细胞来源的核酸疫苗的制备
通过在400g离心5分钟收集微米粒子1激活的2000万个混合抗原提呈细胞，与2000万个E.G7-OVA癌细胞混合，然后使用含有蛋白酶抑制剂的4℃磷酸盐缓冲溶液(PBS)洗涤混合细胞两遍，将细胞重悬在PBS水中后在4℃低功率(20W)超声2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5000g离心10分钟后收集上清液，将上清液通过0.22μm的膜反复挤出，将挤出液使用高压均质机(10000bar)处理3分钟，然后在16000g离心40分钟后弃去上清液收集沉淀，将沉淀在PBS中重悬后与30mg步骤(3)制备的核酸递送前体微米粒子2共孵育10分钟后，使用3μm的滤膜反复共挤出，将挤出液在9000g离心25分钟后弃去上清液收集沉淀部分，将沉淀部分使用4％海藻糖重悬后冷冻干燥48小时后即得微米疫苗6，微米疫苗6的粒径为2.50μm，表面电位为-6mV。每1mg PLGA约负载20μg mRNA组分，每1mgPLGA负载poly(I:C)、CpG2395和CpG1018免疫佐剂各20μg，负载KALA多肽150μg；每1mg PLGA约负载190μg膜组分。
通过在400g离心5分钟收集微米粒子1激活的2000万个混合抗原提呈细胞，与2000万个E.G7-OVA癌细胞混合，然后使用含有蛋白酶抑制剂的4℃磷酸盐缓冲溶液(PBS)洗涤混合细胞两遍，将细胞重悬在PBS水中后在4℃低功率(20W)超声2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5000g离心10分钟后收集上清液，将上清液通过0.22μm的膜反复挤出，将挤出液使用高压均质机(10000bar)处理3分钟，然后在16000g离 心40分钟后弃去上清液收集沉淀，将沉淀在PBS中重悬后与30mg步骤(3)制备的核酸递送前体微米粒子3共孵育10分钟后，使用3μm的滤膜反复共挤出，将挤出液在9000g离心25分钟后弃去上清液收集沉淀部分，将沉淀部分使用4％海藻糖重悬后冷冻干燥48小时后即得微米疫苗7，微米疫苗7的粒径为2.50μm，表面电位为-10mV。每1mg PLGA约负载20μg mRNA组分，每1mgPLGA负载poly(I:C)、CpG1585、CpG2336免疫佐剂各20μg，负载KALA多肽150μg；每1mg PLGA约负载190μg膜组分。
通过在400g离心5分钟收集微米粒子1激活的2000万个混合抗原提呈细胞，与2000万个E.G7-OVA癌细胞混合，然后使用含有蛋白酶抑制剂的4℃磷酸盐缓冲溶液(PBS)洗涤混合细胞两遍，将细胞重悬在PBS水中后在4℃低功率(20W)超声2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5000g离心10分钟后收集上清液，将上清液通过0.22μm的膜反复挤出，将挤出液使用高压均质机(10000bar)处理3分钟，然后在16000g离心40分钟后弃去上清液收集沉淀，将沉淀在PBS中重悬后与30mg步骤(3)制备的核酸递送前体微米粒子4共孵育10分钟后，使用3μm的滤膜反复共挤出，将挤出液在9000g离心25分钟后弃去上清液收集沉淀部分，将沉淀部分使用4％海藻糖重悬后冷冻干燥48小时后即得微米疫苗8，微米疫苗8的粒径为2.50μm，表面电位为-10mV。每1mg PLGA约负载20μg mRNA组分，每1mgPLGA负载poly(I:C)20μg、负载CpG1018免疫佐剂40μg，负载KALA多肽150μg；每1mg PLGA约负载190μg膜组分。
通过在400g离心5分钟收集微米粒子1激活的2000万个混合抗原提呈细胞，与2000万个E.G7-OVA癌细胞混合，然后使用含有蛋白酶抑制剂的4℃磷酸盐缓冲溶液(PBS)洗涤混合细胞两遍，将细胞重悬在PBS水中后在4℃低功率(20W)超声2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5000g离心10分钟后收集上清液，将上清液通过0.22μm的膜反复挤出，将挤出液使用高压均质机(10000bar)处理3分钟，然后在16000g离心40分钟后弃去上清液收集沉淀，将沉淀在PBS中重悬后与30mg步骤(3)制备的对照空白微米粒子5共孵育10分钟后，使用3μm的滤膜反复共挤出，将挤出液在9000g离心25分钟后弃去上清液收集沉淀部分，将沉淀部分使用4％海藻糖重悬后冷冻干燥48小时后即得微米疫苗9，微米疫苗9的粒径为2.50μm，表面电位为-6mV。每1mg PLGA负载poly(I:C)、CpG2395和CpG1018免疫佐剂各20μg，负载KALA多肽150μg；每1mg PLGA约负载190μg膜组分。
(7)微米疫苗用于癌症的预防
选取6-8周的雌性C57BL/6为模型小鼠制备荷瘤小鼠。在小鼠接种肿瘤前第-35、第-28天、第-21天、第-14天和第-7天分别给小鼠注射100μLPBS或者1mg的微米疫苗(微米疫苗5，微米疫苗6，或者微米疫苗7)，在第0天给每只小鼠皮下接种5×105个E.G7-OVA细胞。小鼠肿瘤体积及生存期监测方法同上。
(8)实验结果
如图13所示，图13中PBS control、Microvaccine 6、Microvaccine 7、Microvaccine 8、 Microvaccine 9依次为PBS对照组、微米疫苗6、微米疫苗7、微米疫苗8和微米疫苗9。结果显示，与PBS对照组相比，微米疫苗处理的小鼠，其肿瘤生长速度明显变慢且小鼠生存期明显延长。微米疫苗6、微米疫苗7和微米疫苗8的效果都好于微米疫苗9，说明疫苗内部负载核酸能显著提高疫苗的疗效。而且，微米疫苗6的效果好于微米疫苗7和微米疫苗8，说明核酸递送粒子(疫苗)中含有两种CpG(B类和C类)与poly(I:C)混合佐剂的效果好于核酸递送微米粒子(疫苗)中含有两种A类CpG与poly(I:C)混合佐剂的效果，也好于核酸递送微米粒子(疫苗)中含有一种B类CpG与poly(I:C)混合佐剂的效果；说明使用poly(I:C)+两种不同的CpG(含有至少一种B类CpG)能达到更佳的效果。由此可见，本公开所述的微米疫苗可被用于预防或者治疗癌症。而且混合佐剂的使用有助于提高疫苗效果。
实施例13疫苗用于结肠癌的治疗
本实施例以小鼠结肠癌为癌症模型来说明如何使用负载结肠癌全细胞抗原的抗原递送纳米粒子激活的抗原提呈细胞与负载核酸的核酸递送前体纳米粒子共作用后所制备的纳米疫苗用于治疗结肠癌。本实施例中，首先使用10％辛基葡萄糖苷水溶液裂解结肠癌肿瘤组织并溶解裂解组分，然后，以PLGA为骨架材料，以Poly(I:C)、CpG2336和CpG2006为佐剂，以NH4HCO3为增加溶酶体逃逸物质，制备抗原递送纳米粒子，使用该抗原递送纳米粒子激活抗原提呈细胞后将抗原提呈细胞后将来自抗原提呈细胞的细胞外囊泡与负载核酸的核酸递送前体纳米粒子共作用后制备成纳米疫苗，并使用该纳米疫苗治疗癌症。
(1)肿瘤组织裂解物组分及细菌外囊泡组分的制备
收集肿瘤组织时先在每只C57BL/6小鼠背部皮下接种2×106个MC38-OVA结肠癌细胞，在肿瘤长到体积分别为约1000mm3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10％辛基葡萄糖苷水溶液裂解肿瘤组织并溶解裂解后组分。以上即为制备纳米粒子系统的抗原原料来源。
将鼠李糖乳杆菌在5000g离心30分钟，然后弃去沉淀后收集上清液，将上清液使用1μm的滤膜过滤，在4℃下使用20W超声处理5分钟，然后在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膜组分，然后使用10％辛基葡萄糖苷水溶液裂解和溶解细菌外囊泡膜组分。
(2)抗原递送粒子(纳米粒子1)系统的制备
本实施例中抗原递送纳米粒子1采用复乳法制备。纳米粒子1内部负载肿瘤组织裂解物组分、细菌细胞外囊泡组分、mRNA、免疫佐剂和增加溶酶体逃逸物质。纳米粒子的制备材料PLGA分子量为7KDa-17KDa，所使用的mRNA为编码OVA的mRNA,以Poly(I:C)和两种CpG为佐剂，以KALA多肽(WEAKLAKALAKALAKHLAKALAKALKACEA)为增加溶酶体逃逸物质，且佐剂和KALA多肽负载于纳米粒子内；制备时肿瘤组织裂解物组分：细菌外囊泡组分：mRNA的质量比为4:4:1。制备方法如前所述，在制备过程中首先在纳米粒子内部负载肿瘤组织裂解液组分、细菌细胞外囊泡裂解物组分、增加溶酶体逃逸物质和佐剂， 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后备用；该纳米粒子平均粒径为260nm左右，每1mg PLGA纳米粒子约负载90μg蛋白质和多肽组分，负载编码OVA的mRNA为10μg,每1mg PLGA纳米粒负载poly(I:C)、CpG2336和CpG2006各0.02mg，负载KALA多肽0.12mg。
(3)负载mRNA的核酸递送前体粒子(纳米粒子2,纳米粒子3，纳米粒子4和纳米粒子5)的制备
本实施例中负载mRNA的纳米粒子2所负载的mRNA可以编码OVA抗原。纳米粒子2同时负载有免疫佐剂poly(I:C)、两种B类CpG和KALA多肽(WEAKLAKALAKALAKHLAKALAKALKACEA)。负载mRNA的纳米粒子的制备材料和制备方法同步骤(2)，首先将mRNA、poly(I:C)、CpG7909(B类)、CpG1018(B类)和KALA多肽在水中混合，然后采用复乳法将上述混合物负载于纳米粒子内部，然后将100mg PLGA纳米粒子在9000g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。纳米粒子2平均粒径为210nm左右，每1mg PLGA纳米粒子约负载20μg mRNA组分，每1mgPLGA纳米粒子负载poly(I:C)、CpG7909和CpG1018免疫佐剂各20μg，负载KALA多肽140μg。
本实施例中负载mRNA的纳米粒子3所负载的mRNA可以编码OVA抗原。纳米粒子3同时负载有免疫佐剂poly(I:C)、两种A类CpG和KALA多肽(WEAKLAKALAKALAKHLAKALAKALKACEA)。负载mRNA的纳米粒子的制备材料和制备方法同步骤(2)，首先将mRNA、poly(I:C)、CpG1585(A类)、CpG2336(A类)和KALA多肽在水中混合，然后采用复乳法将上述混合物负载于纳米粒子内部，然后将100mg PLGA纳米粒子在9000g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。纳米粒子3平均粒径为210nm左右，每1mg PLGA纳米粒子约负载20μg mRNA组分，每1mgPLGA纳米粒子负载poly(I:C)、CpG1585和CpG2336免疫佐剂各20μg，负载KALA多肽140μg。
本实施例中负载mRNA的纳米粒子4所负载的mRNA可以编码OVA抗原。纳米粒子4同时负载有免疫佐剂poly(I:C)和KALA多肽(WEAKLAKALAKALAKHLAKALAKALKACEA)。负载mRNA的纳米粒子的制备材料和制备方法同步骤(2)，首先将mRNA、poly(I:C)和KALA多肽在水中混合，然后采用复乳法将上述混合物负载于纳米粒子内部，然后将100mg PLGA纳米粒子在9000g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。纳米粒子4平均粒径为210nm左右，每1mg PLGA纳米粒子约负载20μg mRNA组分，每1mgPLGA纳米粒子负载poly(I:C)60μg，负载KALA多肽140μg。
本实施例中负载mRNA的纳米粒子5所负载的mRNA可以编码OVA抗原。纳米粒子5同时负载有免疫佐剂两种B类CpG和KALA多肽(WEAKLAKALAKALAKHLAKALAKALKACEA)。负载mRNA的纳米粒子的制备材料和 制备方法同步骤(2)，首先将mRNA、CpG7909(B类)、CpG1018(B类)和KALA多肽在水中混合，然后采用复乳法将上述混合物负载于纳米粒子内部，然后将100mg PLGA纳米粒子在9000g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。纳米粒子5平均粒径为210nm左右，每1mg PLGA纳米粒子约负载20μg mRNA组分，每1mgPLGA纳米粒子负载CpG7909和CpG1018免疫佐剂各30μg，负载KALA多肽140μg。
本实施例中负载mRNA的纳米粒子6所负载的mRNA可以编码OVA抗原。纳米粒子6同时负载有免疫佐剂poly(I:C)、两种B类CpG。负载mRNA的纳米粒子的制备材料和制备方法同步骤(2)，首先将mRNA、poly(I:C)、CpG7909(B类)、CpG1018(B类)在水中混合，然后采用复乳法将上述混合物负载于纳米粒子内部，然后将100mg PLGA纳米粒子在9000g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。纳米粒子6平均粒径为210nm左右，每1mg PLGA纳米粒子约负载20μg mRNA组分，每1mgPLGA纳米粒子负载poly(I:C)、CpG7909和CpG1018免疫佐剂各20μg，不负载KALA多肽。
(4)抗原提呈细胞的制备
本实施例使用BMDC和B作为抗原提呈细胞。BMDC制备方法同实施例1。B细胞来自小鼠外周血PBMC，制备方法同上。将BMDC与B细胞按照数量比1:1混合后即为混合抗原提呈细胞。
(5)抗原提呈细胞的激活
将1mg的抗原递送纳米粒子1与BMDC(1000万个)及B细胞(1000万个)在15mL高糖DMEM完全培养基中共孵育48小时(37℃，5％CO2)；孵育体系中含有IL-2(500U/mL)、IL-7(500U/mL)、IL-12(200U/mL)和GM-CSF(200U/mL)。
(6)负载抗原提呈细胞的膜组分以及癌细胞细胞外囊泡膜组分的纳米疫苗的制备
收集与纳米粒1孵育后的混合抗原提呈细胞2000万个，在400g离心5分钟，弃去沉淀后收集上清液，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中重悬后即得被激活的抗原提呈细胞的细胞外囊泡。
收集培养的MC38-OVA细胞2000万个，在400g离心5分钟，弃去沉淀后收集上清液，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的细胞外囊泡。
将收集的被激活的抗原提呈细胞的细胞外囊泡与癌细胞细胞外囊泡混合，然后在4℃低功率(20W)超声2分钟，然后使用0.22μm的滤膜反复共挤出，将挤出液与步骤(3)制备的核酸递送前体纳米粒子(纳米粒子2、纳米粒子3、或者纳米粒子4、或者纳米粒子5、或者纳米粒子6)混合后使用高压均质机(10000bar)处理1分钟，然后使用0.22μm的滤膜反复共挤出，然后在15000g离心30分钟后弃去上清液收集沉淀，将沉淀在4％的海藻糖水溶液中重悬后冷冻干燥48小时后即得纳米疫苗。其中，使用纳米粒子1激活的抗原提呈细胞外囊泡膜组分与纳米粒子2共作用所制备的纳米疫苗为纳米疫苗7，粒径为220纳米，表面电位为-6mV；每1mg PLGA约负载20μg mRNA组分，每1mgPLGA负载poly(I:C)、CpG7909和 CpG1018免疫佐剂各20μg，负载KALA多肽140μg；每1mg PLGA约负载120μg膜组分。使用纳米粒子1激活的抗原提呈细胞外囊泡膜组分与纳米粒子3共作用所制备的纳米疫苗为纳米疫苗8，粒径为220纳米，表面电位为-6mV；每1mg PLGA约负载20μg mRNA组分，每1mgPLGA负载poly(I:C)、CpG1585和CpG2336免疫佐剂各20μg，负载KALA多肽140μg；每1mg PLGA约负载120μg膜组分。使用纳米粒子1激活的抗原提呈细胞外囊泡膜组分与纳米粒子4共作用所制备的纳米疫苗为纳米疫苗9，粒径为220纳米，表面电位为-6mV；每1mg PLGA约负载20μg mRNA组分，每1mgPLGA负载ppoly(I:C)60μg，负载KALA多肽140μg；每1mg PLGA约负载120μg膜组分。使用纳米粒子1激活的抗原提呈细胞外囊泡膜组分与纳米粒子5共作用所制备的纳米疫苗为纳米疫苗10，粒径为220纳米，表面电位为-6mV；每1mg PLGA约负载20μg mRNA组分，每1mgPLGA负载CpG7909和CpG1018免疫佐剂各30μg，负载KALA多肽140μg；每1mg PLGA约负载120μg膜组分。使用纳米粒子1激活的抗原提呈细胞外囊泡膜组分与纳米粒子6共作用所制备的纳米疫苗为纳米疫苗11，粒径为220纳米，表面电位为-6mV；每1mg PLGA约负载20μg mRNA组分，每1mgPLGA负载poly(I:C)、CpG7909和CpG1018免疫佐剂各20μg，不负载KALA多肽；每1mg PLGA约负载120μg膜组分。
(7)纳米疫苗用于治疗癌症
选取6-8周的雌性C57BL/6为模型小鼠制备结肠癌小鼠。在第0天给每只小鼠背部右下方皮下接种2×106个MC38-OVA细胞。在接种结肠癌细胞后第6天、第9天、第12天、第15天、第20天和第25天分别皮下注射0.4mg纳米疫苗(纳米疫苗7、或者纳米疫苗8、或者纳米疫苗9、或者纳米疫苗10、或者纳米疫苗11)或者100μL的PBS。小鼠肿瘤生长和生存期监测方法同上。
(8)实验结果
如图14所示，图14中PBS control、Nanovaccine 7、Nanovaccine 8、Nanovaccine 9、Nanovaccine 10、Nanovaccine 11依次为PBS对照组、纳米疫苗7、纳米疫苗8、纳米疫苗9、纳米疫苗10、纳米疫苗11。结果显示，与PBS对照组相比，纳米疫苗处理小鼠后其肿瘤生长速度明显变慢且小鼠生存期明显延长。而且，纳米疫苗7治疗效果好于纳米疫苗8、纳米疫苗9、纳米疫苗10和纳米疫苗疫苗11。由此可见，本公开所述的纳米疫苗对癌症具有优异治疗效果。
本公开实施例中的纳米粒子、微米粒子、纳米疫苗或者微米疫苗中均未使用任何靶头，在实际应用中也可以根据需要添加甘露糖、CD32抗体、甘露聚糖、CD205抗体、CD19抗体等任何具有靶向靶细胞能力的靶头到纳米粒子、微米粒子、纳米疫苗或者微米疫苗表面。
本公开实施例中说明未使用钙化、硅化等技术处理纳米粒子或微米粒子，在实际可以使用钙化、硅化或其他生物矿化技术、交联、凝胶化等处理或修饰粒子。
本公开实施例中所递送的核酸为mRNA，在实际应用中也可以为DNA或者其他RNA。
本公开中涉及的氨基酸序列如下所示：
R8多肽，RRRRRRRR(SEQ ID NO:1)
B16-M20抗原多肽，FRRKAFLHWYTGEAMDEMEFTEAESNM(SEQ ID NO:2)
B16-M24抗原多肽，TAVITPPTTTTKKARVSTPKPATPSTD(SEQ ID NO:3)
B16-M46抗原多肽，NHSGLVTFQAFIDVMSRETTDTDTADQ(SEQ ID NO:4)
TRP2:180-188抗原多肽，SVYDFFVWL(SEQ ID NO:5)
蜂毒肽，GIGAVLKVLTTGLPALISWIKRKRQQ(SEQ ID NO:6)
KALA多肽，WEAKLAKALAKALAKHLAKALAKALKACEA(SEQ ID NO:7)
RALA多肽，WEARLARALARALARHLARALARALRACEA(SEQ ID NO:8)
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他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公开创造的保护范围之中。

Claims (23)

  1. 一种核酸递送粒子，其具有由粒子材料形成的骨架结构，所述骨架结构的内部负载有核酸，所述骨架结构的表面负载有生物膜组分；
    其中，所述生物膜组分含有来源于激活的抗原提呈细胞的膜组分和/或由所述激活的抗原提呈细胞分泌的细胞外囊泡的膜组分；所述激活的抗原提呈细胞由抗原提呈细胞与抗原递送粒子共作用后获得，所述抗原递送粒子具有由粒子材料形成的骨架结构，以及负载于所述骨架结构的内部和/或表面负载有抗原组分。
  2. 根据权利要求1所述的核酸递送粒子，其中，所述核酸递送粒子进一步负载至少一种如下所示的组分：
    (iii)免疫佐剂；
    (iv)带正电荷的物质，其选自带正电荷的氨基酸、带正电荷的多肽、带正电荷的脂质、带正电荷的蛋白质、带正电荷的聚合物，和/或带正电荷的无机物；
    优选地，所述免疫佐剂包括如下至少一种：模式识别受体类激动剂、卡介苗(BCG)、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、咪喹莫特、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QIO、左旋咪唑、聚胞苷酸、白细胞介素、干扰素、聚肌苷酸、聚腺苷酸、明矾、磷酸铝、羊毛脂、植物油、细胞因子、mRNA、MF59、双链RNA、双链DNA、单链DNA、铝佐剂、锰佐剂、钙佐剂、STING激动剂、内毒素佐剂、脂质体佐剂、CAF01、人参有效成分、黄苠有效成分；
    优选地，所述免疫佐剂包括Toll样受体3激动剂和Toll样受体9激动剂中的至少一种；更优选包括Poly(I:C)、Poly ICLC、A类CpG-OND、B类CpG-OND和C类CpG-OND中的至少一种；
    优选地，所述带正电荷的物质包括蜂毒肽、RALA多肽、KALA多肽、R8多肽、精氨酸、组氨酸、赖氨酸、聚精氨酸、聚赖氨酸、聚组氨酸和NH4HCO3中的任意一种或任意组合。
  3. 根据权利要求1或2所述的核酸递送粒子，其中，所述核酸递送粒子中，所述粒子材料、核酸与生物膜组分的质量比(mg:μg:μg)为1：(1-100)：(10-300)；
    优选地，所述核酸递送粒子进一步包括免疫佐剂和带电荷的物质，所述粒子材料、核酸、免疫佐剂、带正电荷的物质与所述生物膜组分的质量比(mg:μg:μg:μg:μg)为1：(1-100)：(1-200)：(10-500)：(10-300)。
  4. 根据权利要求1-3任一项所述的核酸递送粒子，所述生物膜组分进一步含有至少一种如下所示的组分：
    (a)来源于全细胞裂解物的癌细胞膜组分；其中，所述全细胞裂解物组分来源于肿瘤组织和/或肿瘤细胞；
    (b)来源与细胞外囊泡裂解物的细胞外囊泡膜组分，所述细胞外囊泡由细菌或肿瘤细胞分泌；
    (c)来源于细菌裂解物的细菌膜组分；
    优选地，所述全细胞裂解物包括如下的一种或两种：水溶性抗原的裂解物和非水溶性抗原的溶解物；
    优选地，所述细菌包括如下至少一种：卡介苗、大肠杆菌、长双歧杆菌、短双歧杆菌、乳双歧杆菌、嗜酸乳杆菌、格式乳杆菌、罗伊氏乳杆菌、鼠李糖乳杆菌。
  5. 根据权利要求1-4任一项所述的核酸递送粒子，其中，所述激活抗原提呈细胞的抗原递送粒子所负载的抗原组分包含如下的至少一种：
    (i)具有免疫原性的蛋白和/或多肽；
    (ii)编码抗原的核酸；
    优选地，所述具有免疫原性的蛋白和/或多肽来源于如下(1)，和任选存在的(2)-(3)中的至少一种；
    (1)全细胞裂解物；其中，所述全细胞裂解物组分来源于肿瘤组织和/或肿瘤细胞；
    (2)细菌裂解物；
    (3)细胞外囊泡裂解物；其中，所述细胞外囊泡由细菌或肿瘤细胞分泌；
    优选地，所述全细胞裂解物包括如下的一种或两种：水溶性抗原的裂解物和非水溶性抗原的溶解物；
    优选地，所述细菌包括如下至少一种：卡介苗、大肠杆菌、长双歧杆菌、短双歧杆菌、乳双歧杆菌、嗜酸乳杆菌、格式乳杆菌、罗伊氏乳杆菌、鼠李糖乳杆菌。
  6. 根据权利要求4或5所述的核酸递送粒子，其中，所述细菌裂解物和/或细胞外囊泡裂解物是由细菌和/或细胞外囊泡经含有裂解剂的裂解液裂解得到；
    优选地，所述裂解剂选自尿素、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、Triton、吐温、多肽、氨基酸、糖苷和胆碱中的一种或多种。
  7. 根据权利要求4-6任一项所述的核酸递送粒子，其中，所述非水溶性抗原、所述细菌裂解物或所述细胞外囊泡裂解物彼此独立地溶解于包含如下至少一种溶解剂的溶解液中：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、Triton、吐温、多肽、氨基酸、糖苷和胆碱。
  8. 根据权利要求5-7任一项所述的核酸递送粒子，其中，所述抗原递送粒子进一步负载如下至少一种：
    (iii)免疫佐剂；
    (iv)带正电荷的物质，其选自带正电荷的氨基酸、带正电荷的多肽、带正电荷的脂质、带正电荷的蛋白质、带正电荷的聚合物，和/或带正电荷的无机物；
    可选地，所述免疫佐剂包括如下至少一种：模式识别受体类激动剂、卡介苗(BCG)、 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、咪喹莫特、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QIO、左旋咪唑、聚胞苷酸、白细胞介素、干扰素、聚肌苷酸、聚腺苷酸、明矾、磷酸铝、羊毛脂、植物油、细胞因子、mRNA、MF59、双链RNA、双链DNA、单链DNA、铝佐剂、锰佐剂、钙佐剂、STING激动剂、内毒素佐剂、脂质体佐剂、CAF01、人参有效成分、黄苠有效成分；
    优选地，所述免疫佐剂包括Toll样受体3激动剂和Toll样受体9激动剂中的至少一种；更优选包括Poly(I:C)、Poly ICLC、A类CpG-OND、B类CpG-OND和C类CpG-OND中的至少一种；
    所述带正电荷的物质包括蜂毒肽、RALA多肽、KALA多肽、R8多肽、精氨酸、组氨酸、赖氨酸、聚精氨酸、聚赖氨酸、聚组氨酸和NH4HCO3中的任意一种或任意组合。
  9. 根据权利要求1-8任一项所述的核酸递送粒子，其中，所述抗原提呈细胞选自树突状细胞、B细胞和巨噬细胞中的任意一种或两种以上的组合；
    优选地，所述抗原提呈细胞选自树突状细胞、B细胞和巨噬细胞中的任意两种或三种。
  10. 根据权利要求1-9任一项所述的核酸递送粒子，其中，所述抗原提呈细胞与所述抗原递送粒子的共作用体系中还包含细胞因子组分和/或抗体组分；优选地，所述细胞因子组分包括IL-2、IL-4、IL-7、IL10、IL-12、IL-15、IL-33、GM-CSF、M-CSF中的至少一种；所述抗体组分包括CD80抗体、CD86抗体中的至少一种；
    可选地，所述孵育体系中还包括如下至少一种：
    (1)全细胞裂解物；
    (2)细菌裂解物；
    (3)细胞外囊泡裂解物；其中，所述细胞外囊泡由细菌或癌细胞分泌。
  11. 根据权利要求1-10任一项所述的核酸递送粒子，其中，所述核酸递送粒子由核酸递送前体粒子与生物膜组分共作用得到；
    优选地，所述核酸递送前体粒子进一步负载如下至少一种：
    (iii)免疫佐剂；
    (iv)带正电荷的物质，其选自带正电荷的氨基酸、带正电荷的多肽、带正电荷的脂质、带正电荷的蛋白质、带正电荷的聚合物，和/或带正电荷的无机物。
  12. 根据权利要求1-11任一项所述的核酸递送粒子，其中，所述粒子材料选自天然高分子材料、合成高分子材料和/或无机材料。
  13. 根据权利要求1-12任一项所述的核酸递送粒子，其中，所述的核酸递送粒子和抗原递送粒子的形状为任意形状，包括但不限于球形、椭球形、桶形、多角形、棒状、片状、线形、蠕虫形、方形、三角形、蝶形、圆盘形、囊泡形等；
    可选地，所述核酸递送粒子由核酸递送前体粒子与生物膜组分共作用得到，所述核酸 递送前体粒子的形状为任意形状，包括但不限于球形、椭球形、桶形、多角形、棒状、片状、线形、蠕虫形、方形、三角形、蝶形、圆盘形、囊泡形等。
  14. 根据权利要求1-13任一项所述的核酸递送粒子，其中，所述核酸递送粒子和所述抗原递送粒子彼此独立地选自纳米粒子或微米粒子；
    所述纳米粒子的粒径为1nm-1000nm；优选为50-500nm；更优选为100-400nm；
    所述微米粒子的粒径为1μm-1000μm；优选为1-10μm；更优选为1-5μm。
  15. 根据权利要求1-14任一项述的核酸递送粒子，其中，所述核酸递送粒子和/或所述抗原递送粒子的表面带有负电荷。
  16. 一种核酸递送系统，其中，所述核酸递送系统包括如权利要求1-15任一项所述的核酸递送粒子。
  17. 一种药物组合物，其中，所述药物组合物包括如权利要求1-15任一项所述的核酸递送粒子，或如权利要求16所述的核酸递送系统；
    可选地，所述药物组合物还包括一种或多种药学上可接受的载体。
  18. 一种核酸疫苗，其中，所述核酸疫苗包括如权利要求1-15任一项所述的核酸递送粒子，或如权利要求16所述的核酸递送系统。
  19. 根据权利要求1-15任一项所述的核酸递送粒子，根据权利要求16所述的核酸递送系统，或根据权利要求17所述的药物组合物在如下(1)-(3)至少一项中的用途：
    (1)预防或治疗疾病，或制备用于预防或治疗疾病的药物；
    (2)在受试者中诱导免疫应答，或制备用于在受试者中诱导免疫应答的药物；
    (3)作为或用于制备核酸疫苗；
    可选地，所述疾病为癌症或肿瘤；
    可选地，所述癌症或肿瘤为实体肿瘤或者血液瘤。
  20. 一种核酸递送粒子的制备方法，其中，所述制备方法包括如下步骤：
    将抗原提呈细胞与抗原递送粒子共作用，得到激活的抗原提呈细胞；其中，抗原递送粒子的表面和/或内部负载有抗原组分；尔后制备含有来源于激活的抗原提呈细胞和/或其细胞外囊泡的生物膜组分；
    制备内部负载核酸的核酸递送前体粒子；
    将生物膜组分负载于所述核酸递送前体粒子的表面，得到核酸递送粒子。
  21. 根据权利要求20所述的制备方法，其中，所述抗原递送粒子的制备步骤包括：
    分别制备负载水溶性抗原的递送粒子，以及负载非水溶性抗原的溶解物的递送粒子，混合两种递送粒子，得到所述抗原递送粒子；
    或者，混合所述水溶性抗原和非水溶性抗原的溶解物，得到混合后的抗原组分；利用所述混合后的抗原组分，制备所述抗原递送粒子；
    或者，使用含有溶解剂的溶解液同时溶解水溶性抗原和非水溶性抗原，并利用所述混合抗原组分制备所述抗原递送粒子。
  22. 根据权利要求20或21所述的制备方法，其中，所述将生物膜组分负载于所述核 酸递送前体粒子的表面的步骤包括：
    将所述激活的抗原呈递细胞和/或其细胞外囊泡机械破碎后，收集生物膜组分；
    将所述生物膜组分与所述核酸递送前体粒子共作用，使所述将生物膜组分负载于所述核酸递送前体粒子的表面。
  23. 一种预防或治疗疾病的方法，其中，所述方法包括向受试者施用预防或治疗有效量的如权利要求1-15任一项所述的核酸递送粒子，如权利要求16所述的核酸递送系统，如权利要求17所述的药物组合物，或如权利要求18所述的核酸疫苗。
PCT/CN2023/081489 2022-07-19 2023-03-15 基于激活的抗原提呈细胞的核酸递送粒子、核酸递送系统及制备方法 WO2024016688A1 (zh)

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